Host factors regulating retroviral replication

by interactions with viral RNA and DNA

Gary Zhe Wang

Submitted in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy

under the Executive Committee

of the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY

2016

© 2016

Gary Zhe Wang

All Rights Reserved

ABSTRACT

Host factors regulating retroviral replication

by interactions with viral RNA and DNA

Gary Zhe Wang

Retroviruses are capable of infecting diverse vertebrates, and successful infection requires

intimate interaction between virus and the host cell. During an infection, retroviral particles must

bind specifically to cell surface receptors on the target cell, cross the plasma membrane, reverse-

transcribe their RNA genome into double stranded DNA, find their way to the nucleus, enter the

nucleus and integrate its DNA into host chromosomes. Following integration, expression of viral

mRNA ensues, followed by viral mRNA export into the cytoplasm, translation of viral mRNA

into proteins, and assembly of new virions that will egress from the host cell. We now appreciate

that at many steps of this complex process, the virus must hijack the cellular machinery to

replicate. At the same time, the host cell mobilizes a variety of cellular defense mechanisms to

suppress viral infection. This thesis investigates various aspects of virus-host interactions. I will

first describe the involvement of cellular transcriptional repressor protein ErbB3 binding protein

1 (EBP1) in facilitating transcriptional shutdown of Moloney murine leukemia virus (MLV) gene

expression in mouse embryonic cells. Next, I describe a novel means of regulating the activity of

Yin Yang 1 (YY1), a cellular transcription factor regulating retroviral gene expression, through

post-translational modifications. I show that YY1 is a target of tyrosine phosphorylation by Src

family kinases. Phosphorylation of YY1 impairs its ability to bind DNA and RNA, thereby

downregulating its activity as a transcription factor on retroviral and cellular promoters. Apart

from studying retroviral gene expression, I have also investigated intrinsic cellular defenses

against retroviral infection. This is exemplified by our finding that mouse cells are intrinsically

resistant to infection by betaretroviruses such as Mason-Pfizer monkey virus (M-PMV). The

block against M-PMV occurs after reverse transcription and prior to viral nuclear entry. Finally, I

will present ongoing work examining the fate of viral DNAs following infection, focusing on the

kinetics of its association with cellular core histones and viral structural proteins. Together, this

work provides critical insights into numerous aspects of the virus-host interactions.

i

TABLE OF CONTENTS

LIST OF FIGURES ...................................................................................................................... ii

ACKNOWLEDGEMENTS ........................................................................................................ iii

CHAPTER 1 - INTRODUCTION ............................................................................................... 1

Overview of the retroviral life cycle ......................................................................................................... 1

Yin Yang 1 (YY1) .................................................................................................................................. 14

Cross-species restriction of retroviruses ................................................................................................. 17

Concluding remarks – setting the stage for projects addressing virus-host interactions at multiple steps

of the early viral life cycle ...................................................................................................................... 19

CHAPTER 2 – EBP1, A NOVEL HOST FACTOR INVOLVED IN PRIMER BINDING

SITE-DEPENDENT RESTRICTION IN EMBRYONIC CELLS ........................................ 20

CHAPTER 3 – REGULATION OF YIN YANG 1 BY TYROSINE PHOSPHORYLATION

....................................................................................................................................................... 25

CHAPTER 4 – POSTENTRY RESTRICTION OF MASON-PFIZER MONKEY VIRUS

IN MOUSE CELLS .................................................................................................................... 36

CHAPTER 5 – HISTONES ARE RAPIDLY LOADED ONTO UNINTEGRATED

RETROVIRAL DNAS SOON AFTER NUCLEAR ENTRY ................................................. 43

CHAPTER 6 - DISCUSSION .................................................................................................... 81

EBP1, a novel host factor involved in primer binding site-dependent restriction of moloney murine

leukemia virus in embryonic cells .......................................................................................................... 81

Regulation of Yin Yang 1 by Tyrosine Phosphorylation ........................................................................ 84

Postentry restriction of Mason-Pfizer monkey virus in mouse cells ....................................................... 88

Histones are rapidly loaded onto unintegrated retroviral DNAs soon after nuclear entry ...................... 90

REFERENCES ............................................................................................................................ 94

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LIST OF FIGURES Figure 1-1: The genome architecture of retroviruses .................................................................................... 2

Figure 1-2: The retroviral life cycle .............................................................................................................. 3

Figure 1-3: Modes of viral entry into the host cell ....................................................................................... 5

Figure 1-4: Retroviral reverse transcription reaction .................................................................................... 6

Figure 1-5: Modes of viral nuclear entry ...................................................................................................... 8

Figure 1-6: Viral integration reaction ........................................................................................................... 9

Figure 1-7: Mechanism of MLV silencing in mouse embryonic cells ........................................................ 13

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ACKNOWLEDGEMENTS

First and foremost, I would like to thank my advisor, Dr. Stephen Goff, for his mentorship,

guidance and support. Steve is a phenomenal scientist, a creative thinker, a role model and

someone who truly cares about his trainees. When I first joined the lab, I was a naïve,

overzealous graduate student who wants to work on a new project every day. Steve gave me the

freedom to pursue my ideas. When experiments didn’t work as I envisioned, I would knock on

his door and say “Hey boss, you busy, or can we talk about some science?”, and the answer

would always be “sure, come on in”. This open-door policy saved my butt time and time again. I

have learned so much from him over the past four years. As I write this thesis, I no longer want

to work on a new project every day. I now know the importance of positive and negative controls.

I am now a lot more critical of my own data as well as the data I see. I now know when to

keeping chasing the unknown and when to ditch the project. I now know to “blow off” 2-fold

effects as noise. I now understand the need to address the same question from multiple angles. I

now know when to “emphasize!!!” key findings and when to be cautious and modest about what

I say. I could go on and on. Most importantly, I now know that I love science and I want to be a

researcher for the rest of my life.

I want to thank my thesis advisory committee, Dr. Richard Baer, Dr. Carol Prives, Dr.

Vincent Racaniello for their time and expertise. I also would like to thank Dr. Uttiya Basu and

Dr. Paul Bieniasz from Rockefeller University for their time and input in my thesis defense.

Thank you all for holding my hand during what I think is the toughest degree of my life.

I had a blast being a member of the Goff lab. I could not have done what I have done

without their intellectual input, sharing reagents, and a fun working environment. Thanks to Dr.

Sharon Schlesinger for introducing me to the world of epigenetics and mouse embryonic cells.

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Thanks to my bench-mate Yossi Sabo for all the stimulated discussions, inappropriate jokes and

advice about raising kids. Thanks to Dr. Yiping Zhu for giving me all the tips on how to make

biochemistry look easy. Thanks to Dr. Daniel Griffin for all the clinical insights and stories about

being a sailor. Thanks to Dr. Angela Erazo for helpful discussions and making “Taco Tuesdays”

a Goff lab tradition. Thanks to Dr. Michael Metzger for advice on PCR/cloning/old school

molecular biology, and recommendations on “must-do’s” when visiting Seattle. Thanks to Dr.

Oya Cingӧz for introducing me to the world of innate immunity. Thanks to all my fellow

students in the lab (Andreia Lee, Cheng Wang, Sedef Onal, Marina Ermakova, Marlene Arroyo)

for student-to-student advice. Thanks to Kenia de los Santos, Martha de los Santos and Helen

Wang for putting up with my excessive lab orders and reagent requests. Thanks to Martine

Lecorps for helping me with all the administrative hassles during graduate school.

Thanks to Dr. Ron Liem, Dr. Michael Shelanski, Dr. Steven Reiner, Dr. Patrice Spitalnik,

Mrs. Zaia Sivo and everyone in the MD/PhD program for giving me a chance to train at

Columbia and live in New York City during the prime time of my life.

Thanks to my father Jim Wang, my mother Helen Liu for their love and understanding

throughout my life. Thanks to my wife Ying Wang and my daughter Emily Wang for endless

support and joy. This work is dedicated to my family.

