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Cellular factors involved in HIV-1 replication

Rits, M.A.N.

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Citation for published version (APA):Rits, M. A. N. (2009). Cellular factors involved in HIV-1 replication.

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Cellular Factors Involved in HIV-1

Replication

Maarten Rits

CELLULAR FACTORS INVOLVED IN

HIV-1 REPLICATION

Colofon

© Maarten Rits, 2009. All rights reserved. No part of this publication may be

reproduced, stored in a retrieval system, or transmitted, in any form or by any

means, electronic or, mechanical, photocopying, recording or otherwise, without

prior permission of the author.

Published papers were reprinted with permission from the publisher.

The publication of this thesis is financially supported by:

University of Amsterdam

CELLULAR FACTORS INVOLVED IN

HIV-1 REPLICATION

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties

ingestelde commissie,

in het openbaar te verdedigen in de Agnietenkapel

op dinsdag 23 juni 2009, te 12:00 uur

door

Maarten Alexander Nicolaas Rits geboren te Nijmegen

Promotiecommissie

Promotor prof. dr. H. Schuitemaker

Co-promotor dr. N.A. Kootstra

Overige leden prof. dr. L.A. Aarden

prof. dr. B. Berkhout

prof. dr. F. Miedema

prof. dr. C. Münk

dr. A.J. Verhoeven

dr. A.B. Meijer

Faculteit der Geneeskunde

The research described in this thesis was performed at the department of Clinical Viro-

Immunology, Sanquin research and in the Laboratory of Viral Immuno-Pathogenesis of the

department of Experimental Immunology in the Academic Medical Center, Center for

Infection and Immunity Amsterdam (CINIMA), University of Amsterdam, Amsterdam, The

Netherlands.

This work was performed as part of the Amsterdam Cohort Studies on HIV-1 infection and

AIDS.

The studies were financially supported by the Landsteiner Foundation for blood transfusion

Research (LSBR grant 0435).

Table of Contents

1 General introduction

2 Efficient transduction of simian cells by

HIV-1-based lentiviral vectors that

contain mutations in the capsid protein

Mol Ther 2007;15:930-937

3 Interaction of Cyclophilin A and Trim5

with the HIV-1 capsid

Submitted for publication

4 The effect of Trim5 polymorphisms on the

clinical course of HIV-1 infection

PLoS Pathog 2008;4(2):e18

5 Polymorphisms in the regulatory region of

the cyclophilin A gene influence the

susceptibility for HIV-1 infection

PLoS ONE 2008;3(12):e3975

6 The effect of type I interferons on HIV-1

replication in monocyte derived

macrophages


7 General discussion

Summary

Samenvatting

Nawoord

9

37

61

77

101

119

139

155

161

167

Chapter 1

General Introduction

Chapter 1 11

Retroviruses are enveloped positive strand RNA viruses that have the

ability to reverse transcribe their genomic RNA into double stranded

(ds)DNA. Another unique feature of retroviruses is that the viral dsDNA

integrates into the host genome and can be passed on to subsequent

daughter cells after cell division. If infection occurs in the germ line cell

progenitor, then the viral genome may be inherited in a mendelian

manner as an endogenous retrovirus.

Retroviruses can be divided into two categories according to their

genetic make-up: simple and complex. All retroviruses identified to date

contain at least three major coding regions that hold the genetic

information for the virion proteins; Group-specific Antigen (Gag) codes

for the core and structural proteins, Envelope (Env) codes for the coat

proteins and Polymerase (Pol) codes for the reverse transcriptase,

protease and integrase. Complex retroviruses additionally carry genes

that code for accessory proteins that aid in full infectivity and

pathogenesis (figure 1).

Seven major retroviral subfamilies can be distinguished based on

sequence relatedness 1-3 The different genera show very little sequence

similarity and even within subfamilies, genomic variation is extensive

(figure 2). From the lentivirus subfamily, five groups have been

recognized, infecting primates (Human Immune-deficiency Virus (HIV)

and Simian Immune-deficiency Virus (SIV)), sheep and goat (Maedi-

Visna Virus (MVV)), horses (Equine Infectious Anemia Virus (EIAV)),

cats (Feline Immune-deficiency Virus (FIV)) and cattle (Bovine Immune-

deficiency Virus (BIV)), causing immune deficiencies in their respective

hosts.

HIV-1 originates from a zoonotic transfer

HIV-1 can be subdivided in three groups, Main (M), Outlier (O) and Not-

M Not-O (N) that are thought to represent three individual zoonotic

transfers of SIV from primates to humans. HIV-1 from M-group

originated from simian immunodeficiency virus (SIVcpz) in chimpanzee

(Pan troglodytes troglodytes) 4,5 and it has been estimated that the


introduction of HIV-1 in the human lineage occurred in ~1931 6,7. M-

group HIV-1 is responsible for the current pandemic and is observed in

over 90% of infections worldwide. Phylogenetic analysis identified

different subgroups within the M-group, being subtypes A-K 3.

Additionally, circulating recombinant forms (CRF) have been identified

that most probably results from recombination after either co-infection

or super-infection. O-group HIV-1 originated from an independent

transfer from gorilla and is largely confined to West-Central Africa. The

N-group was discovered in 1998 and is very rare. Besides HIV-1, HIV-2

has arisen from transmissions of SIV from sooty mangabees to human

and is largely confined to West-Africa 8.

Disease and HIV-specific immunity

HIV-1 was isolated in 1983 from biopsies from the lymph nodes of

individuals who suffered from the acquired immune deficiency syndrome

(AIDS) 9,10 and HIV-1 could indeed be linked to the disease. In 2007, an

estimated 33 million people were living with HIV-1 while approximately

2.7 million new infections occurred. The estimated number of people

dying from AIDS was 2 million (source: www.who.com). The primary

targets of HIV-1 are cells of the immune system that express CD4 and a

chemokine coreceptor, (CCR5 and/or CXCR4), such as memory T cells

and macrophages 11-13.

Transmission of HIV-1 can occur horizontally, through unprotected

sexual intercourse with an HIV-1 positive partner, needle sharing among

HIV-1 positive drug users, or receiving contaminated blood products.

Vertical transmission from HIV-1 positive mother to a child can also

occur during pregnancy, birth or breast feeding. Most primary infections

are initiated by CCR5-using viruses which persist throughout the

infection 14-18.

http://www.who.com/

Chapter 1 13

Figure 1: Genomic organization of HIV-1 and virion composition. The

coding genes are flanked by long terminal repeats (LTR), which contain

binding sites for many transcription factors like NFAT, NFkB and sp1. Reverse

transcription initiates from the primer-binding site (PBS), resulting in

transcripts that subsequently undergo differential splicing. Rev interacts with

the Rev Response Element (RRE) to promote the export of unspliced mRNA.

The major coding regions Gag, Pol, and Env are post-translationally cleaved,

resulting in the individual building blocks of the virion. The accessory proteins

Vif, Vpu, Vpr, Tat and Nef are involved in many cellular processes, ensuring

optimal replication.


In approximately 50% of HIV-1 infected individuals, HIV-1 variants that

can additionally use CXCR4 as a co-receptor emerge during the course

of infection 13,19,20. Emergence of CXCR4 using HIV-1 variants in the

natural course of infection is associated with a subsequent accelerated

CD4+ cell decline and more rapid disease progression 21.

After acquiring HIV-1, the clinical course of infection can be divided in an

acute and chronic phase (figure 3). During the acute phase of infection,

the viral load is very high 22,23 and as a result of immune activation, T

cells start proliferating 24. During the first weeks of infection, most of the

memory T cell pool in the gut is depleted 25,26, which may lead to loss of

integrity of gut mucosa resulting in microbial translocation which may

drive aberrant immune activation, affecting the subsequent chronic

phase.

Figure 2: Phylogeny of retroviruses. Adapted from http://www.

retrovirology.com/ The discovery of endogenous retroviruses. Retrovirology. 2006

Oct 3;3:67. Review. PMID 17018135.

Chapter 1 15

A relatively stable viral load characterizes the chronic phase while CD4+

cell numbers are gradually declining 27-30. Although the viral load is

relatively stable during this period, the turnover of the HIV-1 quasi

species is high. Indeed, every day 109 – 1010 new viral particles are

regenerated and cleared 31,32. CD4+ T cells are depleted at great pace,

due to several factors, which include direct killing of CD4+ T cells by the

virus, CTL-mediated death of infected T cells and large scale apoptosis

caused by the aberrant immune activation 33-35. In the absence of

Figure 3: Clinical course of HIV-1 infection. During primary infection, the viral

load is generally very high (solid line), which declines in response to immune pressure. The viral setpoint is predictive for the subsequent clinical latent phase. CD4+ T cells quickly decline as a result of viral replication (dashed line) during the acute phase of HIV-1 infection. During the subsequently clinically latent or chronic phase of infection, CD4+ T cell numbers slowly decline, which ultimately results in generalized immune deficiency and the occurrence of opportunistic infections like candidasis, Pneumocystis pneumonia, and

Toxoplasmosis.


antiretroviral therapy, the continuing insult on the immune system leads

to exhaustion of the immune system 36, the occurrence of opportunistic

infections as a result of immune dysfunction and ultimately death of the

host.

During many viral infections the adaptive immune response consisting of

cytotoxic T-lymphocytes and antibody mediated responses usually is

very effective at neutralizing the infection. Unfortunately, the process of

viral reverse transcription is highly error-prone by a lack of proofreading

activity, resulting in the generation of an HIV-1 quasi-species that is no

longer recognized by the immune system and thus escapes from

immune surveillance 37,38.

HIV-1 replication cycle

The life cycle of HIV-1 is highly dependent on the action of multiple host

proteins that are recruited by HIV-1 to accomplish several essential

processes like entry, reverse transcription (RT), nuclear transport,

integration, transcription, assembly and budding (figure 4).

HIV-1 tropism and cellular entry

Cellular tropism of HIV-1 is predominantly determined by the availability

of the primary HIV-1 receptor CD4 and the chemokine coreceptors CCR5

and CXCR4 that mediate viral entry 39-41. CCR5 and CXCR4 are

expressed on subpopulations of CD4 memory T cells, explaining why

these cells are major targets for HIV-1. In contrast to CCR5 using

variants (R5), the CXCR4 using variants (X4) can additionally infect and

kill naïve CD4 T cells 39-42. This may interfere directly with T cell

ontogeny which might explain the accelerated CD4 cell decline in the

presence of X4 HIV-1 43-48.

The HIV-1 envelope glycoprotein (gp120) interacts with the CD4

receptor on the cellular membrane, which results in a conformational

Chapter 1 17

change in gp120 exposing the variable domains essential for co-receptor

binding 49,50. After binding of gp120 to the co-receptor CCR5 or CXCR4,

gp41 will interact with the cellular membrane, which results in fusion of

the virus envelope with the cellular membrane and release of the viral

core in the cytoplasm.

Active transport of core particles

After fusion with the target cell, the viral core is delivered in the

cytoplasm. RT initiates within the core, however some uncoating of the

core appears to be needed for efficient progress of RT. Uncoating can be

measured by the loss of capsid proteins 51,52 and results in the formation

of an RT complex (RTC). The RTC consists of the RNA dimer associated

with viral proteins necessary for reverse transcription, nuclear targeting

and integration.

When the viral RNA genome is reversed transcribed into the double

stranded proviral DNA, the RTC is integration-competent and is called a

pre-integration complex (PIC) that docks at nucleopore complexes 53. In

contrast to MLV where the capsid protein remains associated with the

PIC, during HIV-1 infection the capsid protein is found in RTCs early

after entry but is at later time-points hardly detectable 53. In addition to

viral proteins, cellular proteins like barrier to auto-integration (BAF),

high-mobility group proteins (HMGs), Ku, lamina-associated peptide

(LAP2a), and lens-epithelium-derived growth factor (LEDGF) have been

found to be associated with retroviral PICs 54-59. These cellular proteins

are recruited to aid in several aspects of the viral replication cycle.

The cytoplasm of the cell is dense and transport of the RTC by diffusion

seems unlikely since the complex is relatively large (~50nm in diameter) 53. Instead, transport to the nucleus is an active process, mediated by

actin- and microtubulin-dependent mechanisms 60-64. Indeed, studies

using fluorescently labeled RTCs have indicated that core protein

complexes accumulate at microtubule-organizing centers (MTOC) 61,

which are located close to the nuclear periphery.


Nuclear entry and integration

Simple retroviruses like MLV are dependent on cell proliferation for their

replication and the PIC of simple retroviruses can only cross the nuclear

membrane during mitosis 65-67. In contrast, more complex retroviruses

like HIV-1 are able to infect non-dividing cells like macrophages,

microglial cells, and artificially arrested cells 68 and therefore the PICs of

complex retroviruses must be able to cross an intact nuclear membrane.

Figure 4: Schematic representation of the HIV-1 replication cycle of HIV-1.

The indicated restriction factors Trim5α, Apobec3G and CD317 can inhibit the

replication cycle at different stages of the replication cycle.

Chapter 1 19

The nuclear membrane consists of a two lipid bilayers 69, which are

joined by nuclear pore complexes (NPCs) that allow trafficking between

the cytoplasm and nucleus. The ability to infect non-dividing cells

implies that the PIC must carry karyophilic signals that direct transport

across the nuclear membrane 70. Both viral and cellular factors have

been implicated in this process 68,71. In the viral matrix and Vpr, two

nuclear localization signals (NLS) reside 72-74. Moreover, IN is recognized

by members of the importin/karyophilin-a family via a NLS 75,76.

Importin7 and Nup98 have been implicated in the nuclear import of HIV-

1 77,78.

Upon entry in the nucleus, the viral cDNA is ligated to the chromosomal

DNA of the host with the aid of the viral integrase protein (IN) and host

factors like LEDGF that functions to tether the cDNA to the host DNA 79,80. For HIV-1 it was shown that integration occurs preferably in

transcriptionally active regions 81-83 where the chromosomal DNA has an

open structure and access is relatively easy.

Transcription, assembly and budding

An integrated provirus can have different transcriptional activity states,

allowing evasion from the immune system through latency. It can

however readily be activated to initiate progeny virion production 84,85.

Viral transcription is initiated by binding of transcription factors to the

LTR, in which the binding sites for several transcription factors like NFAT,

NF-kB and Sp1 are located. Transcription is enhanced by the viral Tat

protein, which functions as a cofactor for RNA polymerase II 86,87.

Transcription generates multiple spliced and unspliced mRNAs, which

are translated into structural proteins for the formation of new virions.

The viral Rev protein accommodates the nuclear export of unspliced and

partially spliced viral RNA by binding to the Rev Response Element

(RRE). Rev interacts with multiple cofactors like Rab/hRIP and carries a

nuclear export signal, which is recognized by Crm1, the main nuclear

export receptor for RNA 88-90 91-93.


Assembly of progeny virions and budding is promoted by small Gag

encoded motifs, called ‘late domains’ that interact with components of

the cellular Endosomal Sorting Complex Required for Transport (ESCRT)

and ubiquitination machinery. For example, HIV-1 PTAP late domains

recruit ESCRT through binding tumor susceptibility gene 101 (TSG101) 94,95. However, when this late domain is rendered non-functional, HIV-1

employs an additional domain that recruits ESCRT through ALG-2

interacting protein X (Alix) 96,97. The ESCRT complex is essential for

membrane budding process, including vesicle formation at the

multivesicular body (MVB)/late endosome and retrovirus budding 98-100.

The assembly and incorporation of HIV-1 envelope glycoproteins into

virions is regulated by an interaction between matrix and the envelope

gp41 cytoplasmic domain 101-103. The matrix protein is myristoylated,

and this fatty acid group targets the Gag precursor to the membrane 104-

107 and lipid rafts 108. During assembly and budding, maturation of the

core particle takes place by cleavage of Gag protein by the viral

Protease. In the virion the Matrix protein remains attached to the viral

lipid membrane, the nucleocapsid associates with the viral RNA genome

and capsid condenses around the ribonucleoprotein complex to form the

shell of the mature core.

Host barriers to retroviral infection

The sheer number of retroviral sequences found in the mammalian

genome indicates a long-lasting evolutionary battle between

transposable sequences and the host. Approximately 8% of the human

genome consists of retroviral sequences 109. Given the extensive co-

evolution of retroviruses with mammalian hosts and the potent biological

effects on the host, it is not hard to imagine that the host has acquired

factors that interfere at crucial steps of the viral lifecycle. Several

recently identified host gene products called restriction factors, have

been identified and shown to dominantly restrict infection by

retroviruses 110-115. These mechanisms include retention of newly

assembled virions to the host cell membrane by CD317 116,117, mutation

Chapter 1 21

of the newly generated cDNA by apolipoprotein B mRNA editing enzyme,

catalytic polypeptide-like (Apobec) 3G 118,119 and recognition of the

capsid protein by tri-partite containing motif (Trim) 5α leading to an

early block to infection 120.

Restriction factors inhibit retroviruses in a species and virus specific

manner and as a consequence of evolution, complex retroviruses like

HIV-1 harbor factors that counteract these inhibitions.

The release of retroviruses and several other enveloped viruses like

Ebola-, Lassa- and Marburg viruses is dominantly inhibited by CD317 121,122. CD317 consists of a transmembrane domain, a GPI anchor and an

extra-cellular domain that has the ability to dimerize 116,117. Newly

assembled virions are tethered by dimerization of the extra-cellular

domain which restricts virion release by retaining the assembled

particles 116. The HIV-1 accessory protein Vpu interferes with this

process by reducing the surface expression of CD317 123.

Apobec3G is a member of the family of cytidine deaminases that

mediates a potent block on viral infection of HIV-1, SIV, HTLV-1, many

type C-retroviruses and hepatitis B virus but also retro-elements 124.

Apobec3G can be incorporated in the virion during assembly and has the

ability to deaminate cytosines in the nascent minus-strand DNA 125-127.

The viral Vif protein binds Apobec3G and counteracts encapsidation by

promoting the proteasome-mediated degradation of Apobec3G 128,129.

Additionally, cellular Apobec3G restricts HIV-1 infection at a post-entry

step in resting CD4+ T cells 130, monocytes and macrophages 131,132.

Capsid specific restrictions

Over 50 years ago, Charlotte Friend observed species-specific

differences in susceptibility to infection with the gamma-retrovirus MLV 133. She described a transmittable, leukemia-like illness in mice of which

the etiologic agent was dependent on the strain of mice examined. The

mouse gene that controls the phenotype was termed Friends virus

susceptibility factor (Fv) 134. The gene product was identified as an


endogenous retroviral capsid protein that blocks a step between virus

entry and proviral integration 135. The viral determinant of restriction

was located in the capsid protein and a single amino acid change at

residue 110 in the capsid protein was sufficient to change the phenotype

of the virus from susceptible to resistant 135.

An analogous restriction to EIAV and N-MLV, but not B-MLV was

observed in human cells 136,137. However, no closely related sequences

to Fv-1 were identified in the genome of humans. Therefore, the

observed restriction was denoted restriction factor-1 (ref-1). Similarly,

many non-human primate cells from New and Old world monkeys can

restrict the infection by various retroviruses, including HIV-1 138-140. The

factor that was responsible was termed lentivirus susceptibility factor-1

(Lv-1) 141,142.

Similar as Fv-1, the viral determinant of ref-1 and Lv-1 mediated

restriction was located in the capsid protein and blocks infection before

reverse transcription 142-146. The restriction could be abrogated efficiently

by saturating amounts of incoming viral particles 135,136,147-149.

Interestingly, even distantly related viruses with very little amino acid

similarity have the capacity to saturate the restriction factor. These

findings suggest that a structurally conserved feature of the assembled

capsid is recognized. In support of this notion, comparisons of divergent

retroviral capsid indicate that the overall structure is relatively

conserved 150-155.

Trim5α

In an effort to identify the gene that was responsible for the restriction

of HIV-1 replication in simain cells, a screen using cDNA from a gene

library that conferred resistance in otherwise permissive human cells

was employed 120. This revealed that the Tri-partite motif protein (Trim)

5α was responsible for the Lv-1 mediated restriction. Soon after its

identification it was demonstrated that ref-1 was a species-specific

variant of Trim5α 149,156-158. The human orthologue was responsible for a

Chapter 1 23

potent restriction of N-MLV and EIAV but displayed only intermediate

activity against HIV-1.

Over 70 Trim proteins have been identified in the human genome 159,160.

Trim family members are found in the genome of metazoan species and

expanded in number during evolution. Although they all derive from a

single ancestor, the different members are implicated in diverse

processes, such as transcriptional regulation, apoptosis, inflammation,

cell polarity determination, and anti-viral activities 159.

The Trim5 gene resides in a cluster of closely related Trim genes on

chromosome 11. Besides Trim5α, a number of other Trims have been

reported to display antiviral activities. Primate Trim1 restricts N-MLV,

but not HIV-1 161, Trim32 can interact with the activation domain of Tat

proteins from HIV-1, HIV-2 and EIAV with high affinity, suggesting a

role in transcriptional regulation of the viral genome 162, and trim22 has

been reported to attenuate transcription directed by the LTR of HIV-1 163,164. It can be expected that more Trim proteins are involved in anti-

viral activities, suggesting that Trim proteins represent a new family of

proteins involved in the innate immune response. This is substantiated

by the fact that Trim expression can be regulated by interferons 165-168, a

hallmark cytokine for innate immunity.

Members of the Trim family contain a RING (really interesting new gene)

domain, one or two B-boxes and a Coiled Coil region, Trim5α

additionally contains a C-terminal B30.2 domain 159,160. Trim proteins

form discrete bodies or speckles in the cytoplasm, mediated by

oligomerization through the Coiled Coil domain 159,169. Indeed, Trim5α

was demonstrated to oligomerize by dimerization or trimerization 170-172.

Trim5α levels in the cell are maintained by a continuous synthesis and

rapid proteasome mediated degradation 173. Imbalances in the synthesis

or processing of Trim5α results in oligomerization and formation of pre-

aggresomal structures, which are called ‘cytoplasmic bodies’ 173.

Deletion studies have demonstrated that all domains of Trim5

contribute to the restriction of retroviruses 174, however the B30.2

domain is essential and specifies whether or not restriction commences


of a broad range of retroviruses 161,174-177. Trim5 orthologues are

identified in the genome of many mammalian species and have been

subject to extensive selection pressure, particularly in the exons that

encode the B30.2 domain 178-180.

HIV-1 is potently restricted by simian Trim5 but is much less affected

by human Trim5α. Substitution of the positively charged arginine at

position 332 for a proline, which is found in simian Trim5α, or any other

negatively charged amino acid, confers potent restrictive activity on

HIV-1 161. Remarkably, this amino acid change also leads to potent

inhibition of SIVmac, while the rhesus macaque Trim5α does not restrict

this virus. This suggests that Trim5α indeed is highly species-specific

and that complex determinants in both capsid and Trim5α specify

potential to inhibit infection.

In Owl Monkey cells, the Lv-1 mediated restriction is dependent on the

ability of incoming viral particles to bind Cyclophilin A (CypA). Cloning of

the Owl Monkey Trim5 gene revealed the presence of a retro transposed

CypA pseudogene within the Trim5 gene, resulting in a fusion protein

TrimCYP 181. The HIV-1 restricting activity present in Owl monkeys, can

be pharmacologically inhibited by cyclosporin A, a competitive inhibitor

of CypA. CsA treated Owl Monkey Kidney cells are a 100-fold more

susceptible to infection with HIV-1 182.

The antiviral mechanism by which Trim5α mediates restriction is

incompletely understood. Binding assays have indicated that the affinity

of Trim5α for a capsid protein largely dictates the potency of restriction 183. In assays designed to analyze the fate of incoming viral cores by

measuring particle density, it was shown that Trim5α mediates

destabilization of core particles 183, specifically inducing the degradation

of capsid proteins 184. The presence of a RING domain implies an

involvement of the proteasome in restriction. However, deletion of

functionally important cysteine residues or deletion of the RING domain

altogether does not result in a loss of restriction 169.

Chapter 1 25

Cyclophilin A

An important cofactor that is recruited by the capsid protein of HIV-1 is

the peptidyl-prolyl isomerase CypA 185-187. Within the hydrophobic pocket

of CypA, proline-containing proteins can bind, which are a substrate for

cis-trans isomerization 188. Mutational analysis and structural studies

demonstrated that the binding region in HIV-1 capsid was located from

amino acid residues 87-92 (HAGPIA), which is part of a proline-rich

region that forms a flexible loop in the N-terminal domain (NTD) of

capsid. The interaction of CypA with the CypA binding region can lead to

a conformational change in capsid proteins in vitro 189,190.

The incorporation of CypA in virions does not appear to impact

substantially on Gag polyprotein processing, RNA packaging, virion

structure, or virion protein content and thus far no function for virion-

associated CypA has been described. The interaction of CypA with the

capsid protein was demonstrated to impact on virion infectivity in the

target cell rather than the producer cell, indicating that an early post-

entry step in the replication cycle is affected by CypA binding 145,182,191.

The interaction can be disrupted by competitive inhibition of CypA with

cyclosporin A (CsA) and derivatives thereof 192, or by mutating key

residues in the CypA binding region. CsA treatment or mutation of these

key residues in the CypA binding region attenuates infectivity in several

human cell lines 182,186,191,193,194.

An interaction between CypA and capsid proteins of SIVcpz, FIV, and

SIVagmTAN has also been demonstrated while the gag proteins of other

lentiviruses like HIV-2, SIVmac and SIVagmGRI are unable to interact

with CypA 187. This indicates that CypA dependent replication is virus

specific and might change after adaptation into a new host.

Scope of this thesis

The studies described in this thesis focus on the role of cellular factors

that specifically interact with the capsid protein in HIV-1 replication.

Previously, it was observed that Trim5 mediated restriction of HIV-1


replication in cells from simian species can be circumvented by

mutations in the CypA-binding region in the viral Gag protein 145,195,196.

In Chapter 2, a further delineation of the specific residues that are

involved in the observed resistance to the Trim5α mediated restriction is

described. In Chapter 3, a biochemical analysis is employed to explore

the physical interactions between Trim5α, CypA and the HIV-1 capsid

protein.

In Chapters 4 and 5, the role of Trim5α and CypA in HIV-1 replication in

vivo was studied. The effects of polymorphisms in the Trim5 gene and

the CypA gene on disease progression was investigated in HIV-1

infected participants of the Amsterdam cohort studies on HIV-1 infection

and AIDS.

Expression of several Trim proteins is regulated by interferons. In

Chapter 6, we analyzed whether Trim proteins were involved in the

interferon-induced blockade in HIV-1 replication in primary macrophages.

In Chapter 7, the findings described in this thesis are discussed in light

of the current views on the role of CypA and Trim5α mediated restriction

in HIV-1 replication.

References

1 Coffin JM. Genetic variation in AIDS viruses. Cell 1986; 46: 1-4.

2 Coffin JM. HIV population dynamics in vivo: implications for genetic variation,

pathogenesis, and therapy. Science 1995; 267: 483-9.

3 Coffin JM. HIV viral dynamics. AIDS 1996; 10 Suppl 3: S75-S84.

4 Gao F, Bailes E, Robertson DL et al. Origin of HIV-1 in the chimpanzee Pan

troglodytes troglodytes. Nature 1999; 397: 436-41.

5 Keele BF, Giorgi EE, Salazar-Gonzalez JF et al. Identification and characterization

of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc

Natl Acad Sci U S A 2008; 105: 7552-7.

6 Korber B, Muldoon M, Theiler J et al. Timing the ancestor of the HIV-1 pandemic

strains. Science 2000; 288: 1789-96.

7 Sharp PM, Bailes E, Chaudhuri RR et al. The origins of acquired immune

deficiency syndrome viruses: where and when? Philos Trans R Soc Lond B Biol Sci

2001; 356: 867-76.

8 Reeves JD, Doms RW. Human immunodeficiency virus type 2. J Gen Virol 2002;

83: 1253-65.

Chapter 1 27

9 Barre-Sinoussi F, Chermann JC, Rey R et al. Isolation of a T-lymphotropic

retrovirus from a patient at risk for acquired immune deficiency syndrome.

Science 1983; 220: 868-71.

10 Popovic M, Sarngadharan MG, Read E, Gallo RC. Detection, isolation and

continuous production of cytopathic retroviruses ( HTLV-III ) from patients with

AIDS and pre-AIDS. Science 1984; 224: 497.

11 Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D. Effects of CCR5 and CD4

cell surface concentrations on infections by macrophage tropic isolates of human

immunodeficiency virus type 1. J Virol 1998; 72: 2855-64.

12 Schuitemaker H, Kootstra NA, De Goede REY et al. Monocytotropic human

immunodeficiency virus 1 (HIV-1) variants detectable in all stages of HIV infection

lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture. J

Virol 1991; 65: 356-63.

13 Alkhatib G, Broder CC, Berger EA. Cell type-specific fusion cofactors determine

human immunodeficiency virus type 1 tropism for T-cell lines versus primary

macrophages. J Virol 1996; 70: 5487-94.

14 Zhu T, Mo H, Wang N et al. Genotypic and phenotypic characterization of HIV-1 in

patients with primary infection. Science 1993; 261: 1179-81.

15 Schuitemaker H, Kootstra NA, Koppelman MHGM et al. Proliferation dependent

HIV-1 infection of monocytes occurs during differentiation into macrophages. J

Clin Invest 1992; 89: 1154-60.

16 Schuitemaker H, Kootstra NA, Groenink M et al. Differential tropism of clinical

HIV-1 isolates for primary monocytes and promonocytic-cell lines. AIDS Res Hum

Retroviruses 1992; 8: 1679-82.

17 Van 't Wout AB, Kootstra NA, Mulder-Kampinga GA et al. Macrophage-tropic

variants initiate human immunodeficiency virus type 1 infection after sexual,

parenteral and vertical transmission. J Clin Invest 1994; 94: 2060-7.

18 Zhang L, Huang Y, He T, Cao Y, Ho DD. HIV-1 subtype and second-receptor use.

Nature 1996; 383: 768.

19 Tersmette M, De Goede REY, Al BJM et al. Differential syncytium-inducing

capacity of human immunodeficiency virus isolates: frequent detection of

syncytium-inducing isolates in patients with acquired immunodeficiency syndrome

(AIDS) and AIDS-related complex. J Virol 1988; 62: 2026-32.

20 Koot M, Vos AHV, Keet RPM et al. HIV-1 biological phenotype in long term

infected individuals, evaluated with an MT-2 cocultivation assay. AIDS 1992; 6:

49-54.

21 Koot M, Van Leeuwen R, De Goede REY et al. Conversion rate towards a

syncytium inducing (SI) phenotype during different stages of HIV-1 infection and

prognostic value of SI phenotype for survival after AIDS diagnosis. J Infect Dis

1999; 179: 254-8.

22 Clark JC, Saag MS, Decker WD et al. High titers of cytopathic virus in plasma of

patients with symptomatic primary HIV-1 infection. N Engl J Med 1991; 324:

954-60.

23 Daar ES, Moudgil T, Meyer RD, Ho DD. Transient high levels of viremia in patients

with primary human immunodeficiency virus type 1 infection. N Engl J Med 1991;

324: 961-4.

24 Roos MThL, De Leeuw NASM, Claessen FAP et al. Viro-immunological studies in

acute HIV-1 infection. AIDS 1994; 8: 1533-8.


25 Brenchley JM, Schacker TW, Ruff LE et al. CD4+ T cell depletion during all stages

of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 2004;

200: 749-59.

26 Guadalupe M, Reay E, Sankaran S et al. Severe CD4+ T-cell depletion in gut

lymphoid tissue during primary human immunodeficiency virus type 1 infection

and substantial delay in restoration following highly active antiretroviral therapy.

J Virol 2003; 77: 11708-17.

27 Mellors JW, Rinaldo CR, Jr., Gupta P et al. Prognosis in HIV-1 infection predicted

by the quantity of virus in plasma. Science 1996; 272: 1167-70.

28 De Wolf F, Spijkerman I, Schellekens PThA et al. AIDS prognosis based on HIV-1

RNA, CD4+ T cell count and function: Markers with reciprocal predictive value over

time after seroconversion. AIDS 1997; 11: 1799-806.

29 Douek DC, Betts MR, Hill BJ et al. Evidence for increased T cell turnover and

decreased thymic output in HIV infection. J Immunol 2001; 167: 6663-8.

30 Douek DC, Picker LJ, Koup RA. T cell dynamics in HIV-1 infection. Annu Rev

Immunol 2003; 21: 265-304.

31 Bonhoeffer S, May RM, Shaw GM, Nowak MA. Virus dynamics and drug therapy.

Proc Natl Acad Sci U S A 1997; 94: 6971-6.

32 Ho DD, Neumann AU, Perelson AS et al. Rapid turnover of plasma virions and

CD4 lymphocytes in HIV-1 infection. Nature 1995; 373: 123-6.

33 Aukrust P, Liabakk N, Muller F et al. Serum levels of tumor necrosis factor-alpha

(TNF alpha) and soluble TNF receptors in human immunodeficiency virus type 1

infection:correlations to clinical,immunologic, and virologic parameters. J Infect

Dis 1994; 169: 1186-7.

34 Bass H, Nishanian P, Hardy W et al. Immune changes in HIV-1

infection:significant correlations and differences in serum markers and lymphoid

phenotypic antigens. Clin Immunol Immunopathol 1992; 64: 63-70.

35 Mahalingam M, Peakman M, Davies E et al. T cell activation and disease severity

in HIV infection. Clin Exp Immunol 1993; 93: 337-43.

36 Sabin CA, Devereux H, Phillips AN et al. Course of viral load throughout HIV-1

infection. J Acquir Immune Defic Syndr 2000; 23: 172-7.

37 Preston BD, Poiesz BJ, Loeb LA. Fidelity of HIV-1 reverse transcriptase. Science

1988; 242: 1168-71.

38 Roberts JD, Bebenek K, Kunkel TA. The accuracy of reverse transcriptase from

HIV-1. Science 1988; 242: 1171-3.

39 Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional cDNA

cloning of a seven-transmembrane, G protein-coupled receptor. Science 1996;

272: 872-7.

40 Dragic T, Litwin V, Allaway GP et al. HIV-1 entry into CD4+ cells is mediated by

the chemokine receptor CC-CKR-5. Nature 1996; 381: 667-73.

41 Deng HK, Liu R, Ellmeier W et al. Identification of the major co-receptor for

primary isolates of HIV-1. Nature 1996; 381: 661-6.

42 Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors:

roles in viral entry, tropism, and disease. Ann Rev Immunol 1999; 17: 657-700.

43 Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR. Change in coreceptor

use correlates with disease progression in HIV-1-infected individuals. J Exp Med

1997; 185: 621-8.

44 Blaak H, Van 't Wout AB, Brouwer M et al. In vivo HIV-1 infection of

CD45RA+CD4+ T cells is established primarily by syncytium-inducing variants and

Chapter 1 29

correlates with the rate of CD4+ T cell decline. Proc Natl Acad Sci USA 2000; 97:

1269-74.

45 Schuitemaker H, Koot M, Kootstra NA et al. Biological phenotype of human

immunodeficiency virus type 1 clones at different stages of infection: progression

of disease is associated with a shift from monocytotropic to T-cell-tropic virus

populations. J Virol 1992; 66: 1354-60.

46 Koot M, Keet IPM, Vos AHV et al. Prognostic value of human immunodeficiency

virus type 1 biological phenotype for rate of CD4+ cell depletion and progression

to AIDS. Ann Intern Med 1993; 118: 681-8.

47 Tersmette M, Gruters RA, De Wolf F et al. Evidence for a role of virulent human

immunodeficiency virus (HIV) variants in the pathogenesis of acquired

immunodeficiency syndrome: studies on sequential HIV isolates. J Virol 1989; 63:

2118-25.

48 Richman DD, Bozzette SA. The impact of the syncytium-inducing phenotype of

human immunodeficiency virus on disease progression. J Infect Dis 1994; 169:

968-74.

49 Sattentau QJ, Moore JP. Conformational changes induced in the human

immunodeficiency virus envelope glycoprotein by soluble CD4 binding. J Exp Med

1991; 174: 407-15.

50 Sullivan N, Sun Y, Sattentau Q et al. CD4-induced conformational changes in the

human immunodeficiency virus type 1 gp120 glycoprotein: Consequences for

virus entry and neutralization. J Virol 1998; 72: 4694-703.

51 Bukrinsky MI, Sharova N, McDonald TL et al. Association of integrase, matrix, and

reverse transcriptase antigens of human immunodeficiency virus type 1 with viral

nucleic acids following acute infection. Proc Natl Acad Sci USA 1993; 90: 6125-9.

52 Fassati A, Goff SP. Characterization of intracellular reverse transcription

complexes of human immunodeficiency virus type 1. J Virol 2001; 75: 3626-35.