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CHAPTER 1 - INTRODUCTION

Overview of the retroviral life cycle

Retroviruses are single-stranded positive-sense RNA viruses capable of infecting many

vertebrates. Infection typically results in life-long chronic viremia. The key to their persistence is

the integration of proviral DNA into the genome of the host’s somatic and germ-line cells.

Infection can cause cell death and lead to depletion of the target cell population; or more often

can eventually lead to leukemia by insertional mutagenesis.

A retroviral virion contains two copies of the single-stranded RNA genome, which

encodes the viral proteins required for productive infection. All known retroviruses contain three

major genes called “gag’, “pol”, and “env’, which encode structural proteins (gag), enzymes

such as reverse transcriptase and protease (pol), as well as surface and transmembrane proteins

(env) (Fig. 1-1). The viral genes in the context of the integrated viral DNA are flanked on either

end by repeated nucleic acid sequences termed long terminal repeats (LTR). LTRs play

indispensable roles in the retroviral life cycle, including viral integration into the host genome

and regulation of viral gene expression.

The life cycle of retroviruses can be divided into two phases (Fig. 1-2). The early phase

begins with binding of virions to cell surface receptors and ends with integration of viral DNA

into the host chromosomes. The late phase begins with the expression of viral genes and ends

with the maturation and release of new virions from the cell surface.

In the first step of the retroviral life cycle, viral envelope glycoproteins interact with

cognate cellular surface receptors. For example, ecotropic Moloney murine leukemia virus

(MLV) utilizes a ubiquitously expressed mouse amino acid transporter (mCAT) to gain entry

into many cell types (Kim et al, 1991; Wang et al, 1991), whereas human immunodeficiency

2

Figure 1-1: The genome architecture of retroviruses

The representative genome of Moloney murine leukemia virus (MLV) is shown. Viral genes,

gag, pol, and env are flanked on both ends by LTR sequences.

Diargram adopted from http://viralzone.expasy.org/all_by_species/67.html.

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Figure 1-2: The retroviral life cycle

Retroviral life cycle can be divided into two phases. Early phase begins with virion binding to

cell surface receptors and ends with integration. Late phase begins with transcription of viral

genes in the nucleus and ends with viral maturation and budding.

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virus-1 (HIV-1) utilizes the CD4 protein of T-lymphocytes as the receptor (Maddon et al, 1986).

The presence or absence of a particular cell surface receptor defines the virus’s cell-type tropism.

Receptor attachment is followed by entry of the virus into the host cell. Different viral envelopes

mediate virus entry into the target cell via distinct mechanisms (Fig. 1-3). For example, the

glycoprotein from vesicular stomatitis virus (VSV-G) promotes viral entry via endocytosis, while

amphotropic (ampho) MLV envelope and HIV-1 envelope facilitate direct fusion with the

plasma membrane (McClure et al, 1990). In the case of ecotropic (eco) MLV envelope, there

exists evidence consistent with both endocytosis (i.e. pH dependence) and direct cell fusion (i.e.

pH independence) (Mothes et al, 2000; Nussbaum et al, 1993).

Following cell entry, the viral core, containing the RNA genome and its associated

proteins, is released into the cytoplasm of the target cell and undergoes partial uncoating to give

rise to a ribonucleoprotein complex termed the reverse transcription complex (RTC). With the

help of the viral-encoded reverse transcriptase enzyme, the single-stranded viral RNA genome is

converted into double-stranded viral DNA genome in a stepwise manner (Fig. 1-4). First, cellular

tRNA bound to the primer binding site (PBS) of the viral genomic RNA functions to prime DNA

synthesis by reverse transcriptase. This is followed by strand extension and strand transfer

reactions, leading to the generation of full length viral DNA genomes. What triggers the

initiation of reverse transcription is not completely understood. One possibility is that exposure

of RTC to the presence of deoxyribonucleotides in the cytoplasm of the host cell may be

sufficient to drive reverse transcription (Goff, 2001).

Following reverse transcription, viral DNA genomes undergo trafficking toward the

nucleus of the infected cell. Given that the cellular cytoplasm is densely populated with proteins

and organelles, random diffusion of viral particles toward the nucleus may be an inefficient mean

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Figure 1-3: Modes of viral entry into the host cell

Different viral envelopes enter the cell by distinct mechanisms. The glycoprotein of vesicular

stomatitis virus (VSV-G) enters the cell by first binding to cell surface receptors. Endocytosis

and subsequent exposure of the virus to the low endosomal pH triggers the fusion of the viral

membrane with the endosomal membrane, this is termed pH-dependent entry. In contrast, the

envelope protein of amphotropic MLV, or HIV-1 can bind to cell surface receptors and directly

fuse with the cell membrane, this is termed pH-independent entry.

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Figure 1-4: Retroviral reverse transcription reaction

1. Using host tRNA as a primer, minus-strand DNA synthesis proceeds until the 5’ end of the

genomic RNA is reached. This short DNA intermediate is termed minus-strand strong-stop DNA

(sss-DNA).

2. RNase-H degrades the RNA strand of the RNA-sssDNA duplex. sssDNA undergoes strand

transfer and anneals to the 3’ end of the viral genomic RNA.

3. Minus-strand DNA synthesis resumes, while RNase H digestion of the RNA template strand

occurs. Note that all of the RNA template is degraded by RNase H with the exception of a

short stretch of RNA called polypurine tract (PPT).

4. The remaining PPT portion of the viral RNA genome serves to prime plus-strand DNA

synthesis.

5. RNase H removes the primer tRNA, and sssDNA anneals to the complementary PBS

segment in minus-strand DNA, this process is called second strand transfer.

6. Plus and minus-strand synthesis is complete.

Diagram is adopted from (Coffin et al, 1997).

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of reaching the target. Consequently, retroviruses have evolved means to hijack cellular

cytoskeletal machinery to facilitate purposeful movements toward the cell nucleus. For

example, HIV-1 infection induces the formation of stable microtubules in the infected cell,

which functions as specialized tracks for HIV-1 particle transport toward the nucleus (Sabo et

al, 2013).

The nuclear envelope of infected cells poses a formidable barrier to retroviral nuclear

translocation (Fig. 1-5). Indeed, retroviruses such as MLV are unable to penetrate intact nuclei of

infected cells, and must therefore depend on breakdown of the nuclear envelope during mitosis

to gain nuclear entry (Roe et al, 1993). In contrast, lentiviruses such as HIV-1 are able to actively

transverse the nuclear envelope of non-dividing cells such as resting T-lymphocytes and

macrophages (Weinberg et al, 1991). Here, certain HIV-1 proteins interact with

nucleocytoplasmic shuttling receptors called importins to gain nuclear entry in a manner similar

to cellular proteins.

Once inside the nucleus, the viral encoded integrase enzyme catalyzes the insertion of

linear viral DNAs into the host chromosomes (Fig. 1-6). The integration reaction proceeds in two

steps. First, integrase catalyzes the removal of two nucleotides from the 3’ terminus of the viral

LTR, termed 3’-end processing (Brown et al, 1989; Roth et al, 1989). The resultant viral DNA

substrate is then joined to the target DNA on host chromosomes through a strand transfer

reaction (Craigie et al, 1990; Engelman et al, 1991). Structurally, the catalytic pocket of integrase

from many different retroviruses contain a centrally located D-D(35)-E motif. For MLV,

mutations in the D-D(35)-E motif (e.g. D184A) results in the loss of all catalytic activity, making

it a useful tool to study integration independent viral processes (Lai et al, 2001).

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Figure 1-5: Modes of viral nuclear entry

Different viruses enter the nucleus of the infected cell by different mechanisms. To gain access

to host chromosomes, MLV requires the dissolution of nuclear envelope during cell division. In

contrast, HIV-1 can be actively imported into the nucleus by interaction with nuclear pore

complexes, rendering them capable of infecting non-dividing cells.