53 Miller MD, Farnet CM, Bushman FD. Human immunodeficiency virus type 1

preintegration complexes: studies of organization and composition. J Virol 1997;

71: 5382-90.

54 Farnet CM, Haseltine WA. Determination of viral proteins present in the HIV-1

preintegration complex. J Virol 1991; 65: 1910-5.

55 Llano M, Delgado S, Vanegas M, Poeschla EM. Lens epithelium-derived growth

factor/p75 prevents proteasomal degradation of HIV-1 integrase. J Biol Chem

2004; 279: 55570-7.

56 Vandegraaff N, Devroe E, Turlure F, Silver PA, Engelman A. Biochemical and

genetic analyses of integrase-interacting proteins lens epithelium-derived growth

factor (LEDGF)/p75 and hepatoma-derived growth factor related protein 2 (HRP2)

in preintegration complex function and HIV-1 replication. Virology 2006; 346:

415-26.

57 Li L, Yoder K, Hansen MS et al. Retroviral cDNA integration: stimulation by HMG I

family proteins. J Virol 2000; 74: 10965-74.

58 Lee R, Kaushik N, Modak MJ, Vinayak R, Pandey VN. Polyamide nucleic acid

targeted to the primer binding site of the HIV-1 RNA genome blocks in vitro HIV-1

reverse transcription. Biochemistry 1998; 37: 900-10.

59 Lin CW, Engelman A. The barrier-to-autointegration factor is a component of

functional human immunodeficiency virus type 1 preintegration complexes. J Virol

2003; 77: 5030-6.


60 Bukrinskaya AG, Ghorpade A, Heinzinger NK et al. Phosphorylation-dependent

human immunodeficiency virus type 1 infection and nuclear targeting of viral DNA.

Proc Natl Acad Sci USA 1996; 93: 367-71.

61 McDonald D, Vodicka MA, Lucero G et al. Visualization of the intracellular behavior

of HIV in living cells. J Cell Biol 2002; 159: 441-52.

62 Anderson JL, Hope TJ. Intracellular trafficking of retroviral vectors: obstacles and

advances. Gene Ther 2005; 12: 1667-78.

63 Goff SP. Intracellular trafficking of retroviral genomes during the early phase of

infection: viral exploitation of cellular pathways. J Gene Med 2001; 3: 517-28.

64 Taunton J. Actin filament nucleation by endosomes, lysosomes and secretory

vesicles. Curr Opin Cell Biol 2001; 13: 85-91.

65 Temin HM, RUBIN H. Characteristics of an assay for Rous sarcoma virus and Rous

sarcoma cells in tissue culture. Virology 1958; 6: 669-88.

66 Temin HM, RUBIN H. A kinetic study of infection of chick embryo cells in vitro by

Rous sarcoma virus. Virology 1959; 8: 209-22.

67 Temin HM. Studies on carcinogenesis by avian sarcoma viruses. VI. Differential

multiplication of uninfected and of converted cells in response to insulin. J Cell

Physiol 1967; 69: 377-84.

68 Fassati A. HIV infection of non-dividing cells: a divisive problem. Retrovirology

2006; 3: 74.

69 Gruenbaum Y, Margalit A, Goldman RD, Shumaker DK, Wilson KL. The nuclear

lamina comes of age. Nat Rev Mol Cell Biol 2005; 6: 21-31.

70 Fouchier RA, Malim MH. Nuclear import of human immunodeficiency virus type-1

preintegration complexes. Adv Virus Res 1999; 52: 275-99.

71 Suzuki Y, Craigie R. The road to chromatin - nuclear entry of retroviruses. Nat

Rev Microbiol 2007; 5: 187-96.

72 Bukrinsky MI, Haggerty S, Dempsey MP et al. A nuclear localization signal within

HIV-1 matrix protein that governs infection of non-dividing cells. Nature 1993;

365: 666-9.

73 Heinzinger NK, Bukrinsky MI, Haggerty SA et al. The vpr protein of human

immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids

in nondividing cells. Proc Natl Acad Sci USA 1994; 91: 7311-5.

74 von Schwedler U, Kornbluth RS, Trono D. The nuclear localization signal of the

matrix protein of human immunodeficiency virus type 1 allows the establishment

of infection in macrophages and quiescent T lymphocytes. Proc Natl Acad Sci USA

1994; 91: 6992-6.

75 Gallay P, Stitt V, Mundy C, Oettinger M, Trono D. Role of the karyopherin pathway

in HIV-1 nuclear import. J Virol 1996; 70: 1027-32.

76 Gallay P, Hope T, Chin D, Trono D. HIV-1 infection of nondividing cells through

the recognition of integrase by the importin/karyopherin pathway. Proc Natl Acad

Sci USA 1997; 94: 9825-30.

77 Zaitseva L, Cherepanov P, Leyens L et al. HIV-1 exploits importin 7 to maximize

nuclear import of its DNA genome. Retrovirology 2009; 6: 11.

78 Ebina H, Aoki J, Hatta S, Yoshida T, Koyanagi Y. Role of Nup98 in nuclear entry of

human immunodeficiency virus type 1 cDNA. Microbes Infect 2004; 6: 715-24.

79 Hombrouck A, De RJ, Hendrix J et al. Virus evolution reveals an exclusive role for

LEDGF/p75 in chromosomal tethering of HIV. PLoS Pathog 2007; 3: e47.

80 Raghavendra NK, Engelman A. LEDGF/p75 interferes with the formation of

synaptic nucleoprotein complexes that catalyze full-site HIV-1 DNA integration in

Chapter 1 31

vitro: implications for the mechanism of viral cDNA integration. Virology 2007;

360: 1-5.

81 Schroder AR, Shinn P, Chen H et al. HIV-1 integration in the human genome

favors active genes and local hotspots. Cell 2002; 110: 521-9.

82 Crise B, Li Y, Yuan C et al. Simian immunodeficiency virus integration preference

is similar to that of human immunodeficiency virus type 1. J Virol 2005; 79:

12199-204.

83 MacNeil A, Sankale JL, Meloni ST et al. Genomic sites of human immunodeficiency

virus type 2 (HIV-2) integration: similarities to HIV-1 in vitro and possible

differences in vivo. J Virol 2006; 80: 7316-21.

84 Han Y, Siliciano RF. Keeping quiet: microRNAs in HIV-1 latency. Nat Med 2007;

13: 1138-40.

85 Bisgrove D, Lewinski M, Bushman F, Verdin E. Molecular mechanisms of HIV-1

proviral latency. Expert Rev Anti Infect Ther 2005; 3: 805-14.

86 Cullen BR. trans-Activation of human immunodeficiency virus occurs via a

bimodal mechanism. Cell 1986; 46: 973-82.

87 Kao SY, Calman AF, Luciw PA, Peterlin BM. Anti-termination of transcription within

the long terminal repeat of HIV-1 by tat gene product. Nature 1987; 330: 489-93.

88 Fornerod M, Ohno M, Yoshida M, Mattaj IW. CRM1 is an export receptor for

leucine-rich nuclear export signals. Cell 1997; 90: 1051-60.

89 Stade K, Ford CS, Guthrie C, Weis K. Exportin 1 (Crm1p) is an essential nuclear

export factor. Cell 1997; 90: 1041-50.

90 Ossareh-Nazari B, Bachelerie F, Dargemont C. Evidence for a role of CRM1 in

signal-mediated nuclear protein export. Science 1997; 278: 141-4.

91 Stutz F, Neville M, Rosbash M. Identification of a novel nuclear pore-associated

protein as a functional target of the HIV-1 Rev protein in yeast. Cell 1995; 82:

495-506.

92 Stutz F, Izaurralde E, Mattaj IW, Rosbash M. A role for nucleoporin FG repeat

domains in export of human immunodeficiency virus type 1 Rev protein and RNA

from the nucleus. Mol Cell Biol 1996; 16: 7144-50.

93 Fritz CC, Green MR. HIV Rev uses a conserved cellular protein export pathway for

the nucleocytoplasmic transport of viral RNAs. Curr Biol 1996; 6: 848-54.

94 Bache KG, Brech A, Mehlum A, Stenmark H. Hrs regulates multivesicular body

formation via ESCRT recruitment to endosomes. J Cell Biol 2003; 162: 435-42.

95 Bache KG, Raiborg C, Mehlum A, Stenmark H. STAM and Hrs are subunits of a

multivalent ubiquitin-binding complex on early endosomes. J Biol Chem 2003;

278: 12513-21.

96 Strack B, Calistri A, Craig S, Popova E, Gottlinger HG. AIP1/ALIX is a binding

partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 2003; 114:

689-99.

97 von Schwedler UK, Stuchell M, Muller B et al. The protein network of HIV budding.

Cell 2003; 114: 701-13.

98 Morita E, Sundquist WI. Retrovirus budding. Annu Rev Cell Dev Biol 2004; 20:

395-425.

99 Carlton JG, Martin-Serrano J. Parallels between cytokinesis and retroviral budding:

a role for the ESCRT machinery. Science 2007; 316: 1908-12.

100 Hurley JH, Emr SD. The ESCRT complexes: structure and mechanism of a

membrane-trafficking network. Annu Rev Biophys Biomol Struct 2006; 35: 277-

98.


101 Freed EO, Myers DJ, Risser R. Characterization of the fusion domain of the human

immunodeficiency virus type 1 envelope glycoprotein gp41. Proc Natl Acad Sci

USA 1993; 87: 4650-4.

102 Freed EO, Martin MA. Virion incorporation of envelope glycoproteins with long but

not short cytoplasmic tails is blocked by specific, single amino acid substitutions

in the human immunodeficiency virus type 1 matrix. J Virol 1995; 69: 1984-9.

103 Hourioux C, Brand D, Sizaret PY et al. Identification of the glycoprotein 41(TM)

cytoplasmic tail domains of human immunodeficiency virus type 1 that interact

with Pr55Gag particles. AIDS Res Hum Retroviruses 2000; 16: 1141-7.

104 Gheysen D, Jacobs E, de FF et al. Assembly and release of HIV-1 precursor

Pr55gag virus-like particles from recombinant baculovirus-infected insect cells.

Cell 1989; 59: 103-12.

105 Jacobs E, Gheysen D, Thines D, Francotte M, De Wilde M. The HIV-1 Gag

precursor Pr55gag synthesized in yeast is myristoylated and targeted to the

plasma membrane. Gene 1989; 79: 71-81.

106 Ono A, Huang M, Freed EO. Characterization of human immunodeficiency virus

type 1 matrix revertants: effects on virus assembly, Gag processing, and Env

incorporation into virions. J Virol 1997; 71: 4409-18.

107 Ono A, Freed EO. Binding of human immunodeficiency virus type 1 Gag to

membrane: role of the matrix amino terminus. J Virol 1999; 73: 4136-44.

108 Ono A, Freed EO. Plasma membrane rafts play a critical role in HIV-1 assembly

and release. Proc Natl Acad Sci U S A 2001; 98: 13925-30.

109 Belshaw R, Pereira V, Katzourakis A et al. Long-term reinfection of the human

genome by endogenous retroviruses. Proc Natl Acad Sci U S A 2004; 101: 4894-

9.

110 Wolf D, Goff SP. Host restriction factors blocking retroviral replication. Annu Rev

Genet 2008; 42: 143-63.

111 Takeuchi H, Matano T. Host factors involved in resistance to retroviral infection.

Microbiol Immunol 2008; 52: 318-25.

112 Baumann JG. Intracellular restriction factors in mammalian cells--An ancient

defense system finds a modern foe. Curr HIV Res 2006; 4: 141-68.

113 Lehmann-Che J, Saib A. Early stages of HIV replication: how to hijack cellular

functions for a successful infection. AIDS Rev 2004; 6: 199-207.

114 Goff SP. Retrovirus restriction factors. Mol Cell 2004; 16: 849-59.

115 Bieniasz PD. Intrinsic immunity: a front-line defense against viral attack. Nat

Immunol 2004; 5: 1109-15.

116 Neil SJ, Zang T, Bieniasz PD. Tetherin inhibits retrovirus release and is

antagonized by HIV-1 Vpu. Nature 2008; 451: 425-30.

117 Van DN, Goff D, Katsura C et al. The interferon-induced protein BST-2 restricts

HIV-1 release and is downregulated from the cell surface by the viral Vpu protein.

Cell Host Microbe 2008; 3: 245-52.

118 Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that

inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 2002;

418: 646-50.

119 Bishop KN, Holmes RK, Sheehy AM, Malim MH. APOBEC-mediated editing of viral

RNA. Science 2004; 305: 645.

120 Stremlau M, Owens CM, Perron MJ et al. The cytoplasmic body component

TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 2004; 427:

848-53.

Chapter 1 33

121 Kaletsky RL, Francica JR, grawal-Gamse C, Bates P. Tetherin-mediated restriction

of filovirus budding is antagonized by the Ebola glycoprotein. Proc Natl Acad Sci U

S A 2009; 106: 2886-91.

122 Jouvenet N, Neil SJ, Zhadina M et al. Broad-spectrum inhibition of retroviral and

filoviral particle release by tetherin. J Virol 2009; 83: 1837-44.

123 Miyagi E, Andrew AJ, Kao S, Strebel K. Vpu enhances HIV-1 virus release in the

absence of Bst-2 cell surface down-modulation and intracellular depletion. Proc

Natl Acad Sci U S A 2009; 106: 2868-73.

124 Goila-Gaur R, Strebel K. HIV-1 Vif, APOBEC, and intrinsic immunity. Retrovirology

2008; 5: 51.

125 Harris RS, Liddament MT. Retroviral restriction by APOBEC proteins. Nat Rev

Immunol 2004; 4: 868-77.

126 Lecossier D, Bouchonnet F, Clavel F, Hance AJ. Hypermutation of HIV-1 DNA in

the absence of the Vif protein. Science 2003; 300: 1112.

127 Mangeat B, Turelli P, Caron G et al. Broad antiretroviral defence by human

APOBEC3G through lethal editing of nascent reverse transcripts. Nature 2003;

424: 99-103.

128 Mehle A, Goncalves J, Santa-Marta M, McPike M, Gabuzda D. Phosphorylation of a

novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes

APOBEC3G degradation. Genes Dev 2004; 18: 2861-6.

129 Stopak K, de NC, Yonemoto W, Greene WC. HIV-1 Vif blocks the antiviral activity

of APOBEC3G by impairing both its translation and intracellular stability. Mol Cell

2003; 12: 591-601.

130 Chiu YL, Soros VB, Kreisberg JF et al. Cellular APOBEC3G restricts HIV-1 infection

in resting CD4+ T cells. Nature 2005; 435: 108-14.

131 Stopak KS, Chiu YL, Kropp J, Grant RM, Greene WC. Distinct patterns of cytokine

regulation of APOBEC3G expression and activity in primary lymphocytes,

macrophages, and dendritic cells. J Biol Chem 2007; 282: 3539-46.

132 Thielen BK, Klein KC, Walker LW et al. T cells contain an RNase-insensitive

inhibitor of APOBEC3G deaminase activity. PLoS Pathog 2007; 3: 1320-34.

133 FRIEND C. Leukemia of adult mice caused by a transmissible agent. Ann N Y Acad

Sci 1957; 68: 522-32.

134 Lilly F. Fv-2: identification and location of a second gene governing the spleen

focus response to Friend leukemia virus in mice. J Natl Cancer Inst 1970; 45:

163-9.

135 Goff SP. Operating under a Gag order: a block against incoming virus by the Fv1

gene. Cell 1996; 86: 691-3.

136 Hatziioannou T, Cowan S, Goff SP, Bieniasz PD, Towers GJ. Restriction of multiple

divergent retroviruses by Lv1 and Ref1. EMBO J 2003; 22: 385-94.

137 Towers G, Bock M, Martin S et al. A conserved mechanism of retrovirus restriction

in mammals. Proc Natl Acad Sci U S A 2000; 97: 12295-9.

138 Shibata R, Sakai H, Kawamura M, Tokunaga K, Adachi A. Early replication block of

human immunodeficiency virus type 1 in monkey cells. J Gen Virol 1995; 76 ( Pt

11): 2723-30.

139 Himathongkham S, Luciw PA. Restriction of HIV-1 (subtype B) replication at the

entry step in rhesus macaque cells. Virology 1996; 219: 485-8.

140 Hofmann W, Schubert D, LaBonte J et al. Species-specific, postentry barriers to

primate immunodeficiency virus infection. J Virol 1999; 73: 10020-8.

141 Besnier C, Takeuchi Y, Towers G. Restriction of lentivirus in monkeys. Proc Natl

Acad Sci U S A 2002; 99: 11920-5.


142 Cowan S, Hatziioannou T, Cunningham T et al. Cellular inhibitors with Fv1-like

activity restrict human and simian immunodeficiency virus tropism. Proc Natl

Acad Sci U S A 2002; 99: 11914-9.

143 Owens CM, Yang PC, Gottlinger H, Sodroski J. Human and simian

immunodeficiency virus capsid proteins are major viral determinants of early,

postentry replication blocks in simian cells. J Virol 2003; 77: 726-31.

144 Owens CM, Song B, Perron MJ et al. Binding and susceptibility to postentry

restriction factors in monkey cells are specified by distinct regions of the human

immunodeficiency virus type 1 capsid. J Virol 2004; 78: 5423-37.

145 Kootstra NA, Munk C, Tonnu N, Landau NR, Verma IM. Abrogation of postentry

restriction of HIV-1-based lentiviral vector transduction in simian cells. Proc Natl

Acad Sci U S A 2003; 100: 1298-303.

146 Munk C, Brandt SM, Lucero G, Landau NR. A dominant block to HIV-1 replication

at reverse transcription in simian cells. Proc Natl Acad Sci U S A 2002; 99:

13843-8.

147 Duran-Troise G, Bassin RH, Rein A, Gerwin BI. Loss of Fv-1 restriction in Balb/3T3

cells following infection with a single N tropic murine leukemia virus particle. Cell

1977; 10: 479-88.

148 Sayah DM, Luban J. Selection for loss of Ref1 activity in human cells releases

human immunodeficiency virus type 1 from cyclophilin A dependence during

infection. J Virol 2004; 78: 12066-70.

149 Yap MW, Nisole S, Lynch C, Stoye JP. Trim5alpha protein restricts both HIV-1 and

murine leukemia virus. Proc Natl Acad Sci U S A 2004; 101: 10786-91.

150 Shi J, Aiken C. Saturation of TRIM5 alpha-mediated restriction of HIV-1 infection

depends on the stability of the incoming viral capsid. Virology 2006; 350: 493-

500.

151 Gamble TR, Yoo S, Vajdos FF et al. Structure of the carboxyl-terminal

dimerization domain of the HIV-1 capsid protein. Science 1997; 278: 849-53.

152 Gitti RK, Lee BM, Walker J et al. Structure of the amino-terminal core domain of

the HIV-1 capsid protein. Science 1996; 273: 231-5.

153 Jin Z, Jin L, Peterson DL, Lawson CL. Model for lentivirus capsid core assembly

based on crystal dimers of EIAV p26. J Mol Biol 1999; 286: 83-93.

154 Khorasanizadeh S, Campos-Olivas R, Summers MF. Solution structure of the

capsid protein from the human T-cell leukemia virus type-I. J Mol Biol 1999; 291:

491-505.

155 Khorasanizadeh S, Campos-Olivas R, Clark CA, Summers MF. Sequence-specific

1H, 13C and 15N chemical shift assignment and secondary structure of the HTLV-

I capsid protein. J Biomol NMR 1999; 14: 199-200.

156 Hatziioannou T, Perez-Caballero D, Yang A, Cowan S, Bieniasz PD. Retrovirus

resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha. Proc

Natl Acad Sci U S A 2004; 101: 10774-9.

157 Keckesova Z, Ylinen LM, Towers GJ. The human and African green monkey

TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities. Proc

Natl Acad Sci U S A 2004; 101: 10780-5.

158 Perron MJ, Stremlau M, Song B et al. TRIM5alpha mediates the postentry block to

N-tropic murine leukemia viruses in human cells. Proc Natl Acad Sci U S A 2004;

101: 11827-32.

159 Reymond A, Meroni G, Fantozzi A et al. The tripartite motif family identifies cell

compartments. EMBO J 2001; 20: 2140-51.

Chapter 1 35

160 Nisole S, Stoye JP, Saib A. TRIM family proteins: retroviral restriction and

antiviral defence. Nat Rev Microbiol 2005; 3: 799-808.

161 Yap MW, Nisole S, Stoye JP. A single amino acid change in the SPRY domain of

human Trim5alpha leads to HIV-1 restriction. Curr Biol 2005; 15: 73-8.

162 Fridell RA, Harding LS, Bogerd HP, Cullen BR. Identification of a novel human zinc

finger protein that specifically interacts with the activation domain of lentiviral Tat

proteins. Virology 1995; 209: 347-57.

163 Tissot C, Mechti N. Molecular cloning of a new interferon-induced factor that

represses human immunodeficiency virus type 1 long terminal repeat expression.

J Biol Chem 1995; 270: 14891-8.

164 Bouazzaoui A, Kreutz M, Eisert V et al. Stimulated trans-acting factor of 50 kDa

(Staf50) inhibits HIV-1 replication in human monocyte-derived macrophages.

Virology 2006; 356: 79-94.

165 Asaoka K, Ikeda K, Hishinuma T et al. A retrovirus restriction factor TRIM5alpha is

transcriptionally regulated by interferons. Biochem Biophys Res Commun 2005;

338: 1950-6.

166 Chelbi-Alix MK, Pelicano L, Quignon F et al. Induction of the PML protein by

interferons in normal and APL cells. Leukemia 1995; 9: 2027-33.

167 Carthagena L, Parise MC, Ringeard M et al. Implication of TRIM alpha and

TRIMCyp in interferon-induced anti-retroviral restriction activities. Retrovirology

2008; 5: 59.

168 Rajsbaum R, Stoye JP, O'Garra A. Type I interferon-dependent and -independent

expression of tripartite motif proteins in immune cells. Eur J Immunol 2008; 38:

619-30.

169 Perez-Caballero D, Hatziioannou T, Zhang F, Cowan S, Bieniasz PD. Restriction of

human immunodeficiency virus type 1 by TRIM-CypA occurs with rapid kinetics

and independently of cytoplasmic bodies, ubiquitin, and proteasome activity. J

Virol 2005; 79: 15567-72.

170 Mische CC, Javanbakht H, Song B et al. Retroviral restriction factor TRIM5alpha is

a trimer. J Virol 2005; 79: 14446-50.

171 Langelier CR, Sandrin V, Eckert DM et al. Biochemical characterization of a

recombinant TRIM5alpha protein that restricts human immunodeficiency virus

type 1 replication. J Virol 2008; 82: 11682-94.

172 Kar AK, az-Griffero F, Li Y, Li X, Sodroski J. Biochemical and biophysical

characterization of a chimeric TRIM21-TRIM5alpha protein. J Virol 2008; 82:

11669-81.

173 Diaz-Griffero F, Li X, Javanbakht H et al. Rapid turnover and polyubiquitylation of

the retroviral restriction factor TRIM5. Virology 2006; 349: 300-15.

174 Perez-Caballero D, Hatziioannou T, Yang A, Cowan S, Bieniasz PD. Human

tripartite motif 5alpha domains responsible for retrovirus restriction activity and

specificity. J Virol 2005; 79: 8969-78.

175 Stremlau M, Perron M, Welikala S, Sodroski J. Species-specific variation in the

B30.2(SPRY) domain of TRIM5alpha determines the potency of human

immunodeficiency virus restriction. J Virol 2005; 79: 3139-45.

176 Perron MJ, Stremlau M, Sodroski J. Two surface-exposed elements of the

B30.2/SPRY domain as potency determinants of N-tropic murine leukemia virus

restriction by human TRIM5alpha. J Virol 2006; 80: 5631-6.

177 Nakayama EE, Miyoshi H, Nagai Y, Shioda T. A specific region of 37 amino acid

residues in the SPRY (B30.2) domain of African green monkey TRIM5alpha


determines species-specific restriction of simian immunodeficiency virus SIVmac

infection. J Virol 2005; 79: 8870-7.

178 Sawyer SL, Wu LI, Emerman M, Malik HS. Positive selection of primate

TRIM5alpha identifies a critical species-specific retroviral restriction domain. Proc

Natl Acad Sci U S A 2005; 102: 2832-7.

179 Song B, Gold B, O'hUigin C et al. The B30.2(SPRY) domain of the retroviral

restriction factor TRIM5alpha exhibits lineage-specific length and sequence

variation in primates. J Virol 2005; 79: 6111-21.

180 Si Z, Vandegraaff N, O'hUigin C et al. Evolution of a cytoplasmic tripartite motif

(TRIM) protein in cows that restricts retroviral infection. Proc Natl Acad Sci U S A

2006; 103: 7454-9.

181 Nisole S, Lynch C, Stoye JP, Yap MW. A Trim5-cyclophilin A fusion protein found

in owl monkey kidney cells can restrict HIV-1. Proc Natl Acad Sci U S A 2004;

101: 13324-8.

182 Towers GJ, Hatziioannou T, Cowan S et al. Cyclophilin A modulates the sensitivity

of HIV-1 to host restriction factors. Nat Med 2003; 9: 1138-43.

183 Stremlau M, Perron M, Lee M et al. Specific recognition and accelerated uncoating

of retroviral capsids by the TRIM5alpha restriction factor. Proc Natl Acad Sci U S A

2006; 103: 5514-9.

184 Chatterji U, Bobardt MD, Gaskill P et al. Trim5alpha accelerates degradation of

cytosolic capsid associated with productive HIV-1 entry. J Biol Chem 2006; 281:

37025-33.

185 Luban J, Bossolt KL, Franke EK, Kalpana GV, Goff SP. Human immunodeficiency

virus type 1 Gag protein binds to cyclophilins A and B. Cell 1993; 73: 1067-78.

186 Franke EK, Yuan HEH, Luban J. Specific incorporation of cyclophilin A into HIV-1

virions. Nature 1994; 372: 359-62.

187 Lin TY, Emerman M. Cyclophilin A interacts with diverse lentiviral capsids.

Retrovirology 2006; 3: 70.

188 Fanghanel J, Fischer G. Insights into the catalytic mechanism of peptidyl prolyl

cis/trans isomerases. Front Biosci 2004; 9: 3453-78.

189 Bosco DA, Eisenmesser EZ, Pochapsky S, Sundquist WI, Kern D. Catalysis of

cis/trans isomerization in native HIV-1 capsid by human cyclophilin A. Proc Natl

Acad Sci U S A 2002; 99: 5247-52.

190 Bosco DA, Kern D. Catalysis and binding of cyclophilin A with different HIV-1

capsid constructs. Biochemistry 2004; 43: 6110-9.

191 Thali M, Bukovsky A, Kondo E et al. Functional association of cyclophilin A with

HIV-1 virions. Nature 1994; 372: 363-5.

192 Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW. Cyclophilin: a

specific cytosolic binding protein for cyclosporin A. Science 1984; 226: 544-7.

193 Hatziioannou T, Perez-Caballero D, Cowan S, Bieniasz PD. Cyclophilin interactions

with incoming human immunodeficiency virus type 1 capsids with opposing

effects on infectivity in human cells. J Virol 2005; 79: 176-83.

194 Sokolskaja E, Sayah DM, Luban J. Target cell cyclophilin A modulates human

immunodeficiency virus type 1 infectivity. J Virol 2004; 78: 12800-8.

195 Chatterji U, Bobardt MD, Stanfield R et al. Naturally occurring capsid substitutions

render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-

resistant in Owl monkey cells. J Biol Chem 2005; 280: 40293-300.

196 Ikeda Y, Ylinen LM, Kahar-Bador M, Towers GJ. Influence of gag on human

immunodeficiency virus type 1 species-specific tropism. J Virol 2004; 78: 11816-

22.

Chapter 2

Efficient Transduction of Simian

Cells by HIV-1 based Lentiviral

Vectors that Contain Mutations in

the Capsid Protein

Maarten AN Rits1,2, Karel A van Dort1,2, Carsten Münk3,

Alexander B Meijer4 and Neeltje A Kootstra1,2

1. Department of Clinical Viro Immunology, Sanquin Research,

Landsteiner Laboratory, Amsterdam, The Netherlands

2. Center of Infection and Immunity Amsterdam (CINIMA),

University of Amsterdam, Amsterdam, The Netherlands

3. Department of Medical Biotechnology, Paul-Ehrlich-Institute,

Langen, Germany

4. Department of Plasma Proteins, Sanquin Research, Amsterdam,

The Netherlands

Mol Ther 2007;15:930-937

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Chapter 2 39

Abstract

Recently, the cyclophilin A (CyPA)-binding region of the HIV-1 capsid

protein was identified as a viral determinant involved in the post-entry

restriction in Old World monkey cells. Here, we constructed a panel of

HIV-1-based lentiviral vectors (LVs) that contain either mutations in the

CyPA-binding region or the CyPA-binding region of the related viruses

HIV-1 group O and HIV-2. We demonstrated that amino-acid changes in

the CyPA-binding region of the capsid can alter the phenotype of the

virus resulting in CyPA-independent infection in human cells and non-

restricted infection in simian cells. Combining these data with the

available structural data, we speculate that reduced affinity of the capsid

for CyPA is associated with an unrestricted infection of simian cells. In

addition, we observed that primary rhesus macaque peripheral blood

mononuclear cells could be transduced efficiently by the LV that

contained the CyPA-binding region of HIV-2. Therefore, this LV might be

very useful for long-term safety studies in large animal models like

rhesus macaques.

Introduction The ability of the HIV-1-based lentiviral vector (LV) to deliver genes into

hematopoietic stem cells holds great promise for the treatment of

infectious diseases like acquired immune deficiency syndrome (AIDS),

and hereditary hematopoietic disorders like severe combined immune

deficiency. The main potential risk of gene transfer using integrating

viral vectors is insertional mutagenesis resulting from random

integration into the host cell genome. This became evident after the

recent development of T-cell leukemia in three patients with X-linked

severe combined immune deficiency in a gene therapy trial using a

retroviral vector.1,2,3 These studies indicate that extensive preclinical

experiments in large animal models are required to evaluate the safety

of the transfer vector and the therapeutic transgene during a long-term

follow-up.

Old World monkeys like rhesus macaque would be an adequate model

for gene therapy safety studies using HIV-1-based LVs. However, it was

observed that Old World monkeys posses an antiviral defense

mechanism that restricts retroviruses.4,5,6,7,8 The cellular factor involved

in this restriction was initially named Lv-1, and later identified as the

tripartite interaction motif 5α (Trim5α).9 This antiviral activity appeared

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40 Transduction of simian cells by HIV-1 based vectors

to be species-specific and was shown to block infection of a broad range

of retroviruses.9,10,11,12,13,14 Trim5α obtained from different Old World

monkey species were particularly effective in restricting HIV-1.

Another cellular factor involved in early post-entry steps in HIV-1

infection is Cyclophilin A (CyPA). CyPA interacts specifically with the

CyPA-binding region of the HIV-1 group M capsid protein and is

encapsidated into the virions.15,16,17,18 However, HIV-1 virions produced

in the absence of CyPA are infectious, demonstrating that packaging of

CyPA is not required for virus replication. This indicates that CyPA

present in the target cells is sufficient to support infection.19,20 Although

most group M HIV-1 viruses require CyPA for replication, naturally

occurring variants carrying amino-acid substitutions in the CyPA-binding

region that render replication in human cells independent of CyPA have

been identified.19,21

HIV-1 group O viruses are phylogenetically and distantly related to HIV-

1 group M viruses, and it is thought that they originate from a separate

zoonosis of simian immunodeficiency virus (SIV)CPZ.22 Similar to HIV-1

group M viruses, HIV-1 group O viruses incorporate CyPA; however,

infection of these viruses is not dependent on CyPA.23 Another lentivirus

reported to be able to replicate independent of CyPA is HIV-2.24 HIV-2

originates from a cross-species transmission of SIVsmm25 and unlike HIV-

1 the capsid protein is unable to interact with CyPA.24

The mechanism by which CyPA supports HIV-1 infection remains

unclear. Some reports suggest that CyPA protects HIV-1 from antiviral

factors like Trim5α,20,26,27,28,29 whereas others have suggested that CyPA

facilitates disassembly of the viral capsid.30,31,32

Previously, we and others demonstrated that the CyPA-binding region of

the HIV-1 capsid protein is the viral determinant involved in the post-

entry restriction in Old World monkey cells and that mutations in this

region increased the transduction efficiency in these cells.19,33,34,35

Furthermore, we observed that escape from the restriction coincided

with CyPA-independent transduction in human cells.19 Here, we

constructed HIV-1-based LVs that can efficiently transduce Old World

monkey cells. We studied the amino acids of the CyPA-binding region of

HIV-1 group M, group O, and HIV-2 viruses that are required for

efficient transduction of simian cells and analyzed the role of CyPA in the

observed restriction in simian cells. We observed that amino-acid

changes in the CyPA-binding region that have previously been shown to

































Chapter 2 41

reduce CyPA-binding affinity were able to overcome the restriction in

simian cells.

Results

Resistance of HIV-1-based LV post-entry restriction in simian cells by a

single amino-acid change in the CyPA-binding region of capsid

The HIV-1-based LV is derived from an HIV-1 group M virus.36,37,38

Recently, we demonstrated that substitution of the CyPA-binding region

of the LV capsid protein by that of the macrophage tropic HIV-1 Ba-L

(gB) was able to escape the post-entry restriction in simian cells.19 The

CyPA-binding region of the HIV-1 Ba-L variant contains three amino-acid

differences (glutamine 87, proline 88, and valine 91) as compared with

the wild-type (WT) HIV-1 HxB2. Here, we analyzed which of these

amino-acid residues is responsible for the observed resistance of HIV-1-

based LVs to restriction in simian cells. We constructed three additional

mutant LVs (Table 1) that contained a valine at position 91 (HM.1), a

glutamine at position 87 and a valine at position 91 (HM.2), or a proline

at position 88 (HM.3). Green fluorescent protein (GFP)-expressing

mutant LV were produced and their infectivity was analyzed in human

293T cells, African green monkey kidney cell line CV-1, and the rhesus

macaque fibroblast line FrhL2. Similar to our previous observations,19 we

observed that infection with low dose of WT was restricted in the simian

cell lines CV-1 and FrhL2, and that restriction could be saturated by

infection with high amounts of virus. Moreover, gB was not restricted in

simian cells and efficiently infected CV-1 and FrhL2 cells (Figure 1a).

HM.1, HM.2, and HM.3 were able to infect 293T cells efficiently,

although some variation in the infectious virus titers was observed.

HM.1 showed consistently a slight increase in the infectious titer per

nanogram p24, whereas HM.2 showed a slight decrease in the infectious

titer compared with WT and gB. Similar to WT, HM.1 was restricted in

simian cells (Figure 1a), indicating that a valine at position 91 is not

sufficient for overriding the post-entry restriction in simian cells. A

phenotype similar to gB was observed for HM.2 and HM.3. These were

not restricted in CV-1 and FrhL2 (Figure 1a), indicating that either a

glutamine at position 87 or proline at position 88 results in efficient

transduction of simian cells.





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Table 1. Mutations in the CyPA-binding region of HIV-1 based LV

CyPA binding region AA position

86 87 88 89 90 91 92 93

WT V H A G P I A P

gB . Q P . . V . .

HM.1 . . . . . V . .

HM.2 . Q . . . V . .

HM.3 . . P . . . . .

HO.1 P A M G P L P P

HO.2 V . . . . . . .

HO.3 V . P . . . . .

H2.1 — I P G P L P P

H2.2 — . A . . . . .

H2.3 — H . . . . . .

H2.4 — H A . . . . .

H2.5 V H A . . . . .

To analyze whether the restriction observed for HM.1 and WT in simian

cells was caused by the same cellular factor, coinfection experiments

using high concentration of WT to saturate the restriction factor were

performed. FrhL2 cells were infected with high concentrations (10 ng

p24) of LacZ-expressing WT and coinfected with increasing amounts of

HM.1-expressing GFP. As demonstrated in Figure 1b, we observed that

coinfection with WT increased GFP expression from HM.1 in FrhL2 cells,

indicating that restriction of WT and HM.1 in simian cells was indeed

caused by the same restriction factor. Coinfection experiments using WT

and HM.1 in CV-1 cells confirmed these data (data not shown).


Chapter 2 43

Figure 1. Transduction of human and simian cells by the HIV-1-based LVs

that contain mutations in the CyPA-binding region. (a) 293T ( ), CV-1 ( ),

and FrhL2 ( ) cells were inoculated with increasing concentrations of LV and the

efficiency of infection was analyzed by FACS. Results are representative of three

independent experiments. (b) FrhL2 were inoculated with increasing amounts of

GFP expressing HM.1 alone ( ) or in combination with 10 ng p24 of WT

expressing LacZ ( ). The infectivity of HM.1 was analyzed by FACS. (c) 293T

cells were inoculated with 5 ng p24 of LV in the absence or presence of CsA

(1 µg/ml). Five days after inoculation, transduction efficiencies were analyzed by

FACS. Results are given as percentage of the untreated control. Results are

representative of three independent experiments.