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Figure 1-6: Viral integration reaction

The integration reaction proceeds in two distinct steps. First, integrase catalyzes the removal of

two nucleotides from the 3’ terminus of the viral LTR, termed 3’-end processing. This exposes a

3’ hydroxyl group (OH), which acts as a nucleophile to attack target DNA during the joining

reaction. Joining reaction generates a DNA gap with 5’ 2-nucleotide overhangs, which can then

be removed, and the gap repaired to complete integration.

Diagram is adopted from (Coffin et al, 1997).

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Do retroviruses integrate randomly or at preferred sites in the host genome? Initial in

vitro studies demonstrated that proviral insertion occurs irrespective of DNA sequences but is

influenced by the chromatin structure (Pryciak et al, 1992). Gammaretroviruses such as MLV

prefers to integrate near transcription start sites, as well as in the vicinity of strong enhancers and

active promoters (De Ravin et al, 2014; LaFave et al, 2014; Wu et al, 2003). Lentiviruses

including HIV-1 prefer to integrate inside the bodies of active genes, especially genes that were

activated in cells following HIV-1 infection (Schroder et al, 2002). Thus, such strong bias in

integration site selection may promote robust retroviral gene expression.

Following integration, the viral promoter encoded in the LTR region functions to drive

the transcription of viral genes in a manner similar to cellular genes. The resultant viral RNA can

then be exported into the cytoplasm and be translated by host ribosomes to generate viral

proteins. Alternatively, full length viral RNA can also be packaged directly into progeny virions.

After assembly of viral RNA and proteins, new immature virions bud from the plasma

membrane and the Gag and Gag-Pol precursor proteins undergo proteolytic cleavage by the viral

protease, generating mature virions capable of infecting new cells.

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Transcriptional regulation of MLV in embryonic cells

MLV is unable to replicate in mouse embryonic stem (ES) or embryonic carcinoma (EC)

cells (Barklis et al, 1986; Teich et al, 1977). In embryonic cells, reverse transcription and

proviral integration proceed normally, but viral transcription is repressed, and hence no viral

gene products can be detected. Several mechanisms are likely involved in the transcriptional

silencing, including 1) the absence of enhancer proteins recognizing binding sites in the viral

LTR (Hilberg et al, 1987; Linney et al, 1984); 2) recruitment of trans-acting transcriptional

repressors (Akgun et al, 1991; Flanagan et al, 1989; Tsukiyama et al, 1989), and 3) de novo

DNA methylation (Niwa et al, 1983). One critical site for transcriptional silencing, termed the

repressor binding site (RBS), shows extensive overlap (17/18 bps) with the primer binding site

(PBSpro) of MLV (Barklis et al, 1986; Feuer et al, 1989; Loh et al, 1987), which normally

functions during the priming of reverse transcription by host proline tRNA. The RBS by itself is

sufficient to induce potent transcriptional repression of reporter constructs in EC cells,

irrespective of its orientation or position (Loh et al, 1990; Petersen et al, 1991). In addition,

electrophoretic mobility shift assays (EMSAs) using RBS as the probe demonstrate the presence

of stem-cell specific nuclear factors. A single base-pair mutation in the RBS, termed the B2

mutation, is sufficient to abolish nuclear factor binding, and thereby restore viral gene expression

(Loh et al, 1990; Petersen et al, 1991). These findings have allowed the characterization of the

stem-cell specific trans-acting transcriptional repressor complex as containing Trim28 (also

known as KAP-1 or Tif1-beta), a known transcriptional co-repressor (Wolf et al, 2008a; Wolf &

Goff, 2007; Wolf et al, 2008b), and Krüppel associated box (KRAB) zinc finger protein 809

(ZFP809) (Wolf & Goff, 2009), a DNA binding transcription factor. The current model for PBS-

mediated silencing consists of the binding of PBSpro by ZFP809, and the subsequent recruitment

of Trim28 to the provirus via the ZFP809 KRAB box domain. This association permits Trim28-

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dependent recruitment of additional chromatin modifiers such as the histone H3K9

methyltransferase SETDB1 (Schultz et al, 2002), heterochromatin-associated protein gamma

(HP1gamma) (Wolf et al, 2008a), the NURD histone deacetylases complex (Schultz et al, 2001)

and EBP1 (Wang et al, 2014). Together, the ensuing histone and DNA methylation of the

proviral DNA ensures transcriptional silencing and genomic stability (Fig. 1-7).

Beside the ZFP809/Trim28 complex, another zinc finger protein, Yin Yang 1 (YY1), also

binds to the LTR of MLV in embryonic cells, augmenting the recruitment of Trim28 and

downstream chromatin modifiers to the proviral DNA (Schlesinger et al, 2013). YY1 binding to

the viral LTR occurs rapidly following infection, leading to the initiation of MLV promoter

silencing. Indeed, retroviral gene expression is significantly upregulated upon MLV infection of

embryonic cells devoid of YY1, or by infecting wild-type embryonic cells with MLV LTR

mutant that no longer bind YY1. Therefore, both ZFP809 and YY1 are capable of binding

separate DNA sequences on the viral LTR and act to cooperatively recruit Trim28 and its

downstream epigenetic modifiers to turn off viral gene expression.

13

Figure 1-7: Mechanism of MLV silencing in mouse embryonic cells

(A) In embryonic cells, two zinc finger transcription factors, YY1 and ZFP809 binds to unique

DNA sequences in the MLV LTR upon infection. Together, this leads to the recruitment of

Trim28, a host transcriptional repressor, as well as chromatin modifiers such as histone

methyltransferase SETDB1, NuRD histone deacetylases complex, HP1 and EBP1 to the proviral

promoter. These chromatin modifiers are responsible for the deposition of repressive histone

marks such as H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3) to

initiate MLV silencing. Ultimately, complete MLV silencing is achieved and maintained by

DNA methylation. (B) ZFP809 is not expressed in differentiated cells, hence no MLV silencing

is observed.

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Yin Yang 1 (YY1)

Yin Yang 1 (YY1) is a ubiquitously expressed DNA/RNA binding transcription factor

that takes its name from its dual activity on the adeno-associated virus (AAV) P5 promoter,

where the presence of adenoviral protein E1A converts YY1 from a transcriptional repressor to

an activator (Shi et al, 1991). In mice, targeted deletion of YY1 results in peri-implantation

lethality, indicative of an essential role in embryogenesis (Donohoe et al, 1999). YY1 binds to

DNA through the recognition of a specific consensus sequence, leading to the activation or

repression of many cellular genes including c-Myc, c-Fos, -actin, IFN-, CREB, Sp1 and others

(Gordon et al, 2006; Shi et al, 1997). Moreover, YY1 has been shown to physically interact with

numerous proteins required for cell growth, apoptosis and cancer progression, including p53,

Mdm2, Ezh2, and Rb (Gordon et al, 2006; Shi et al, 1997). In Xenopus oocytes, YY1 has been

demonstrated to associate with structurally divergent RNA species (Belak et al, 2008), raising

the possibility that YY1 can bind both DNA and RNA. Highlighting this dual-binding property,

YY1 was shown to tether Xist, a noncoding RNA (lncRNA), to DNA regions on the X

chromosome, leading to X chromosome inactivation in maternal cells (Jeon & Lee, 2011).

Therefore, it appears that the zinc finger domain of YY1 can simultaneously bind different

nucleic acid motifs in Xist RNA and DNA (Jeon & Lee, 2011).