In our previous study,19 we observed that WT infection of human cells

was inhibited by cyclosporine A (CsA) treatment, indicating a

dependence on CyPA for early post-entry steps in human cells.

Furthermore, we observed that resistance to the inhibitory factor in

simian cells coincided with CyPA-independent infection in human cells.

To analyze whether the infection competence of mutant LVs in simian

cells coincided again with CyPA dependence in human cells, 293T cells

were infected with an inoculum of 5 ng p24 of WT, gB, or HM.1–3 in the

presence or absence of CsA (1 µg/ml). Compared to WT, HM.1 was

sensitive to CsA treatment and an inhibition of approximately 50% in

the infectivity was observed (Figure 1c). HM.2 and HM.3 were relatively

insensitive to CsA treatment and showed comparable infectivity to gB

(Figure 1c). These observations demonstrated that unrestricted infection

of HIV-1 group M LVs in simian cells coincides with CypA-independent

infection in human cells.

CyPA-independent infection of chimeric HIV-1 group O LV is not

associated with unrestricted infection of simian cells

To analyze whether CyPA-independent infection of HO.1 virus was

associated with unrestricted infection of simian cells, we constructed a

chimeric HIV-1 group O LV containing the CyPA-binding region of the

HIV-1 group O variant MVP-5180 (HO.1) (Table 1). First, we confirmed

that HO.1 was indeed able to infect human cells independent of CyPA by

showing a lack of effect of the CyPA inhibitor CsA (1 µg/ml) on infection

of 293T cells (Figure 2a). Next, infectivity of HO.1 was analyzed in

simian CV-1 and FrhL2 cells. In contrast to our previous observations in

HIV-1 group M LVs, the CyPA-independent phenotype of HO.1 was not

associated with unrestricted infection in simian cells and reduced

infectivity was observed in CV-1 and FrhL2 cells (Figure 2b). To analyze

whether infection of simian cells with HO.1 was restricted by the same

cellular factor as WT, coinfection experiments were performed to

saturate the inhibitory cellular factor using WT or HO.1-expressing LacZ.

We observed that GFP expression from both HO.1 and WT could be

enhanced by coinfection with WT or HO.1 in FrhL2 (Figure 2c) and CV-1

cells, respectively (data not shown), indicating that the same cellular

factor is involved in the post-entry restriction of both LVs in simian cells.








Chapter 2 45

Figure 2. Transduction of human and simian cells by the HIV-1-based LVs

that contain mutations in the CyPA-binding region derived from HO.1

viruses. (a) 293T cells were inoculated with increasing concentrations of HO.1 in

the presence ( ) or absence ( ) of CsA (1 µg/ml) and infectivity was analyzed by

FACS. (b) 293T ( ), CV-1 ( ), and FrhL2 ( ) were inoculated with increasing

concentrations of HO.1, and the efficiency of infection was analyzed by FACS. (c)

Left panel: FrhL2 cells were inoculated with increasing amounts of GFP expressing

HO.1 alone ( ) or in combination with 10 ng p24 of WT expressing LacZ ( ).

Right panel: FrhL2 cells were inoculated with increasing amounts of GFP

expressing WT alone ( ) or in combination with 10 ng p24 of HO.1 expressing

LacZ ( ). Infection efficiency was analyzed by FACS. Results are representative

of three independent experiments. (d) 293T ( ), CV-1 ( ), and FrhL2 ( ) were

inoculated with increasing concentrations of HO.2 and HO.3, and the efficiency of

infection was analyzed by FACS. Results are representative of three independent

experiments.


In this study, we observed that amino acids proximal to the CyPA-

binding residues (glycine at position 89 and proline at position 9016,18)

are critical in the observed restriction in simian cells and that amino-acid

changes at these positions can circumvent restriction. To analyze

whether the post-entry restriction in simian cells of HO.1 in simian cells

could also be reversed by mutations at these positions, additional

mutant LVs were constructed. When the proline at position 86 of HO.1

was replaced by a valine (HO.2) (Table 1), we observed that this LV was

still restricted in simian cells comparable to the parental LV (Figure 2d).

However, when we additionally replaced methionine at position 88 with

a proline (HO.3) (Table 1), the LV became resistant to the inhibitory

factor and were able to infect CV-1 and FrhL2 efficiently (Figure 2d).

Resistance of HIV-1-based LVs from restriction in simian cells by

the CyPA-binding region of HIV-2

To analyze whether the CyPA-independent replication of HIV-2 was

associated with an unrestricted infection of simian cells, the region in

the capsid protein of HIV-2ROD corresponding to the CyPA-binding region

was placed into the HIV-1-based LV (H2.1) (Table 1). H2.1 had reduced

infectivity in human cells compared with WT. This reduced infectivity

was not due to the presence of restricting cellular factors present in

293T cells, as coinfection experiments using high concentrations of H2.1

or WT-expressing LacZ did not increase GFP expression from H2.1 (data

not shown). When 293T cells were infected with increasing

concentration of H2.1 in the presence of CsA, we indeed observed that

infection was independent of CyPA (Figure 3a). Next, we analyzed

whether H2.1 was able to infect simian cells efficiently. 293T, CV-1, and

FrhL2 cells were infected with increasing concentrations of H2.1, and

GFP expression was analyzed at day 5. H2.1 efficiently infected simian

cells (Figure 3b), indicating that the LV containing the CyPA-binding

region of HIV-2 escapes the restriction in simian cells.










Chapter 2 47 Figure 3. Transduction of

human and simian cells by

the HIV-1-based LVs that

contain mutations in the

CyPA-binding region derived

from HIV-2 viruses. (a) 293T

cells were inoculated with

increasing concentrations of

H2.1 in the presence ( ) or

absence ( ) of CsA (1 µg/ml)

and infectivity was analyzed by

FACS. (b) 293T ( ), CV-1 ( ),

and FrhL2 ( ) were inoculated

with increasing concentrations

of H2.1 and the efficiency of

infection was analyzed by

FACS. Results are

representative of three

independent experiments. (c)

293T ( ), CV-1 ( ), and FrhL2

( ) were inoculated with

increasing concentrations of

H2.2, H2.3, H2.4, and H2.5 and

the efficiency of infection was

analyzed by FACS. (d) Left

panel: FrhL2 cells were

inoculated with increasing

amounts of GFP expressing

H2.5 alone ( ) or in

combination with 10 ng p24 of

WT expressing LacZ ( ). Right

panel: FrhL2 cells were

inoculated with increasing

amounts of GFP expressing WT

alone ( ) or in combination with

10 ng p24 of H2.5 expressing

LacZ ( ). Infection efficiency

was analyzed by FACS. Results

are representative of three

independent experiments.


To analyze whether mutations at amino-acid positions proximal to the

CyPA-binding site (position 89 and 90) were critical in the escape of

H2.1 from post-entry restriction in simian cells, additional mutations

were introduced in the CyPA-binding region of the capsid protein (H2.2,

H2.3, H2.4, and H2.5) (Table 1) and infectivity was analyzed in 293T,

CV-1, and FrhL2 cells. When the proline at position 88, or the isoleucine

at position 87 were replaced respectively by an alanine (H2.2) or a

histidine (H2.3), the phenotype of the LV did not change and high

infectivity for both mutants was observed in CV-1 and FrhL2 (Figure 3c).

Even when both amino acids were replaced simultaneously (H2.4), CV-1

and FrhL2 cells were infected with efficiency comparable to infection of

293T cells (Figure 3c). The HIV-2 CyPA-binding region lacks one amino

acid compared with WT. To analyze whether the length of the CyPA-

binding region is important for the non-restricted phenotype observed

for H2.1–H2.4, an additional valine was introduced (H2.5). When H2.5

was used to infect 293T cells, we observed that the virus had increased

infectivity and infectious titer compared with HO.1 and HO.2.

Furthermore, we observed that H2.5 infection was independent of CyPA,

as CsA treatment did not affect infectivity in 293T cells (data not

shown). Next, we analyzed whether H2.5 was able to infect simian cells.

Compared with infection of 293T cells, infection of CV-1 and FrhL2 was

reduced (Figure 3c), indicating that H2.5 was restricted in simian cells.

To confirm that H2.5 was restricted by the same cellular factor

restricting WT, coinfection experiments were performed. We observed

that GFP expression from either H2.5 or WT was enhanced by

coinfection with respectively WT or H2.5 in FrhL2 (Figure 3d) and CV-1

cells (data not shown), indicating that the similar cellular factor is

responsible for the observed post-entry restriction in simian cells.

Role of CyPA in the post-entry restriction of LVs in simian cells

Here, we observed that the CyPA-binding region of the capsid protein is

involved in the post-entry restriction observed in simian cells. To

analyze whether CyPA plays a role in post-entry restriction, we analyzed

whether CsA treatment had an effect on viral infectivity in simian cells.

FrhL2 and CV-1 cells were treated with CsA (1 µg/ml) and subsequently

infected with increasing concentrations of WT, HO.1, HO.2, H2.5, or the

non-restricted H2.1. Fluorescence-activated cell sorter (FACS) analysis

revealed that CsA treatment of FrhL2 cells increased infection of WT and






Chapter 2 49

HO.1, but the block in infection could not be overcome (Figure 4).

However, CsA treatment of FrhL2 cells was able to overcome the

restriction for HO.2 and H2.5, indicating that CyPA might be involved in

the post-entry restriction observed in simian cells. No effect of CsA

treatment was observed on the infectivity of H2.1. Similar results were

observed in CV-1 cells (data not shown).

Figure 4. Effect of CsA treatment on LV transduction of simian cells. FrhL2

cells were inoculated with increasing concentrations of WT, HO.1, H2.1, HO.2, and

H2.5 in the presence ( ) or absence ( ) of CsA (1 µg/ml) and infectivity was

analyzed by FACS. Results are representative of three independent experiments.


50 Transduction of simian cells by HIV-1 based vectors Efficient transduction of rhesus macaque peripheral blood

mononuclear cell (PBMC) by the HIV-1-based LV containing the CyPA-

binding region of HIV-2

We observed efficient transduction of simian cells by several HIV-1-

based vectors in FrhL2 and CV-1 cells. To analyze whether these mutant

LV were also able to transduce primary rhesus macaque cells, the

transduction efficiency of gB, HM.2, and H2.1 in PBMC obtained from

rhesus macaque and human was compared. PBMCs obtained from two

rhesus macaques and two human donors were inoculated at a

multiplicity of infection of 10 for gB and HM.2 and 5 for H2.1. For

comparison, rhesus macaque PBMC and human PBMC were inoculated

with WT and HO.1 at a multiplicity of infection of 10. At day 6 post-

inoculation, FACS analysis revealed that all HIV-1-based LVs efficiently

transduced human PBMC (Figure 5). As expected, rhesus macaque

PBMC were resistant to transduction with WT and HO.1. However, gB

and HM.2, which were able to transduce FrhL2 and CV1 cells efficiently,

were restricted in primary rhesus macaque PBMC (Figure 5). In contrast,

transduction of H2.1 in rhesus macaque PBMC was not restricted (Figure

5), indicating that this vector is able to circumvent the antiviral activity

present in primary rhesus macaque cells.

Figure 5. Transduction of primary rhesus macaque PBMC by LVs. PBMC

obtained from two human donors and two rhesus macaques were inoculated with

WT, gB, HM.2, HO.1, and H2.1 and infectivity was analyzed by FACS. Results are

representative of two independent experiments.






Chapter 2 51

Discussion

HIV-1-based LVs have previously been demonstrated to be restricted in

simian cells,4,5,6,7,8 and this limits the use of Old World monkey models

for long-term safety studies of the vector. Recently, it was observed that

mutations in the CyPA-binding region of the gag capsid protein increases

the transduction efficiency of HIV-1-based LVs in simian cells.19,33,34,35 In

this study, we analyzed the effect of mutations in the CyPA-binding

region of HIV-1 group M, HO.1, or HIV-2 in the background of a HIV-1

group M-based LV on infectivity in simian cells and sensitivity to CsA

treatment of human cells. Structural analysis of the CyPA–capsid

proteins containing the WT CyPA-binding region demonstrated that the

central proline at position 90 together with the glycine at position 89 are

critical for CyPA binding.16,18,39 In addition, residues in close proximity to

the central proline contribute to the binding interaction.31,40,41 Based on

our results, mutant LVs could be divided into three groups (Table 2). WT

and HM.1 belong to the first group that are sensitive to CsA treatment of

human cells and are restricted in simian cells. According to the available

structural data, these LVs have a high affinity for CyPA. Replacement of

the isoleucine at position 91 by a valine, as in HM.1, had no effect on

the CyPA–capsid interaction.40

LVs in the second group (Table 2) are not restricted in simian cells and

are able to infect human cells in a CyPA-independent manner. In gB,

HM.2, and HO.3, the histidine at position 87 is replaced with a glutamine

or an alanine. Computational and theoretical studies have suggested

that the histidine at position 87 makes energetically favorable contacts

with CyPA.42 Therefore, replacement of this residue might interfere with

the CyPA–capsid interaction. Indeed, a fivefold reduction in the affinity

for CyPA was observed when the histidine was replaced by a glutamine

or an alanine.40 HM.3 still contains the histidine at position 87; however,

the alanine at position 88 is replaced by a proline. The methyl group of

the alanine at position 88 also makes energetically favorable contacts

with CyPA and replacement of this alanine reduced the affinity for

CyPA.40 Also belonging to this group are LVs that contain the CyPA-

binding region of HIV-2 (H2.1–4), which is one amino acid shorter

compared with WT. The length of the CyPA-binding loop has previously

been described to be critical for the interaction between the capsid

protein and CyPA, and the lack of a single amino acid might interfere























with the capsid–CyPA interaction.43 This is in agreement with the

observation that HIV-2 virions are unable to incorporate CyPA.23 These

data imply that the CyPA-binding region in the second group of

recombinant LVs share a low binding affinity for CyPA.

Table 2 - Mutant LV can be divided into three different groups

based on the infectivity in simian cells and CsA-treated human

cells.

Group 1 Group 2 Group 3

Restriction in simian cells Yes No Yes

CyPA dependence Yes No No

Capsid/CyPA interaction High40

Low40,43

Intermediate40

CyPA-binding loop WT: VHAGPIAP gB: VQPGPVAP HO.1: PAMGPLPP

HM.1: VHAGPVAP HM.2: VQAGPVAP HO.2: VAMGPLPP

HM.3: VHPGPIAP H2.5: VHAGPLPP

HO.3: VAPGPLPP

H2.1: -IPGPLPP

H2.2: -IAGPLPP

H2.3: -HPGPLPP

H2.4: -HAGPLPP

CsA, cyclosporine A; CyPA, cyclophilin A; LV, lentiviral vector; WT, wild type.

The third group has an intermediate phenotype and these LVs are

insensitive to CsA treatment of human cells, but are still restricted in

simian cells. The CyPA-binding region of HO.1 virus is very distinct from

WT. Although the central glycine and proline are still present, the

surrounding amino acids differ from the WT sequence of the CyPA-

binding region. Previously, it was demonstrated that the HO.1 CyPA-

binding region was able to interact with CyPA although the binding was

somewhat less tight compared with WT.31,40 HO.2 and H2.5 behave

similarly in the infection experiments compared with HO.1; therefore,

we predict that these mutant LVs also have an intermediated binding

affinity for CyPA. Based on the available structural data, we speculate

that a reduction in the binding affinity of capsid for CyPA is associated

with non-restricted infection of simian cells (Table 2).



https://literatuur.amc.nl/http/www.nature.com/mt/journal/v15/n5/fig_tab/6300091t2.html#figure-title






Chapter 2 53

Interestingly, we observed that HO.2 and H2.5 were able to escape

restriction completely in simian cells by CsA treatment. This suggests a

direct role for CyPA in Trim5α-mediated inhibition in simian cells, which

is in agreement with earlier reports.28,29,44,45 In these studies, it was

observed that CyPA downregulation using siRNA or disruption of the

CyPA–capsid interaction by CsA enhanced infectivity of HIV-1 in simian

cells. Concomitantly, we observed that reduction of binding affinity of

the HIV-1 capsid for CyPA is associated with unrestricted infection of

simian cells, which also indicates that CyPA is required for restriction of

HIV-1 in Old World monkey cells.

CsA-dependent virus replication has been described previously.17,43,46

Braaten et al. observed that replication of HIV-1 in which the alanine at

position 92 was substituted by a glutamic acid or when glycine at

position 94 was replaced by an aspartic acid became CsA-dependent in

CEM and Hela CD4 cells.17,4646,47 A similar observation was made by

Bukovsky et al.43 for SIVmac that contained the HIV-1 CyPA-binding

region up to position 90. The described CsA-dependent mutants

contained mutations in the region distal to the proline at position 90 and

it was suggested that mutation of amino-acid residues involved in the

formation of the type II tight turn results in CsA-dependent virus

replication.17,43,46 The HO.2 and H2.5 LV also contain mutations distal to

the proline at position 90. However, CsA-dependent infection in simian

cells was not observed when an additional proline was present proximal

to the proline at position 90 (HO.1 and HO.3) or when the CyPA-binding

region was one amino acid shorter (H2.1-4). This indicates that amino

acids proximal to the CyPA-binding region can also influence CsA-

dependent infection.

Recently, it was observed that HIV-2 was restricted by Trim5 from

rhesus macaques.8,14 We observed that the HIV-2 CyPA binding loop was

able to convert the HIV-1 group M LV from a sensitive to a resistant

virus in rhesus macaque and African green monkey cells. Although our

data suggest that the CyPA binding region is involved in the observed

restriction of group M HIV-1 in simian cells, it is not the primary

determinant involved in the restriction of HIV-2. We observed that

mutations in the CyPA-binding region can affect the infectious titer of

LVs in human cells. In particular, reduced infectivity was observed for

LVs that contains a HIV-2-like CyPA-binding region (H2.1-H2.4), which


















is one amino acid shorter compared with the other LVs. The reduced

infectivity of these LVs was not due to the presence of saturable

inhibitory factors as coinfection experiments did not increase the

transduction efficiency. Therefore, it is more likely to assume that the

short CyPA-binding region might affect the packaging and assembly

process.

The transduction efficiency of gB, HM.2, and H2.1 was also evaluated in

primary rhesus macaque PBMC. In contrast to our observations in FrhL2

and CV1 cells, only H2.1 was able to transduce rhesus macaque PBMC at

levels comparable to human PBMC. Previously, we observed that the gB

mutant LV was able to transduce hematopoietic stem cells obtained from

baboon efficiently,19 whereas this LV was restricted in primary rhesus

macaque PBMC. Therefore, it cannot be excluded that other species-

specific differences in cellular factors or cellular factors whose

expression is dependent on the differentiation state of the cell

population might be involved in the observed restriction of gB and HM.2

in rhesus macaque PBMC.

A major obstacle for gene therapy studies in non-human primates is the

inefficient transduction owing to the presence of inhibitory factors like

Trim5 . Therefore, the LVs in this study might be very useful to conduct

long-term gene therapy studies in non-human primate models like

rhesus macaques.

Materials and Methods

Cell cultures and production of HIV-1-based LVs. Human 293T

cells, the African green monkey kidney cell line CV-1, and the rhesus

macaque fibroblast line FrhL2 were cultured in Dulbecco's modified

Eagle's medium supplemented with 10% fetal bovine serum (Hyclone,

Logan, UT), antibiotic/antimycotic (Life Technologies, Rockville, MD).

The HIV-1-based LV pseudotyped with vesicular stomatitis virus

glycoprotein envelope was used to analyze the effect of mutations in the

capsid protein on the infectivity in 293T, CV-1, and FrhL2 cells. The LV

production system consists of four constructs: LV-construct, which


Chapter 2 55

produces the viral RNA containing the cDNA sequence of GFP or LacZ

under control of a cytomegalovirus promoter; the packaging construct

encoding for gag/pol (packaging construct); the vesicular stomatitis

virus glycoprotein-expressing construct (pcytomegalovirus–vesicular

stomatitis virus glycoprotein); the HIV-1 rev-expressing construct

(pRous sarcoma virus–rev).37 LVs were produced by transient

transfection in 293T cells using the calcium phosphate method, as

described previously.37 Infectious LV was harvested at 48 and 72 h post-

transfection and filtered through 0.22-µm-pore cellulose acetate filters.

The infectious LVs were concentrated by ultra centrifugation (2 h at

50,000 g) and subsequently purified by ultra centrifugation on a 20%

sucrose gradient (2 h at 46,000 g).

Vector concentrations were analyzed using an immunocapture p24-gag

enzyme-linked immunosorbent assay (Alliance; DuPont-NEN). To

determine the infectious titer, 293T cells were plated in 24-well plates at

a density of 1×105 cells/well and transduced with serial dilutions of LV.

Four days after inoculation, transduction efficiency was analyzed by

FACS.

Construction of HIV-1-based LVs containing mutant capsid

proteins.Capsid mutants were created by a site-directed mutagenesis

(Stratagene, La Jolla, CA) of the packaging construct. The mutant

constructs were subsequently sequenced to confirm the presence of

mutations and exclude introduction of unwanted mutations. Mutant

packaging constructs were used to produce LVs as described above.

Infection assays. 293T, CV-1, and FrhL2 cells were plated in 24-well

plates at a density of 1×104 cells/well 24 h before inoculation. Cells

were inoculated with different amounts of LV as indicated in the results.

The number of infected cells was quantified by FACS analysis 4–6 days

after inoculation. Where indicated, cells were treated with CsA (1 µg/ml;

Sigma, St Louis, MO) starting 1 h before inoculation. In the coinfection

experiments, 293T, CV-1, and FrhL2 cells were inoculated with 10 ng

p24 of the LacZ-expressing LV containing the WT or mutant capsid

protein and indicated concentrations of GFP-expressing LV containing

WT or mutant capsid proteins. The effect of the coinfection on the

infectivity of the WT or mutant LV was analyzed by FACS 4–6 days after

inoculation.




PBMC were isolated from rhesus macaque blood or human buffy coat

using ficoll density gradient centrifugation. PBMC were inoculated with

GFP-expressing LV at a multiplicity of infection of 5 or 10 for 16 h.

Subsequently, cells were washed and cultured in Iscove's modified

Dulbecco's medium supplemented with 10% fetal calf serum (Hyclone,

Logan, UT), 1 µg/ml phytohemagglutinin, antibiotic/antimycotic (Life

Technologies, Rockville, MD) at a density of 1×105 per 100 µl. The

percentage of GFP-expressing cells was determined by FACS analysis 6

days after inoculation.

Acknowledgements

NAK is supported by The Netherlands Organization for Scientific

Research (NWO VENI Grant no. 916.36.024). MANR is supported by the

Landsteiner foundation of blood transfusion research (LSBR Grant no.

0435). This work was initiated in the lab of Professor Inder Verma at the

Salk Institute and we are very grateful for his support and critical

reading of the manuscript. We thank Nina Tonnu for excellent technical

support and Professor Hanneke Schuitemaker for helpful discussions and

critical reading of this paper.

References

1. Hacein-Bey-Abina, S, von Kalle, C, Schmidt, M, Le Deist, F, Wulffraat, N and

McIntyre, E et al. (2003). A serious adverse event after successful gene therapy

for X-linked severe combined immunodeficiency. N Engl J Med 348: 255–256.

2. Hacein-Bey-Abina, S, Von Kalle, C, Schmidt, M, McCormack, MP, Wulffraat, N and

Leboulch, P et al. (2003). LMO2-associated clonal T cell proliferation in two

patients after gene therapy for SCID-X1. Science 302: 415–419.

3. Woods, NB, Bottero, V, Schmidt, M, von Kalle, C and Verma, IM (2006). Gene

therapy: therapeutic gene causing lymphoma. Nature 440: 1123.

4. Hofmann, W, Schubert, D, LaBonte, J, Munson, L, Gibson, S and Scammell, J et

al. (1999). Species-specific, postentry barriers to primate immunodeficiency virus

infection. J Virol 73: 10020–10028.

5. Besnier, C, Takeuchi, Y and Towers, G (2002). Restriction of lentivirus in

monkeys. Proc Natl Acad Sci USA 99: 11920–11925. |

6. Cowan, S, Hatziioannou, T, Cunningham, T, Muesing, MA, Gottlinger, HG and

Bieniasz, PD (2002). Cellular inhibitors with Fv1-like activity restrict human and

simian immunodeficiency virus tropism. Proc Natl Acad Sci USA 99: 11914–

11919.

Chapter 2 57

7. Münk, C, Brandt, S M, Lucero, G and Landau, NR (2002). A dominant block to

HIV-1 replication at reverse transcription in simian cells. Proc Natl Acad Sci USA

99: 13843–13848.

8. Hatziioannou, T, Cowan, S, Goff, SP, Bieniasz, PD and Towers, GJ (2003).

Restriction of multiple divergent retroviruses by Lv1 and Ref1. EMBO J 22: 385–

394.

9. Stremlau, M, Owens, C M, Perron, M J, Kiessling, M, Autissier, P and Sodroski, J

(2004). The cytoplasmic body component TRIM5 alpha restricts HIV-1 infection in

Old World monkeys. Nature 427: 848–853.

10. Hatziioannou, T, Perez-Caballero, D, Yang, A, Cowan, S and Bieniasz, P D (2004).

Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5

alpha. Proc Natl Acad Sci USA 101: 10774–10779.

11. Keckesova, Z, Ylinen, L M and Towers, G J (2004). The human and African green

monkey TRIM5 alpha genes encode Ref1 and Lv1 retroviral restriction factor

activities. Proc Natl Acad Sci USA 101: 10780–10785.

12. Perron, M J, Stremlau, M, Song, B, Ulm, W, Mulligan, RC and Sodroski, J (2004).

TRIM5alpha mediates the postentry block to N-tropic murine leukemia viruses in

human cells. Proc Natl Acad Sci USA 101: 11827–11832.

13. Yap, MW, Nisole, S, Lynch, C and Stoye, JP (2004). Trim5 protein restricts both

HIV-1 and murine leukemia virus. Proc Natl Acad Sci USA 101: 10786–10791.

14. Ylinen, LM, Keckesova, Z, Wilson, SJ, Ranasinghe, S and Towers, GJ (2005).

Differential restriction of human immunodeficiency virus type 2 and simian

immunodeficiency virus SIVmac by TRIM5 alpha alleles. J Virol 79: 11580–

11587.

15. Luban, J, Bossolt, KL, Franke, EK, Kalpana, GV and Goff, SP (1993). Human

immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. Cell 73:

1067–1078.

16. Franke, EK, Yuan, HE and Luban, J (1994). Specific incorporation of cyclophilin A

into HIV-1 virions. Nature 372: 359–362.

17. Braaten, D, Aberham, C, Franke, EK, Yin, L, Phares, W and Luban, J (1996).

Cyclosporine A-resistant human immunodeficiency virus type 1 mutants

demonstrate that Gag encodes the functional target of cyclophilin A. J Virol 70:

5170–5176.

18. Braaten, D, Franke, EK and Luban, J (1996). Cyclophilin A is required for an early

step in the life cycle of human immunodeficiency virus type 1 before the initiation

of reverse transcription. J Virol 70: 3551–3560.

19. Kootstra, NA, Munk, C, Tonnu, N, Landau, NR and Verma, IM (2003). Abrogation

of postentry restriction of HIV-1-based lentiviral vector transduction in simian

cells. Proc Natl Acad Sci USA 100: 1298–1303.

20. Towers, GJ, Hatziioannou, T, Cowan, S, Goff, SP, Luban, J and Bieniasz, PD

(2003). Cyclophilin A modulates the sensitivity of HIV-1 to host restriction

factors. Nat Med 9: 1138–1143.

21. Chatterji, U, Bobardt, MD, Stanfield, R, Ptak, RG, Pallansch, LA and Ward, PA et

al. (2005). Naturally occurring capsid substitutions render HIV-1 cyclophilin A

independent in human cells and TRIM-cyclophilin-resistant in Owl monkey cells. J

Biol Chem 280: 40293–40300.

22. Gao, F, Bailes, E, Robertson, DL, Chen, Y, Rodenburg, CM and Michael, SF et al.

(1999). Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature

397: 436–441.


23. Braaten, D, Franke, EK and Luban, J (1996). Cyclophilin A is required for the

replication of group M human immunodeficiency virus type 1 (HIV-1) and simian

immunodeficiency virus SIV(CPZ)GAB but not group O HIV-1 or other primate

immunodeficiency viruses. J Virol 70: 4220–4227.

24. Billich, A, Hammerschmid, F, Peichl, P, Wenger, R, Zenke, G and Quesniaux, V et

al. (1995). Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin

A analog with activity against human immunodeficiency virus (HIV) type 1:

interference with HIV protein-cyclophilin A interactions. J Virol 69: 2451–2461.

25. Gao, F, Yue, L, White, AT, Pappas, PG, Barchue, J and Hanson, AP et al. (1992).

Human infection by genetically diverse SIVSM-related HIV-2 in west Africa.

Nature 358: 495–499.

26. Sayah, DM and Luban, J (2004). Selection for loss of Ref1 activity in human cells

releases human immunodeficiency virus type 1 from cyclophilin A dependence

during infection. J Virol 78: 12066–12070.

27. Sokolskaja, E, Sayah, DM and Luban, J (2004). Target cell cyclophilin A

modulates human immunodeficiency virus type 1 infectivity. J Virol 78: 12800–

12808.

28. Sokolskaja, E, Berthoux, L and Luban, J (2006). Cyclophilin A and TRIM5alpha

independently regulate human immunodeficiency virus type 1 infectivity in human

cells. J Virol 80: 2855–2862.

29. Keckesova, Z, Ylinen, LM and Towers, GJ (2006). Cyclophilin A renders human

immunodeficiency virus type 1 sensitive to Old World monkey but not human

TRIM5 alpha antiviral activity. J Virol 80: 4683–4690.

30. Bosco, DA, Eisenmesser, EZ, Pochapsky, S, Sundquist, WI and Kern, D (2002).

Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A.

Proc Natl Acad Sci USA 99: 5247–5252.

31. Howard, BR, Vajdos, FF, Li, S, Sundquist, WI and Hill, CP (2003). Structural

insights into the catalytic mechanism of cyclophilin A. Nat Struct Biol 10: 475–

481.

32. Bosco, DA and Kern, D (2004). Catalysis and binding of cyclophilin A with

different HIV-1 capsid constructs. Biochemistry 43: 6110–6119.

33. Ikeda, Y, Ylinen, LM, Kahar-Bador, M and Towers, GJ (2004). Influence of gag on

human immunodeficiency virus type 1 species-specific tropism. J Virol 78:

11816–11822.

34. Hatziioannou, T, Cowan, S, Von Schwedler, UK, Sundquist, WI and Bieniasz, PD

(2004). Species-specific tropism determinants in the human immunodeficiency

virus type 1 capsid. J Virol 78: 6005–6012.

35. Owens, CM, Song, B, Perron, MJ, Yang, PC, Stremlau, M and Sodroski, J (2004).

Binding and susceptibility to postentry restriction factors in monkey cells are

specified by distinct regions of the human immunodeficiency virus type 1 capsid. J

Virol 78: 5423–5437.

36. Naldini, L, Blomer, U, Gallay, P, Ory, D, Mulligan, R and Gage, FH et al. (1996). In

vivo gene delivery and stable transduction of nondividing cells by a lentiviral

vector. Science 272: 263–267.

37. Dull, T, Zufferey, R, Kelly, M, Mandel, RJ, Nguyen, M and Trono, D et al. (1998).

A third-generation lentivirus vector with a conditional packaging system. J Virol

72: 8463–8471.

Chapter 2 59

38. Zufferey, R, Dull, T, Mandel, RJ, Bukovsky, A, Quiroz, D and Naldini, L et al.

(1998). Self-inactivating lentivirus vector for safe and efficient in vivo gene

delivery. J Virol 72: 9873–9880.

39. Gamble, TR, Vajdos, FF, Yoo, S, Worthylake, DK, Houseweart, M and Sundquist,

WI et al. (1996). Crystal structure of human cyclophilin A bound to the amino-

terminal domain of HIV-1 capsid. Cell 87: 1285–1294.

40. Yoo, S, Myszka, DG, Yeh, C, McMurray, M, Hill, CP and Sundquist, WI (1997).

Molecular recognition in the HIV-1 capsid/cyclophilin A complex. J Mol Biol 269:

780–795.

41. Vajdos, FF, Yoo, S, Houseweart, M, Sundquist, WI and Hill, CP (1997). Crystal

structure of cyclophilin A complexed with a binding site peptide from the HIV-1

capsid protein. Protein Sci 6: 2297–2307.

42. Agarwal, PA (2004). Cis/Trans isomerization in HIV-1 capsid protein catalyzed by

cyclophlin A: insights from computational and theoretical studies. Proteins:

Structure, Function, and Bioinformatics 56: 449–463.

43. Bukovsky, AA, Weimann, A, Accola, MA and Gottlinger, HG (1997). Transfer of

the HIV-1 cyclophilin-binding site to simian immunodeficiency virus from Macaca

mulatta can confer both cyclosporin sensitivity and cyclosporin dependence. Proc

Natl Acad Sci USA 94: 10943–10948.

44. Berthoux, L, Sebastian, S, Sokolskaja, E and Luban, J (2005). Cyclophilin A is

required for TRIM5 {alpha}-mediated resistance to HIV-1 in Old World monkey

cells. Proc Natl Acad Sci USA 102: 14849–14853.

45. Stremlau, M, Song, B, Javanbakht, H, Perron, M and Sodroski, J (2006).

Cyclophilin A: An auxiliary but not necessary cofactor for TRIM5 alpha restriction

of HIV-1. Virology 351: 112–120.

46. Luban, J (1996). Absconding with the chaperone: essential cyclophilin–Gag

interaction in HIV-1 virions. Cell 87: 1157–1159.

47. Aberham, C, Weber, S and Phares, W (1996). Spontaneous mutations in the

human immunodeficiency virus type 1 gag gene that affect viral replication in the

presence of cyclosporins. J Virol 70: 3536–3544.

Chapter 3

Interaction of Cyclophilin A and

Trim5 with the HIV-1 capsid

Maarten A.N. Rits, Karel A van Dort, Linaida Pisas, Corrine

Beugeling and Neeltje A. kootstra


Chapter 3 63

Background: Trim5α is a host restriction factor that is able to block a broad range of retroviruses in a species specific manner. HIV-1 can be efficiently blocked by Trim5α from old world monkeys, whereas human Trim5α only partially inhibits HIV-1 replication. The viral determinant

involved in susceptibility of HIV-1 to Trim5α mediated inhibition is located in the CypA binding region of capsid and mutations in this region are able to abrogate the restriction. Here, we analyzed whether mutations in the CypA binding region of the HIV-1 capsid had an effect

on the interaction between capsid and the cellular factors CypA and Trim5α. Results: Our data demonstrated that in vitro produced HIV-1 capsid/nucleocapsid (CA/NC) protein complexes were able to interact with the cellular factors CypA and Trim5α. Mutations in the CypA binding region of capsid (H87Q, A88P and I91V) that were associated with CypA

independent replication and a reduced sensitivity to Trim5α mediated inhibition did not interfere with binding of CypA and Trim5α. However, a decrease in the affinity of the mutant capsid protein for CypA was observed when intracellular CypA concentrations were limited by

addition of cyclosporin A. Using a so called ‘fate of capsid’ assay, we observed that mutations in the CypA binding region or addition of cyclosporin A prevented destabilization of viral core particles within the first 24 hours after

inoculation.

Conclusions: Our data suggests that CypA is involved in HIV-1 core destabilization, thereby playing an important role in the uncoating

process. Virus particles with mutations in the CypA binding region in the HIV-1 capsid protein that modulate CypA binding are less sensitive to CypA mediated destabilization.

Background The ability of the human immuno deficiency virus (HIV) 1 to efficiently establish an infection is dependent on many host factors that are required during different stages of the viral life cycle. Cyclophilin A (CypA) is an important cofactor for HIV-1 and specifically interacts with residues in a loop between the 4th and 5th alpha helices of the capsid protein,[1,2]. CypA is recruited into the virion during assembly at a

stoichiometry of ~1:10 [3-5]. The interaction between CypA and capsid is most significant for infectivity of the virus in a newly infected cell [6-9].