Apart from its function on cellular promoters, YY1 plays a critical role in the

transcriptional regulation of retroviral promoters. For example, YY1 binds to and represses the

HIV-1 promoter, thereby maintaining the virus in a latent state (Bernhard et al, 2013; Margolis et

al, 1994). As discussed above, in the case of MLV, YY1 directly binds to the negative control

region (NCR) in the viral LTR (Flanagan et al, 1992), leading to transcriptional silencing of

MLV in mouse embryonic cells (Schlesinger et al, 2013). Consistent with its dual role in

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transcriptional activation, YY1 potentiates expression from the Human T-lymphotropic Virus 1

(HTLV-1) promoter, likely through association with the HTLV-1 RNA (Wang GZ and Goff SP,

unpublished observations).

The sequence-specific DNA binding activity of YY1 is mediated by four C2H2-type zinc

finger motifs (amino acid 298-414) located in the C-terminus (Hariharan et al, 1991). Besides

their role in DNA binding, portions of the zinc finger motifs (amino acid 333-397), together with

a region rich in alanine and glycine (amino acid 157-201) contributes to transcriptional

repression (Bushmeyer et al, 1995; Galvin & Shi, 1997). The N-terminal region contains several

unusual features also required for transcriptional activation. This includes two stretches of acidic

residues (amino acids 16-29 and amino acid 43-53), followed by a glycine rich region (amino

acid 54-69), 11 consecutive histidine residues (amino acid 70-80), and sequences rich in proline

and glutamine (amino acid 80-100) (Austen et al, 1997; Bushmeyer et al, 1995; Lee et al, 1995;

Lee et al, 1994).

Despite the wealth of knowledge garnered about YY1 interacting proteins and target

promoters regulated by YY1, much less is known about YY1 regulation. Based on previous

studies, YY1 appears to be a target of several posttranslational modifications, some of which

contribute to changes in YY1 activity. Sumoylation of YY1 by PIASy on lysine 288 negatively

affects the transcriptional activity of YY1 (Deng et al, 2007), while O-linked glycosylation of

YY1 disrupts its interaction with Rb, leading to enhanced DNA binding (Hiromura et al, 2003).

Moreover, YY1 also undergoes a complex acetylation/deacetylation cycle that changes its

affinity for DNA and capacity to interact with histone deacetylases (Yao et al, 2001). In prostate

cancer cells, nitric oxide (NO) has been shown to facilitate S-nitrosylation of YY1 on cysteine

residues, resulting in decreased DNA binding and upregulation of Fas expression (Hongo et al,

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2005). Serine/threonine phosphorylation of YY1 occurs during the cell cycle. Specifically,

threonine 39 is a target of Polo-like kinase 1 (Plk1), but the functional importance of this

modification remains to determined (Rizkallah et al, 2011b). Phosphorylation of serine 180 and

184 by Aurora B kinase has been shown to have effects on DNA binding and YY1’s ability to be

acetylated by histone acetyltransferase p300 (Kassardjian et al, 2012). Finally, phosphorylation

of threonine 348 and 378 in the DNA binding domain of YY1 by an unknown kinase leads to

decreased DNA binding (Rizkallah & Hurt, 2009).

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Cross-species restriction of retroviruses

Successful retroviral infection requires cellular factors to support each phase of the

retroviral life cycle. Therefore, retroviral tropism is partly dictated by whether the infected cell

possesses the necessary co-factors to assist viral replication. For example, HIV-1 infects human

T cells and macrophages efficiently but is unable to infect mouse cells. Multiple steps in the viral

life cycle are blocked in mouse cells. First, the mouse homologues of the human T cell receptors

and coreceptors CD4, CXCR4 and CCR5 (Atchison et al, 1996; Landau et al, 1988) cannot bind

the HIV env-encoded gp120, preventing HIV-1 from entering the cell. This entry block can be

bypassed in engineered mouse cell lines expressing the human T-cell receptors (Browning et al,

1997; Clayton et al, 1988), or alternatively, by pseudotyping HIV-1 virions with foreign viral

envelope proteins such as the glycoprotein G of the vesicular stomatitis virus (VSV-G). Upon

establishment of the provirus in mouse cells, the late phase of the HIV-1 life cycle is hindered by

additional cellular blocks. Transcription initiated in the Long Terminal Repeat (LTR) is defective

due to the incompatibility of the HIV-1 Tat transactivator with mouse cyclin T1 (Fujinaga et al,

1999; Garber et al, 1998; Kwak et al, 1999; Wei et al, 1998). In addition, viral assembly is

defective due to the absence of necessary factors required for late stages of the retroviral life

cycle, a phenotype that can be rescued by the fusion of uninfected human cells with infected

mouse cells (Bieniasz & Cullen, 2000; Mariani et al, 2001; Mariani et al, 2000). Study of blocks

to HIV-1 replication in mouse cells has led to the identification and characterization of critical

cellular factors necessary for infection.

Species-specific retroviral tropism is also governed by the presence of species-specific

restriction factors. For example, human and primates are known to be resistant to N-tropic MLV

infection (Besnier et al, 2003; Towers et al, 2000). The human protein mediating this restriction

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was identified as TRIM5 (Perron et al, 2004). In addition to restricting N-tropic MLV, rhesus

macaque TRIM5 has the remarkable ability to restrict HIV-1 infection (Stremlau et al, 2004;

Yap et al, 2004). This observation was later attributed to species-specific variations in the

TRIM5 sequence (Sawyer et al, 2005; Stremlau et al, 2005; Yap et al, 2005), supporting the

notion that species-specific restriction factors are a major determinant of retroviral tropism.

Mechanistically, TRIM5 has been proposed to interact with the viral capsid (CA) (Sebastian &

Luban, 2005), leading to premature dissociation of viral cores (Stremlau et al, 2006), and

terminating infection prior to nuclear entry (Stremlau et al, 2004). In mice, the Friend virus

susceptibility factor 1 (Fv1) gene functions similarly to TRIM5. Fv1 encodes a gag-like protein

with homologies to the ERV-L family of endogenous retroviruses (Best et al, 1996). There are

two major naturally occurring alleles of Fv1. NIH Swiss mice carry the Fv1n allele, which

restricts the replication of B-tropic MLVs but not N-tropic MLVs. In contrast, BALB/c mice

carry the Fv1b allele, which confers resistance to N-tropic MLV infection but not B-tropic MLVs

(Goff, 1996; Stoye, 1998). A few laboratory mice and some wild mice carry a third Fv1 allele,

Fv1nr

, which restricts the replication of B-tropic MLVs and a subset of N-tropic MLVs (Kozak,

1985; Pincus et al, 1971).

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Concluding remarks – setting the stage for projects addressing virus-host interactions at

multiple steps of the early viral life cycle

For the next few chapters, I will share my published work on different facets of the retroviral life

cycle. Chapter 2 and 3 focus on the regulation of gene expression from the MLV promoter in

mouse embryonic cells. In chapter 2, I will describe a novel ZFP809 interacting protein, EBP1,

and its involvement in potentiating MLV silencing in mouse embryonic cells. In chapter 3, I

discuss regulation of YY1 activity through tyrosine phosphorylation, and its implications in the

control of viral and cellular promoters. Chapters 4 and 5 will move away from retroviral gene

expression and focus on other early events of infection. In chapter 4, I describe the ability of

mouse cells to block infection by betaretroviruses such as M-PMV at the step of viral nuclear

entry. In chapter 5, I describe the most recent project examining the “state” of the viral DNAs

following infection, focusing on the association of viral DNAs with cellular histones during early

infection.