64 Capsid specific restrictions

The interaction of CypA with the HIV-1 capsid can be inhibited by cyclosporine A (CsA), a compound that competitively interacts with the hydrophobic binding pocket present in CypA, or by mutation of critical residues in the CypA binding region. Blocking the CypA-capsid

interaction results in a reduction of HIV-1 infectivity in most cell lines [8-10]. CypA has gained attention when it was identified as a cofactor that

potentiated restriction by the tripartite containing motif (Trim) 5α from some simian species [11-13]. Most Trim proteins contain a RING, a B-box and a Coiled coil domain, while Trim5α additionally encodes a B30.2 domain [14,15]. Trim5α restricts HIV-1 at an early post-entry

step [16] and it was demonstrated that Trim5α physically interacts with the capsid protein via the C-terminal B30.2 domain [17]. Trim5α exerts its effects in a virus and host specific manner [6,8] and species specific

variation in the carboxyl terminal B30.2 domain largely accounts for the differences in the potency of the restriction of a specific retrovirus [18-21]. Binding assays have demonstrated that the ability of Trim5α orthologues from different species, to associate with the HIV-1 capsid,

correlates with the ability to restrict virus replication [17]. For example, Old world monkey Trim5α strongly associates with the capsid of HIV-1, whereas human Trim5α which moderately restricts HIV-1 replication, only weakly associates with the HIV-1 capsid.

Recently, it was demonstrated that the viral determinant in Trim5α mediated restriction was located in the CypA binding region of the capsid protein of HIV-1 and that mutations in this region can abrogate the restriction [7,13,22-24]. This suggests that there might be a role

for CypA in Trim5α mediated inhibition. Indeed, the interaction between the HIV-1 capsid and CypA was required for Trim5α mediated inhibition of HIV-1 replication in old world monkey cells [11,13,25]. In contrast,

CypA supports HIV-1 replication in human cells and it has been suggested that HIV-1 has recruited CypA to protect itself against the anti-viral activity of human Trim5α. More recent studies using siRNA to knock-down CypA and Trim5α indicated that Trim5α and CypA in

human cells independently regulated viral infectivity [11,26]. However, mutations in the CypA binding region of the HIV-1 capsid that are associated with CypA independent replication also mediate protection from human Trim5α [24].

In this report, we studied the association between HIV-1 capsid and the cellular factors CypA and Trim5α using in vitro produced and assembled capsid-like particles and pull-down assays.

Chapter 3 65

Results Binding of CypA and Trim5α to HIV-1 Capsid Previously it has been demonstrated that substitutions in the CypA

binding region of the capsid protein render HIV-1 replication independent of CypA. In addition, these mutations may reduce the sensitivity of the virus to Trim5α mediated inhibition [7,13,22-24]. Here we analyzed whether mutations in the CypA binding region might

influence the interaction between the viral capsid and the cellular proteins CypA and Trim5α. CA/NC complexes that contained either the wild type CypA binding region or a mutant CypA binding region with 3

amino acid substitutions (H87Q, A88P and I91V), were covalently coupled to CNBr-activated sepharose and used to pull down CypA and Trim5α from cellular lysates of 293T cells.

Endogenous CypA was pulled down from the cell lysates by both wild type and mutant CA/NC complexes (figure 1). The interaction between CypA and the CA/NC complexes was specific and the amount of capsid bound CypA could be reduced by addition of Cyclosporin A (CsA) (figure

1). The addition of CsA to the lysates inhibited the interaction of CypA with mutant CA/NC more efficiently than the interaction with wild type CA/NC, suggesting that the reduced affinity of the mutant CA/NC for CypA becomes apparent only when the amount of CypA is reduced.

Figure 1: CypA interacts with wild type and mutant HIV-1 CA/NC complexes.

In vitro produced CA/NC complexes that contained either the wild type CypA binding region or mutant CypA binding region with 3 amino acid substitutions (H87Q, A88P, I91V), were incubated with lysates of 293T cells in the presence or absence of CsA (1 μg/ml). The interaction of CypA and the CA/NC complexes was analyzed by SDS-PAGE and Western blotting.

Next we analyzed whether mutations in the CypA binding region of capsid interfered with the interaction of capsid and Trim5α. In vitro produced wild type and mutant CA/NC complexes that were coupled to


CNBr-activated sepharose beads were incubated with cell lysates of 293T cells expressing HSV-tagged Trim5α. Trim5α efficiently interacted with both wild type and mutant CA/NC. When wild type and mutant CA/NC complexes were incubated with cell lysates expressing myc-

tagged Trim5γ, no detectable levels of Trim5γ were pulled down by either wild type or mutant CA/NC complexes (figure 2). This indicates that Trim5α is specifically pulled down by wild type and mutant CA/NC complexes and confirms previous observations that the B30.2 domain of

Trim5α is required for the interaction with CA/NC complexes [17].

Figure 2: Trim5α interacts with wild type and mutant HIV-1 CA/NC complexes through the B30.2 domain. In vitro produced CA/NC complexes that contained

either the wild type CypA binding region or mutant CypA binding region with 3 amino acid substitutions (H87Q, A88P, I91V), were incubated with 293T cell lysates expressing HSV-tagged Trim5α, myc-tagged Trim5γ or both. The ability of wild type and mutant CA/NC complexes to pull down Trim5α and Trim5γ from the cell lysate was analyzed by SDS-PAGE and Western blotting.

Trim5γ over-expression was reported to reduce Trim5α mediated restriction of HIV-1 replication [16]. Co-expression of HSV-tagged Trim5α and myc-tagged Trim5γ had no effect on Trim5α binding to wild type or mutant CA/NC in the pull-down assay (figure 2), suggesting that inhibition of Trim5α mediated restriction by Trim5γ is not due to reduced binding of Trim5α to the HIV-1 capsid protein in the presence of Trim5γ.

Chapter 3 67

Low competitive binding of CypA and Trim5α to CA/NC Recently it has been demonstrated that mutations in the CypA binding region of the HIV-1 capsid reduce the sensitivity of the virus to Trim5α mediated inhibition [7,13,22-24]. This suggests that Trim5α and CypA

might compete for binding to the HIV-1 capsid. Therefore we tested whether the interaction between Trim5α and CA/NC complexes could be modulated by the addition of CsA which prevents CypA binding to the CA/NC complexes. In vitro produced wild type and mutant CA/NC

complexes were incubated with lysates of 293T cells expressing HSV-tagged Trim5α in the presence or absence of 1 μg/ml CsA. An efficient interaction between Trim5α and both wild type and mutant CA/NC complexes was observed. Addition of CsA only resulted in a slight

increase in Trim5α (monomeric and dimeric) binding to wild type as well as mutant CA/NC complexes (figure 3). This suggests that there is no strong competition between CypA and Trim5α for binding to wild type

and mutant CA/NC complexes, albeit that CypA may be able to slightly hinder recognition of the capsid protein by Trim5α.

Figure 3: CypA does not interfere with the interaction between Trim5α and HIV-1 capsid. In vitro produced CA/NC complexes that contained either the wild

type CypA binding region or mutant CypA binding region with 3 amino acid substitutions (H87Q, A88P, I91V), were incubated with 293T cell lysates expressing HSV-tagged Trim5α in the presence or absence of CsA (1 μg/ml). The ability of wild type and mutant CA/NC complexes to pull down Trim5α in the presence or absence of CsA was analyzed by SDS-PAGE and Western blotting.


CypA enhances disassembly of HIV-1 cores The stability of a retroviral capsid during cellular entry, in response to cellular factors such as Trim5α or CypA, is very important for efficient infection [12,30]. Previously, it was demonstrated that binding of

Trim5α to the retroviral capsid of a restriction sensitive virus induced destabilization of the viral core [17,30]. Moreover, substitution of the histidine for a glutamine at position 87 in capsid resulted in higher stability of the core particle. Here, we analyzed the role of CypA in the

destabilization of wild type and mutant viral cores using the previously described fate of capsid assay [17]. HeLa cells were inoculated with VSV-G pseudotyped wild type and mutant lentiviral vectors (LV; 37.5 μg p24 in 15×106 cells) in the presence or absence of 1μg/ml CsA. Twenty-

four hours after infection, cells were harvested and washed 3 times with PBS. Cytosolic extracts were prepared and intracellular core particles were pelleted through a 50% sucrose cushion and analyzed by Western

blotting. High concentrations of capsid proteins of the mutant LV could be detected in the pellet, whereas only limited amounts of wild type capsid proteins were detected even after overexposure of the blots (figure 4).

This indicated that mutant viral cores are more resistant to destabilization as compared to wild type cores, confirming previous observations [17].

Figure 4: Effect of CypA on the stability of wild type and mutant HIV-1 core particles after infection. HeLa cells were inoculated with wild-type LV or mutant

LV that contained 3 amino acid substitutions (H87Q, A88P, I91V) in the CypA binding region in the presence or absence of CsA (1 μg/ml). 24 hours later, the cells were lyzed and sedimented through a 55% sucrose gradient to pellet the intracellular particulate capsid. The pellet was resuspended in sample buffer and analyzed for the presence of capsid proteins by SDS-PAGE and western blotting using an anti-p24 antibody.

Chapter 3 69

Moreover, when CsA was added to the cultures during infection, an increased amount of mutant capsid was observed in the pellet fraction (figure 4). After overexposure of the blot, a similar increase in the amount of capsid was observed in cells inoculated with the wild type LV

in the presence of CsA. This data indicate that CypA is involved in the destabilization of the viral core early after virus entry and that mutations in the CypA binding region of the capsid protein make the virus less sensitive to CypA mediated destabilization.

As a control we added ammonium chloride (30mM NH4Cl) to the cells to inhibit endosome acidification and prevent subsequent entry of the VSV-G pseudotyped HIV-1 into the cytoplasm of the cells. FACS analysis of NH4Cl treated cells demonstrated complete inhibition of infection (data

not shown). However, we did observe capsid protein in the pelleted NH4Cl treated cells, which could be the result of endosome rupture during cell lysis. Notably, equal amounts of wild-type and mutant core

particles could be detected in the pellet fraction of the NH4Cl treated cells, indicating that the particles do not differ in intrinsic stability.

Discussion Trim5α is a cellular restriction factor that targets the capsid of incoming retroviruses in a species-specific manner [16,31,32]. The CypA binding region of the HIV-1 capsid protein has been identified as an important viral determinant in Trim5α mediated inhibition and amino acid substitutions in this region modulate sensitivity to Trim5α mediated

inhibition [6-9,11-13,22-24]. This also suggested that CypA might be involved in Trim5α mediated inhibition. Indeed, CypA was demonstrated to be involved in Trim5α mediated inhibition of retroviruses in old world monkey cells [11,13,25]. In human cells, Trim5α and CypA were shown

to be independent regulators of viral infectivity [11,26]. However, mutations in the CypA binding region of the HIV-1 capsid also seem to interfere with sensitivity of the virus to inhibition by Trim5α in human cells [24]. Here we analyzed whether the interaction between the viral

capsid and the cellular proteins Trim5α and CypA is affected by mutations in the CypA binding loop (H87Q, A88P and I91V). CypA was able bind efficiently to both wild-type and mutant CA/NC complexes, and addition of CsA could prevent this interaction. The

interaction between the mutant CA/NC and CypA appeared to be more sensitive to CsA treatment indicating that the mutations in the CypA binding region of capsid might be associated with decreased affinity for

CypA. This is in agreement with computational and theoretical studies which have suggested that a histidine at position 87 makes favourable contacts with CypA [33]. Indeed, a five-fold reduction in affinity was observed when this residue was replaced by a glutamine [34].


Trim5α was also able to interact with both the wild type and mutant CA/NC complexes, indicating the mutations in the CyPA binding loop do not abrogate Trim5α binding. Furthermore we observed that wild type and mutant CA/NC structures were unable to interact with Trim5γ, a

Trim5 isoform that lacks the B30.2 domain through alternative splicing. This confirms previous observations that the interaction between Trim5α and HIV-1 capsid is dependent on the B30.2 domain of Trim5α [17]. Previously it was demonstrated that substitutions in the CypA binding

region of capsid reduced sensitivity to Trim5α mediated restriction of viral replication [7,22-24,31,35,36]. However, no difference in Trim5α binding between wild type and mutant CA/NC particles could be demonstrated in our assay, indicating that the Trim5α binding site on

the viral capsid protein is not disrupted by the amino acid substitutions. This also implies that binding of Trim5α to capsid particles does not necessarily correlate with restricted infection which is in agreement with

previous studies that demonstrated that binding is necessary but not sufficient for restriction of a susceptible retrovirus [37]. Although we did not observe differences between the interaction of wild type or mutant capsid particles and Trim5α, it cannot be excluded that the amino acid

substitutions in the CypA binding region might induce structural changes in the capsid protein that influence the relative resistance to Trim5α mediated inhibition. Furthermore we analyzed the effect of CypA on wild type and mutant

core particles shortly after infection. LV carrying wild type or mutant capsid proteins were used to infect HeLa cells and the fate of the wild type and mutant core particles in the cytoplasm of the cells was analyzed using a previously described assay [17]. We observed that

viral cores of wild type LV were lost rapidly after infection. Viral cores of LV carrying mutations in the CypA binding region that are associated with CypA independent replication and a reduced affinity for CypA were

more stable, and high concentrations of core particles could be detected in the cytoplasm at 24 hours after infection. In addition, we observed that destabilization of the viral core upon infection could be inhibited by CsA. These data suggest that CypA is involved in destabilization of the

viral core and that mutations in the CypA binding region that reduce the affinity of the capsid protein for CypA can reduce destabilization. This is in agreement with previous observations where CypA after fusion to a trimerization domain was able to restrict retroviruses very efficiently.

This restriction was similarly associated with an increased destabilization of retroviral core particles [38,39]. Differences in the CypA binding affinity of the HIV-1 capsid have previously been demonstrated to be

associated with a decreased sensitivity to Trim5α mediated inhibition cells from simian species [7,13,18,22-24]. Here, we observed that the reduced affinity of the viral capsid for CypA was associated with an increased stability of the viral core during infection. However, in human

Chapter 3 71

cells, CypA and Trim5α are independent regulators of HIV-1 infectivity [11,26]. Therefore, it is likely that the reported cis-trans isomerise activity of CypA is responsible for the observed increase in destabilization of the viral core [40,41]. Additional research is required

to unravel the role of CypA in the uncoating process of HIV-1 and why HIV-1 maintains the ability to bind CypA even though CypA independent HIV-1 variants have been isolated from HIV-1 infected individuals [22,24].

Materials and methods Cell cultures and virus production 293T cells were cultured in Dulbecco Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and penicillin (100

U/ml) (Invitrogen) and streptomycin (100 μg/ml) (Invitrogen). HeLa cells were cultured in IMDM supplemented with 10% FCS and penicillin (100U/ml) and streptomycin (100 μg/ml). HIV-1 based lentiviruses expressing the green fluorescent protein (GFP)

were produced by calcium phosphate transfection of 293T cells with the lentiviral vector (LV) construct, the packaging construct for gag/pol, vesicular stomatitis virus glycoprotein envelope (pCMV-VSV-G) and rev (pRSV-rev) as described previously [27]. Infectious LVs were

harvested at 48 and 72 hour after transfection and filtered through a 0.22μm filter. The LVs were concentrated by ultracentrifugation (2 hours at 46000×g). Vector concentrations were determined using an in-house p24 specific ELISA [28]. The mutant HIV-1 based LV has been described previously [7] and contains 3 amino acid substitutions in CypA binding region (H87Q, A88P and I91V) of the packaging construct. Plasmids The cDNA for Trim5α and Trim5γ was obtained by RT-PCR amplification from HeLa cells with primers Trim5 Fw: 5’-GCT TGG TAC CGA TGG CTT CTG GAA TCC TGG-3’, and Trim5α Rv 5’-AGA GCT TGG TGA GCA CAG AG-3’ and trim5γ Rv 5’ AGC TTG GTA CCT AAG GAG GGG TAA GTT ATG TG-3’. The amplified fragments were digested with KpnI and EcoRI and ligated in the EcoRI and KpnI restriction sites in the eukaryotic expression vector. Trim5α was cloned into pcdna3.1(+) that contains a HSV-tag (Invitrogen) and Trim5γ was cloned into pcdna3.1(A) that contains a Myc-tag (Invitrogen).

To express HIV-1 capsid-nucleocapsid (CA/NC) proteins in E. coli, HIV-1 CA/NC genes of the wild type and mutant packaging constructs were amplified by PCR with primers CA/NC Fw: 5’-CAT AAT CAT AAT GCC TAT


AGT GCA GAA CAT CC-3’, and CA/NC Rv: 5’-GTA TCG AGG TAC CGG ACG AGG GGT CGC TGC C-3’. The amplified products were digested with NdeI and KpnI and ligated in the NdeI and KpnI digested pET43.1 expression vector (Novagen, Madison, USA) that contains a histidine

tag. In vitro production of CA/NC complexes

E. coli BL21(DE3)pLysS cells were transformed with the pET43.1 CA/NC expression vector and grown to O.D600 of ~0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a concentration of 1 mM and incubated for 12 hours at 37’C in a shaking incubator. The

induced cells were harvested by centrifugation and lysed by Bugbuster reagent (Novagen, Madison, USA) to release proteins and processed as described by the manufacturer. The lysates were incubated with

washed Ni-NTA beads (Novagen, Madison, USA) for 1hr at 4’C in a rotator and loaded onto columns for 4 sequential washing steps with increasing amounts of imidazole (20, 30, 40 and 50 mM) in the wash buffer (50 mM NaH2PO4; 300 mM NaCl). CA/NC was eluted from the Ni-

NTA beads with wash buffer containing 250 mM imidazole, subsequently desalted using a PD-10 column (GE Healthcare) and finally eluted with PBS. Total CA/NC yield was measured by p24 specific ELISA as described previously [28].

CA/NC complexes were assembled in the presence of viral RNA that contains a packaging signal. Viral RNA was transcribed from an EcoRI digested lentiviral construct [29] in a total volume of 100 μl by T3 RNA polymerase according to the manufacturers recommendation

(Promega, Leiden, Netherlands). For CA/NC assembly, 100 μg CA/NC was combined with 100 μl of the produced viral RNA, adjusted to 0.5 M NaCl to a total volume of 1ml and allowed to assemble overnight at 4˚C. Capsid pull-down assay Assembled CA/NC complexes were coupled to Cyanogen bromide (CNBr)

activated-sepharose beads (Amersham). 100 mg CNBr beads were swollen by incubation in 14 ml of 1 mM HCl for 15 min. Next the beads were washed once in ice-cold 1 mM HCl and twice in binding buffer (0.1 M NaHCO3 (pH8.5); 0.5 M NaCl). Excess binding buffer was

removed and assembled CA/NC complexes were added to the beads and incubated for at least 24 hrs. Subsequently, the beads were washed 3 times with binding buffer and used to pull down cellular proteins from cell lysates.

Cell lysates were prepared from untransfected 293T cells and 293T cells that were transfected with pcDNA-Trim5α-HSV or pcDNA-Trim5γ-myc. Transfected and untransfected 293T cells were lysed in NP40 lysis buffer

Chapter 3 73

(50mM Tris-HCl (pH7.5); 150mM NaCl; 0,05% sodium deoxycholaat) containing protease inhibitor cocktail (Roche, Woerden, The Netherlands). Precipitates were resolved by SDS-PAGE (NuPAGE, Invitrogen, Breda,

The Netherlands) and Western blotted on polyvinylideenfluoride (PVDF) membranes (Immobilon-P, Millipore). Proteins that were specifically pulled down by wild type and mutant CA/NC complexes were visualized using monoclonal mouse antibodies recognizing HSV-tagged proteins

(Novagen, Madison, USA) and myc-tagged proteins (Invitrogen, Breda, The Netherlands). Binding of endogenous CypA was visualized using a polyclonal rabbit antibody that recognizes CypA (Affinity Bio-reagents, Rockford, USA). Specific recognition of the tagged proteins was

visualized by horseradish peroxidase labeled secondary antibody recognizing mouse or rabbit immunoglobulins (Amersham, Roosendaal, The Netherlands).

Fate of Capsid assay The ‘fate of capsid assay’ has been described previously [17]. In brief,

HeLa cells were inoculated with wild type LV and mutant LV for 24 hrs to ensure substantial entry. Next, the cells were detached and washed three times with ice-cold PBS, and subsequently the cells were lyzed in a hypotonic lysis buffer (10 mM Tris-HCl; 10 mM KCl; 1 mM EDTA) for 15

min and a freeze-thawing step. Following brief centrifugation to pellet insoluble material, cleared supernatants were layered on a 50% sucrose cushion and centrifuged at 4ºC for 2 hrs at 25,000 rpm (rotor type sw40.1). The supernatant was removed and the pellet was

resuspended in 500 μl RIPA buffer. Next, capsid proteins were immune precipitated with anti-p24 antibodies, washed three times with RIPA buffer and analyzed by polyacryl amide electrophoresis and Western blotting (Invitrogen, Breda, The Netherlands).

Competing interests. The authors declare that they have no competing interests.

Author Contributions. MR, CB and LP performed the experiments. NK, MR conceived and designed the experiments, analyzed the data and wrote the paper.

Acknowledgements. The authors like to acknowledge Bin Zheng and Rianne Borsje for technical assistance and Hanneke Schuitemaker for critical reading of the manuscript. This work was supported by the

Landsteiner Foundation for Blood Transfusion Research (grant 0435), Netherlands Organization for scientific Research (NWO grant number 96.36.024) and the Dutch AIDS fund (grant 2004062).


References 1. Bukovsky AA, Weimann A, Accola MA, Gottlinger HG: Transfer of the HIV-1

cyclophilin-binding site to simian immunodeficiency virus from Macaca mulatta can confer

both cyclosporin sensitivity and cyclosporin dependence. Proc Natl Acad Sci U S A

1997, 94:10943-10948.

2. Gamble TR, Vajdos FF, Yoo S, Worthylake DK, Houseweart M, Sundquist WI, Hill CP:

Crystal structure of human cyclophilin A bound to the amino-terminal domain of

HIV-1 capsid. Cell 1996, 87:1285-1294.

3. Thali M, Bukovsky A, Kondo E, Rosenwirth B, Walsh CT, Sodroski J, Gottlinger HG:

Functional association of cyclophilin A with HIV-1 virions. Nature 1994, 372:363-365.

4. Colgan J, Yuan HE, Franke EK, Luban J: Binding of the human immunodeficiency

virus type 1 Gag polyprotein to cyclophilin A is mediated by the central region of capsid

and requires Gag dimerization. J Virol 1996, 70:4299-4310.

5. Franke EK, Yuan HEH, Luban J: Specific incorporation of cyclophilin A into HIV-1

virions. Nature 1994, 372:359-362.

6. Towers GJ, Hatziioannou T, Cowan S, Goff SP, Luban J, Bieniasz PD: Cyclophilin A

modulates the sensitivity of HIV-1 to host restriction factors. Nat Med 2003,

9:1138-1143.

7. Kootstra NA, Munk C, Tonnu N, Landau NR, Verma IM: Abrogation of postentry

restriction of HIV-1-based lentiviral vector transduction in simian cells. Proc Natl Acad Sci

U S A 2003, 100:1298-1303.

8. Hatziioannou T, Perez-Caballero D, Cowan S, Bieniasz PD: Cyclophilin interactions

with incoming human immunodeficiency virus type 1 capsids with opposig effects on

infectivity in human cells. J Virol 2005, 79:176-183.

9. Sokolskaja E, Sayah DM, Luban J: Target cell cyclophilin A modulates human

immunodeficiency virus type 1 infectivity. J Virol 2004, 78:12800-12808.

10. Franke EK, Luban J: Inhibition of HIV-1 replication by cyclosporine A or related

compounds correlates with the ability to disrupt the Gag-cyclophilin A interaction.

Virology 1996, 222:279-282.

11. Keckesova Z, Ylinen LM, Towers GJ: Cyclophilin A renders human

immunodeficiency virus type 1 sensitive to Old World monkey but not human TRIM5

alpha antiviral activity. J Virol 2006, 80:4683-4690.

12. Stremlau M, Song B, Javanbakht H, Perron M, Sodroski J: Cyclophilin A: an

auxiliary but not necessary cofactor for TRIM5alpha restriction of HIV-1. Virology

2006, 351:112-120.

13. Rits MA, van Dort KA, Munk C, Meijer AB, Kootstra NA: Efficient transduction of

simian cells by HIV-1-based lentiviral vectors that contain mutations in the capsid

protein. Mol Ther 2007, 15:930-937.

14. Reymond A, Meroni G, Fantozzi A, Merla G, Cairo S, Luzi L, Riganelli D, Zanaria E,

Messali S, Cainarca S et al.: The tripartite motif family identifies cell

compartments. EMBO J 2001, 20:2140-2151.

15. Nisole S, Stoye JP, Saib A: TRIM family proteins: retroviral restriction and

antiviral defence. Nat Rev Microbiol 2005, 3:799-808.

16. Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J: The

cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World

monkeys. Nature 2004, 427:848-853.

17. Stremlau M, Perron M, Lee M, Li Y, Song B, Javanbakht H, Diaz-Griffero F,

Anderson DJ, Sundquist WI, Sodroski J: Specific recognition and accelerated

uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc Natl

Acad Sci U S A 2006, 103:5514-5519.

18. Ohkura S, Yap MW, Sheldon T, Stoye JP: All three variable regions of the

TRIM5alpha B30.2 domain can contribute to the specificity of retrovirus restriction. J

Virol 2006, 80:8554-8565.

Chapter 3 75

19. Sawyer SL, Wu LI, Emerman M, Malik HS: Positive selection of primate

TRIM5alpha identifies a critical species-specific retroviral restriction domain. Proc Natl

Acad Sci U S A 2005, 102:2832-2837.

20. Song B, Gold B, O'hUigin C, Javanbakht H, Li X, Stremlau M, Winkler C, Dean M,

Sodroski J: The B30.2(SPRY) domain of the retroviral restriction factor TRIM5alpha

exhibits lineage-specific length and sequence variation in primates. J Virol 2005, 79:6111-

6121.

21. Yap MW, Nisole S, Stoye JP: A single amino acid change in the SPRY domain of

human Trim5alpha leads to HIV-1 restriction. Curr Biol 2005, 15:73-78.

22. Chatterji U, Bobardt MD, Stanfield R, Ptak RG, Pallansch LA, Ward PA, Jones MJ,

Stoddart CA, Scalfaro P, Dumont JM et al.: Naturally occurring capsid substitutions

render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in

Owl monkey cells. J Biol Chem 2005, 280:40293-40300.

23. Ikeda Y, Ylinen LM, Kahar-Bador M, Towers GJ: Influence of gag on human

immunodeficiency virus type 1 species-specific tropism. J Virol 2004, 78:11816-

11822.

24. Kootstra NA, Navis M, Beugeling C, van Dort KA, Schuitemaker H: The presence of

the Trim5alpha escape mutation H87Q in the capsid of late stage HIV-1 variants is

preceded by a prolonged asymptomatic infection phase. AIDS 2007, 21:2015-2023.

25. Berthoux L, Sebastian S, Sokolskaja E, Luban J: Cyclophilin A is required for

TRIM5{alpha}-mediated resistance to HIV-1 in Old World monkey cells. Proc Natl Acad

Sci U S A 2005, 102:14849-14853.

26. Sokolskaja E, Berthoux L, Luban J: Cyclophilin A and TRIM5alpha independently

regulate human immunodeficiency virus type 1 infectivity in human cells. J Virol 2006,

80:2855-2862.

27. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L: A third-

generation lentivirus vector with a conditional packaging system. J Virol 1998,

72:8463-8471.

28. Tersmette M, Winkel IN, Groenink M, Gruters RA, Spence P, Saman E, van

der Groen G, Miedema F, Huisman JG: Detection and subtyping of HIV-1 isolates with a

panel of characterized monoclonal antibodies to HIV-p24 gag. Virology 1989, 171:149-

155.

29. Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L: Gene transfer by lentiviral

vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat

Genet 2000, 25:217-222.

30. Chatterji U, Bobardt MD, Gaskill P, Sheeter D, Fox H, Gallay PA: Trim5alpha

accelerates degradation of cytosolic capsid associated with productive HIV-1 entry. J

Biol Chem 2006, 281:37025-37033.

31. Hatziioannou T, Perez-Caballero D, Yang A, Cowan S, Bieniasz PD: Retrovirus

resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha. Proc

Natl Acad Sci U S A 2004, 101:10774-10779.

32. Lee K, KewalRamani VN: In defense of the cell: TRIM5alpha interception of

mammalian retroviruses. Proc Natl Acad Sci U S A 2004, 101:10496-10497.

33. Agarwal PK: Cis/trans isomerization in HIV-1 capsid protein catalyzed by

cyclophilin A: insights from computational and theoretical studies. Proteins 2004, 56:449-

463.

34. Yoo S, Myszka DG, Yeh C, McMurray M, Hill CP, Sundquist WI: Molecular

recognition in the HIV-1 capsid/cyclophilin A complex. J Mol Biol 1997, 269:780-795.

35. Owens CM, Yang PC, Gottlinger H, Sodroski J: Human and simian

immunodeficiency virus capsid proteins are major viral determinants of early,

postentry replication blocks in simian cells. J Virol 2003, 77:726-731.

36. Owens CM, Song B, Perron MJ, Yang PC, Stremlau M, Sodroski J: Binding and

susceptibility to postentry restriction factors in monkey cells are specified by

distinct regions of the human immunodeficiency virus type 1 capsid. J Virol 2004,

78:5423-5437.


37. Sebastian S, Grutter C, Strambio de CC, Pertel T, Olivari S, Grutter M, Luban J: An

Invariant Surface Patch on the TRIM5{alpha} PRYSPRY Domain is Required for

Retroviral Restriction but Dispensable for Capsid-Binding. J Virol 2009.

38. Javanbakht H, az-Griffero F, Yuan W, Yeung DF, Li X, Song B, Sodroski J: The

ability of multimerized cyclophilin A to restrict retrovirus infection. Virology 2007,

367:19-29.

39. Yap MW, Dodding MP, Stoye JP: Trim-cyclophilin A fusion proteins can restrict

human immunodeficiency virus type 1 infection at two distinct phases in the viral life cycle.

J Virol 2006, 80:4061-4067.

40. Bosco DA, Eisenmesser EZ, Pochapsky S, Sundquist WI, Kern D: Catalysis of

cis/trans isomerization in native HIV-1 capsid by human cyclophilin A. Proc Natl Acad Sci U

S A 2002, 99:5247-5252.

41. Bosco DA, Kern D: Catalysis and binding of cyclophilin A with different HIV-1 capsid

constructs. Biochemistry 2004, 43:6110-6119.

Chapter 4

The Effect of Trim5 Polymorphisms

on the Clinical Course of HIV-1

Infection Daniëlle van Manen1,2¤, Maarten A. N. Rits1,2¤, Corrine Beugeling1,2,

Karel van Dort1,2¤, Hanneke Schuitemaker1,2,3, Neeltje A. Kootstra1,2¤* 1 Department of Clinical Viro Immunology, Sanquin Research, Landsteiner

Laboratory, University of Amsterdam, Amsterdam, The Netherlands, 2 Center of

Infection and Immunity Amsterdam (CINIMA), University of Amsterdam,

Amsterdam, The Netherlands, 3 Department of Experimental Immunology,

Amsterdam Medical Center, Amsterdam, The Netherlands

¤ Current address: Department of Experimental Immunology, Amsterdam Medical

Center, Amsterdam, The Netherlands

PLoS Pathog 2008;4(2):e18

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http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.0040018#cor1

Chapter 4 79

Abstract

The antiviral factor tripartite interaction motif 5α (Trim5α) restricts a

broad range of retroviruses in a species-specific manner. Although

human Trim5α is unable to block HIV-1 infection in human cells, a

modest inhibition of HIV-1 replication has been reported. Recently two

polymorphisms in the Trim5 gene (H43Y and R136Q) were shown to

affect the antiviral activity of Trim5α in vitro. In this study, participants

of the Amsterdam Cohort studies were screened for polymorphisms at

amino acid residue 43 and 136 of the Trim5 gene, and the potential

effects of these polymorphisms on the clinical course of HIV-1 infection

were analyzed. In agreement with the reported decreased antiviral

activity of Trim5α that contains a Y at amino acid residue 43 in vitro, an

accelerated disease progression was observed for individuals who were

homozygous for the 43Y genotype as compared to individuals who were

heterozygous or homozygous for the 43H genotype. A protective effect

of the 136Q genotype was observed but only after the emergence of

CXCR4-using (X4) HIV-1 variants and when a viral load of 104.5 copies

per ml plasma was used as an endpoint in survival analysis.

Interestingly, naive CD4 T cells, which are selectively targeted by X4

HIV-1, revealed a significantly higher expression of Trim5α than memory

CD4 T cells. In addition, we observed that the 136Q allele in

combination with the −2GG genotype in the 5′UTR was associated with

an accelerated disease progression. Thus, polymorphisms in the Trim5

gene may influence the clinical course of HIV-1 infection also

underscoring the antiviral effect of Trim5α on HIV-1 in vivo.

Author Summary

The clinical course of HIV-1 infection is highly variable between

individuals, and host genetic variations may at least account for part of

these differences. Recently two single nucleotide polymorphisms in the

tripartite interaction motif 5 gene (Trim5) have been reported to affect

the antiviral activity of the Trim5α protein. Here we analyzed the effect

of these polymorphisms on the clinical course of HIV-1 infection in

participants of the Amsterdam Cohort studies. We observed an

accelerated disease progression for individuals who were homozygous

80 Polymorphisms in Trim5

for the 43Y genotype that has been associated with a decreased antiviral

activity of Trim5α in vitro. The 136Q genotype has in vitro been

associated with a slightly higher anti-HIV-1 activity. We observed a

protective effect of the 136Q genotype only after the emergence of

CXCR4-using HIV-1 variants using viral load above 104.5 copies per ml

plasma as an endpoint in survival analysis. These results suggest that

genetic variations in the Trim5 gene may influence the clinical course of

HIV-1 infection and confirm a role of Trim5α on HIV-1 in vivo.

Introduction

The susceptibility to HIV-1 infection and subsequent disease progression

is highly variable between individuals. Host genetic variations have

previously been demonstrated to account for at least part of these

differences. Polymorphisms in chemokine receptors that serve as HIV-1

coreceptors, or in their natural ligands, have been associated with

reduced susceptibility to infection as well as disease progression [1–4].

Furthermore, certain HLA types have been correlated with the clinical

course of infection [5–8].

Variations in genes involved in innate immunity may also contribute to

the differential susceptibility of humans to HIV-1 infection and the highly

variable outcome of the disease. Recently, the tripartite interaction motif

5α (Trim5α) has been identified as part of the intrinsic immunity that

protects human and non-human primates against retroviral infection

[9,10]. Trim5α targets the capsid of the incoming retrovirus in the

cytoplasm directly after entry and interferes with viral replication at an

early post-entry step most likely at the poorly understood uncoating

process [11–19]. Species-specific variations in Trim5α account for the

restriction pattern of specific retroviruses [20–26]. For example, HIV-1

replication is blocked efficiently by Trim5α of rhesus macaques and

African green monkeys, whereas SIV-mac is only restricted by Trim5α

from African green monkey. Human Trim5α efficiently blocks N-tropic

MLV and equine infectious anaemia virus, but is much less efficient in

restricting HIV-1 replication. This indicates that HIV-1 has at least

partially adapted to the human variant of this restriction factor.

Recently, we observed that Trim5α escape variants developed late in

infection in a proportion of the HIV-1 infected individuals [27]. The

emergence of the escape variants was preceded by a prolonged

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Chapter 4 81

asymptomatic phase, indicating that Trim5α mediated suppression of

viral replication indeed plays a role in HIV-1 pathogenesis.

The potential role of polymorphisms within the Trim5 gene on HIV-1

susceptibility has recently been studied [28–31]. Of the eight

nonsynonymous polymorphisms that have been identified in the Trim5

gene, two have been reported to have functional consequences with

regard to the antiviral activity of Trim5α (H43Y and R136Q) [28,30]. The

H43Y is located in the RING domain of Trim5α and may impair its

putative E3 ligase activity [28,30]. Indeed, the 43Y variant of Trim5α

was less efficient in restricting HIV-1 replication in vitro [28,30]. The

R136Q polymorphism has been associated with a slightly higher anti-

HIV-1 activity of Trim5α [30]. In agreement, the R136Q polymorphism

was more frequently observed in high risk seronegative as compared to

HIV-1 infected individuals in a cohort of African Americans [30],

although not confirmed in other study populations [29,30]. So far no

significant association between Trim5 polymorphisms and HIV-1 disease

progression have been demonstrated [29,31,32].

Here we studied the effect of the Trim5α H43Y and R136Q

polymorphisms on the clinical course of HIV-1 infection in participants of

the Amsterdam Cohort studies. In addition, we analyzed whether the

R136Q genotype in combination with a SNP in the 5′UTR of Trim5

(−2G/C) was associated with susceptibility to HIV-1 infection or disease

progression.