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CHAPTER 2 – EBP1, A NOVEL HOST FACTOR INVOLVED IN PRIMER BINDING

SITE-DEPENDENT RESTRICTION IN EMBRYONIC CELLS

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CHAPTER 3 – REGULATION OF YIN YANG 1 BY TYROSINE PHOSPHORYLATION

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CHAPTER 4 – POSTENTRY RESTRICTION OF MASON-PFIZER MONKEY VIRUS

IN MOUSE CELLS

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CHAPTER 5 – HISTONES ARE RAPIDLY LOADED ONTO UNINTEGRATED

RETROVIRAL DNAS SOON AFTER NUCLEAR ENTRY

Histones are rapidly loaded onto unintegrated retroviral DNAs

soon after nuclear entry

Gary Z. Wang1,2

, Ying Wang3, Stephen P. Goff#

4,5,6

1Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University, New

York, NY 10032, USA

2Medical Scientist Training Program, Columbia University College of Physicians and Surgeons,

New York, NY 10032, USA

3 Division of Nephrology, Department of Medicine, Columbia University Medical Center, New

York, NY 10032, USA

4Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY

10032, USA

5Department of Microbiology and Immunology, Columbia University, New York, NY 10032,

USA

6Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA

Running title: Histone association with retroviral DNAs

Address correspondence to Stephen P. Goff

spg1@cumc.columbia.edu

Abstract word count: 334

Text word count: 45208

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ABSTRACT

In the early stages of retroviral infection, a DNA copy of the viral genome is formed by

reverse transcription of the RNA genome. The course of DNA synthesis take place in the

cytoplasm in the context of a large structure closely resembling the virion core. The product of

this reaction is a fully double-stranded linear DNA contained within a pre-integration complex

(PIC), which retains many copies of the viral capsid (CA) and nucleocapsid (NC) proteins. The

linear viral DNA is delivered into the nucleus by routes that differ among virus strains. A portion

of the linear DNA then gives rise to circular double-stranded DNAs containing either one or two

copies of the Long Terminal Repeat (LTR) sequences, and a portion is inserted into the host

genome to form the integrated provirus. The proviral DNA eventually becomes covered with

nucleosomes, and depending on the cell type, the histone tails of the nucleosomes are modified

by the addition of histone marks that regulate the expression of the viral DNA. The timing of

nucleosome loading onto the viral DNA and, in particular, whether nucleosomes are loaded onto

unintegrated viral DNAs, has not been determined. In this study, we show that unintegrated viral

DNAs of Moloney murine leukemia virus rapidly associate with core histones. Loading of

nucleosomes on the viral DNA requires nuclear entry but does not require viral DNA integration.

Moreover, we observe that histone loading is correlated with unloading of viral structural

proteins from the viral DNA. The histones associated with unintegrated DNAs are marked by

covalent modifications, but the appearance of the modified histones is delayed relative to the

time of core histone loading. Expression from the unintegrated DNA can be enhanced by

modulation of the histone modifying machinery. Together, our data show that histones are

loaded on unintegrated DNAs very rapidly after nuclear entry and not only after formation of the

integrated provirus.

KEYWORDS: Retrovirus/Nucleosome/Histone/Epigenetics

45

INTRODUCTION

DNA of eukaryotic chromosomes is condensed into chromatin by the association with

nucleosomes, each consisting of an octomeric complex of the four core histones. Nucleosomes

are normally loaded onto chromosomal DNA during the course of DNA replication

(Ramachandran & Henikoff, 2015) . As a replication fork moves along the DNA, nucleosomes

are displaced from the unreplicated DNA ahead of the fork and then must reassociate onto both

daughter branches behind the growing fork. The current models suggest that the relocalization of

the nucleosomes is a highly dynamic process in which the “old” nucleosomes are distributed to

both daughter branches, and “new” nucleosomes are added to restore normal nucleosome density.

During this process, epigenetic marks on the old chromatin must be preserved or reestablished on

the new DNAs to ensure maintenance of the appropriate pattern of gene expression (Zhu &

Reinberg, 2011). New nucleosomes may also be loaded at sites of DNA damage, perhaps under

the direction of the PCNA factor present at the sites. The loading of nucleosomes is thought to be

promoted by an array of dedicated chaperones, including CAF-1, ATRX, and DAXX (Burgess &

Zhang, 2013). In contrast to these normal settings of chromatin formation, there are unusual

circumstances when naked DNA, or DNA previously free of nucleosomes, enters a cell and must

acquire a nucleosomal array de novo. In these situations there are no “old” or preexisting

nucleosomes to seed or facilitate the loading of new nucleosomes onto the DNA. One such

circumstance occurs in the course of retroviral infection.

Retroviruses are single-stranded RNA viruses with a complex life cycle. The core of the

mature virion particle consists of a protein shell made up of the viral capsid protein (CA)

enclosing two copies of the RNA genome tightly associated with the viral nucleocapsid protein

(NC). The viral NC protein is a small, highly basic protein that is tightly bound to the viral RNA

46

and condenses while the CA protein forms a hexameric lattice that encapsidates the viral RNA.

Following viral entry into the target cell, the single-stranded viral RNA genome is copied by

reverse transcriptase (RT) to generate a linear double-stranded DNA version of the genome,

contained within a large complex termed the preintegration complex (PIC). Following the

completion of reverse transcription, the double-stranded viral DNA and its associated proteins

are trafficked into the nucleus of the infected cell by one of a variety of mechanisms that vary

among different viral genera. For example, gammaretroviruses such as Moloney murine

leukemia virus (MLV) are unable to enter the intact nucleus of a nondividing cell and depend on

nuclear membrane breakdown during cell division to gain entry (Roe et al, 1993). In contrast,

lentiviruses such as HIV-1 are actively transported across the nuclear membrane even in non-

dividing cells such as macrophages and resting T lymphocytes (Weinberg et al, 1991). The

distinct requirements for nuclear entry also serve as a point of restriction for cross-species

transmission of retroviruses. For example, betaretroviruses such as Mason-Pfizer monkey virus

(M-PMV) are unable to enter the nucleus of mouse cells, defining a potent cross-species block at

this step of the viral life cycle (Wang & Goff, 2015).

Once inside the nucleus, viral encoded integrase (IN) enzyme catalyzes the insertion of

linear viral DNAs into the host chromosomes. The integration reaction proceeds in two steps.

First, IN catalyzes the removal of two nucleotides from the 3’ terminus of the viral long terminal

repeat (LTR), termed 3’-end processing (Brown et al, 1989; Roth et al, 1989). The resultant viral

DNA substrate is then joined to the target DNA on host chromosomes through a strand transfer

reaction (Craigie et al, 1990; Engelman et al, 1991). Structurally, the catalytic pocket of IN from

many different retroviruses contain a centrally located D-D(35)-E motif. For MLV, mutations in

47

the D-D(35)-E motif (e.g. D184A) results in the loss of all catalytic activities, making it a useful

tool to study integration independent viral processes (Lai et al, 2001).

Homologous recombination of linear viral DNAs at the LTR leads to the formation of 1-

LTR circles. 2-LTR circles, containing full length viral DNA and both sets of LTRs arranged

back to back, are products of non-homologous end joining (NHEJ) DNA repair pathways in the

nucleus. This property makes 2-LTR circles a useful marker of viral nuclear import, as the

unique nature of the LTR-LTR junction allow their levels to be readily quantified by PCR

(Butler et al, 2001).

In permissive cells, following integration, the 5’ LTR of the proviral DNA serves as the

promoter to generate full-length genomic viral mRNAs necessary for viral gene expression and

genome replication. However, certain cell types possess the ability to block this step of the

retroviral life cycle. For example, MLV is unable to replicate in mouse embryonic stem (ES) or

embryonic carcinoma (EC) cells (Akgun et al, 1991; Hilberg et al, 1987; Linney et al, 1984). In

embryonic cells, reverse transcription and proviral integration proceed normally, but viral

transcription is repressed, and hence no viral gene products can be detected. One major

mechanism in the transcriptional silencing of MLV in embryonic cells involves the recruitment

of trans-acting transcriptional repressors such as ZFP809, Trim28, YY1 and Ebp1 (Rowe et al,

2010; Schlesinger et al, 2013; Wang et al, 2014; Wolf & Goff, 2007; Wolf & Goff, 2009) to

DNA binding sites on the viral LTR. This results in the recruitment of chromatin modifiers such

as SETDB1 (Matsui et al, 2010), which deposits repressive histone marks such as H3K9

trimethylation (H3K9me3) en route to complete promoter silencing.