Results

Distribution of H43Y and R136Q Trim5 Genotypes

The prevalence of Trim5α polymorphisms H43Y and R136Q was studied

in 327 HIV-1 positive participants of the Amsterdam Cohort studies. For

the H43Y polymorphism a minor allele frequency of 0.115 was observed.

Of the 327 HIV-1 positive participants, 61 (18.7%) were heterozygous

and 7 (2.1%) were homozygous for the 43Y allele. The R136Q

polymorphism was observed at a minor allele frequency of 0.379. Of the

















327 participants, 156 (47.7%) were heterozygous and 46 (14.1%) were

homozygous for the 136Q allele. Six mutually exclusive haplotypes were

observed for the combination of R136Q and H43Y polymorphisms:

43HH/136RR (n = 88), 43HH/136RQ (n = 125), 43HH/136QQ (n = 46),

43HY/136RQ (n = 31), 43HY/136RR (n = 30) and 43YY/136RR (n = 7).

The 43Y polymorphism was not observed in the group homozygous for

the 136QQ genotype and the 136Q polymorphism was never observed

in combination with a homozygous 43YY genotype, also confirming that

the 43Y polymorphism and the 136Q polymorphism are not located on

the same allele [29,31]. No significant differences in the H43Y or R136Q

minor allele frequencies were observed between the HIV-1 seropositive

individuals and healthy controls (allele frequencies of 0.106 and 0.389

for H43Y and R136Q, respectively).

Effect of the H43Y Trim5 Genotype on the Clinical Course of HIV-1

Infection

Kaplan Meier and Cox Proportional Hazard analysis with clinical AIDS

(Definition CDC 1987 and 1993), CD4 T cell counts below 200 cells/μl

blood, and plasma viral RNA load above 104.5 copies per ml plasma were

used as end points to determine the effect of polymorphisms in the

Trim5 gene on disease progression. We observed an accelerated disease

progression in the group homozygous for the 43Y allele relative to the

43 HH wild type genotype, with a relative hazard (RH) of 3.1 (p =

0.006) and 2.8 (p = 0.007) for AIDS diagnosis according to the 1987 or

1993 CDC definition, respectively (Figure 1A and 1B; Table 1). An

accelerated progression rate was also observed when CD4 T cell counts

below 200 cells per μl blood were used as end point (Figure 1C; Table

1). The median viral RNA load of participants of the Amsterdam cohort

progressing to AIDS has previously been determined at 104.5 copies HIV-

1 RNA per ml plasma [1]. When viral RNA load above 104.5 copies per ml

plasma was used as endpoint in the survival analysis, we again observed

an accelerated disease progression for individuals homozygous for the

43Y genotype (Figure 1D; Table 1). The heterozygous genotype (43HY)

was not associated with delayed disease progression (Figure 1).



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Chapter 4 83

Figure 1: Survival Analysis for the H43Y Genotype . Kaplan Meier analysis for

time in years from seroconversion to AIDS according to the CDC 1987 definition

(A), to AIDS according to the CDC 1993 definition (B), to CD4 count below 200

cells/μl blood (C), to viral RNA load above 104.5

copies per ml plasma (D), and to

first detection of X4-variants (E) based on the H43Y genotype. Bold lines indicate

individuals with the wild type genotype (43HH); dashed black lines indicate

individuals heterozygous for the 43Y genotype (43HY); thin black lines indicate

individuals homozygous for the 43Y genotype (43YY).

Development of CXCR4 using HIV-1 variants (X4-variants) has

previously been associated with an accelerated disease progression

[33]. The ability of HIV-1 variants to use CXCR4 as a coreceptor and

replicate in MT2 cells was analyzed routinely during follow-up in the

cohort studies in 319 of 327 individuals from our study population.



During the course of infection X4-variants developed in 126 individuals.

No association between the prevalence of X4-variants and the H43Y

genotype could be observed (data not shown). However, X4-variants did

develop more rapidly in individuals who were homozygous for the 43Y

genotype (Figure 1E; Table 1).

Our study population consisted of 130 participants who seroconverted

for HIV-1 antibodies during follow-up and 197 seroprevalent participants

with an imputed seroconversion date [34]. Inclusion of seroprevalent

participants in our analysis did not bias our data and Cox regression

analysis stratifying for seroconvertors and seroprevalent participants

gave similar results (data not shown).

Table 1: Cox Regression Analysis for Progression to AIDS, CD4 Cells <200

Cells/μl, and X4-Variant Development

Effect of the R136Q Trim5 Genotype on the Clinical Course of HIV-1

Infection

Next we examined a potential role for the R136Q polymorphism in Trim5

on the clinical course of HIV-1 infection. Using clinical AIDS (Definition

CDC 1987 and 1993), CD4 T cell counts below 200 cells/μl blood or viral

RNA load above 104.5 copies per ml plasma as endpoint in Kaplan Meier




Chapter 4 85

and Cox proportional hazard analysis, no significant associations

between the 136RQ or 136QQ genotype and the clinical course of

infection were revealed (data not shown). The R136Q polymorphism

also had no effect on the time to first detection of X4-variants (data not

shown). In addition, the prevalence of CXCR4 using HIV-1 variants was

not associated with the R136Q genotype.

Next we analyzed whether a potential effect of the R136Q Trim5

genotype was dependent on the coreceptor usage of the virus present.

The R136Q genotype had no significant effect on the clinical course of

infection when only CCR5 using HIV-1 variants (R5-variants) were

present irrespective the end point used in the survival analysis (data not

shown). However, a significant protective effect on disease progression

associated with the R136Q genotype was observed after X4-variant

development using the median viral RNA load of participants of the

Amsterdam cohort progressing to AIDS (104.5 copies per ml) as an end

point in survival analysis, with a RH of 0.44 (p = 0.008) and 0.26 (p =

0.030) for the heterozygous 136RQ and homozygous 136QQ genotype,

respectively as compared to the 136RR genotype (Figure 2; Table 2). At

the moment of X4-development the viral load of individuals who were

homozygous (QQ), heterozygous (RQ) or wild type (RR) for the amino

acid residue at position 136 was similar (data not shown). The R136Q

polymorphism was not associated with disease progression after X4-

variant development using clinical AIDS or CD4 cell counts below 200

cells μl blood as end points (Table 2).

Figure 2: Survival Analysis for R136Q Genotype after Emergence of X4-

Variants. Kaplan Meier analysis for

time in years from the moment of

first detection of X4-variants to viral

RNA load above 104.5

copies per

ml plasma based on the R136Q

genotype. Bold black lines indicate

individuals with the wild type

genotype (136RR); dashed black

lines indicate individuals

heterozygous for the 136Q

genotype (136RQ); thin black lines

indicate individuals homozygous

for the 136Q genotype (136QQ).




86 Polymorphisms in Trim5 Table 2: Cox Regression Analysis for Progression to AIDS, CD4 Cells <200

Cells/μl, or Viral RNA Load >104.5

/ml after X4-Variant Development

Trim5α mRNA Expression Levels in Naïve and Memory CD4 T Cells

The protective effect of the 136Q Trim5α variant on disease progression

only after emergence of X4 variants may imply that Trim5α has a

stronger effect on X4 variants than on R5 variants. Previously we

demonstrated that R5 and X4 HIV-1 variants partially reside in different

CD4 T cell compartments due to differential expression of coreceptors

CCR5 and CXCR4 [35,36]. R5 variants were selectively isolated from

CD4 memory T cells, whereas X4 variants were isolated from memory

and naïve CD4 T cells. Here we analyzed whether differences of the

Trim5α mRNA levels in naïve and memory CD4 T cell populations could

contribute to the differential effect of the 136Q variant on X4 and R5

variants in vivo. Naïve and memory CD4 T cells were isolated from PBMC

from 12 healthy controls by FACS sorting based on CD45RO and CD27

expression and Trim5α mRNA levels were analyzed by quantitative real

time PCR. To correct for differences in input, Trim5α mRNA levels were

normalized for β-actin mRNA levels. Trim5α mRNA levels were

significantly higher in naïve (CD45RO−CD27+) CD4 T cells as compared

to memory (CD45RO+) CD4 cells (p = 0.019) (Figure 3A).

In addition, Trim5α mRNA levels in naïve and memory CD4 T cells were

analyzed in HIV-1 positive individuals early and late in the course of




Chapter 4 87

infection (23 PBMC samples from 11 individuals). Although no

differences in Trim5α mRNA levels were observed during the course of

infection, we again observed a significantly higher Trim5α mRNA level in

naïve CD4 T cells as compared to memory CD4 T cells (p = 0.003)

(Figure 3B).

Figure 3: Analysis of Trim5α mRNA Levels in Naïve and Memory CD4 T Cells.

(A) Trim5α mRNA levels in naïve CD4 T cells (CD45RO-CD27+) and memory CD4

T cells (CD45RO+) obtained from healthy controls. (B) Average Trim5α mRNA

levels in naïve and memory CD4 T cells during the course of infection from HIV-1

infected individuals. Trim5α mRNA levels are normalized for β-actin mRNA levels.

Different symbols represent Trim5α mRNA levels of naïve and memory CD4 T cells

from the different individuals.

Effect of Combined R136Q and −2G/C Genotypes on Susceptibility to

HIV-1 Infection and Disease Progression

Recently a G to C polymorphism at position −2 in the 5′UTR of the

Trim5 gene (−2G/C; rs3824949) in combination with the R136Q

polymorphism has been associated with enhanced susceptibility to HIV-1

infection (136Q/−2G haplotype) and accelerated disease progression

(136R/−2G haplotype) [29]. To analyze whether the combined R136Q

and −2G/C genotype was also associated with HIV-1 susceptibility or

disease progression in our study population, we genotyped our study

population for the G to C polymorphism at position −2 (−2G/C). The




−2C allele frequency was 0.486 and 0.418 in our HIV-1 positive

individuals and healthy controls, respectively. When the −2G/C

genotype was analyzed in combination with the R136Q genotype no

significant differences in the distribution of the combined genotypes was

observed between the HIV-1 infected individuals and the healthy

controls. The −2GG genotype was not observed in combination with the

homozygous 136Q genotype in both study populations.

Figure 4: Survival Analysis for the −2G/C Genotype in Combination with the

136Q Genotype. Kaplan Meier analysis for time in years from seroconversion to

AIDS according to the CDC 1993 definition. Bold black lines indicate individuals

with the −2GG genotype; dashed black lines indicate individuals with the −2GC

genotype; thin black lines indicate individuals with the −2CC genotype.

Next we analyzed the effect of the combined R136Q and −2G/C

genotypes on disease progression using clinical AIDS (Definition CDC

1993) as an end point. Individuals carrying the 136Q allele (136RQ and

136QQ) in combination with the −2GG genotype showed an accelerated

progression to disease in comparison to the −2GC (p = 0.009; RH 2.6;

95% CI 1.3–5.2) and −2CC genotype (p = 0.056; RH 2.1; 95% CI 1.0–

4.3) (Figure 4). In contrast to the study by Speelmon et al. [29], no



Chapter 4 89

significant effect of the −2G/C genotype on disease progression was

observed in the group with the 136RR genotype.

AIDS Incidence in Relation to H43Y Genotype and Other Progression

Markers

Uni- and multivariate relative hazard analysis were used to determine

the predictive value of the Trim5 H43Y genotype (43YY) in combination

with previously established prediction markers such as CD4 T cell count,

plasma viral RNA load, the presence of X4-variants, and CCR5-genotype

[1]. Univariate analysis indicated that homozygosity for the H43Y

polymorphism, CD4 T cell counts below 500 cells per μl, viral RNA load

above 104.5 copies per ml plasma and the presence of X4-variants at

18–30 months after seroconversion were predictive for more rapid

progression to AIDS, whereas heterozygosity for the CCR5-Δ32

genotype had a protective effect (Table 3). Multivariate analysis at 2

years after seroconversion indicated that CD4 T cell counts below 500

cells per μl blood, viral RNA load above 104.5 copies per ml plasma, the

presence of X4-variants and homozygosity for the Trim5 H43Y genotype

(43YY) were independent predictors for progression to AIDS (Table 3).

In our study population the homozygous H43Y genotype was not

observed in combination with a heterozygous CCR5-Δ32 genotype

excluding this parameter from the multivariate analysis.

Discussion

Old World monkey Trim5α very efficiently blocks HIV-1 infection at an

early step in the viral replication cycle, immediately after cellular entry.

Human Trim5α is also able to interfere with HIV-1 infection, albeit less

efficiently. Although this may suggest that HIV-1 has at least partially

adapted to human Trim5α, we have recently provided the first evidence

that Trim5α might still play a role in HIV-1 pathogenesis [27]. We

observed that HIV-1 variants containing a H87Q mutation in the

cyclophilin A binding region of capsid, which has previously been

associated with escape from Trim5α [11–16], developed during the late

phase of infection in a proportion of the HIV-1 infected individuals [27].









The emergence of these Trim5α escape variants was preceded by a

prolonged asymptomatic phase implying that Trim5α contributed to

control of virus replication in vivo and concomitantly selected for Trim5α

resistant variants.

Two genetic polymorphisms in the human Trim5 gene have recently

been described to affect the antiviral activity of Trim5α on HIV-1. The

H43Y polymorphism has been associated with an impaired anti-HIV-1

activity of Trim5α in vitro [28,30,32]. In agreement, we here observed

that a 43YY homozygous genotype was predictive for an accelerated

progression to AIDS, independent of CD4 cell counts, viral RNA load in

plasma, and HIV-1 coreceptor usage at 18–30 months after

seroconversion.

In previous studies, the H43Y genotype had no effect on HIV-1 disease

progression [29–32]. Speelmon et al. observed no significant difference

in viral RNA load in the period from 100 days until 2 years post infection

between individuals who were homozygous (YY), heterozygous (HY) or

wild type (HH) for amino acid residue 43 [29]. In agreement, we also

did not observe a difference in viral RNA load at 2 years after

seroconversion between the H43Y genotypic groups (data not shown).

However, the studies by Speelmon et al. and by Goldschmidt et al. did

also not show significant differences in CD4 cell decline between the

H43Y genotypic groups [29,31]. Due to the low minor allele frequency

for H43Y in the study population by Speelmon et al., which had a size of

only 90 individuals, the number of individuals homozygous for the 43Y

genotype might have been to low to observe significant difference

between the genotypes. The discrepancy between our results and those

of Goldschmidt et al. may lie in their relatively short average follow-up

time of 3.2 years as compared to the average follow-up of 7.9 years on

patients in our study. Nakayama et al. observed similar frequencies of

homozygous 43YY and heterozygous 43HY genotypes in progressors and

LTNP in a study population of Japanese HIV-1 infected individuals [32].

In our Amsterdam cohort however, none of the individuals homozygous

for the 43YY genotype had an asymptomatic follow up of 10 years or

more. Javanbakht et al. observed no significant differences in

progression to CD4 T cell counts below 200 cells per μl blood, AIDS

defining events or AIDS related deaths associated with the different

H43Y genotypes in two large cohorts of African Americans and European










Chapter 4 91

Americans [30]. In the African American population the frequency of

individuals homozygous for the 43YY genotype is however very low

which might account for the discrepancy with our data. However, the

minor allele frequency for H43Y in the European American cohort is

similar to the frequency in the Amsterdam cohort (0.114 and 0.115

respectively). Unfortunately, lack of details on their European American

cohort and their analyses make it difficult to bring up potential

explanations for the inconsistency in results.

The R136Q polymorphism has been associated with a slight increase of

the anti-HIV-1 activity of Trim5α in vitro [30], although not confirmed

by others [28,29,31]. In agreement with earlier reports [29,31], we did

not observe an effect of the R136Q polymorphism on disease

progression. A protective effect of the 136Q variant was only evident in

the phase of infection when X4-variants were present, where a delayed

rise in viral load above the median load during progression to disease

(104.5 copies per ml) was observed in individuals who were homozygous

(QQ) or heterozygous (RQ) for the 136Q genotype as compared to

individuals with the wild type genotype (RR).

The protective effect of the 136Q Trim5α variant on disease progression

only after the emergence of X4-variants, may imply that Trim5α in vivo

affects replication of X4-variants more efficiently than R5-variants. X4-

variants develop in 50% of the HIV-1 infected individuals during the

natural course of infection after which R5- and X4-variants coexist [35–

37]. While co-existing R5- and X4-variants infect memory CD4 T cells

that co-express CCR5 and CXCR4, X4-variants have the unique ability to

additionally infect naive CD4 T cells that selectively express CXCR4

[35,36]. Here we observed that naïve CD4 T cells expressed higher

levels of Trim5α as compared to memory CD4 T cells. It is tempting to

speculate that high Trim5α expression levels in naive T cells in

combination with a more potent antiviral activity associated with the

136Q polymorphism provide prolonged control of HIV-1 replication in

carriers of X4-variants.

Recently a G to C polymorphism at position −2 in the 5′UTR of the

Trim5 gene (−2G/C; rs3824949) in combination with the R136Q

polymorphism has been associated with HIV-1 susceptibility and disease

progression [29]. Speelmon et al. observed an enrichment of the














136Q/−2G haplotype in the HIV-1 positive population [29], however we

were unable to confirm this and observed an equal distribution of the

−2G/C polymorphism in combination with the 136Q allele between the

HIV-1 positive population and the control group. However, the 136Q

allele in combination with the −2GG genotype was associated with

accelerated disease progression in our study population. Speelmon et al.

observed an association between a faster CD4 T cell decline and the

136R/−2G haplotype [29], but we were unable to confirm this. In our

study population we observed a −2C allele frequency of 0.486 and

0.418 in the HIV-1 positive study population and the control group,

respectively, which is similar to the frequencies in the European

American population reported by Javanbakht et al. [30]. However the

frequencies for the −2C allele frequencies in the study populations of

Speelmon et al. were much lower (0.38 in exposed seronegatives and

0.31 in HIV-1 infected population) [29]. Therefore, it cannot be excluded

that differences in the distribution of the −2G/C polymorphism in

different study populations account for the observed discrepancies in

results.

Our data confirm a role of Trim5α in the clinical course of infection. In

addition, they show that different genetic variants in Trim5α are

associated with a differential clinical course of infection. Overall, these

results may encourage exploiting the possibility of using Trim5α or alike

derivatives in antiviral strategies.

Materials and methods

Study participants.

The study population, 364 Caucasian, homosexual men enrolled in the

Amsterdam Cohort studies (ACS) on the natural history of HIV-1

infection between October 1984 and March 1986, was previously

described [1]. The censor date of our study was set at the first day of

effective antiretroviral therapy of the participant. Of the 364

participants, 131 seroconverted during the study. The remaining 233

men were positive for HIV-1 antibodies at entry between October 1984

and April 1985. In previous epidemiological studies, the time since

seroconversion of these prevalent cases has been estimated based on






Chapter 4 93

the incidence of HIV-1 infection amongst homosexual participants of the

Amsterdam Cohort and was on average 1.5 years before entry into the

cohort studies [34]. For analysis, we combined the 131 participants with

documented seroconversion and 233 seroprevalent participants with an

imputed seroconversion date as one study group, since previous studies

have not revealed differences in AIDS-free survival between the two

groups [4].

The ACS has been conducted in accordance with the ethical principles

set out in the declaration of Helsinki and written informed consent is

obtained prior to data collection. The study was approved by the

Amsterdam Medical Center institutional medical ethics committee.

Trim5 genotyping.

DNA samples of 327 out of 364 participants of the Amsterdam Cohort

studies were available for Trim5 genotyping. For analysis of the Trim5

R136Q polymorphism (rs10838525), DNA samples were amplified by

PCR using Taq DNA polymerase (Invitrogen) and primer pair Trim5-F

(5′-ATGGCTTCTGGAATCCTGGTTAATG-3′) and Trim5-R136Q-R (5′-

CCCGGGTCTCAGGTCTATCATG-3′). The following amplification cycles

were used: 5min 95°C; 35 cycles of 30s 95°C, 30s 50°C, 90s 72°C;

5min 72°C. Subsequently PCR products were purified and subjected to a

restriction digest with 1U Ava1 (1.5 hour 37°C; NEB) and analyzed on a

1% agarose gel. A PCR product containing an R at position 136 will

result in digestion of the PCR product into a 405bp and 121bp product. A

PCR product containing a Q at position 136 will result in a 526bp

(undigested) product. For conformation, 15 samples (5 homozygous

136R, 5 homozygous 136Q and 5 heterozygous 136QR) have been

sequenced with the ABI prism BigDue Terminator kit V1.1 (Applied

Biosystems) using primers Trim5-F and Trim5-R136Q-R). Sequences

were analyzed on an ABI 3130XL Genetic Analyzer.

For analysis of the Trim5 H43Y polymorphism (rs3740996), DNA

samples were amplified by PCR using Taq DNA polymerase (Invitrogen)

and primer pair Trim5-F and Trim5-H43Y-R (5′-

GGCTGGTAACTGATCCGGCAC-3′). For analysis of the −2GC

polymorphism (rs3824949), DNA samples were amplified by PCR using

Taq DNA polymerase (Invitrogen) and primer pair Tr5−2GC (5′-




GCAGGGATCTGTGAACAAGAGG-3′) and Trim5-H43Y-R. The following

amplification cycles were used: 5min 95°C; 35 cycles of 30s 95°C, 30s

55°C, 90s 72°C; 5min 72°C. Subsequently PCR products were purified

and sequenced with the ABI prism BigDue Terminator kit V1.1 (Applied

Biosystems) using primers Trim5-F and Trim5-H43Y-R for H43Y and

Tr5−2GC and Trim5-H43Y-R for −2GC. Sequences were analyzed on an

ABI 3130XL Genetic Analyzer.

FACS sorting naïve and memory CD4 T cells.

Cryopreserved PBMC were stained with antibodies against CD4 (tricolor

conjugated; Caltag Laboratories), CD45RO (FITC conjugated; BD

Biosciences) and CD27 (phycoerythrin conjugated; Caltag Laboratories),

and sorted using a MoFlo cell sorter (Cytomation Inc.). Cells were sorted

in two different cell populations: naïve (CD45RO-CD27+) CD4 T cells

and memory (CD45RO+) CD4 T cells.

RNA isolation and quantitative PCR.

Total RNA was isolated from naïve and memory CD4 cells from HIV-1

infected individuals or healthy donors, using the RNeasy mini kit

(Qiagen, Hilden, Germany). Subsequently, cDNA was prepared using the

SuperScript™ First-Strand Synthesis System for RT-PCR (In Vitrogen).

Trim5α mRNA levels were analyzed by SYBR green qPCR using the

LightCycler (Roche). The reaction mix contained 20 mM Tris-HCl (pH

8.4), 50 mM KCl, 3 mM MgCl2, 200 μM dNTP, 250 μg/ml BSA, 500 nM

primers, SYBR green I nucleic acid gel stain 40,000× diluted in water,

and 0.6 U platinum Taq DNA polymerase (In Vitrogen). The following

primer sets were used for detection of Trim5α cDNA: Trim5α -RNA-F 5′-

ccaggatagttccttccatac-3′ and Trim5α-R 5′-agagcttggtgagcacagagtc-3′.

Serial dilution of plasmid DNA containing cDNA of Trim5α were used as a

standard curve. To correct for differences in the cDNA input, levels of β-

actin cDNA were analyzed by a SYBR green qPCR using the following

primer set: BA-RNA-F 5′-ggcccagtcctctcccaagtccac-3′ and BA-RNA-R 5′-

ggtaagccctggctgcctccacc-3′. A serial dilution of 8E5 cells was used as a

standard curve for β-actin. SYBR green qPCR was performed using the

following program on the LightCycler: (1) preincubation and

denaturation: 50°C for 2 min, 95°C for 2 min; (2) amplification and

Chapter 4 95

quantification: 45 cycles of 95°C for 5 sec, 55°C for 15 sec, 72°C for 15

sec; (3) melting curve: 95°C for 0 sec, 65°C for 15 min, 95°C for 0 sec

with a temperature transition rate of 0.1°C/sec. Specificity of the PCR

products measured using the SYBR green method was confirmed by a

melting curve.

Statistical analysis.

Kaplan Meier and Cox proportional hazard analysis were performed to

study the relation between the R136Q and H43Y polymorphisms in the

Trim5 gene and disease progression. The following endpoints were

considered for analysis: (1) AIDS according to the 1987 Centers for

Disease Control and Prevention (CDC) definition [38]; (2) AIDS

according to the 1993 CDC definition [39]; (3) CD4 T cell counts below

200 cells/μl blood; (4) viral RNA load above 104.5 copies per ml blood

plasma; (5) detection of X4-variants by coculture of patient PBMC and

MT2 cells [37]. Fisher's exact test was used to analyze an association

between the R136Q, H43Y polymorphisms and prevalence of X4-

variants. Sequential Bonferroni correction (Simes-Hochberg method)

was used to correct for multiple comparisons [40,41].

Univariate and multivariate relative hazards were calculated at 2 years

after seroconversion for the H43Y genotype, CCR5 genotype, presence

of X4-variants at 18–30 months after seroconversion, CD4 T cells at 18–

30 months after seroconversion and viral RNA load at 18–30 months

after seroconversion.

Trim5α mRNA levels in naïve and memory CD4 T cells were compared

using the Students T test.

Acknowledgements

This study was performed as part of the Amsterdam Cohort Studies

(ACS) on HIV infection and AIDS, a collaboration between the

Amsterdam Health Service, the Academic Medical Centre of the

University of Amsterdam, Sanquin Blood Supply Foundation, and the







University Medical Centre Utrecht

(http://www.amsterdamcohortstudies.org/). The ACS are part of the

Netherlands HIV Monitoring Foundation and financially supported by the

Netherlands National Institute for Public Health and the Environment.

We are greatly indebted to all cohort participants for their continuous

participation. The authors thank Judith Burger and Brigitte Boeser-

Nunnink for excellent technical assistance. The authors thank Ronald

Geskus for helpful discussions.

Author Contributions

HS and NAK conceived and designed the experiments and wrote the

paper. DvM, MANR, CB, and KvD performed the experiments. DvM and

NAK analyzed the data.

References

1. de Roda Husman AM, Koot M, Cornelissen M, Keet IP, Brouwer M, et al.

(1997) Association between CCR5 genotype and the clinical course of HIV-1

infection. Ann Intern Med 127: 882–890.

2. Smith MW, Dean M, Carrington M, Winkler C, Huttley GA, et al. (1997)

Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection

and disease progression. Hemophilia Growth and Development Study

(HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia

Cohort Study (MHCS), San Francisco City Cohort (SFCC), ALIVE Study.

Science 277: 959–965.

3. Winkler C, Modi W, Smith MW, Nelson GW, Wu X, et al. (1998) Genetic

restriction of AIDS pathogenesis by an SDF-1 chemokine gene variant. ALIVE

Study, Hemophilia Growth and Development Study (HGDS), Multicenter AIDS

Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San

Francisco City Cohort (SFCC). Science 279: 389–393.

4. van Rij RP, de Roda Husman AM, Brouwer M, Goudsmit J, Coutinho RA, et al.

(1998) Role of CCR2 genotype in the clinical course of syncytium-inducing

(SI) or non-SI human immunodeficiency virus type 1 infection and in the time

to conversion to SI virus variants. J Infect Dis 178: 1806–1811.

5. Kaslow RA, Carrington M, Apple R, Park L, Munoz A, et al. (1996) Influence of

combinations of human major histocompatibility complex genes on the course

of HIV-1 infection. Nat Med 2: 405–411.

6. Klein MR, van der Burg SH, Hovenkamp E, Holwerda AM, Drijfhout JW, et al.

(1998) Characterization of HLA-B57-restricted human immunodeficiency virus

http://www.amsterdamcohortstudies.org/

Chapter 4 97

type 1 Gag- and RT-specific cytotoxic T lymphocyte responses. J Gen Virol

79(Pt 9): 2191–2201.

7. Flores-Villanueva PO, Yunis EJ, Delgado JC, Vittinghoff E, Buchbinder S, et al.

(2001) Control of HIV-1 viremia and protection from AIDS are associated with

HLA-Bw4 homozygosity. Proc Natl Acad Sci U S A 98: 5140–5145.

8. Mann DL, Garner RP, Dayhoff DE, Cao K, Fernandez-Vina MA, et al. (1998)

Major histocompatibility complex genotype is associated with disease

progression and virus load levels in a cohort of human immunodeficiency

virus type 1-infected Caucasians and African Americans. J Infect Dis 178:

1799–1802.

9. Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, et al. (2004) The

cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old

World monkeys. Nature 427: 848–853.

10. Sayah DM, Sokolskaja E, Berthoux L, Luban J (2004) Cyclophilin A

retrotransposition into TRIM5 explains owl monkey resistance to HIV-1.

Nature 430: 569–573.

11. Kootstra NA, Munk C, Tonnu N, Landau NR, Verma IM (2003) Abrogation of

postentry restriction of HIV-1-based lentiviral vector transduction in simian

cells. Proc Natl Acad Sci U S A 100: 1298–1303.

12. Ikeda Y, Ylinen LM, Kahar-Bador M, Towers GJ (2004) Influence of gag on

human immunodeficiency virus type 1 species-specific tropism. J Virol 78:

11816–11822.

13. Hatziioannou T, Cowan S, Von Schwedler UK, Sundquist WI, Bieniasz PD

(2004) Species-specific tropism determinants in the human immunodeficiency

virus type 1 capsid. J Virol 78: 6005–6012.

14. Owens CM, Song B, Perron MJ, Yang PC, Stremlau M, et al. (2004) Binding

and susceptibility to postentry restriction factors in monkey cells are specified

by distinct regions of the human immunodeficiency virus type 1 capsid. J Virol

78: 5423–5437.

15. Stremlau M, Perron M, Lee M, Li Y, Song B, et al. (2006) Specific recognition

and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction

factor. Proc Natl Acad Sci U S A 103: 5514–5519.

16. Rits MAN, Van Dort KA, Münk C, Meijer AB, Kootstra NA (2007) Efficient

transduction of simian cells by HIV-1 based lentiviral vectors that contain

mutations in the capsid protein. Mol Ther 15: 930–937.

17. Perez-Caballero D, Hatziioannou T, Zhang F, Cowan S, Bieniasz PD (2005)

Restriction of human immunodeficiency virus type 1 by TRIM-CypA occurs

with rapid kinetics and independently of cytoplasmic bodies, ubiquitin, and

proteasome activity. J Virol 79: 15567–15572.

18. Perez-Caballero D, Hatziioannou T, Yang A, Cowan S, Bieniasz PD (2005)

Human tripartite motif 5alpha domains responsible for retrovirus restriction

activity and specificity. J Virol 79: 8969–8978.

19. Javanbakht H, Diaz-Griffero F, Stremlau M, Si Z, Sodroski J (2005) The

contribution of RING and B-box 2 domains to retroviral restriction mediated

by monkey TRIM5alpha. J Biol Chem 280: 26933–26940.

20. Keckesova Z, Ylinen LM, Towers GJ (2004) The human and African green

monkey TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor

activities. Proc Natl Acad Sci U S A 101: 10780–10785.


21. Hatziioannou T, Perez-Caballero D, Yang A, Cowan S, Bieniasz PD (2004)

Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of

TRIM5alpha. Proc Natl Acad Sci U S A 101: 10774–10779.

22. Perron MJ, Stremlau M, Song B, Ulm W, Mulligan RC, et al. (2004)

TRIM5alpha mediates the postentry block to N-tropic murine leukemia viruses

in human cells. Proc Natl Acad Sci U S A 101: 11827–11832.

23. Si Z, Vandegraaff N, O'Huigin C, Song B, Yuan W, et al. (2006) Evolution of a

cytoplasmic tripartite motif (TRIM) protein in cows that restricts retroviral

infection. Proc Natl Acad Sci U S A 103: 7454–7459.

24. Song B, Javanbakht H, Perron M, Park DH, Stremlau M, et al. (2005)

Retrovirus restriction by TRIM5alpha variants from Old World and New World

primates. J Virol 79: 3930–3937.

25. Yap MW, Nisole S, Lynch C, Stoye JP (2004) Trim5alpha protein restricts both

HIV-1 and murine leukemia virus. Proc Natl Acad Sci U S A 101: 10786–

10791.

26. Ylinen LM, Keckesova Z, Wilson SJ, Ranasinghe S, Towers GJ (2005)

Differential restriction of human immunodeficiency virus type 2 and simian

immunodeficiency virus SIVmac by TRIM5alpha alleles. J Virol 79: 11580–

11587.

27. Kootstra NA, Navis M, Beugeling C, van Dort KA, Schuitemaker H (2007) The

presence of the Trim5alpha escape mutation H87Q in the capsid of late stage

HIV-1 variants is preceded by a prolonged asymptomatic infection phase. Aids

21: 2015–2023.

28. Sawyer SL, Wu LI, Akey JM, Emerman M, Malik HS (2006) High-frequency

persistence of an impaired allele of the retroviral defense gene TRIM5alpha in

humans. Curr Biol 16: 95–100.

29. Speelmon EC, Livingston-Rosanoff D, Li SS, Vu Q, Bui J, et al. (2006) Genetic

association of the antiviral restriction factor TRIM5alpha with human

immunodeficiency virus type 1 infection. J Virol 80: 2463–2471.

30. Javanbakht H, An P, Gold B, Petersen DC, O'Huigin C, et al. (2006) Effects of

human TRIM5alpha polymorphisms on antiretroviral function and

susceptibility to human immunodeficiency virus infection. Virology 354: 15–

27.

31. Goldschmidt V, Bleiber G, May MT, Martinez R, Ortiz M, et al. (2006) Role of

common human TRIM5alpha variants in HIV-1 disease progression.

Retrovirology 3: 54.

32. Nakayama EE, Carpentier W, Costagliola D, Shioda T, Iwamoto A, et al.

(2007) Wild type and H43Y variant of human TRIM5alpha show similar anti-

human immunodeficiency virus type 1 activity both in vivo and in vitro.

Immunogenetics 59: 511–515.

33. Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, et al. (1993) Prognostic

value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion

and progression to AIDS. Ann Intern Med 118: 681–688.

34. Geskus RB (2000) On the inclusion of prevalent cases in HIV/AIDS natural

history studies through a marker-based estimate of time since

seroconversion. Stat Med 19: 1753–1769.

35. Blaak H, van't Wout AB, Brouwer M, Hooibrink B, Hovenkamp E, et al. (2000)

In vivo HIV-1 infection of CD45RA(+)CD4(+) T cells is established primarily

Chapter 4 99

by syncytium-inducing variants and correlates with the rate of CD4(+) T cell

decline. Proc Natl Acad Sci U S A 97: 1269–1274.

36. van Rij RP, Blaak H, Visser JA, Brouwer M, Rientsma R, et al. (2000)

Differential coreceptor expression allows for independent evolution of non-

syncytium-inducing and syncytium-inducing HIV-1. J Clin Invest 106: 1569.

37. Koot M, Vos AH, Keet RP, de Goede RE, Dercksen MW, et al. (1992) HIV-1

biological phenotype in long-term infected individuals evaluated with an MT-2

cocultivation assay. Aids 6: 49–54.

38. Center for Disease Control.Revision of the CDC surveillance case definition for

acquired immunodeficiency syndrome. MMWR. 36: 1S–15S.

39. Center for Disease Control.1993 revised classification system for HIV infection

and expanded surveillance case definition for AIDS among adolescentes and

adults. MMWR. 41(RR-17): pp. 1–19.

40. Simes JR (1986) An improved Bonferroni procedure for multiple tests of

significance. Biometrika 73: 75–754.

41. Hochberg Y (1988) A sharper Bonferroni procedure for multiple tests of

significance. Biometrika 75: 800–802.

Chapter 5

Polymorphisms in the Regulatory

Region of the Cyclophilin A Gene

Influence the Susceptibility for

HIV-1 Infection

Maarten A. N. Rits, Karel A. van Dort, Neeltje A. Kootstra*

Department of Experimental Immunology, Sanquin Research,

Landsteiner Laboratory, and Center for Infectious Diseases and

Immunity Amsterdam (CINIMA), Academic Medical Center, University of

Amsterdam, Amsterdam, the Netherlands

PLoS ONE 2008;3(12):e3975

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Chapter 5 103

Abstract

Background Previous studies have demonstrated an association

between polymorphisms in the regulatory regions of Cyclophilin A

(CypA) and susceptibility to both HIV-1 infection and disease

progression. Here we studied whether these polymorphisms are

associated with susceptibility to HIV-1 infection and disease progression

in the Amsterdam Cohort on HIV-1 infection and AIDS (ACS) in a group

of men having sex with men (MSM) and drug users (DU).

Methodology/Principal Findings We screened participants of the ACS

for the C1604G and A1650G polymorphisms in the regulatory regions of

CypA. The prevalence of the 1650G allele was significantly higher in high

risk seronegative MSM than in HIV-1 infected MSM. However, C1604G or

A1650G were not associated with the clinical course of infection in MSM

of the ACS. Interestingly, participants of the ACS-DU who carried the

1604G allele showed a significantly accelerated progression when viral

RNA load above 104.5 copies per ml plasma was used as an endpoint in

survival analysis.