Several fundamental aspects of retroviral replication remain unexplored. Given that

reverse transcription generates de novo foreign DNAs in the host cell, this raises the question of

48

what is the histone state of these retroviral DNAs following infection. Are retroviral DNAs a

target of histone loading? If so, when, where and on what forms of viral DNA does this occur?

Following reverse transcription, do retroviral DNAs remain associated with their cognate

structural proteins such as NC and CA? When and where does NC and CA dissociate from the

viral DNA?

In this study, using MLV as a model, we describe the kinetics of histone association with

the viral DNA genomes. We show that unintegrated linear viral DNAs and 2-LTR circles

undergo rapid core histone loading in a nuclear entry-dependent and integration-independent

manner. Moreover, histone loading appears to be correlated with the removal of viral structural

proteins such as NC and CA from the viral DNA. In contrast to core histone loading, loading of

epigenetically modified histones occurs after a delay, thereby postponing host cell regulation of

the viral promoter. Finally, we show that unintegrated viral DNAs do give rise to a low level of

gene expression, and that this expression can be increased by changing the activity of chromatin

modifiers. Together, our data provide insights into fundamental aspects of retroviral biology.

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RESULTS

Core histone loading onto retroviral DNAs occurs rapidly following infection

To investigate histone loading onto viral DNA genomes following infection, we utilized a

single-round MLV reporter genome in which viral genes were replaced with the GFP gene

(MLV-GFP). Virions capable of infecting a wide range of cell lines were prepared by collecting

culture medium after transfection of 293T cells with plasmids encoding the MLV-GFP reporter

genome, the MLV gag-pol protein, and the VSV-G envelope protein. This reporter virus is

capable of carrying out all the early events of a single round of infection, including reverse

transcription, nuclear entry, integration, and reporter gene expression. Kinetic studies

characterizing the formation of viral DNA replication intermediates following infection were

first performed. Mouse embryonic fibroblasts (MEFs) were transduced with MLV-GFP reporter

virus, total DNA was harvested at 12, 24 h or 6 days post infection, and levels of viral DNA

replication intermediates was monitored by quantitative real-time PCR using primers specific for

GFP (to detect total viral DNA) or 2-LTR circles (a marker of nuclear entry) (Fig. 1A). To

control for potential plasmid DNA carryover in the viral preparation, transduction of MEFs with

heat inactivated (HI) virus were carried out in parallel. Total viral DNA, which includes linear

DNA, circular DNA, and integrated proviral DNA, appeared at 12 h post infection, rose to a peak

at 24 h, and then decreased to a stable copy number of approximately 3200 copies by 6 days (Fig

1A). The 2-LTR circles appeared later, rose to a peak at 24 h post infection, and was completely

undetectable by 6 days, indicative of their short half-life in the nucleus (Fig. 1B). Of note, the

maximum amount of 2-LTR circles at early time points accounts for 10-20% of total viral DNAs.

As negative controls, MEFs transduced with heat-inactivated virus yielded no PCR signal,

indicating minimal contaminating plasmid DNA carryover from the virus preparation.

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We next monitored the association of nucleosomal histone H3 with retroviral DNAs by

Chromatin immunoprecipitation (ChIP). Infected MEF cells were formaldehyde cross-linked at

12, 24 h, and 6 days post infection, and processed to yield chromatin of appropriate size (200-

800 base pairs). Chromatin immunoprecipitation (ChIP) assays were performed with antibodies

specific for histone H3 or non-specific IgG control, followed by quantitative real-time PCR

analysis of immunoprecipitated DNA using primers targeting GFP (total viral DNA) or 2-LTR

circles. As a positive control, PCR primers targeting the promoter region of endogenous GAPDH

gene were used to represent fully chromatinized cellular DNA. Approximately 10% of the

GAPDH promoter sequences were specifically immunoprecipitated with the anti-histone H3

antibody, but not the non-specific IgG control antibody (Fig. 1B). Assuming that the GAPDH

promoter is fully chromatinized at all times, these results suggests that our ChIP recovery

efficiency is approximately 10%. In the case of viral DNAs, we detected rapid association of

histone H3 with total viral DNA to levels comparable to that of the cellular GAPDH gene by 24

h post infection (Fig. 1B). At 6 days, the observed ChIP signal with the GFP probe (total viral

DNA) remained high, indicative of fully chromatinized proviral DNA. In the case of 2-LTR

circles, histone H3 loading reached similar levels to that of the cellular GAPDH gene by 12 h

post infection (Fig. 1B). Of note, the signal of chromatin-bound DNA in the total DNA was too

high to be accounted for only by the circular DNAs, thusthe majority of the signal must be

derived from the linear form. Similar kinetics of histone loading onto viral DNAs were observed

in MLV-GFP infected F9 embryonic carcinoma cells (Fig. 1C and 1D). Together, these findings

suggest that both total and circular retroviral DNA forms undergo rapid chromatinization

following infection.

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Histone loading of retroviral DNAs occurs independently of viral integration

To investigate whether viral integration is needed for efficient histone loading, two

independent approaches were taken. First, MLV-GFP reporter viruses were packaged using wild-

type (WT) or catalytically inactive MLV integrase (IN) mutant (D184A) (Lai et al, 2001). The

infectivity of WT or D184A-IN virions was confirmed using flow cytometry analysis of infected

NIH-3T3 fibroblasts 48 h post infection (Fig. 2B). Compared to WT virus, a 70-fold reduction in

the infectivity of D184A-IN virions was observed (Fig. 2B). Moreover, cells infected with

D184A-IN virions contained slightly higher levels of total viral DNA (GFP) and 2-LTR circles at

12 and 24 h post infection, but no viral DNA was detected at 6 days post infection (Fig. 2C).

This suggests that the D184A mutation has no adverse effects on reverse transcription and viral

nuclear entry, but is defective in viral integration. Compared to WT virus, histone H3 ChIP of

cells infected with D184A-IN virus showed comparable accumulation of histone H3 on both

total viral DNA (GFP) and 2-LTR circles (Fig. 2D). This observation suggests that viral

integration is not required for histone loading and that most of the chromatinized viral DNA

early on likely represent unintegrated linear and circular DNA forms.

Alternatively, similar ChIP experiments were performed on MEF cells infected with WT-

virus in presence or absence of the integrase inhibitor raltegravir (Fig. 2E). As expected,

raltegravir treatment prevented the formation of GFP+ cells (Fig. 2F). Analogous to the case for

D184A-IN virus, raltegravir treatment caused a 2-fold increase in the levels of 2-LTR circles

without affecting total viral DNA (GFP) levels (Fig. 2G). Compared to mock treated cells, ChIP

analysis using raltegravir treated cells revealed normal accumulation of histone H3 on both total

viral DNA (GFP) and 2-LTR circles (Fig. 2H), further highlighting the non-requirement for

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integration. Taken together, our data suggests that early during infection, chromatinization of

total viral DNA occurs irrespective of viral integration.