Conclusion/Significance The results obtained in this study suggest that

the A1650G polymorphism in the regulatory region of the CypA gene

may be associated with protection from HIV-1 infection, while the 1604G

allele may have a weak association with the clinical course of infection in

DU.

Introduction

The peptidyl prolyl isomerase Cyclophilin A (CypA) is a cytoplasmic

factor that is essential for efficient infection of HIV-1 [1]–[3]. It is

specifically incorporated into the HIV-1 virion which is mediated through

an interaction with the capsid protein of which an exposed loop between

helices 4 and 5 mimic a natural ligand for CypA [4], [5]. Although CypA

is incorporated in the virion, the presence of CypA in the target cell has

the more significant effect on virus replication [6]–[9]. The molecular

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104 Polymorphisms in cyclophilin A

mechanism by which CypA enhances infection is largely unknown. CypA

can catalyze the cis/trans isomerization of prolyl-peptide bonds in the

HIV-1 capsid protein [10], which suggests that CypA has a possible role

in uncoating of the viral core following entry into the cytoplasm.

Recently, CypA has gained a lot of interest when it was identified as a

cofactor for the tri-partite containing motif (Trim)5α in several simian

species [11]–[14]. Trim5α is a retroviral restriction factor that

associates with the capsid protein and blocks HIV-1 infection at an early

step following entry of the viral core into the cytoplasm [15]. Moreover,

Trim5-CypA fusion proteins have been discovered in several simian

species [16], [17], suggesting that CypA in some instances can function

as a capsid recognition domain for antiretroviral proteins.

Polymorphisms in human genes that serve as HIV-1 cofactors or

restriction factors have been described to influence susceptibility to HIV-

1 and disease progression. For example, polymorphisms in chemokine

receptors that serve as HIV-1 coreceptors and their natural ligands have

been associated with susceptibility to infection as well as disease

progression [18]–[21]. Recently, polymorphisms in cellular factors like

Apobec3G, CUL5 and Trim5α, that are directly or indirectly involved in

innate immunity have also been demonstrated to have an effect on the

clinical course of infection [22]–[24].

Previously 11 polymorphisms in the CypA gene have been identified

none of which were located in the coding region of the gene [25]. Two of

these SNPs (A1604G and C1650G) might affect CypA expression levels

based on their location in the promoter region of the CypA gene and

these polymorphism have been demonstrated to affect susceptibility to

HIV-1 infection and disease progression [25], [26]. Here we studied the

role of SNPs in the regulatory region of CypA gene on HIV-1

susceptibility and course of HIV-1 infection in participants of the

Amsterdam Cohort Studies on HIV-1 infection and AIDS (ACS).

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Chapter 5 105

Results

Distribution of the regulatory polymorphisms C1604G and A1650G in

the CypA gene and the effect on susceptibility to HIV-1 infection

The haplotype frequency of polymorphisms C1604G and A1650G in the

regulatory region of the CypA gene was studied in three groups: HIV-1

positive MSM of the ACS (n = 334), MSM of the ACS who remained

seronegative despite reported high-risk behavior (High-risk

seronegatives, HRSN; n = 68), and HIV-1 negative blood donors

(controls) (n = 104). For all groups, genotype distributions and minor

allele frequencies are shown in Table 1. Although a higher allele

frequency of C1604G was observed in the HIV-1 infected MSM as

compared to the HRSN participants and controls, this difference was not

statistically significant indicating that the C1604G is not associated with

susceptibility to HIV-1 infection (Table 1).

Table 1: Genotype distributions of the CypA SNPs C1604G and A1650G and risk for

HIV-1 infection.

The minor allele frequency of the A1650G polymorphism in the HRSN

was significantly increased as compared to HIV-1 positive MSM,

suggesting that the 1650G allele may be associated with a decreased

susceptibility to HIV-1 infection in participants of the ACS.

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Effects of regulatory CypA polymorphisms on disease progression

Next, we examined the influence of polymorphisms C1604G and A1650G

on the clinical course of infection in HIV-1 positive MSM of the ACS.

Kaplan Meier and Cox proportional hazard analyses were used to test for

potential differences in rates of progression to CD4+ T cells below 200

cells per µl, clinical AIDS according to the CDC definition of 1987 and

1993 [27], [28] or plasma viral RNA load above 104.5 copies per ml

between individuals who carried the major or minor allele (dominant

model). The C1604G and the A1650G polymorphisms were not

associated with survival time among MSM of the ACS, independent of

the endpoints used for analysis (Table 2). In addition, both

polymorphisms were also not associated with CD4+ T cell number or

plasma viral RNA load at setpoint in HIV-1 positive MSM (data not

shown).

Similar analyses were performed in the ACS of drug users (DU, n =

120). In this cohort, 9% of the individuals were heterozygous for

C1604G (minor allele frequency of 0.046) and 25.4% were heterozygous

for A1650G (minor allele frequency of 0.129). None of the individuals

was homozygous for either 1604G or 1650G. The minor allele frequency

for the C1604G and A1650G polymorphism among DU was similar to the

frequencies observed in the controls (Table 1).

Table 2. Effects of CypA SNPs C1604G and A1650G on disease progression in

the Amsterdam cohort of MSM.

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Chapter 5 107

Table 3. Effects of CypA SNPs C1604G and A1650G on disease progression in

DU of the Amsterdam cohort.

Also in participants of the ACS-DU, the minor alleles of C1604G and

A1650G were not associated with progression to a first CD4+ T cell

count below 200 cells per µl blood, or clinical AIDS (table 3). However,

carriers of the minor allele of C1604G showed a significantly accelerated

progression towards a viral RNA load above 104.5 copies per ml plasma

(log rank p = 0.007, RH 2.55, CI95% 1.252–5.171; p = 0.01) (Figure 1,

table 3). DU carrying the A1650G minor allele tended to have a

somewhat slower progression towards viral RNA load above 104.5 copies

per ml plasma, albeit not statistically significant (Figure 1, table 3). No

significant association between CD4+ T-cells or RNA setpoint and the

C1604G or A1650G polymorphism was observed (data not shown).


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Figure 1. Survival analysis for C1604G and A1650G in DU of the ACS. Kaplan

Meier analysis for time in months from seroconversion to viral RNA load above 10

4.5 copies per ml plasma in DU of the ACS based on the C1604G genotype (left

panel) or A1650G genotype (right panel). Black lines indicate individuals homozygous for the major allele (left panel: 1604CC; right panel: 1650AA); Gray lines indicate individuals heterozygous for the minor allele (left panel: 1604CG; right panel: 1650AG).

Effect of regulatory polymorphisms in CypA on CypA expression levels

and HIV-1 replication

Next we analyzed whether the C1604G and A1650G polymorphisms in

the regulatory regions of the CypA gene were associated with altered

CypA expression levels. To this end, we performed quantitative RT-PCR

on CypA mRNA levels in peripheral blood mononuclear cells (PBMC) of

28 healthy blood donors genotyped for the C1604G and A1650G

polymorphism. We observed a reduced CypA expression in PBMC that

were heterozygous for the 1604G minor allele as compared to PBMC that

were homozygous for the major allele (1604C) (Unpaired T-test; P =

0.023) (figure 2). No differences in CypA expression were observed in

PBMC heterozygous for the 1650G minor allele compared to PBMC

homozygous for the 1650A major allele.


Chapter 5 109

Next, we analyzed the replicative capacity of HIV-1 in PHA-stimulated

PBMC from healthy individuals with known genotypes for the C1604G

and A1650G polymorphism. PHA-stimulated PBMC were inoculated with

NL4-3 Ba-L (2×103 TCID50 per 5×106 cells). Subsequently, virus

replication was assessed by measuring p24 antigen levels in the culture

supernatant every other day for a period of two weeks. No significant

differences in p24 production were observed at any time point during

the culture period, irrespective of the genotype of the regulatory region

of the CypA gene (figure 3).

Figure 2. Analysis of CypA expression levels. CypA mRNA

levels in PBMC obtained from healthy controls that were homozygous for the 1604 and 1650 major alleles (1604CC/1650AA), heterozygous for the 1604 minor allele (1604CG), or heterozygous for the 1650 minor allele (1650AG). CypA mRNA levels were normalized for β-actin mRNA levels.

Figure 3. Analysis of HIV-1

replication in genotyped PBMC.

Replication of NL4-3 Ba-L in PHA

stimulated PBMC from healthy

donors homozygous for the 1604

and 1650 major allele

(1604CC/1650AA), heterozygous

for the 1604 minor allele

(1604CG), or heterozygous for

the 1650 minor allele (1650AG).

Virus replication was analyzed by

measuring p24 production in

culture supernatant. P24

production at day 12 after

infection is shown.



Discussion

CypA has been identified as a cofactor for HIV-1, leading to enhanced

infection by a largely unknown mechanism [2]. The identification of

Trim5-CypA fusion proteins as restriction factors [16], [17] and the

dependency of Rhesus Trim5α on CypA has renewed the interest in this

ubiquitously expressed cellular protein [11]–[14]. Here we analyzed the

effects of polymorphisms in the promoter region of the CypA gene on

susceptibility for HIV-1 infection and disease progression in participants

of the ACS.

In a previous study, 1604G was observed to be associated with a more

rapid CD4+ T-cell decline in African Americans and a trend towards

more rapid progression to AIDS was observed in European Americans

[25]. In our cohort of HIV-1 positive Caucasian MSM, no association

between 1604G and progression to AIDS or any other endpoint was

observed. This is in agreement with observations in the Swiss cohort of

HIV-1 infected individuals who are also all Caucasian [26].

In the ACS on HIV-1 infected drug users, we could also not demonstrate

an association between polymorphisms in the regulatory region of the

CypA gene and survival to CD4+ T-cell counts below 200 cells per µl or

AIDS according to the 1987 and 1993 definitions. In this cohort,

however, time to plasma viral RNA load above 104.5 copies per ml was

significantly reduced in 1604G carriers. These observations suggest only

a minor effect of polymorphisms in the regulatory region of CypA which

may become apparent only during the late stages of infection when viral

load is increasing.

A second polymorphism in the regulatory region of the CypA promoter

involves position 1650. In a Swiss Caucasian cohort, 1650G was

reported to be associated with a rapid CD4+ T cell loss and enhanced

progression to disease [26]. An association between the presence of

1650G and delayed disease progression was also observed in a cohort of

HIV-1 infected African Americans and a trend towards a delayed

progression to AIDS was observed in European Americans [25]. We

were unable to confirm these results in our cohort of MSM. However, we

did observe an enrichment of the 1650G allele in MSM who have

remained seronegative despite high risk behavior (HRSN) as compared

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Chapter 5 111

to HIV-1 infected MSM which might indicate that the 1650G allele is

associated with a reduced susceptibility to HIV-1 infection. This

contradicts findings of an earlier study where a reduced prevalence of

the 1650G allele was observed in European Americans who remained

seronegative despite high risk behavior as compared to a seroconverter

cohort [25]. Furthermore, no effect of the 1650G allele on the HIV-1

susceptibility and replication was observed in an in vitro replication

assay using PHA stimulated PBMC from donors who were homozygous

for the 1650A allele or heterozygous for the 1650G allele. However, in

vitro infection of PHA stimulated PBMC may be not reflective of in vivo

transmission conditions.

The observed differences between our present study and earlier reports

[25], [26] may be explained by differences in the study populations.

Interactions with other sequence variations that are distributed

differently between the populations, immunological differences, different

HIV-1 transmission routes, and differences in the circulating virus

populations may account for the variable outcomes of these studies.

Variations in natural history of HIV-1 infection among different HIV-1

infected cohorts have been reported before. For example, heterozygosity

for the 32 basepair deletion in the CCR5 gene has a strong protective

effect in the cohort of HIV-1 infected MSM of the ACS [18] whereas no

effect on disease progression was observed in HIV-1 infected DU of the

ACS [29].

Polymorphisms in the promoter region of CypA may have an effect on

the CyPA expression levels and recently it was demonstrated that the

1604G allele enhances binding of transcription factors such as SP1,

resulting in increased transcription of the CypA gene [25], [30].

Seemingly in contrast, we here observed a reduction in CypA expression

in PBMC isolated from healthy C1604G heterozygous blood donors when

compared to PBMC from carriers of the major allele. However, another

polymorphism in the regulatory region of CypA, (C1575G), which is in

100% linkage disequilibrium with the C1604G polymorphism, was shown

to reduce nuclear factor binding and decrease transcription of CypA

[25]. Our results may thus indicate that the effect of these

polymorphisms on CypA transcription is dominated by 1575G allele.

However, the reduction in CypA expression levels had no effect on HIV-1





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replication in vitro using PHA stimulated PBMC from donors who were

homozygous or heterozygous for the C1604G genotype.

Overall, we here show that polymorphisms in the regulatory region of

the CypA are only weakly associated with the clinical course of infection

in the ACS. However, considering the important role of this protein in

HIV-1 replication, manipulation of CypA activity by therapeutic

intervention should be seriously considered.

Materials and Methods

Study participants

The study population, 364 Caucasian, MSM enrolled in the Amsterdam

Cohort studies (ACS) on the natural history of HIV-1 infection between

October 1984 and March 1986, was previously described [18]. The

censor date of our study was set at the first day of effective

antiretroviral therapy of the participant or end of follow-up. Of the 364

participants, 131 seroconverted during the study. The remaining 233

men were positive for HIV-1 antibodies at entry between October 1984

and April 1985. In previous epidemiological studies, the time since

seroconversion of these prevalent cases has been estimated based on

the incidence of HIV-1 infection amongst MSM of the Amsterdam Cohort

and was on average 1.5 years before entry into the cohort studies [31].

For analysis, we combined the 131 participants with documented

seroconversion and 233 seroprevalent participants with an imputed

seroconversion date as one study group, since previous studies have not

revealed differences in AIDS-free survival between the two groups [21].

From 334 participants DNA samples were available for analysis.

Sixty-eight MSM with high-risk behavior that persistently remained HIV-

seronegative (HRSN) were retrospectively selected from the ACS. They

remained seronegative during a follow up of 5 years before January

1996 and had reported unprotected receptive anal intercourse with at

least 2 different partners before January 1996 (introduction of highly

active antiretroviral therapy (HAART)).


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Chapter 5 113

A second study population consists of 120 HIV-1 infected intravenous

drug users (DU) that participate in the Amsterdam Cohort Studies on

HIV and AIDS among intravenous drug users which started enrolment in

1985. Asymptomatic men and women who were living in the Amsterdam

area and who reported intravenous drug use in the preceding 6 months

were enrolled in a prospective study on the prevalence and incidence of

HIV-1 infection and risk factors for AIDS. In our study, we included 84

individuals who seroconverted for HIV antibodies during active follow-

up. The median time between the last negative test and the first positive

test for this group of prospective seroconverters was 3.9 months

(interquartile range, 3.7–5.1). At entry in the cohort study 37

individuals were positive for HIV antibodies but had a negative test

result before entry (i.e., retrospective seroconverters). Negative test

results were obtained from samples taken for hepatitis B virus serology.

The median interval between the last negative and first positive tests

was 4.1 years (interquartile range, 1.8–5.4) for these seroconverters.

For all 120 HIV infected DU (76 men and 44 women) used for our

present analysis, the imputed seroconversion date [31] was calculated

and used for further analysis.

Healthy volunteer blood donors (n = 104) were used as HIV-1 negative

control group. The ACS has been conducted in accordance with the

ethical principles set out in the declaration of Helsinki and written

informed consent is obtained prior to data collection. The study was

approved by the Academic Medical Center institutional medical ethics

committee.

Genotyping for regulatory polymorphisms in CypA

DNA samples of 334 HIV+ MSM, 68 HRSN, 120 HIV+ DU and 104

healthy controls were available for genotyping. For analysis of the

polymorphism in the promoter region of CypA, DNA samples were

amplified by PCR using Taq DNA polymerase (Invitrogen) in the

presence of 3 mM MgCl with primer pair C1604G-F (5′-

GCACTGTCACTCTGGCGAAGTCGCAGAC-3′) and P4H-R (5′-

GCCGAGCACGTGCGTCGGACAGGAC-3′). The following amplification

cycles were used: 5 min 95°C; 40 cycles of 30 s 95°C, 45 s 65°C, 60 s

72°C; 10 min 72°C. Subsequently PCR products were subjected to a

restriction digest with 1U BstN1 (1 hour 60°C; New England Biolabs) to

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detect the C1604G polymorphism or with 10U Rsa1 (1 hour 37°C; New

England Biolabs) to detect the A1650G polymorphism and analyzed on a

1.25% agarose gel. A PCR product containing a C at position 1604 or a

G at position 1650 will result in a 524 bp fragment (undigested) product.

A PCR product containing a C at position 1604 will result in digestion of

the PCR product by BstN1 into a 423 bp and 101 bp fragments. A PCR

product containing an A at position 1650 will be digested with Rsa1

resulting in a 369 bp and 55 bp fragments.

CypA expression analysis and in vitro replication assay

Cells from 28 healthy blood donors were isolated, genotyped for C1604G

and A1650G and the CypA expression levels were measured by real time

Light cycler PCR. RNA was isolated by Trizol (Invitrogen) according to

the manufacturer's protocol. Subsequently, cDNA was prepared using

the SuperScript™ First-Strand Synthesis System for RT-PCR

(Invitrogen). CypA mRNA levels were analyzed by SYBR green qPCR

using the LightCycler (Roche). The reaction mix contained 20 mM Tris-

HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 200 µM dNTP, 250 µg/ml BSA,

500 nM primers, SYBR green I nucleic acid gel stain 40.000× diluted in

water, and 0.6 U platinum Taq DNA polymerase (Invitrogen). The

following primer sets were used for detection of CypA cDNA: CypA-2F:

5′-CTTCATCCTAAAGCATACGG-3′ and CypA-2R: 5′-

CTTCTTGCTGGTCTTGCC-3′. Serial dilutions of plasmid DNA containing

cDNA of CypA were used as a standard curve. To correct for differences

in the cDNA input, levels of β-actin cDNA were analyzed by a SYBR

green qPCR using the following primer set: BA-RNA-F 5′-

GGCCCAGTCCTCTCCCAAGTCCAC-3′ and BA-RNA-R 5′-

GGTAAGCCCTGGCTGCCTCCACC-3′. A serial dilution of 8E5 cells was

used as a standard curve for β-actin. SYBR green qPCR was performed

using the following program on the LightCycler: (1) preincubation and

denaturation: 50°C for 2 min, 95°C for 2 min; (2) amplification and

quantification: 45 cycles of 95°C for 5 sec, 55°C for 15 sec, 72°C for 15

sec; (3) melting curve: 95°C for 0 sec, 65°C for 15 min, 95°C for 0 sec

with a temperature transition rate of 0.1°C/sec. Specificity of the PCR

products measured using the SYBR green method was confirmed by a

melting curve.

Chapter 5 115

The replicative capacity of HIV-1 in PBMC from healthy blood donors

that were genotyped for C1604G and A1650G was investigated. PBMC

were isolated from buffy coats obtained from healthy seronegative blood

donors by Ficoll-isopaque density gradient centrifugation. Cells

(5×106/ml) were stimulated for 2 days in Iscove's modified Dulbecco

medium (Whitaker) supplemented with 10% fetal calf serum (Hyclone),

penicillin (Gibco Brl) (100 U/ml), streptomycin (Gibco Brl) (100 µg/ml)

and phytohemagglutinin (Welcome) (5 µg/ml). Cells were inoculated

with 2000 TCID50 NL4-3(Ba-L) and replication was assessed by

measuring virus production in culture supernatants by in-house p24

antigen capture enzyme-linked immunosorbent assay (ELISA) [32].

Statistical analysis

Statistical analysis was performed employing SPSS (version 16). The

genetic effects of SNPs on HIV-1 susceptibility were assessed by

comparing the allelic and genotypic frequencies between susceptibility

groups using the Pearson chi square test. The minor allele frequencies

were compared to the most common genotype as a reference group.

Kaplan Meier and Cox proportional hazard analysis were performed to

study the genotypic association and relative hazard (RH) between the

C1604G and A1650G polymorphisms in the regulatory regions of the

CypA gene and disease progression. The following endpoints were

considered for analysis: 1) AIDS according to the 1987 Centers for

Disease Control and Prevention (CDC) definition [27]; 2) AIDS according

to the 1993 CDC definition [28]; 3) CD4+ T cell counts below 200

cells/µl blood and 4. Viral RNA load above 104.5 copies per ml plasma.

CypA expression and differences in replicative capacity of HIV-1 in

genotyped donor PBMC were compared using the student T-test. A

result was considered significant if the P-value was <0.05.

Acknowledgements

This study was performed as part of the Amsterdam Cohort Studies

(ACS) on HIV-1 infection and AIDS, a collaboration between the

Amsterdam Health Service, the Academic Medical Center of the

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University of Amsterdam, Sanquin Blood Supply Foundation, and the

University Medical Center Utrecht

(http://www.amsterdamcohortstudies.org/). We are greatly indebted to

all cohort participants for their continuous participation. The authors

thank Jodie Kamati for excellent technical assistance and Hanneke

Schuitemaker for helpful discussion and critical reading of the

manuscript.

Author contributions

Conceived and designed the experiments: MAR NAK. Performed the

experiments: MAR KAVD. Analyzed the data: MAR NAK. Wrote the

paper: MAR NAK.

References

1. Braaten D, Franke EK, Luban J (1996) Cyclophilin A is required for an early step in

the life cycle of human immunodeficiency virus type 1 before the initiation of

reverse transcription. J Virol 70: 3551–3560.

2. Luban J, Bossolt KL, Franke EK, Kalpana GV, Goff SP (1993) Human

immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. Cell 73:

1067–1078.

3. Franke EK, Yuan HEH, Luban J (1994) Specific incorporation of cyclophilin A into

HIV-1 virions. Nature 372: 359–362.

4. Gamble TR, Vajdos FF, Yoo S, Worthylake DK, Houseweart M, et al. (1996) Crystal

structure of human cyclophilin A bound to the amino-terminal domain of HIV-1

capsid. Cell 87: 1285–1294.

5. Braaten D, Ansari H, Luban J (1997) The hydrophobic pocket of cyclophilin is the

binding site for the human immunodeficiency virus type 1 Gag polyprotein. J Virol

71: 2107–2113.

6. Hatziioannou T, Perez-Caballero D, Cowan S, Bieniasz PD (2005) Cyclophilin

interactions with incoming human immunodeficiency virus type 1 capsids with

opposing effects on infectivity in human cells. J Virol 79: 176–183.

7. Sokolskaja E, Sayah DM, Luban J (2004) Target cell cyclophilin A modulates human

immunodeficiency virus type 1 infectivity. J Virol 78: 12800–12808.

8. Kootstra NA, Munk C, Tonnu N, Landau NR, Verma IM (2003) Abrogation of

postentry restriction of HIV-1-based lentiviral vector transduction in simian cells.

Proc Natl Acad Sci U S A 100: 1298–1303.

http://www.amsterdamcohortstudies.org/

Chapter 5 117

9. Towers GJ, Hatziioannou T, Cowan S, Goff SP, Luban J, et al. (2003) Cyclophilin A

modulates the sensitivity of HIV-1 to host restriction factors. Nat Med 9: 1138–

1143.

10. Bosco DA, Eisenmesser EZ, Pochapsky S, Sundquist WI, Kern D (2002) Catalysis of


Acad Sci U S A 99: 5247–5252.

11. Berthoux L, Sebastian S, Sokolskaja E, Luban J (2005) Cyclophilin A is required for

TRIM5{alpha}-mediated resistance to HIV-1 in Old World monkey cells. Proc Natl

Acad Sci U S A 102: 14849–14853.

12. Keckesova Z, Ylinen LM, Towers GJ (2006) Cyclophilin A renders human

immunodeficiency virus type 1 sensitive to Old World monkey but not human

TRIM5 alpha antiviral activity. J Virol 80: 4683–4690.

13. Stremlau M, Song B, Javanbakht H, Perron M, Sodroski J (2006) Cyclophilin A: an

auxiliary but not necessary cofactor for TRIM5alpha restriction of HIV-1. Virology

351: 112–120.

14. Rits MA, van Dort KA, Munk C, Meijer AB, Kootstra NA (2007) Efficient transduction

of simian cells by HIV-1-based lentiviral vectors that contain mutations in the

capsid protein. Mol Ther 15: 930–937.

15. Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, et al. (2004) The

cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World

monkeys. Nature 427: 848–853.

16. Sayah DM, Sokolskaja E, Berthoux L, Luban J (2004) Cyclophilin A

retrotransposition into TRIM5 explains owl monkey resistance to HIV-1. Nature

430: 569–573.

17. Nisole S, Lynch C, Stoye JP, Yap MW (2004) A Trim5-cyclophilin A fusion protein

found in owl monkey kidney cells can restrict HIV-1. Proc Natl Acad Sci U S A 101:

13324–13328.

18. De Roda Husman AM, Koot M, Cornelissen M, Brouwer M, Broersen SM, et al.

(1997) Association between CCR5 genotype and the clinical course of HIV-1

infection. Ann Intern Med 127: 882–890.

19. Smith MW, Dean M, Carrington M, Winkler C, Huttley GA, et al. (1997) Contrasting

genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease

progression. Science 277: 959–965.

20. Winkler C, Modi W, Smith MW, Nelson GW, Wu X, et al. (1998) Genetic restriction

of AIDS pathogenesis by an SDF-1 chemokine gene variant. Science 279: 389–

393.

21. van Rij RP, Broersen S, Goudsmit J, Coutinho RA, Schuitemaker H (1998) The role

of a stromal cell-derived factor-1 chemokine gene variant in the clinical course of

HIV-1 infection. AIDS 12: F85–F90.

22. An P, Bleiber G, Duggal P, Nelson G, May M, et al. (2004) APOBEC3G genetic

variants and their influence on the progression to AIDS. J Virol 78: 11070–11076.

23. An P, Duggal P, Wang LH, O'Brien SJ, Donfield S, et al. (2007) Polymorphisms of

CUL5 are associated with CD4+ T cell loss in HIV-1 infected individuals. PLoS Genet

3: e19.

24. van Manen D, Rits MA, Beugeling C, van Dort KA, Schuitemaker H, et al. (2008)

The effect of Trim5 polymorphisms on the clinical course of HIV-1 infection. PLoS

Pathog 4: e18.


25. An P, Wang LH, Hutcheson-Dilks H, Nelson G, Donfield S, et al. (2007) Regulatory

polymorphisms in the cyclophilin A gene, PPIA, accelerate progression to AIDS.

PLoS Pathog 3: e88.

26. Bleiber G, May M, Martinez R, Meylan P, Ott J, et al. (2005) Use of a combined ex

vivo/in vivo population approach for screening of human genes involved in the

human immunodeficiency virus type 1 life cycle for variants influencing disease

progression. J Virol 79: 12674–12680.

27. Centers for disease control (1987) Revision of the CDC surveillance case definition

for acquired immunodeficiency syndrome. MMWR 36: (suppl 1)1S–15S.

28. Centers for disease control (1993) 1993 revised classification system for HIV

infection and expanded surveillance case definition for AIDS among adolescents

and adults. MMWR 41 (RR-17): 1–19.

29. Schinkel J, Langendam M, Coutinho RA, Krol A, Brouwer M, et al. (1999) No

evidence for an effect of the CCR5 Δ32/+ and the CCR2b 64I/+ mutations on

Human Immunodeficiency Virus type 1 (HIV-1) disease progression disease

progression among HIV-1 infected injecting drug users. J Infect Dis 179: 825–831.

30. Palacin M, Rodriguez I, Garcia-Castro M, Ortega F, Reguero JR, et al. (2008) A

search for cyclophilin-A gene (PPIA) variation and its contribution to the risk of

atherosclerosis and myocardial infarction. Int J Immunogenet 35: 159–164.

31. Geskus RB (2000) On the inclusion of prevalent cases in HIV/AIDS natural history

studies through a marker-based estimate of time since seroconversion. Stat Med

19: 1753–1769.

32. Tersmette M, Winkel IN, Groenink M, Gruters RA, Spence P, et al. (1989) Detection

and subtyping of HIV-1 isolates with a panel of characterized monoclonal

antibodies to HIV-p24 gag. Virology 171: 149–155.

Chapter 6

The effect of Type I Interferons on

HIV-1 replication in monocyte

derived macrophages M.A.N. Rits, K.A. van Dort and N.A. Kootstra

Dept Experimental Immunology, Sanquin Research, Landsteiner

Laboratory, and Center for Infectious Diseases and Immunity

Amsterdam (CINIMA) at the Academic Medical Center of the University

of Amsterdam; 1105AZ Amsterdam, the Netherlands.


Chapter 6 121

Abstract

Type I interferons are known regulators of HIV-1 replication. Here we

studied the effects of Type I IFNs (IFN-α and IFN-β) on HIV-1 replication

in monocyte-derived macrophages (MDM). IFN-α treatment of the MDM

resulted in complete inhibition of virus replication. The absence of

reverse transcription (RT) products in these cells indicated that HIV-1

replication was blocked early in the replication cycle. IFN- treated MDM

expressed normal levels of the HIV-1 (co)receptors CD4 and CCR5 and a

similar restriction was observed for HIV-1 pseudotyped with VSV-G,

excluding viral entry as the rate limiting step in IFN- treated MDM. The

block on virus replication in IFN-α treated MDM coincided with increased

Trim5α expression, which can interfere at an early step in the viral life

cycle. This suggests that Trim5α might at least be partially involved in

the restriction.

IFN-β treatment of MDM also resulted in inhibition of virus replication. In

these cells, the presence of completed RT products indicated that entry,

uncoating and RT could proceed normally in IFN-β treated MDM.

However, we observed a strong inhibition of HIV-1 LTR driven

chloramphenicol acetyltransferase (CAT) activity, indicating that IFN-β

treatment can strongly inhibit HIV-1 transcription. IFN-β treatment had

no effect on Trim5 and Trim22 expression, which respectively interfere

with RT and HIV-1 transcription. This implies involvement of other IFN-β

responsive cellular factors in the observed inhibition of HIV-1 replication.

Our data indicate that type I interferons are able to block HIV-1

replication in MDM at different stages of the viral lifecycle.

Introduction

The innate immune system plays an important role in the first line of

defense against viral infection and prevents spread of the virus through

the body. The best studied innate antiviral defense mechanism is the

interferon system 21. Interferons (IFNs) are a family of cytokines that

are rapidly upregulated upon viral infection. IFNs can be subdivided in

type I and type II IFNs, which both can induce an antiviral state in

target cells but act through different cellular receptors. IFN-α and IFN-β

belong to the IFN type I family and are secreted by many cell types,

122 Effects of Interferons on HIV-1 transduction

whereas IFN-γ is a member of the IFN type II family and is produced by

cells of the immune system. IFNs bind their cognate receptor resulting

in signal transduction via the Jak/STAT pathway, which leads to

transcriptional activation of IFN stimulated genes (ISG) and the

induction of a number of genes encoding cellular effector proteins.

Among these effecter proteins are protein Kinase RNA-dependent, 2’5’

oligoadenylate synthetase/RNAseL, and certain Mx proteins 28,30. More

recently other cellular factors, involved in the intrinsic cellular defense

against HIV-1 and other retroviruses, have been identified to be IFN

responsive. These include the apolipoprotein B cytidine deaminase 3

(Apobec3)G 31, CD317 23 and a number of tri-partite containing motif

(Trim) proteins such as Trim5α 1,27 and Trim22 3,7,35.

Apobec3G can be incorporated in the HIV-1 virion in the absence of

sufficient concentrations of the HIV-1 encoded accessory protein Vif and

restricts viral replication through deamination of cytosines in the minus

strand DNA of the virus during reverse transcription 5,31. Additionally,

Apobec3G can restrict HIV-1 infection at an early post-entry step in

resting CD4+ cells by a yet unknown mechanism 10. CD317 blocks viral

replication in the absence of sufficient amounts of the HIV-1 encoded

accessory protein Vpu 23 at the step of budding of the virion from the

infected cell. Newly produced virions are retained at the cell surface and

are degraded after endocytosis. Trim proteins are able to interfere with

HIV-1 replication at different levels of the HIV-1 replication cycle 24.

Trim5α restricts HIV-1 at an early post-entry step 32 by interacting with

the viral capsid protein 17,19,33 and attenuating infection mainly by

disturbing the regulated process of viral uncoating 9,33, reverse

transcription, and/or trafficking of the viral core to the nucleus 4,12,32.

Human Trim5α, as compared to many simian Trim5α orthologues, only

intermediately restricts HIV-1. However, the association between a

genetic polymorphism in the Trim5 gene with a decreased antiviral

activity 16 and an accelerated clinical course of HIV-1 infection suggests

that Trim5α contributes to control of HIV-1 replication in humans in vivo 37. Trim22 interferes with HIV-1 transcription most likely by suppression

of the activity of the HIV-1 LTR 7,35 and inhibition of virus production

through disruption of cellular trafficking of viral proteins 3.

Besides CD4+ T cells, macrophages are critical target cells for HIV-1 and

are among the first cells that become infected during primary infection 8,29,36. In our present study, we analyzed the effects of Type I IFNs on

Chapter 6 123

HIV-1 replication in human monocyte derived macrophages (MDM) and

observed that both IFN-α and IFN-β treatment inhibited HIV-1

replication in MDM. IFN-α treatment resulted in restriction of HIV-1 at an

early phase of the replication cycle most likely before or at reverse

transcription (RT), whereas IFN-β inhibited a phase in HIV-1 replication

probably at the level of viral transcription. The block in HIV-1 replication

in IFN-α treated MDM coincided with an increase in Trim5α expression,

which might indicate that Trim5α is involved in the observed restriction.

IFN-β treatment of MDM had no effect on transcription of known

restriction factors and therefore a novel IFN- induced innate immune

factor may be involved in this antiviral response in MDM.

Materials and methods

Virus

Virus stocks were prepared by Calcium phosphate transfection of 293T

cells as described previously 13 with pNL4-3 Ba-L for replication

competent HIV-1. VSV-G pseudotyped single round luciferase reporter

virus was obtained by transfection of pNL4-3-luc-R-E clone (obtained

through the AIDS Research and Reference reagent program, from Dr

Nathaniel Landau) 11,15. Before storage, the virus stocks were filtered

through a 0.22 μm filter. The infectious titers of the virusstocks were

quantified by determining the 50% tissue culture infectious dose

(TCID50) on PHA stimulated stimulated peripheral blood mononuclear

cells (PBMC) obtained from healthy donors. Replication of NL4-3 Ba-L

was analyzed using an in-house p24 capture ELISA 34. Infection of the

single round reporter virus was determined by luciferase activity.

Luciferase activity was quantified by addition of 25μl of freshly prepared

luciferase substrate (0.83mM ATP, 0.83mM D-luciferine (Duchefa

Biochemie B.V., Haarlem, The Netherlands), 18.7mM MgCl2, 0.78μM

Na2H2P2O7, 38.9mM tris (pH7.8), 0.39% (vol/vol) GLYCEROL, 0.03%

(VOL/VOL) Triton X-100, and 2.6μM dithiotreitol) to the cell culture.

Luminescence was measured for 1 sec per well in a luminometer (Centro

LB 960, Berthold Technologies).


MDM isolation, culture and infection

Monocytes were isolated from peripheral blood mononuclear cells

(PBMC) from healthy blood donors by plastic adherence. In brief, PBMC

were isolated by Ficoll density gradient centrifugation and were

resuspended in endotoxin free Iscove’s modified Dulbecco’s medium

(IMDM) supplemented with 10% human serum, penicillin (100U/ml)

(Invitrogen) and streptomycin (100μg/ml) (Invitrogen) at a density of

5×106 cells per ml. PBMC were incubated in a culture flask for 2 hours at

37˚C in a humidified atmosphere supplemented with 5% CO2 to allow

monocytes to adhere to the culture flask. Unbound cells were removed

by 3 sequential washing steps with PBS. Monocytes were detached from

the culture flask using a cell scraper after 5 minute incubation with

10mM EDTA in PBS. To obtain MDM, the cells were cultured in 24-wells

tissue culture plates at a density of 1×106 cells per ml for 5 days in

endotoxin free IMDM supplemented with 10% human serum

(Invitrogen), penicillin (100U/ml) (Invitrogen) and streptomycin

(100μg/ml) (Invitrogen) at 37˚C in a humidified atmosphere

supplemented with 5% CO2.