Histone loading of retroviral DNAs requires viral nuclear entry

It is well established that 2-LTR circles, products of host cell non-homologous end

joining, are found exclusively in the nucleus, making it a useful marker for viral nuclear entry

(Bukrinsky et al, 1992; Bukrinsky et al, 1991; Shoemaker et al, 1980). Like many nuclear

proteins, histones are first synthesized in the cytosol and later trafficked to the nucleus (Burgess

& Zhang, 2013). Since viral reverse transcription also takes place in the cytoplasm and that

linear viral DNA is able to undergo rapid chromatinization following infection (Fig. 2D and 2H),

we examined the possibility that histone loading onto viral DNAs may occur in the cytoplasm

prior to viral nuclear entry. Previously, it has been shown that in mitotically active cells, nuclear

envelope breakdown facilitates entry of the MLV preintegration complex (PIC) into the nucleus,

while growth arrested cells that fail to undergo cell division block MLV infection at nuclear

entry (Roe et al, 1993). To block nuclear entry, NIH-3T3 cells were treated with or without

aphidicolin, a reversible inhibitor of eukaryotic DNA synthesis for 24 h (Fig. 3A). DNA content

analysis of aphidicolin treated cells using fluorescence activated cell sorting (FACS) revealed

cell cycle arrest in the G1/S phase (Fig. 3B). Growth arrested cells were then transduced with

MLV-GFP reporter viruses for 24 h, after which chromatin from infected cells was subjected to

ChIP using histone H3 antibodies. At 24 h post infection, aphidicolin treatment did not affect

levels of total viral DNA (GFP), hence suggesting that viral entry, uncoating and reverse

transcription proceeded normally (Fig. 3C). In contrast, a 30-fold reduction in the levels of 2-

LTR circles was observed, indicative of defective viral nuclear entry (Fig. 3C). Strikingly,

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despite normal levels of input total viral DNA, ChIP analysis revealed a 10-fold reduction in

histone H3 associated viral DNA in aphidicolin arrested cells, whereas histone H3 coverage of

the cellular GAPDH gene was unaffected by the drug (Fig. 3C).

To further address the nuclear entry requirement for retroviral histone loading, we took

advantage of our earlier finding that mouse cells exhibit a nuclear entry block to Mason-Pfizer

monkey virus (M-PMV) infection (Wang & Goff, 2015). Mouse Ba/F3 cells, a pro-B cell line,

were infected with VSV-G pseudotyped MLV-GFP or M-PMV-GFP (Fig. 3E), and the

efficiency of infection was monitored by flow cytometry analysis 48 h later (Fig. 3F). Compared

to MLV, Ba/F3 cells show a 100-fold block to M-PMV infection (Fig. 3F). Although M-PMV

infected cells showed normal levels of total viral DNA (GFP), a 60-fold reduction in the

formation of 2-LTR circles was observed, highlighting a block in nuclear entry (Fig. 3G).

Importantly, in comparison to MLV, we also observed a 6-fold reduction in histone H3

association with M-PMV total viral DNA (Fig. 3H). Together, our two independent approaches

highlight the nuclear entry requirement for efficient histone loading onto viral DNAs.

Loading of epigenetically modified histones onto viral DNAs is delayed compared to core

histones

To extend our above observation that retroviral DNAs undergo rapid association with

core histone H3 early during infection, we next examined the kinetics of epigenetically modified

histone loading onto retroviral DNAs. To do this, we made use of a previously established MLV

transcriptional silencing phenomenon in F9 mouse embryonic carcinoma cells. Flow cytometry

analysis of F9 cells transduced with MLV-GFP at various times post-infection is depicted in Fig.

4A. At 1 day post infection, modest GFP expression (13%) was observed. At 5 days, less than 1%

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of the cells were GFP+ (Fig. 4A). This data suggest that there is a lag to complete promoter

silencing. Based on this, we hypothesized that the delay in MLV silencing may be due to the

absence of repressive histone marks on the viral DNA. To test this, ChIP analysis from infected

cells were carried out using antibodies against histone H3K9 trimethylation (H3K9me3), a

marker of silenced chromatin, or acetyl-histone H3 (H3Ac), a marker of active chromatin,

followed by quantitative real-time PCR analysis using the indicated primers (Fig. 4B). As

controls, the specificity of these antibodies for ChIP was confirmed using positive control

primers specific for endogenous promoters Polrmt (H3K9me3) and GAPDH (H3Ac). During

early infection, there was very low enrichment of H3K9me3 marks on the viral DNA, consistent

with the modest number of GFP+ cells (Fig. 4B). In contrast, H3K9me3 became highly enriched

on the viral DNA by 6 days post infection (Fig. 4B), consistent with the complete silencing of

the viral promoter (Fig. 4A). Conversely, no enrichment of H3Ac marks on viral DNAs was

observed at all times (Fig. 5C). Together, these results suggest that the kinetics of modified

histone loading is slower than that of core histone loading, thereby contributing to the delay in

MLV silencing (Fig. 5A). As expected, deposition of repressive histone marks onto viral DNAs

is only seen in mouse embryonic cells, as reciprocal experiments in differentiated MEF cells

showed enrichment of H3Ac (Fig. 4E), but not H3K9me3 (Fig. 4D) marks on the viral DNA at 6

days post infection, consistent with high MLV gene expression in these cells.

Characterization of the expression of unintegrated MLV DNA in NIH-3T3 cells

Our findings that unintegrated MLV genomes are rapidly covered with core histones (Fig. 2D

and 2H) together with the observation that unintegrated MLV genomes show poor gene

expression (Fig. 2B) raised the possibility that unintegrated MLV DNA may lack specific

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chromatin modifications that prevent the recruitment of host transcriptional machinery. Histone

acetyltransferases and deacetylases are key components of the eukaryotic transcriptional

machinery that control the activation or silencing of cellular promoters. Acetylation of histone

H3 and H4 on lysine residues decrease the protein’s overall positive charge, thereby leading to

an open chromatin conformation conducive to transcription activation. In contrast, deacetylation

of histone H3 and H4 leads to chromatin condensation and transcriptional silencing. To test the

possibility that unintegrated MLV DNA may be deficient in histone acetylation, NIH-3T3 cells

were first infected with WT or D184A-IN GFP viruses for 24 h, followed by the addition of

Trichostatin A (TSA), a specific inhibitor of histone deacetylases (Fig. 5A). Interestingly,

following TSA treatment, GFP expression from cells infected with D184A-IN virus showed a 4-

fold increase in the percentage of GFP+ cells as well as a 3-fold increase in the mean

fluorescence intensity (Fig. 5B, upper panel). In contrast, TSA addition failed to increase GFP

expression in cells infected with WT-IN viruses (Fig. 5B, lower panel), suggesting that this drug

only affects expression from unintegrated viral DNAs. We next performed ChIP assays using

acetylated histone H3 antibodies before and after the addition of TSA in both WT and D184A-IN

mutant cells. Compared to mock treatment, TSA treatment of D184A-IN infected cells resulted

in a 4-fold increase in H3 acetylation of linear, but not 2-LTR circles (Fig. 5D). This suggests

that only linear, but not circular unintegrated viral DNA forms contribute to gene expression. In

contrast, exposure to TSA did not further increase the extent of histone H3 acetylation in WT-IN

infected cells (Fig. 5D), perhaps because fully integrated proviral DNA already exhibits high

enrichment in acetylated H3 marks. Together, these data suggest that gene expression from

unintegrated DNAs is hampered by the lack of necessary chromatin modifications that promotes

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RNA polymerase recruitment, and that the chromatin environment can be corrected by the

inhibition of histone deacetylases.

Characterization of the kinetics of nucleocapsid (NC) and capsid (CA) association with viral

DNA following reverse transcription

The NC proteins of retroviruses are small, highly basic nucleic acid binding proteins that directly

contacts the RNA genome in the mature virion. Apart from its structural role, proper NC is

indispensable for many steps of viral replication including viral assembly, genomic RNA

packaging, reverse transcription and integration (Mori et al, 2015). Here, we examined the fate of

viral DNAs following infection with respect to its association with NC. ChIP assays using MLV-

NC specific antibodies were performed at different times post infection in infected NIH-3T3

cells, followed by quantitative real time PCR to monitor the extent of NC association with total

viral DNA (GFP) and 2-LTR circles (Fig. 6A). Shown in Fig. 6A, highest association of NC with

the total viral DNA was observed 4 h post infection, followed by a rapid decrease to very low

levels by 48 h. Of note, no association between NC and 2-LTR circles was detected at all times

(Fig. 6A). Similar kinetics of association was observed between CA and total viral DNA (Fig.