Type I Interferons (IFN-α A/D and IFN-β both obtained from Sigma-

Aldrich) were added to the MDM cultures at the indicated concentrations

at day 2 post isolation. At day 5 post isolation, MDM were harvested for

total RNA isolation or inoculated with NL4-3 Ba-L at a multiplicity of

infection (m.o.i.) of 0.01. Twenty-four hours after inoculation,

unabsorbed virus was removed. Culture supernatant samples were

taken every 2–3 days post inoculation and were analyzed for virus

production by an in-house p24 antigen capture ELISA 34. For the

analysis of the process of reverse transcription by PCR, the virus stocks

were treated with 20 Units DNase (Promega) for 45 min and filtered

through a 0.22-µm-pore-size filter before inoculation, to distinguish for

newly synthesized proviral DNA. DNA samples were harvested 24 hours

after inoculation.

When VSV-G psuedotyped single round luciferase reporter viruses were

used, MDM were harvested at day 3 after inoculation and luciferase

activity was analyzed as described above.

Chapter 6 125

RNA and DNA isolation and PCR analysis

Total RNA was isolated using TRIzol reagent according to the

manufacturer (Invitrogen). cDNA was synthesized using the

Superscript™ First-strand synthesis system for RT-PCR (Invitrogen)

using oligo(dT)20. The cDNA was then subjected to LightCycler (Roche)

qPCR using SYBR Green I nucleic acid gel stain (Invitrogen) to analyze

CCR5, Trim5α, Trim22 and β-actin expression levels. The reaction mix

contained 20 mM Tris-HCl (ph8.4), 50 mM KCl, 3mM MgCl2, 200μM

dNTPs, 250μg/ml BSA, 500nM primer, SYBR Green diluted 40.000× in

H2O and 0.6 Units platinum Taq DNA polymerase (Invitrogen). Trim5α

primers: Fw: 5’cca gga tag ttc cca tac3’, Rv: 5’aga gct tgg tga gca cag

agt 3’. Primers used for Trim22: Fw: 5’ gga tgc cag cac gct cat ctc ag 3’

and Rv 5’ ttc agc atc acg tcc acc cag tag t 3’. Primers used for CCR5:

CCR5-S: 5’-gat agg tac ctg gct gtc gtc cat-3’ and CCR5-AS: 5’-acc agc

ccc aag atg act atc t-3’. To correct for differences in cDNA input, levels

of β-actin were measured. The primers for β-actin used were: β-actin-S

5’-ggg tca gaa gga ttc cta tg-3’ and β-actin-AS 5’-ggt ctc aaa cat gat ctg

gg-3’. Specificity of the PCR products measured using the SYBR green

method was confirmed by a melting curve. Real-time PCR was

performed running the following program on the LightCycler (Roche):

(1) preincubation and denaturation: 50°C for 2 min, 95°C for 2 min; (2)

amplification and quantification: 45 cycles of 95°C for 5 sec, 55°C for 15

sec, 72°C for 15 sec; (3) melting curve: 95°C for 0 sec, 65°C for 15

min, 95°C for 0 sec with a temperature transition rate of 0.1°C/sec. A

standard curve for quantification of Trim5α and Trim22 mRNA was

prepared from serial dilution of plasmid DNA containing the cDNA of

Trim5α and Trim22. CCR5 and β-actin standards were prepared by serial

dilutions of 8E5 cells 14.

To determine the reverse transcription efficiency, viral DNA was isolated

from MDM 24 hr after inoculation using the L6 method as described 6.

The DNA was subjected to PCR using primer pairs amplifying early R/U5

and intermediate/late pol reverse transcription products in HIV-1

reverse transcription using 50mM MgCl2 and 25 Units of GO-taq

(Promega). For amplification of the R/U5 region, the primers pair eRT2F

5’-gtg ccc gtc tgt tgt gtg ac-3’ and eRT2R 5’-ggc gcc act gct aga gat tt-3’

was used 22. The following PCR program was used: (1) preincubation

and denaturation: 94°C for 5 min; (2) amplification and quantification:

28 cycles of 94°C for 15 sec, 60°C for 30 sec, 72°C for 45 sec, followed


by an incubation at 72°C for 5 min. The pol region was amplified using

primer pair pol-D Fw 5’ gct aca tga act gct acc agg 3’ and pol-F Rv 5’tta

gtc agt gct gga atc agg 3’. The following PCR program was used: (1)

preincubation and denaturation: 94°C for 5 min; (2) amplification and

quantification: 28 cycles of 94°C for 15 sec, 50°C for 30 sec, 72°C for

45 sec, followed by an incubation at 72°C for 5 min.

To correct for differences in DNA input, β-actin levels were measured

using the primer pair described above. The following PCR amplification

program was used: (1) preincubation and denaturation: 94°C for 5 min;

(2) amplification and quantification: 28 cycles of 94°C for 5 sec, 55°C

for 30 sec, 72°C for 45 sec, followed by an incubation at 72°C for 5 min.

PCR products were visualized using a 1% agarose gels containing SyBr-

Safe according to the manufacturers recommendation (Invitrogen).

HIV-1 DNA standards were prepared by serial dilutions of 8E5 cells,

which carry a single proviral DNA copy per cell 14.

Transfection and CAT assay

293T cells were seeded at a density of 50×103 cells/ml in a 24wells

plate. The next day the cells were transfected by Calcium phosphate

method with DNA constructs that contains the long terminal repeat

(LTR) of HIV-IIIB linked to a chloramphenicol acetyltransferase (LTR-

CAT) construct were cotransfected with the simian virus 40-tat (SV-TAT)

plasmid construct in which the tat gene is under control of the simian

SV40 promotor, to increase the level of LTR driven CAT expression. For

the analysis of specific promoter activity, a DNA construct in which CAT

expression is regulated by NF-κB binding sites linked to the thymidine

kinase promoter was used. After 24 hours later, the medium was

replaced and fresh IFNs were added. Forty-eight hours after

transfection, the cells were harvested and CAT expression was

measured by ELISA as described by the manufacturer (Roche).

FACS analysis

Monocytes were cultured and allowed to differentiate into MDM. At

day.5, type I IFNs (500U/ml) were added to the cultures. The next day,

the cells were washed with PBS and detached by incubation in 10mM

EDTA in PBS for 5min at 37°C. Cells were washed and resuspended in

Chapter 6 127

FACS buffer (5% FCS in PBS supplemented with 10% TNC). For the

FACS staining, 2×105 MDM were incubated with antibodies against CD4

(PerCP conjugated), CCR5 (FITC conjugated) in FACS buffer for 20min

on ice. The cells were washed with FACS buffer and fixed with 2%

paraformaldehyde for 10min. Fluorescence was analyzed on a FACS-

Canto II (Becton Dickinson).

Statistical analysis

Statistical analyses and figures were generated in Graphpad prism.

CCR5, Trim5α and Trim22 mRNA levels were compared using the

Students T-test.

Results

Type I IFNs inhibit HIV-1 replication in MDM

The effect of Type I IFNs on replication of HIV-1 NL4-3 Ba-L in MDM was

analyzed. MDM from 3 different donors were treated with 500 U/ml IFN-

α or IFN-β 72 hours before inoculation with HIV-1 NL4-3 Ba-L (m.o.i.

0.01). Twenty-four hours after inoculation the cells were washed to

remove unabsorbed virus and virus replication in the culture

supernatant was analyzed every 2–3 days in a p24 antigen capture

ELISA. IFN-α treatment of MDM resulted in complete inhibition of virus

replication and p24 levels in the culture supernatant were below the

detection limit of the ELISA of 25ng p24 per ml. IFN-β treatment of MDM

also reduced virus replication albeit to a lesser extent (fig 1). Similar

replication kinetics of NL4-3 Ba-L in MDM were observed when IFN

treatment was performed 24 hours before inoculation (data not shown).

To exclude that IFN treatment of MDM down regulated expression of the

receptor and coreceptor for HIV-1 entry in MDM, CD4 and CCR5

expression levels were analyzed. FACS analysis revealed no difference in

surface expression of CD4 and CCR5 on IFN treated MDM (data not

shown). Quantitative real time PCR demonstrated a modest increase in

CCR5 mRNA levels when MDM were treated with IFN-α or IFN-β (fig 2)

but the differences were not significant. This indicated that IFN

treatment did not negatively affect CD4 and CCR5 expression on the cell


surface of MDM and that the entry receptors were expressed and

available for entry.

FIG 1: Replication of HIV-1

NL4-3 Ba-L in MDM treated

with IFN-α or IFN-β. MDM from

3 healthy donors were treated

with 500 Units/ml IFN-α or IFN-

β for for 72 hours before

inoculation with HIV-1 NL4-3

Ba-L at an m.o.i. of 0.01. Virus

replication was monitored by

measuring p24 production in

the culture supernatants.

Symbols represent: IFN-α

treatment (■) IFN-β treatment

(▲) and medium (♦).

FIG 2: Effect of IFN-α or IFN-β treatment on CCR5 mRNA expression levels in

MDM MDM from 6 healthy donors were cultured in the presence or absence of

500U/ml of IFN-α or IFN-β. Total RNA was isolated and subjected to quantitative

RT-PCR. To correct for differences in cDNA input, CCR5 expression levels were

corrected for β-actin input.

0 5 10 150

50

100

150

Day

ng

p24

None IFN- IFN-10-3

10-2

10-1

100

Treatment

Co

pie

s C

CR

5/

-acti

n

Chapter 6 129

Type I IFNs interfere with early events of the HIV-1 lifecycle in MDM

To further analyze at which step in the viral replication cycle IFN-α and

IFN-β treatment were interfering with HIV-1 replication in MDM, we

infected IFN-α and IFN-β treated MDM from 2 healthy donors with a

single round HIV-1 luciferase reporter virus (NL4-3-luc-R-E-) that was

pseudotyped with the envelope glycoprotein of Vesicular Stomatitis Virus

(VSV-G). MDM were treated with IFN-α or IFN-β (1-31.3U/ml) for 24

hours, a dose dependent inhibition of luciferase activity was observed,

with maximum inhibition to levels below the limit of detection at IFN

concentrations of 500 U/ml. This indicated that both IFN-α and IFN-β

interfered with HIV-1 replication at a post-entry step of the replication

cycle (fig 3).

FIG 3: Inhibition of VSV-G pseudotyped HIV-1 reporter virus in MDM by IFN-α and

IFN-β. MDM were treated with serial dilutions of IFN-α or IFN-β and inoculated with

VSV-G pseudotyped lentiviral vectors encoding the luciferase gene. Two days after

inoculation, relative light units (RLU) were measured using a luminometer. Symbols

represent: IFN-α treatment (■) and IFN-β treatment (▲).

Next we analyzed whether HIV-1 replication in IFN treated MDM was

inhibited at the level of reverse transcription (RT). MDM from 3 healthy

donors were treated with IFN-α (500U/ml) or IFN-β (500U/ml) 72 hours

before inoculation with a DNase treated inoculum of NL4-3 Ba-L

0 10 20 30103

104

105

106

IFN (U/ml)

RL

U


(moi=0.01). One day after inoculation, DNA was extracted and analyzed

for the presence of proviral DNA. We amplified a fragment in the R/U5

region in the LTR and in the pol region representing early and

intermediate/late products of the RT process, respectively. In IFN-α

treated MDM the amount of both early and late RT products was

severely reduced as compared to the untreated control MDM (fig 4). This

indicated that inhibition of HIV-1 replication induced by IFN-α occurred

most likely prior to or during RT. IFN-β treatment of MDM had no or

little effect on the generation of RT products in two out of three donors

(fig 4), indicating that RT proceeded normally in IFN-β treated MDM and

that the block in HIV-1 replication was at a post–RT step. In MDM

obtained from donor 2, decreased levels of pol PCR products were

observed. However, virus replication in MDM cultures of this donor could

be detected one week after inoculation (fig 1), which indicates that IFN-

β treatment might have delayed the process of RT but had not blocked

RT irreversibly.

FIG 4: PCR analysis for the presence of proviral DNA in IFN-α or IFN-β treated

MDM. MDM from 3 healthy donors were treated with 500 Units/ml IFN-α or IFN-β

and subsequently inoculated with HIV-1 NL4-3 Ba-L at an m.o.i. of 0.01. DNA was

isolated at 1 day after inoculation and PCR analysis for the detection of proviral

DNA was performed. R/U5 PCR products represent early products of RT and Pol

PCR products represent intermediate/late products of the RT process. To correct

for differences in DNA input an additional PCR detecting β-actin was performed.

The effect of Type I IFN on HIV-1 transcription

To analyze whether type 1 IFN could interfere with HIV-1 replication at a

transcriptional level, we used a reporter constructs in which the

chloramphenicol acetyl transferase (CAT) gene is under control of an

HIV-1 LTR or an NF-κB responsive promotor. 293T cells were

Chapter 6 131

transfected with the HIV-1 LTR and the NF-κB controlled CAT reporter

constructs. The next day, the medium was refreshed and the cells were

treated with IFN-α (500U/ml) or IFN-β (500U/ml) for one day. At day 2

after transfection, cell lysates were harvested and analyzed for CAT

activity by ELISA. In both IFN-α and IFN-β treated cells that were

transfected with the LTR CAT construct, a strong reduction in CAT

activity was observed while NF-κB driven CAT activity was not affected

by IFNs (fig 5). Notably, a modest increase in CAT activity was observed

when the 293T cells that were transfected with the NF-B controlled CAT

reporter construct were incubated with IFN-α.

FIG 5: Analysis of the effects of IFN-α and IFN-β on HIV-1 LTR or NF-kB

transcriptional activity. 293T cells were transiently transfected with HIV-1 LTR or

NF-κB reporter construct expressing CAT. After 24 hours, the cells were treated

with IFN-α and IFN-β (500U/ml) and subsequently transcriptional activation of the

HIV-1 LTR and NF-kB sites was analyzed by an ELISA detecting CAT.

Regulation of Trim5α and Trim22 expression in MDM by Type I IFN

Expression of a number of cellular proteins that are known to interfere

with HIV-1 replication, are regulated by type I IFNs. Among these

cellular proteins are Trim5α and Trim22, which respectively block HIV-1

replication prior to RT 32 and at a transcriptional level 7,35. To analyze

whether Trim5α was involved in the block in HIV-1 infection prior to RT

observed in IFN-α treated MDM Trim5α mRNA expression levels were

analyzed in IFN treated MDM. MDM obtained from 6 healthy donors were

treated with IFN-α (500U/ml) or IFN-β (500U/ml). Three days after IFN

treatment, total RNA was extracted and the amount of Trim5α mRNA

was assessed by quantitative RT-PCR. A significant increase in Trim5α

NF-B

None IFN- IFN-0

25

50

75

100

Treatment

ng

/ml

LTR-TAT

None IFN- IFN-0

25

50

75

100

Treatment

ng

/ml


mRNA levels was observed when MDM were treated with IFN-α

(p=0.0013), whereas IFN-β treatment did not have an effect on the

Trim5α expression in MDM (fig 6a).

Next we investigated whether Trim22 mRNA levels were altered in IFN

treated MDM. IFN-α treatment resulted in a significant increase of

Trim22 mRNA levels in MDM (p=0.0067) whereas IFN-β treatment had

no effect on Trim22 expression levels (fig 6b).

FIG 6: Effect of IFN-α and IFN-β treatment on Trim5α and Trim22 expression levels

in MDM. MDM from 6 healthy donors were cultured in the presence or absence of

500U/ml IFN-α or IFN-β. Total RNA was isolated and subjected to real-time RT-

PCR to determine Trim5α (a) and Trim22 (b) mRNA expression levels. To correct

for differences in cDNA input, Trim5α and Trim22 expression levels were corrected

for β-actin input.

Discussion

The results obtained in this study demonstrate that IFN-α and IFN-β

block HIV-1 replication in primary MDM confirming previous studies 2,18.

The restriction observed in IFN treated MDM was not due to changes in

entry receptor expression since CCR5 and CD4 expression on the cellular

membrane was not affected by treatment with either IFN-α and IFN-β.

Additionally, when receptor mediated entry was circumvented by the

use of a VSV-G pseudotyped single round reporter virus, a strong

inhibition of infection was observed in MDM treated with IFN-α and IFN-

Chapter 6 133

β, confirming a block to infection at a post-entry level. In agreement

with previous studies 2,18, we observed that HIV-1 infection in IFN-α

treated MDM was blocked at or before the reverse transcription step.

IFN-β treatment of MDM resulted in a donor specific efficiency on the

process of RT, however, detection of p24 production in all cultures

confirmed that RT could proceed upon IFN-β treatment. Therefore it is

likely that IFN-β inhibits HIV-1 replication in MDM at a late step in the

replication cycle, probably at the level of viral transcription. Indeed, IFN-

β treatment inhibited the transcriptional activity of the HIV-1 LTR in

293T cells.

Recently it was demonstrated that expression of restriction factors like

Apobec3G and Trim proteins can be regulated by IFNs in different cells

types 1,25-27. Trim proteins are known to interfere with HIV-1 replication

at different steps of the viral lifecycle 24. Trim5α blocks HIV-1 replication

at an early post-entry step 32, and Trim22 interferes with LTR driven

viral transcription and cellular trafficking or viral proteins 3,7,35. In IFN-α

treated MDM, the block at an early post-entry level coincided with

increased Trim5α levels. Since human Trim5α only intermediately

restricts HIV-1 32, it is highly unlikely that Trim5α alone is responsible

for the strong antiviral activity observed upon IFN-α treatment.

Unfortunately, we were unable to knock down Trim5α in primary MDM

and therefore could not quantify the contribution of Trim5α in the IFN-α

induced restriction in MDM.

Previous studies have indicated that the block in HIV-1 replication in

IFN-α treated MDM was mediated by Trim22 7. Trim22 was

demonstrated to bind the LTR promoter region of HIV-1 and to regulate

viral transcription 35. In agreement, we here observed an increase in

Trim22 expression when MDM were treated with IFN-α. IFN-β treatment

of MDM had no effect on Trim22 expression levels, albeit that also in

IFN- treated MDM LTR mediated transcriptional activity was strongly

reduced. This indicates that additional factors regulated by IFN-β might

be involved in the observed reduction in transcriptional activity of the

HIV-1 LTR.

Here we observed that Trim5α and Trim22 expression levels were

regulated by IFN-α, but not IFN-β. This observation combined with

previous observations that both IFN-α and IFN-β were able to regulate

Trim5α and Trim22 mRNA expression in different cell lines and primary

cells 1,3,7,20,27, indicates that Trim5α expression can be regulated both by


IFN-α and IFN-β, but that the regulation of Trim5 by IFN- is cell type

specific. Type I IFNs have been demonstrated to regulate expression of

many Trim proteins 26 of which Trim5, Trim6, Trim21, Trim22 and

Trim34 have been reported to posses antiviral activity 20,38. However,

only Trim5α and Trim22 have been demonstrated to restrict HIV-1 20.

In conclusion, we have here demonstrated that type I IFNs can block

both early and late steps in the HIV-1 replication cycle in primary MDM.

As we observed an IFN-α mediated increase in the expression of Trim5α

and Trim22 in MDM, it is tempting to speculate that these factors

contribute to the observed restriction in HIV-1 replication. However, the

exact mechanism involved in the type I IFN induced restriction of HIV-1

replication remains to be elucidated. Identification of cellular factors

involved in IFN induced cellular resistance to HIV-1 will increase our

knowledge on virus-host interactions and might provide new potential

targets for antiviral therapy.

Acknowledgements

The authors thank Hanneke Schuitemaker for critical reading of the

manuscript.

This work was supported by the Netherlands Organization for scientific

Research (NWO grant number 96.36.024); the Dutch AIDS fund (grant

2004062) and the Landsteiner Foundation for Blood Transfusion

Research (grant 0435).

References

1. Asaoka, K., K. Ikeda, T. Hishinuma, K. Horie-Inoue, S. Takeda, and S.

Inoue. 2005. A retrovirus restriction factor TRIM5alpha is transcriptionally

regulated by interferons. Biochem. Biophys. Res. Commun. 338:1950-1956.

2. Baca-Regen, L., N. Heinzinger, M. Stevenson, and H. E. Gendelman. 1994.

Alpha interferon-induced antiretroviral activities: restriction of viral nucleic acid

synthesis and progeny virion production in human immunodeficiency virus type 1-

infected monocytes. J. Virol. 68:7559-7565.

3. Barr, S. D., J. R. Smiley, and F. D. Bushman. 2008. The interferon response

inhibits HIV particle production by induction of TRIM22. PLoS. Pathog. 4:e1000007.

4. Besnier, C., Y. Takeuchi, and G. Towers. 2002. Restriction of lentivirus in

monkeys. Proc. Natl. Acad. Sci. U. S. A 99:11920-11925.

Chapter 6 135

5. Bishop, K. N., R. K. Holmes, A. M. Sheehy, and M. H. Malim. 2004. APOBEC-

mediated editing of viral RNA. Science 305:645.

6. Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-

van Dillen, and J. Van der Noordaa. 1991. A rapid and simple method for

purification of nucleic acids. J. Clin. Microbiol. 28:495-503.

7. Bouazzaoui, A., M. Kreutz, V. Eisert, N. Dinauer, A. Heinzelmann, S.

Hallenberger, J. Strayle, R. Walker, H. Rubsamen-Waigmann, R. Andreesen,

and B. H. von. 2006. Stimulated trans-acting factor of 50 kDa (Staf50) inhibits

HIV-1 replication in human monocyte-derived macrophages. Virology 356:79-94.

8. Braathen, L. R., G. Ramirez, R. O. F. Kunze, and H. Gelderblom. 1987.

Langerhans cells as primary target cells for HIV infection. Lancet1094.

9. Chatterji, U., M. D. Bobardt, P. Gaskill, D. Sheeter, H. Fox, and P. A. Gallay.

2006. Trim5alpha accelerates degradation of cytosolic capsid associated with

productive HIV-1 entry. J. Biol. Chem. 281:37025-37033.

10. Chiu, Y. L., V. B. Soros, J. F. Kreisberg, K. Stopak, W. Yonemoto, and W. C.

Greene. 2005. Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells.

Nature 435:108-114.

11. Connor, R. I., B. Kuan Chen, S. Choe, and N. R. Landau. 1995. Vpr is required

for efficient replication of human immunodeficiency virus type-1 in mononuclear

phagocytes. Virology 206:935-944.

12. Cowan, S., T. Hatziioannou, T. Cunningham, M. A. Muesing, H. G. Gottlinger,

and P. D. Bieniasz. 2002. Cellular inhibitors with Fv1-like activity restrict human

and simian immunodeficiency virus tropism. Proc. Natl. Acad. Sci. U. S. A

99:11914-11919.

13. Dull, T., R. Zufferey, M. Kelly, R. J. Mandel, M. Nguyen, D. Trono, and L.

Naldini. 1998. A third-generation lentivirus vector with a conditional packaging

system. J. Virol. 72:8463-8471.

14. Folks, T. M., D. Powell, M. M. Lightfoote, S. Koenig, A. S. Fauci, S. Benn, A.

Rabson, D. Daugherty, H. E. Gendelman, M. D. Hoggan, S. Venkatesan, and

M. A. Martin. 1986. Biological and biochemical characterization of a cloned Leu-3-

cell surviving infection with the acquired immune deficiency syndrome retrovirus. J.

Exp. Med. 164:280-290.

15. He, J., S. Choe, R. Walker, P. Di Marzio, D. O. Morgan, and N. R. Landau.

1995. Human Immunodeficiency Virus Type 1 Viral protein R (Vpr) arrests cells in

the G2 phase of the cell cycle by inhibiting p34/cdc2 activity. J. Virol. 69:6705-

6711.


16. Javanbakht, H., P. An, B. Gold, D. C. Petersen, C. O'hUigin, G. W. Nelson, S.

J. O'Brien, G. D. Kirk, R. Detels, S. Buchbinder, S. Donfield, S. Shulenin, B.

Song, M. J. Perron, M. Stremlau, J. Sodroski, M. Dean, and C. Winkler. 2006.

Effects of human TRIM5alpha polymorphisms on antiretroviral function and

susceptibility to human immunodeficiency virus infection. Virology 354:15-27.

17. Kar, A. K., F. az-Griffero, Y. Li, X. Li, and J. Sodroski. 2008. Biochemical and

biophysical characterization of a chimeric TRIM21-TRIM5alpha protein. J. Virol.

82:11669-11681.

18. Kornbluth, R. S., P. S. Oh, J. R. Munis, P. H. Cleveland, and D. D. Richman.

1989. Interferons and bacterial lipopolysaccharide protect macrophages from

productive infection by human immunodeficiency virus in vitro. J. Exp. Med.

169:1137-1151.

19. Langelier, C. R., V. Sandrin, D. M. Eckert, D. E. Christensen, V.

Chandrasekaran, S. L. Alam, C. Aiken, J. C. Olsen, A. K. Kar, J. G. Sodroski,

and W. I. Sundquist. 2008. Biochemical characterization of a recombinant

TRIM5alpha protein that restricts human immunodeficiency virus type 1 replication.

J. Virol. 82:11682-11694.

20. Li, X., B. Gold, C. O'hUigin, F. az-Griffero, B. Song, Z. Si, Y. Li, W. Yuan, M.

Stremlau, C. Mische, H. Javanbakht, M. Scally, C. Winkler, M. Dean, and J.

Sodroski. 2007. Unique features of TRIM5alpha among closely related human TRIM

family members. Virology 360:419-433.

21. Malmgaard, L. 2004. Induction and regulation of IFNs during viral infections. J.

Interferon Cytokine Res. 24:439-454.

22. Munk, C., S. M. Brandt, G. Lucero, and N. R. Landau. 2002. A dominant block

to HIV-1 replication at reverse transcription in simian cells. Proc Natl Acad Sci U S A

99:13843-13848.

23. Neil, S. J., T. Zang, and P. D. Bieniasz. 2008. Tetherin inhibits retrovirus release

and is antagonized by HIV-1 Vpu. Nature 451:425-430.

24. Nisole, S., J. P. Stoye, and A. Saib. 2005. TRIM family proteins: retroviral

restriction and antiviral defence. Nat. Rev. Microbiol. 3:799-808.

25. Peng, G., K. J. Lei, W. Jin, T. Greenwell-Wild, and S. M. Wahl. 2006. Induction

of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced

anti-HIV-1 activity. J. Exp. Med. 203:41-46.

26. Rajsbaum, R., J. P. Stoye, and A. O'Garra. 2008. Type I interferon-dependent

and -independent expression of tripartite motif proteins in immune cells. Eur. J.

Immunol. 38:619-630.

Chapter 6 137

27. Sakuma, R., A. A. Mael, and Y. Ikeda. 2007. Alpha interferon enhances

TRIM5alpha-mediated antiviral activities in human and rhesus monkey cells. J. Virol.

81:10201-10206.

28. Samuel, C. E. 2001. Antiviral actions of interferons. Clin. Microbiol. Rev. 14:778-

809, table.

29. Schuitemaker, H., M. Koot, N. A. Kootstra, M. W. Dercksen, R. E. Y. De

Goede, R. P. Van Steenwijk, J. M. A. Lange, J. K. M. Eeftink Schattenkerk, F.

Miedema, and M. Tersmette. 1992. Biological phenotype of human

immunodeficiency virus type 1 clones at different stages of infection: progression of

disease is associated with a shift from monocytotropic to T-cell-tropic virus

populations. J. Virol. 66:1354-1360.

30. Sen, G. C. 2001. Viruses and interferons. Annu. Rev. Microbiol. 55:255-281.

31. Sheehy, A. M., N. C. Gaddis, J. D. Choi, and M. H. Malim. 2002. Isolation of a

human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein.

Nature 418:646-650.

32. Stremlau, M., C. M. Owens, M. J. Perron, M. Kiessling, P. Autissier, and J.

Sodroski. 2004. The cytoplasmic body component TRIM5alpha restricts HIV-1

infection in Old World monkeys. Nature 427:848-853.

33. Stremlau, M., M. Perron, M. Lee, Y. Li, B. Song, H. Javanbakht, F. az-

Griffero, D. J. Anderson, W. I. Sundquist, and J. Sodroski. 2006. Specific

recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha

restriction factor. Proc. Natl. Acad. Sci. U. S. A 103:5514-5519.

34. Tersmette, M., I. N. Winkel, M. Groenink, R. A. Gruters, P. Spence, E.

Saman, G. van der Groen, F. Miedema, and J. G. Huisman. 1989. Detection

and subtyping of HIV-1 isolates with a panel of characterized monoclonal antibodies

to HIV-p24 gag. Virology 171:149-155.

35. Tissot, C. and N. Mechti. 1995. Molecular cloning of a new interferon-induced

factor that represses human immunodeficiency virus type 1 long terminal repeat

expression. J. Biol. Chem. 270:14891-14898.

36. Van 't Wout, A. B., N. A. Kootstra, G. A. Mulder-Kampinga, N. Albrecht-van

Lent, H. J. Scherpbier, J. Veenstra, K. Boer, R. A. Coutinho, F. Miedema, and

H. Schuitemaker. 1994. Macrophage-tropic variants initiate human

immunodeficiency virus type 1 infection after sexual, parenteral and vertical

transmission. J. Clin. Invest. 94:2060-2067.

37. van Manen, D., M. A. Rits, C. Beugeling, K. A. van Dort, H. Schuitemaker,

and N. A. Kootstra. 2008. The effect of Trim5 polymorphisms on the clinical

course of HIV-1 infection. PLoS. Pathog. 4:e18.


38. Zhang, F., T. Hatziioannou, D. Perez-Caballero, D. Derse, and P. D. Bieniasz.

2006. Antiretroviral potential of human tripartite motif-5 and related proteins.

Virology 353:396-409.

Chapter 7

General Discussion

Chapter 7 141

Retroviruses including HIV-1 infect primates in a highly species specific

manner and are specifically adapted to utilize the host cell machinery for

their replication. Recently, genome wide siRNA screens have identified

numerous cellular host factors that are essential for replication 1-3.

Primate genomes contain large proportions of retroviral sequences and

transposable elements. This indicates that primate species have

encountered retroviruses on multiple occasions in the past, which might

drive evolution of host genes that interfere with retroviral replication and

zoonotic transfer of retroviruses. In the recent years several inhibitory

factors like Fv1, Apobec, CD317 and the tri-partite containing motif

(Trim) 5α have been identified that restrict retroviruses at multiple steps

in their replication cycle 4-8. Although identified only recently, it is

already firmly established that restriction factors have an important role

in protection against viruses, suggesting that they are part of the

conventional immune system. An important feature is the Interferon

mediated transcriptional regulation. CD317 was identified by its potent

up regulation in response to IFN-α 8 and many Trim family members,

including Trim5α, have been reported to be regulated by IFNs in

immune cells 9 (this thesis), a property that links them directly to innate

immune responses.

Several members of the large Trim family are thought to have a role in

immunity related processes 10,11. Several Trims are involved in antiviral

activity; notably, the anti retroviral activity of Trim1 strongly affects N-

MLV replication 12, Trim22 inhibits HIV-1 replication, although the stage

at which it inhibits is unsure 13-15. Trim28 restricts MLV LTR driven

transcription in murine embryonic cells 16. Additionally, an extensive

screen for antiviral activity among Trim proteins suggested that Trim11,

Trim31 and Trim62 could interfere with various stages of MLV and HIV-1

replication 17.

Besides the IFN mediated regulation of transcription, a number of

additional features suggest a substantial role for Trim5α in anti retroviral

immunity. First, elevated levels of non-synonymous substitutions in

Trim5α relative to other host cellular factors indicate that the pressure

for adaptation is high 18, a feature that is observed more often in host

factors that directly interact with the pathogen. Second and more

elusive, there is remarkable similarity in the ability to interact with

structured pathogen associated complexes, reminiscent of the pattern

recognition receptors, also known as Toll-like receptors (TLR). TLRs are


important mediators of immune responses and are required for full

immunogenic potential 19. Similar as how TLRs recognize particular

patterns specifically associated with pathogens, Trim5α recognizes and

binds the assembled retroviral core complexes. Finally, Trim5α interacts

by association of the B30.2 domain to the core particle. Structural

predictions of the B30.2 domain suggested resemblance to the folding of

immuno globulin, consisting of layered antiparallel β-sheets 20.

The molecular mechanism by which Trim5α mediates restriction is

unknown. Viral cores are subject to increased destabilization when

sufficiently bound by Trim5α 21,22. The premature destabilization or

uncoating could have several effects, including exposure of cDNA to

factors present in the cytoplasm or degradation of the core protein

complex by Trim5α induced proteasome mediated mechanisms.

Evolutionary battles between host and pathogen

For some restriction factors CD317 and Apobec3G, complex retroviruses

have adopted accessory proteins, Vpu and Vif respectively, that interfere

with the imposed restriction. Although the molecular mechanism by

which Vpu interferes with CD317 is largely unknown, it is known that Vif

directly interacts with Apobec3G and targets it for proteasome mediated

degradation 23. On a molecular evolutionary level, ultimately Vif’s goal is

to strengthen the interaction with Apobec3, while Apobec’s ultimate goal

is to escape the inhibition mediated by the Vif protein. Similarly, Trim5α

interacts with the retroviral capsid, the affinity of which largely

determines the strength of inhibition 21. Retroviruses can escape from

Trim5α mediated restriction by sequence variation in capsid, resulting in

a loss of recognition.

On an inter-species level, it is often observed that one or the other has

the advantage. Indeed, a high level of species-specific infection of

retroviruses is observed which is imparted to some extent to the

inhibitory effects of restriction factors. The simian Trim5α interacts with

the HIV-1 capsid with much higher affinity when compared to its human

orthologue 21, and the restricting activity is consequently very high. This

is specified by sequence variation in the variable regions on the B30.2

domain of Trim5α 24. The variation largely specifies the highly species

and virus specific response.

Chapter 7 143

Interestingly, the adaptation of Trim5α to inhibit one retrovirus can lead

to inability to resist other retroviruses. Human ancestors have been

infected by retroviruses for millions of years. In that regard, HIV-1 is a

rather new human pathogen; host defenses have not been selected to

inhibit HIV-1, but are probably adapted to inhibit more ancient retroviral

infections. Notably, the potent restriction of a chimpanzee endogenous

retrovirus, which is absent from the human genome, by the human

Trim5α suggests that Trim5α had an important role in the protection

against this retrovirus 25. Indeed, the adaptation of the human Trim5α to

past retroviral episodes could have selected for Trim5 variants that

have now demonstrated reduced ability to inhibit HIV-1.

In addition to Trim5α, cyclophilin A (CypA) was identified previously as a

binding partner for the HIV-1 capsid 26. CypA is a highly versatile protein,

which results in species specific phenotypes of HIV-1 infection. It acts as

a cofactor for HIV-1 in human cells 26, in which it was suggested to

participate in the regulated process of uncoating, while in some non-

human primate species, CypA functions as a cofactor for Trim5α

mediated inhibition of HIV-1 (chapter 2)27,28.

CypA interacts specifically with the capsid protein of different

lentiviruses, like FIV, SIVagmTAN, SIVcpz, and HIV-1 29, indicating that

CypA binding is a rather common phenomenon among lentiviruses. It is

hard to say whether this property arose independently in these separate

lineages, or whether a common ancestor was able to bind CypA and that

this property was lost in other lentiviruses like SIVs. The ability for a

lentivirus to interact with CypA could also be evolutionary dynamic and

might be changed or lost upon adaptation to a new host.

Cyclophilin A modulates HIV-1 infectivity

The HIV-1 capsid protein between helices 4 and 5 (CypA binding region)

and the residues G89 and P90 are critical for the interaction with CypA 30-32. Disruption of this interaction by cyclosporin A (CsA) or mutation of

critical residues in this loop that inhibits CypA binding, results in

inhibition of HIV-1 replication at an early post entry step 31-38.

It has been suggested that the conformation of the CypA binding region

is important for viral fitness and CypA has been implicated to be

responsible for maintaining the conformation of the CypA binding region


39. However, CypA binding to the HIV-1 capsid catalyzes cis-trans

isomerization and structural changes in the capsid protein have been

observed 40-43. In continuation of these observations, we employed a so

called ‘fate of capsid’ assay, described in chapter 3, to analyze potential

differences in core structure during an infection, regarding the presence

of CypA. We observed that CypA could potently modify the structural

organization of the assembled HIV-1 core, which suggests that CypA can

change core conformation and assembly of the HIV-1 core. This

suggests that CypA is involved in HIV-1 uncoating.

Mutations in the HIV-1 capsid have diverse effects on infectivity

Although most primary HIV-1 isolates require CypA binding for optimal

replication, CypA independent replication of HIV-1 group M variants has

also been reported 44,45. Indeed, naturally occurring variations in the

CypA binding region of capsid have been identified in primary isolates 46,47. All identified virus variants retained the central glycine-proline

motif and variation was observed primarily at amino acid positions V86,

H87, I91, and M96. In addition, other HIV-1 groups and HIV-2 show

sequence diversity in the CypA binding region at positions proximal and

distal to the conserved glycine-proline motif, indicating that sequence

variability is well tolerated in this region.

The naturally occurring substitutions identified in the HIV-1 group M

variants were reduced in affinity for CypA but this did not noticeably

lead to alterations in infectivity (this thesis), suggesting that the CypA

binding loop adopts a particular configuration that allows for CypA

independent HIV-1 infection. The independency was mostly associated

with residues proximal to the central glycine-proline motif in the CypA

binding region. More specifically, an H87Q/P largely reproduced the

CypA independent infectivity phenotype 48 (this thesis), suggesting a

crucial interaction for the histidine at position 87 in CypA modulated

infectivity.

Mutations that render HIV-1 infection CypA independent also appear to

be able to compensate for mutations in the capsid protein that have

been demonstrated to impact the stability of the assembled core particle.

For example, the T110N escape mutation in the TW10 epitope presented

by HLA-B57/5801 is frequently observed in HIV-1 positive carriers of the

HLA-B57/5801 allele and has important consequences on core stability.

Chapter 7 145

This substitution in the capsid protein is often associated with upstream

mutations at residues H87, I93 and M96 in the CypA binding region,

which partially restores the defect induced by T110N mutation 49. The

compensatory mutations reduce the affinity for CypA and consequently

display a higher stability of the core protein complex and regain of

infectivity.

In contrast to the CypA independent infection observed by introduction

of mutations proximal to the CypA binding region, CypA hypersensitivity

has been recognized which mapped to mutations distal from the CypA

binding region 35,50. Mutations in the distal region of the CypA binding

site render HIV-1 replication CsA-dependent. Introduction of these

substitutions have no apparent effect on CypA affinity for the HIV-1

capsid. Given the potent effects of CypA on infectivity of these HIV-1

variants, this suggests that the effects of CypA on these HIV-1 mutants

might have structural consequences. Nuclear magnetic resonance (NMR)

indicated that the flexible proline-rich loop was altered by mutations in

the distal region of the CypA binding region, probably through

perturbation of the Type II tight turn in the CypA binding region 50,51.

This might result in the increased response to CypA by alteration of the

loop flexibility, leading to yet unknown effects on capsid structure and

stability.

Interestingly, substitutions of residues at distant positions in the HIV-1

capsid protein have also been implicated in CypA hyper responsiveness.

The T54A substitution, located in the alpha helix 3 of capsid, also

renders HIV-1 CsA-dependent and thus resembles the phenotypes

observed with HIV-1 mutants altered in the CypA binding region 52. In

addition, consequences of R132K mutation in the KK10 epitope

presented by carriers of the HLA B27 also result in particles that are

non-infectious, but could be rescued by CsA treatment 53.

Combined, these data suggest that CypA can induce substantial

structural changes and has an effect on capsid regions that appear to be

involved in global core stability. Notably, some of the CsA dependent

phenotypes could be rescued to some extent by introduction of

compensatory mutations that render HIV-1 infection CypA independent,

leading to regain of infectivity. It is tempting to speculate that the

affinity of CypA for the capsid protein combined with the CypA

expression levels in the target cell can explain the observed infectivity.


Detailed analysis of HIV-1 infectivity in cells with varying expression of

CypA indicated that the effects of CypA on wild type HIV-1 infection

appear to be bimodal and infectivity phenotypes of mutants having

altered CypA dependence could largely be ascribed to CypA expression

levels 39,54,55. This indicates that there is a fine balance between CypA

expression levels and CypA affinity for capsid in HIV-1 infection; high

expression levels of CypA may increase capsid destabilization resulting

in reduced replication. On the other hand, low levels of CypA might

tighten capsid-capsid interaction thereby preventing proper core

uncoating and reducing virus replication.

The observation that residues at distant positions in the capsid protein

can alter CypA sensitivity indicates that both CypA affinity for the capsid

protein as well as structural consequences upon CypA binding lie at the

heart of CypA induced HIV-1 infectivity, being able to alter core

structure and stability. In addition, the interaction with one particular

host factor could result in altered recognition by yet other host factors.

The hereafter-discussed CypA-enhanced Trim5α mediated restriction

observed in many non-human primates is an example of such a

mechanism.

CypA acts as a Trim5α cofactor in many non-human primates

Many African green monkeys and rhesus macaques are highly refractory

to HIV-1 infection, which can largely be ascribed to the inhibitory

activity of Trim5α from these species 7. Although the exact binding site

for Trim5α in the HIV-1 capsid is not known, the CypA binding region

has been identified to be an important viral determinant in HIV-1

restriction in these cells 44,47 (chapter 2). The CypA enhanced sensitivity

for these non-human primate Trim5α orthologues largely coincided with

the CypA dependence of HIV-1 infection in human cells (chapter 2).

HIV-1 containing mutations proximal to the central glycine-proline motif

can lead to circumvention of Trim5α mediated restriction in simian cells 44,47 (chapter 2). Indeed, HIV-1 sensitivity to Trim5α decreased in

mutants that were able to infect human cells in a CypA independent

manner 27,28,44,56 (chapter 2). CypA most likely promotes Trim5α

mediated restriction in these simian species by effects on the capsid

protein rather than on Trim5α directly, since CypA has no influence on

Chapter 7 147

the Trim5α mediated restriction of retroviruses like MLV that do not

interact with CypA.

Co-infection experiments performed in simian cell lines demonstrated

that CypA independent HIV-1 variants were unable to efficiently saturate

the restriction of wild type HIV-1 in these cells 44,45(this thesis). This

observation suggested that Trim5α did not recognize these mutant viral

capsid proteins. However, Stremlau et al demonstrated that HIV-1 core

particles with the H87Q substitution were still susceptible to Trim5α

mediated destabilization when rhesus macaque Trim5α was

overexpressed 21, indicating that these mutants are still recognized and

sensitive to Trim5α mediated inhibition.

Co-infection experiments performed to establish whether or not

particular virus variants are sensitive to the same restriction must be

interpreted with great care. The effects of mutations on capsid stability,

but also possible differences in recognition by CypA, Trim5α and other

thus far unknown factors that have altered interactions or consequences

induced by the interaction could mystify the interpretation. In this

respect, Forshey et al observed reduced ability to saturate the

restriction in simian cells when the stability of the HIV-1 capsid was

altered 57,58. The mutations that were introduced were not located in the

exposed loops but did impair the stability of the cores.

Speculatively, the particles having altered stability could be impaired in

efficient processing kinetics. Altered processing may also explain part of

the inability for chimeric HIV-1 to saturate for the restriction. We

observed increased amounts of core protein retained in the cytoplasm of

infected cells when CypA was inhibited (chapter 3). The assay we

employed to quantify dissolution of capsid from whole particles fails to

discriminate between uncoating/destabilization and disappearance, e.g.

by proteasome mediated degradation. The retention of large amounts of

particles in the cytoplasm might result in alterations in core processing

kinetics.

The stimulatory effects of CypA on HIV-1 infectivity in many human cells

initially led to the suggestion that CypA protected the HIV-1 core from

the human Trim5α orthologue 38. However, the human Trim5α

orthologue does not appear to respond to the presence of CypA since

knock-down and over expression experiments demonstrated that CypA

and Trim5α influence HIV-1 infectivity independently 28,37,59.


Host genetic variation influences disease progression and susceptibility

to HIV-1

Cellular factors like CypA and Trim5α have a direct effect on HIV-1

replication and therefore genetic variations in these genes might also

have an effect on HIV-1 susceptibility and the clinical outcome of the

disease. Genetic variation in genes that encode for HIV-1 cofactors or

restriction factors can have a strong impact on the outcome of disease.

For example, carriers of a 32bp deletion in the chemokine coreceptor

renders these individuals highly refractory to infection with HIV-1 and

individuals that are heterozygous for this deletion have a slower disease

progression 60-62.

The polymorphisms identified in Trim5α and CypA did not strongly

predict the clinical course of HIV-1 infection, and the variation in clinical

course of HIV-1 infection was observed largely during the late stages of

disease.

Two single nucleotide polymorphisms in the regulatory region of the

CypA gene were analyzed for their effects on HIV-1 susceptibility and

disease progression (chapter 4). C1604G and A1650G are located in the

regulatory region of CypA gene. Increased CypA expression was

observed in purified cells of 1604G, however, no difference in in vitro

replication of HIV-1 was observed in purified cells from genotyped

donors (chapter 4).

Notably, we observed an accelerated increase in viral load in carriers of

the C1604G minor allele in a cohort of injecting drug users (IDU), but

not MSM. However, differential effects of genetic polymorphisms on the

clinical course of infection in IDU and MSM have been described before,

specifically for the effect of a heterozygous CCR5-Δ32 genotype 63.

A future for therapy?

The existence of restriction factors that inhibit the most basic aspect of

the HIV-1 replication cycle sparkles the imagination considering the

application of therapeutic intervention. One could consider the

therapeutic use of a highly restricting simian Trim protein as a transgene

in an ex-vivo gene therapy setting. Inhibitors of the HIV-1 accessory

proteins Vif and Vpu could reduce their capacity to interfere with the

potent inhibition posed by Apobec3G and CD317 respectively.

Chapter 7 149

During the course of our investigation, it became clear that inhibitors for

capsid assembly and disassembly, as well as non-immunogenic CypA

inhibitors such as Debio-025 could be highly beneficial in reducing HIV-1

replication. Some promising candidates are being developed 48,64 that

target CypA and/or capsid and may interfere with the regulated process

of uncoating. Such therapy may prove beneficial in the outcome of the

clinical course of infection for HIV-1 infected individuals.

References

1 Brass AL, Dykxhoorn DM, Benita Y et al. Identification of Host Proteins Required for

HIV Infection Through a Functional Genomic Screen. Science 2008.

2 Konig R, Zhou Y, Elleder D et al. Global analysis of host-pathogen interactions that

regulate early-stage HIV-1 replication. Cell 2008; 135: 49-60.

3 Zhou H, Xu M, Huang Q et al. Genome-scale RNAi screen for host factors required

for HIV replication. Cell Host Microbe 2008; 4: 495-504.

4 FRIEND C. Leukemia of adult mice caused by a transmissible agent. Ann N Y Acad

Sci 1957; 68: 522-32.

5 Lilly F. Fv-2: identification and location of a second gene governing the spleen focus

response to Friend leukemia virus in mice. J Natl Cancer Inst 1970; 45: 163-9.

6 Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that inhibits

HIV-1 infection and is suppressed by the viral Vif protein. Nature 2002; 418: 646-

50.

7 Stremlau M, Owens CM, Perron MJ et al. The cytoplasmic body component

TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 2004; 427:

848-53.

8 Neil SJ, Zang T, Bieniasz PD. Tetherin inhibits retrovirus release and is antagonized

by HIV-1 Vpu. Nature 2008; 451: 425-30.

9 Rajsbaum R, Stoye JP, O'Garra A. Type I interferon-dependent and -independent

expression of tripartite motif proteins in immune cells. Eur J Immunol 2008; 38:

619-30.

10 Reymond A, Meroni G, Fantozzi A et al. The tripartite motif family identifies cell

compartments. EMBO J 2001; 20: 2140-51.


11 Nisole S, Stoye JP, Saib A. TRIM family proteins: retroviral restriction and antiviral

defence. Nat Rev Microbiol 2005; 3: 799-808.

12 Yap MW, Nisole S, Lynch C, Stoye JP. Trim5alpha protein restricts both HIV-1 and

murine leukemia virus. Proc Natl Acad Sci U S A 2004; 101: 10786-91.

13 Barr SD, Smiley JR, Bushman FD. The interferon response inhibits HIV particle

production by induction of TRIM22. PLoS Pathog 2008; 4: e1000007.

14 Tissot C, Mechti N. Molecular cloning of a new interferon-induced factor that

represses human immunodeficiency virus type 1 long terminal repeat expression. J

Biol Chem 1995; 270: 14891-8.

15 Bouazzaoui A, Kreutz M, Eisert V et al. Stimulated trans-acting factor of 50 kDa

(Staf50) inhibits HIV-1 replication in human monocyte-derived macrophages.

Virology 2006; 356: 79-94.

16 Wolf D, Goff SP. TRIM28 mediates primer binding site-targeted silencing of murine

leukemia virus in embryonic cells. Cell 2007; 131: 46-57.

17 Uchil PD, Quinlan BD, Chan WT, Luna JM, Mothes W. TRIM E3 ligases interfere with

early and late stages of the retroviral life cycle. PLoS Pathog 2008; 4: e16.

18 Johnson WE, Sawyer SL. Molecular evolution of the antiretroviral TRIM5 gene.

Immunogenetics 2009; 61: 163-76.

19 Thompson JM, Iwasaki A. Toll-like receptors regulation of viral infection and disease.

Adv Drug Deliv Rev 2008; 60: 786-94.

20 Henry J, Ribouchon MT, Offer C, Pontarotti P. B30.2-like domain proteins: a growing

family. Biochem Biophys Res Commun 1997; 235: 162-5.

21 Stremlau M, Perron M, Lee M et al. Specific recognition and accelerated uncoating of

retroviral capsids by the TRIM5alpha restriction factor. Proc Natl Acad Sci U S A

2006; 103: 5514-9.

22 Chatterji U, Bobardt MD, Gaskill P et al. Trim5alpha accelerates degradation of

cytosolic capsid associated with productive HIV-1 entry. J Biol Chem 2006; 281:

37025-33.

23 Sheehy AM, Gaddis NC, Malim MH. The antiretroviral enzyme APOBEC3G is

degraded by the proteasome in response to HIV-1 Vif. Nat Med 2003; 9: 1404-7.

24 Ohkura S, Yap MW, Sheldon T, Stoye JP. All three variable regions of the

TRIM5alpha B30.2 domain can contribute to the specificity of retrovirus restriction. J

Virol 2006; 80: 8554-65.

Chapter 7 151

25 Kaiser SM, Malik HS, Emerman M. Restriction of an extinct retrovirus by the human

TRIM5alpha antiviral protein. Science 2007; 316: 1756-8.

26 Luban J, Bossolt KL, Franke EK, Kalpana GV, Goff SP. Human immunodeficiency

virus type 1 Gag protein binds to cyclophilins A and B. Cell 1993; 73: 1067-78.

27 Berthoux L, Sebastian S, Sokolskaja E, Luban J. Cyclophilin A is required for

TRIM5{alpha}-mediated resistance to HIV-1 in Old World monkey cells. Proc Natl

Acad Sci U S A 2005; 102: 14849-53.

28 Keckesova Z, Ylinen LM, Towers GJ. Cyclophilin A renders human immunodeficiency

virus type 1 sensitive to Old World monkey but not human TRIM5 alpha antiviral

activity. J Virol 2006; 80: 4683-90.

29 Lin TY, Emerman M. Cyclophilin A interacts with diverse lentiviral capsids.

Retrovirology 2006; 3: 70.

30 Franke EK, Yuan HEH, Luban J. Specific incorporation of cyclophilin A into HIV-1

virions. Nature 1994; 372: 359-62.

31 Franke EK, Luban J. Inhibition of HIV-1 replication by cyclosporine A or related

compounds correlates with the ability to disrupt the Gag-cyclophilin A interaction.

Virology 1996; 222: 279-82.

32 Thali M, Bukovsky A, Kondo E et al. Functional association of cyclophilin A with HIV-

1 virions. Nature 1994; 372: 363-5.

33 Braaten D, Franke EK, Luban J. Cyclophilin A is required for an early step in the life

cycle of human immunodeficiency virus type 1 before the initiation of reverse

transcription. J Virol 1996; 70: 3551-60.

34 Braaten D, Luban J. Cyclophilin A regulates HIV-1 infectivity, as demonstrated by

gene targeting in human T cells. EMBO J 2001; 20: 1300-9.

35 Hatziioannou T, Perez-Caballero D, Cowan S, Bieniasz PD. Cyclophilin interactions

with incoming human immunodeficiency virus type 1 capsids with opposing effects

on infectivity in human cells. J Virol 2005; 79: 176-83.

36 Sokolskaja E, Sayah DM, Luban J. Target cell cyclophilin A modulates human

immunodeficiency virus type 1 infectivity. J Virol 2004; 78: 12800-8.

37 Sokolskaja E, Berthoux L, Luban J. Cyclophilin A and TRIM5alpha independently

regulate human immunodeficiency virus type 1 infectivity in human cells. J Virol

2006; 80: 2855-62.

38 Towers GJ, Hatziioannou T, Cowan S et al. Cyclophilin A modulates the sensitivity of

HIV-1 to host restriction factors. Nat Med 2003; 9: 1138-43.


39 Gatanaga H, Das D, Suzuki Y et al. Altered HIV-1 Gag protein interactions with

cyclophilin A (CypA) on the acquisition of H219Q and H219P substitutions in the

CypA binding loop. J Biol Chem 2006; 281: 1241-50.

40 Bosco DA, Eisenmesser EZ, Pochapsky S, Sundquist WI, Kern D. Catalysis of


Acad Sci U S A 2002; 99: 5247-52.

41 Bosco DA, Kern D. Catalysis and binding of cyclophilin A with different HIV-1 capsid

constructs. Biochemistry 2004; 43: 6110-9.

42 Gamble TR, Vajdos FF, Yoo S et al. Crystal structure of human cyclophilin A bound

to the amino-terminal domain of HIV-1 capsid. Cell 1996; 87: 1285-94.

43 Agresta BE, Carter CA. Cyclophilin A-induced alterations of human

immunodeficiency virus type 1 CA protein in vitro. J Virol 1997; 71: 6921-7.

44 Kootstra NA, Munk C, Tonnu N, Landau NR, Verma IM. Abrogation of postentry

restriction of HIV-1-based lentiviral vector transduction in simian cells. Proc Natl

Acad Sci U S A 2003; 100: 1298-303.

45 Ikeda Y, Ylinen LM, Kahar-Bador M, Towers GJ. Influence of gag on human

immunodeficiency virus type 1 species-specific tropism. J Virol 2004; 78: 11816-22.

46 Kootstra NA, Navis M, Beugeling C, van Dort KA, Schuitemaker H. The presence of

the Trim5alpha escape mutation H87Q in the capsid of late stage HIV-1 variants is

preceded by a prolonged asymptomatic infection phase. AIDS 2007; 21: 2015-23.

47 Chatterji U, Bobardt MD, Stanfield R et al. Naturally occurring capsid substitutions

render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-

resistant in Owl monkey cells. J Biol Chem 2005; 280: 40293-300.

48 Ptak RG, Gallay PA, Jochmans D et al. Inhibition of human immunodeficiency virus

type 1 replication in human cells by Debio-025, a novel cyclophilin binding agent.

Antimicrob Agents Chemother 2008; 52: 1302-17.

49 Brockman MA, Schneidewind A, Lahaie M et al. Escape and compensation from early

HLA-B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency

virus type 1 Gag alter capsid interactions with cyclophilin A. J Virol 2007; 81:

12608-18.

50 Bukovsky AA, Weimann A, Accola MA, Gottlinger HG. Transfer of the HIV-1

cyclophilin-binding site to simian immunodeficiency virus from Macaca mulatta can

confer both cyclosporin sensitivity and cyclosporin dependence. Proc Natl Acad Sci U

S A 1997; 94: 10943-8.

Chapter 7 153

51 Ganser-Pornillos BK, von Schwedler UK, Stray KM, Aiken C, Sundquist WI. Assembly

properties of the human immunodeficiency virus type 1 CA protein. J Virol 2004; 78:

2545-52.

52 Yang R, Aiken C. A mutation in alpha helix 3 of CA renders human

immunodeficiency virus type 1 cyclosporin A resistant and dependent: rescue by a

second-site substitution in a distal region of CA. J Virol 2007; 81: 3749-56.

53 Schneidewind A, Brockman MA, Yang R et al. Escape from the dominant HLA-B27-

restricted cytotoxic T-lymphocyte response in Gag is associated with a dramatic

reduction in human immunodeficiency virus type 1 replication. J Virol 2007; 81:

12382-93.

54 Ylinen LM, Schaller T, Price A et al. Cyclophilin A levels dictate infection efficiency of

human immunodeficiency virus type 1 capsid escape mutants A92E and G94D. J

Virol 2009; 83: 2044-7.

55 Ackerson B, Rey O, Canon J, Krogstad P. Cells with high cyclophilin A content

support replication of human immunodeficiency virus type 1 Gag mutants with

decreased ability to incorporate cyclophilin A. J Virol 1998; 72: 303-8.

56 Stremlau M, Song B, Javanbakht H, Perron M, Sodroski J. Cyclophilin A: an auxiliary

but not necessary cofactor for TRIM5alpha restriction of HIV-1. Virology 2006; 351:

112-20.

57 Forshey BM, von SU, Sundquist WI, Aiken C. Formation of a human

immunodeficiency virus type 1 core of optimal stability is crucial for viral replication.

J Virol 2002; 76: 5667-77.

58 Forshey BM, Shi J, Aiken C. Structural requirements for recognition of the human

immunodeficiency virus type 1 core during host restriction in owl monkey cells. J

Virol 2005; 79: 869-75.

59 Nakayama EE, Shingai Y, Kono K, Shioda T. TRIM5alpha-independent anti-human

immunodeficiency virus type 1 activity mediated by cyclophilin A in Old World

monkey cells. Virology 2008; 375: 514-20.

60 De Roda Husman AM, Koot M, Cornelissen M et al. Association between CCR5

genotype and the clinical course of HIV-1 infection. Ann Intern Med 1997; 127:

882-90.

61 Smith MW, Dean M, Carrington M et al. Contrasting genetic influence of CCR2 and

CCR5 variants on HIV-1 infection and disease progression. Science 1997; 277: 959-

65.

62 Winkler CA, Hendel H, Carrington M et al. Dominant effects of CCR2-CCR5

haplotypes in HIV-1 disease progression. J Acquir Immune Defic Syndr 2004; 37:

1534-8.


63 Schinkel J, Langendam M, Coutinho RA et al. No evidence for an effect of the CCR5

D32/+ and the CCR2b 64I/+ mutations on Human Immunodeficiency Virus type 1

(HIV-1) disease progression disease progression among HIV-1 infected injecting

drug users. J Infect Dis 1999; 179: 825-31.

64 Li J, Tan Z, Tang S et al. Discovery of dual inhibitors targeting both HIV-1 capsid

and human cyclophilin A to inhibit the assembly and uncoating of the viral capsid.

Bioorg Med Chem 2009.

Summary

Summary 157

Summary

Retroviruses such as the human immune deficiency virus (HIV-1) are

adapted to their host and exploit the host cell machinery for different

steps in their replication, such as reverse transcription, integration and

virion assembly. Host cells also express cellular factors that are able to

block virus replication and protect against infection. In this thesis we

studied the role of cellular proteins that specifically interact with the

HIV-1 capsid and thereby influenced HIV-1 replication.

Trim5α is a host restriction factor that is involved in the highly species-

specific tropism of retroviruses. Trim5α specifically recognizes the capsid

proteins of susceptible retroviruses and blocks infection at an early post-

entry level in the viral replication cycle. HIV-1 can be efficiently inhibited

by Trim5α from many non-human primates, but much less efficient by

human Trim5α. The Cyclophilin A (CypA) binding region in the viral

capsid was identified as the viral determinant involved in the restriction

induced by rhesus macaque Trim5α. In chapter 2 we analyzed which

specific amino acid residues in the CypA binding region of HIV-1 or the

closely related HIV-1 group O and HIV-2 were required for the

restriction observed in simian cells. Mutations in the CypA binding region

altered the phenotype of the virus allowing CypA independent infection

in human cells and a non-restricted phenotype in simian cell lines. We

concluded that a reduced affinity of capsid for Cyclophilin A largely

determined the unrestricted phenotype in cell lines derived from rhesus

macaques and African green monkeys. Our results also indicated that

CypA is required for Trim5α mediated inhibition of HIV-1 in simian cells.

This restriction was however not lifted for the CypA-mutant virus in

peripheral blood mononuclear cells (PBMC) from rhesus macaques,

suggesting that in these primary cells additional mechanisms may exist

that restrict HIV-1 replication. In contrast to the combined effect of

CypA and Trim5α on retroviral restriction in simian cells, Trim5α and

CypA are independent regulators of viral infectivity in human cells.

In chapter 3, the effect of mutations in the CypA binding region of the

HIV-1 capsid on the interaction with human Trim5α and CypA was

investigated. We observed that mutations in the CypA binding region of

capsid (H87Q, A88P and I91V) that were associated with CypA

independent replication did not interfere with binding of CypA and

Trim5α. Addition of Cyclosporin A (CsA) did not alter the ability of

158 Summary

human Trim5α to bind to wild type and mutant capsid, suggesting that

CypA and Trim5α do not compete for binding to the capsid protein. We

observed that mutations in the CypA binding region and addition of CsA

increased the stability of the viral core. This suggests that CypA plays a

role in destabilization of the viral core and promotes disassembly of the

core during early events in the replication cycle.

In chapters 4 and 5, the role of CypA and Trim5α in HIV-1 pathogenesis

was studied. Participants of the Amsterdam cohort studies on HIV-1

infection and AIDS (ACS) were screened for polymorphisms in the Trim5

gene (chapter 4) and the CypA gene (chapter 5).

Recently two polymorphisms in the Trim5 gene (H43Y and R136Q) were

shown to affect the antiviral activity of Trim5α in vitro. In agreement with

the reported decreased antiviral activity of Trim5α that contains a Y at

amino acid residue 43 in vitro, an accelerated disease progression was

observed for individuals who were homozygous for the 43Y genotype as

compared to individuals who were heterozygous or homozygous for the

43H genotype. The R136Q polymorphism has been associated with an

increased antiviral activity in vitro. In the ACS however, a protective

effect on the clinical course of infection associated with the 136Q

genotype was only observed after the emergence of CXCR4-using (X4)

HIV-1 variants and only when a viral load of 104.5 copies per ml plasma

was used as an endpoint in survival analysis. Interestingly, naive CD4+

T-cells, which are selectively targeted by X4 HIV-1, revealed a

significantly higher expression of Trim5α than memory CD4+ T-cells.

Thus, polymorphisms in the Trim5 gene may influence the clinical course

of HIV-1 infection also underscoring the antiviral effect of Trim5α on HIV-

1 in vivo.

Previously described C1604G and A1650G polymorphisms in the

regulatory region of CypA were not associated with the clinical course of

infection in the ACS. However, the prevalence of the 1650G allele was

significantly higher in high risk seronegative MSM as compared to HIV-1

infected MSM, suggesting that the A1650G polymorphism may be

associated with protection from HIV-1 infection.

Type I interferons have recently been reported to regulate expression of

several HIV-1 restriction factors. In chapter 6, we studied whether HIV-

1 restriction factors were involved in the Type I IFNs (IFN-α and IFN-β)

mediated block in HIV-1 replication in monocyte-derived macrophages

(MDM). IFN-α treatment of the MDM resulted in complete inhibition of

virus replication at an early step of the replication cycle. The block on

Summary 159

virus replication in IFN-α treated MDM coincided with increased Trim5α

expression, which also interferes with replication at an early step of the

viral life cycle. This suggests that Trim5α might at least be partially

involved in the restriction. IFN-β treatment of MDM also resulted in

inhibition of virus replication mainly at the transcriptional level. IFN-β

treatment had no effect on Trim22 expression, which has previously

been demonstrated to interfere with HIV-1 transcription. This implies

involvement of other IFN-β responsive cellular factors in the observed

inhibition of HIV-1 replication.

In chapter 7, the main findings in this thesis are discussed in light of

current knowledge on the role of CypA and Trim5α mediated inhibition in

HIV-1 replication.

Samenvatting

Samenvatting 163

Samenvatting

Retrovirussen zoals het humaan immuun-deficientie virus (HIV) zijn

aangepast aan de gastheer waarin replicatie plaatsvindt. Voor replicatie

zijn vele gastheer factoren nodig die assisteren bij reverse transcriptie,

integratie, en het assembleren van nieuwe virions. Het grote aantal

endogene retrovirale elementen in het humane genoom is een indicatie

dat er in het verleden veel infecties met retrovirussen zijn geweest. Het

is dan ook niet verwonderlijk dat in de evolutie specifieke mechanismen

zijn ontwikkeld die actief retrovirale infectie kunnen bestrijden om zo

een belangrijke barrière te vormen tegen retrovirale zoonose.

Trim5α is een cytoplasmatisch eiwit wat mede verantwoordelijk is voor

het zeer beperkte gastheer tropisme van retrovirussen. Het capside

eiwit van HIV wordt specifiek herkend door Trim5α en dit resulteert in

een barrière vroeg in de virale replicatie cyclus. In veel niet-humane

primaten wordt HIV-1 zeer sterk geremd door Trim5α. De remming kan

worden opgeheven door mutaties te maken in de cyclofiline A bindings

regio, wat suggereert dat cyclofiline A fungeert als cofactor voor Trim5α

in cellen van deze apen soorten. In hoofdstuk 2 wordt beschreven welke

specifieke aminozuren in het capside eiwit van HIV-1 betrokken zijn bij

de gevoeligheid voor Trim5α. De mutaties hebben een divers effect op

de infectie efficiëntie welke samen lijken te vallen met de affiniteit van

het capside voor cyclofiline A. De mutaties die leiden tot resistentie

tegen Trim5α in apencellen leiden ook tot CypA onafhankelijke infectie

van humane cellen. In tegenstelling tot de CypA-afhankelijke restrictie

in niet-humane primaat cellen, reguleren CypA en Trim5α infectiviteit

van HIV-1 onafhankelijk van elkaar in humane cellen. Hoofdstuk 3

beschrijft een biochemische analyse waarin de invloed van mutaties in

de CypA bindings regio op de binding van in vitro geassembleerde

capsides met humaan Trim5α en cyclofiline A wordt bestudeerd. Hier is

gevonden dat de affiniteit van humaan Trim5α voor het wild type

capside van HIV-1 en het HIV-1 capside met mutaties in de CypA

binding regio gelijk is. Echter, de affiniteit van Cyclofiline A voor capside

met mutaties in de CypA bindings regio was lager. Wanneer de

interactie tussen het capside eiwit en cyclofiline A werd verhinderd door

het toevoegen van cyclosporine A (CsA) zagen we alleen een minimale

toename van de Trim5α binding. Dit geeft aan dat cyclofiline A en

Trim5α niet competeren voor de binding met het HIV-1 capside, maar

dat er wel sprake kan zijn van enige vorm van sterische hinder voor

164 Samenvatting

binding aan het capside. Verder vonden we dat de stabiliteit van het

virale core partikel toe nam als er mutaties in de CypA binding region

waren aangebracht of als er CsA werd toegevoegd. Dit suggereert dat

CypA een belangrijke rol speelt in het destabiliseren van de virale core

in een vroege fase van de replicatie cyclus.

Om inzicht te krijgen in de rol van Trim5α en CypA op het verloop van

HIV-1 infectie in mensen, zijn genetische variaties in Trim5 en CypA

genen bestudeert in deelnemers van de Amsterdamse cohort studies

naar HIV-1 infectie en AIDS (ACS) (hoofdstuk 4 en hoofdstuk 5).

Genetische variatie in genen betrokken bij HIV-1 replicatie kunnen een

invloed hebben op de ontvankelijkheid voor HIV-1 en het klinische

beloop van HIV-1 infectie. In het Trim5 gen zijn 2 genetische variaties

beschreven (H43Y en R136Q) die invloed hebben op de antivirale

activiteit van Trim5α in vitro en deelnemers van de ACS zijn voor deze

varianten gescreend. Deelnemers die homozygoot waren voor het 43Y

genotype, hadden een versnelde progressie naar AIDS, wat

overeenkomt met de eerder beschreven verlaagde antivirale activiteit

van de 43Y Trim5α variant in vitro. Een polymorfisme op positie 136 was

geassocieerd met een licht verhoogde antivirale activiteit van Trim5α in

vitro. In de ACS vonden we dat dit polymorfisme geen invloed had op de

tijd tussen infectie en AIDS. Een beschermend effect van dit

polymorfime was echter wel zichtbaar na het ontstaan van HIV-1

varianten die CXCR4 kunnen gebruiken als coreceptor (X4-varianten)

maar alleen als een virale load hoger dan 104.5 kopieën viraal RNA per

ml plasma als eindpunt in de analyse werd gebruikt. Naive CD4 T cellen,

die specifiek door X4-varianten kunnen worden geinfecteerd, hebben

hogere Trim5α concentraties dan memory CD4 T cellen. Dit zou kunnen

verklaren waarom het sterkere antivirale effect van de 136Q Trim5α

alleen zichtbaar wordt nadat X4-varianten zijn ontstaan. Deze resultaten

laten zien dat Trim5α invloed heeft op het verloop van de HIV-1 infectie.

In de regulatoire regio van het CypA gen liggen twee polymorfismen

(C1604G en A1650G) die mogelijk invloed hebben op de gevoeligheid

voor infectie of het klinische beloop van de infectie. In de ACS vonden

we geen effect van deze polymorfismen op het klinische beloop van de

HIV-1 infectie. Wel bleek het A1650G polymorfisme veel vaker voor te

komen in de groep HIV-1 negatieve homosexuele mannen met hoog

risico gedrag ten opzichte van de HIV-1 geïnfecteerde homosexuele

mannen. Dit suggereert dat het A1650G polymorfisme geassocieerd is

met een verminderde gevoeligheid voor HIV-1 infectie.

Samenvatting 165

Interferonen (IFN) zijn belangrijke cytokines die een rol spelen tijdens

infectie met velerlei pathogenen en recentelijk is aangetoond dat IFN de

expressie van verschillende HIV-1 restrictiefactoren reguleren. In

hoofdstuk 6 hebben we bestudeerd of deze restrictiefactoren

verantwoordelijk zijn voor het remmende effect van IFN op HIV-1

replicatie in macrofagen. Behandeling van macrofagen met IFN-α

resulteerde in een blokkade van de virusreplicatie op een zeer vroeg

niveau in de replicatiecyclus, nog voor het reverse transcriptie proces

wat normaal gesproken initieert vlak na binnenkomst in de cel. De

hoeveelheid Trim5α mRNA was verhoogd na IFN-α behandeling, wat

suggereert dat Trim5α mogelijk een aandeel heeft in het blokkeren van

HIV-1 replicatie in IFN-α behandelde macrofagen. IFN-β heeft ook een

remmend effect op HIV-1 replicatie in macrofagen en blokkeert de

replicatie cyclus tijdens virale transcriptie. Trim22 is eerder beschreven

als een HIV-1 restrictie factor die in staat is replicatie te remmen op het

niveau van transcriptie, maar IFN-β behandeling had geen effect op de

expressie van Trim22 in macrofagen. Dit betekent dat er mogelijk nog

andere restrictie factoren zijn die door IFN-β gereguleerd worden en dat

deze factoren betrokken zijn bij de remming van HIV-1 replicatie in IFN-

β behandelde macrofagen.

In hoofdstuk 7 worden de resultaten beschreven in dit proefschrif

bediscusseerd en wordt de rol van cyclofiline A en Trim5α in HIV-1

replicatie besproken.

Nawoord

Nawoord 169

Veel dank gaat uit naar de deelnemers van de Amsterdam

Cohort Studies, zonder wie veel van de in dit proefschrift beschreven studies niet mogelijk waren geweest. Hanneke en Neeltje, bedankt voor de mogelijkheid om het onderzoek te kunnen doen. Linaida, Corrine, en Karel; bedankt voor de

praktische ondersteuning. Karel, je bent een uitzonderlijk goed analist en het was altijd erg gezellig bij de uitvoering van de experimenten. Ik ben blij dat jij naast me staat als paranimf tijdens de verdediging van dit boekje. Verder wil ik

iedereen bedanken van de afdeling EXIM-LVIP, met name Ad voor de algemene ondersteuning, Matthijs voor interessante discussies, buurman John, Andrea, Diana, Danielle, Evelien,

Marit, Angelique, Marga, Sebastiaan, Judith, Agnes, Irma, en Ellen voor de laatste loodjes. Mijn waardering gaat ook uit naar de studenten die ik heb mogen begeleiden. Bin en Rianne: ik heb minstens even veel

geleerd, het ga jullie goed. Natuurlijk was er ook een thuisfront en ondanks dat het misschien niet altijd volledig duidelijk was waar ik nou mee

bezig was, waren jullie er altijd voor mij. Papa en mama, ik kan jullie niet genoeg bedanken voor de stabiele basis, steun en vertrouwen en dat jullie alles voor mij mogelijk hebben gemaakt. Inge en Ralf, bedankt. Inge, volgens mij ben ik net

zo’n volhouder als jij, fijn dat je mijn paranimf wilt zijn. Hans, jij werd erg ziek en je bent helaas niet meer bij ons om deze dag mee te maken, je wordt enorm gemist. Ans,

bedankt voor je altijd luisterend oor en ondersteuning. Dank aan alle familie en vrienden. Juli, jij was een constante factor in een roerige tijd. Jouw

intelligentie, vermogen tot relativeren en liefde hebben mij erdoorheen gesleept. Dit boekje draag ik op aan jou.

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