6B). In each case, the specificity of the antibodies used for ChIP is confirmed by the lack of non-

specific binding to the cellular GAPDH gene, as well as using cells that were infected with heat

inactivated (HI) virus (Fig. 6A and 6B).

The timing of NC and CA dissociation from the total viral DNAs (Fig. 6A and 6B)

appears to coincide with the rapid accumulation of core histones (Fig. 1B and 1D). Since histone

loading of viral DNAs takes place in the nucleus (Fig. 3D and 3H), this raises the possibility that

upon nuclear entry, core histones may promote the dissociation of NC and CA from the viral

DNAs by direct competition for DNA binding. If this was the case, one would expect that

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preventing nuclear entry may result in the stabilization of NC and CA-DNA complexes. Indeed,

NC and CA ChIP analysis from aphidicolin treated NIH-3T3 cells showed a 3-fold increase in

NC and CA binding to total viral DNAs (Fig. 6C). This implies that histone and viral protein

binding to the viral DNA may be mutually exclusive.

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DISCUSSION

In this study, using MLV as a model for retroviruses, we described for the first time the

state of the viral DNAs following infection. By performing histone H3 ChIP analysis on murine

cells infected with MLV, we show that core histone H3 rapidly associates with total viral DNAs

following infection (Fig. 1B and 1D). Moreover, experiments using either integrase mutant MLV

or raltegravir treated cells demonstrate that histone loading of total viral DNAs occurs

independently of viral integration (Fig. 2D and 2H). Total viral DNA is composed of linear DNA,

a direct product of reverse transcription and a substrate for integration, circular DNA, a marker

of nuclear entry, and fully integrated proviral DNA. 2-LTR circles constitute 10-15% of total

viral DNAs (Fig. 1A and 1C), while the remaining is made up of mostly linear DNA, other forms

of circular DNA, and fully integrated proviral DNA. From this, we deduce that during early

infection, histone loading of linear DNA must account for a fraction of the total ChIP signal (Fig.

1B and 1D). Since linear DNA also serve as precursors to 2-LTR circles, one can envision

several potential mechanisms of histone loading onto 2-LTR circles, including (1) histone

loading onto linear DNA followed by circularization, (2) circularization followed by histone

loading, or (3) histone loading and DNA circularization occurring simultaneously. Our current

data cannot distinguish between these possible modes of histone loading.

The majority, if not all of the histone loading onto viral DNAs occurs in the nucleus. This

is supported by our observation that perturbation of viral nuclear entry, either by infecting

aphidicolin arrested cells with MLV (Fig. 3D), or by infecting mouse cells with M-PMV, a virus

previously shown to be defective in nuclear entry (Fig. 3H), all caused a marked reduction in the

extent of histone H3 association with total viral DNAs.

Using a previously characterized model of MLV promoter silencing in mouse embryonic

cells, we were able to directly measure the timing of epigenetically modified histone association

59

with viral DNAs. In F9 embryonic carcinoma cells, significant core histone loading occurred as

early as 12 h post infection (Fig. 1D). In contrast, modified histone loading, as measured by the

presence of H3K9 trimethylation (H3K9me3), became evident much later at 6 days post

infection (Fig. 4B). The delay in the appearance of modified histones on the viral DNAs

correlates with the delay in the complete transcriptional silencing of the MLV promoter (Fig.

4A). In the case of differentiated cells, where the MLV promoter is fully active, acetylated

histone H3 loading was also found to occur much later at 6 days compared to core histone

loading as early as 12 h (Fig. 4E). Together, these data suggest that the appearance of both

repressive and active histone marks occur after core histone loading, thereby causing a transient

delay in the host cell modulation of viral gene expression. In host cells, DNA replication during

S-phase is associated with rapid reassembly of nucleosomes and inheritance of parental histone

modifications (Zentner & Henikoff, 2013). This is achieved by histones around the parental

DNA strand acting as a template to direct histone modifications of the daughter DNA strand

(Zhu & Reinberg, 2011). Retroviral infection generates de novo foreign DNA that the cell has

never seen before, hence the delay in the appearance of histone marks may reflect the absence of

previously established epigenetic memory, and the timing required to establish it in the first

place.

Using a catalytically inactive IN mutant (D184A), we were able to study gene expression

derived from unintegrated viral DNAs. Under basal conditions, the expression level from

unintegrated viral DNAs was very low compared to integrated proviral DNAs (Fig. 5B),

consistent with previous reports (Nakajima et al, 2001; Poon & Chen, 2003). However, the

addition of HDAC inhibitor TSA following infection caused a marked increase in the expression

of unintegrated, but not integrated viral DNAs (Fig. 5B). The increase in expression from

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unintegrated DNAs appears to be a product of two mechanisms. First, the increase in gene

expression from unintegrated viral DNA was accompanied by an increase in the association of

acetylated histone H3, a marker of actively transcribed genes, with the viral promoter (Fig. 5D),

thus favoring the recruitment of the host transcriptional machinery. Interestingly, such H3Ac

enrichment was only observed on total viral DNA, but not 2-LTR circles, implying that the

enhanced gene expression may be a product of increased transcription from only linear DNA

templates and not circular forms (Fig. 5D). Secondly, TSA appears to increase the stability of

unintegrated, but not integrated DNA forms, thereby increasing the amount of viral template

available for transcription (Fig. 5C). Why this is the case is a topic for future investigations, one

possibility is that TSA treatment may prolong the half-life of unintegrated DNA forms by

reducing the activity of the DNA degradation machinery in the nucleus.

Using antibodies specific for the NC and CA proteins of MLV, we were able to follow

the kinetics of NC and CA dissociation from the viral DNA following infection (Fig. 6A and 6B).

High degree of NC and CA association with the viral DNA early during infection followed by

rapid decay coincides with the timing of histone loading, suggesting that the two events may be

mutually exclusive. Indeed, by blocking nuclear entry and preventing histone loading, we were

able to prolong the association of NC and CA with the viral DNAs (Fig. 6C). One possible

model is that following entry of viral DNAs into the nucleus, core histones may compete with

NC and CA for binding to the viral DNAs, leading to their disassembly (Fig. 7C).

The precise mechanism by which core histones are loaded onto retroviral DNAs remains

a mystery, and likely requires the activity of cellular histone chaperones. Since retroviral

infection generates newly synthesized DNA through reverse transcription, one may predict that

histone loading of viral DNA is analogous to histone loading of newly synthesized cellular DNA

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during DNA replication. For cellular DNA, the Caf1 complex is thought to mediated de novo

histone H3/H4 assembly of newly synthesized DNA during replication (Gaillard et al, 1996;

Kaufman et al, 1995). However, preliminary experiments using Caf1 knockdown cells showed

no impairment in the extent of histone loading onto viral DNAs (data not shown), highlighting

the likely involvement of other chaperones, a topic of future studies. Equally interesting is the

question of whether viral encoded proteins play a role in facilitating histone loading onto its own

DNA.

Future studies will be aimed at elucidating the effect of histone loading on the retroviral

replication potential. For instance, does histone loading protects linear DNA from degradation?

Does histone loading of linear DNA promote efficient integration or integration site selection?

Answering these questions will require an understanding of the cellular machinery responsible

for histone loading onto viral DNAs.

62

MATERIALS AND METHODS

Cell lines and antibodies

Cell lines including human embryonic kidney (HEK)-293 (CRL-1573), F9 embryonic carcinoma

(CRL-1720), Mouse embryonic fibroblasts (MEF) (SCRC-1008) and NIH-3T3 (CRL-1658) cells

were purchased from American Type Culture Collection. All cells were cultured in Dulbecco’s

modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum

(FBS), 2 mM glutamine, 1000 U/mL penicillin and 100 mg/mL streptomycin. Ba/F3 cells were

cultured in RPMI medium supplemented with 10% heat-inactivated FBS and 1.6 ng/mL of

recombinant IL-3 (R&D Systems). All cells were maintained in a 37°C incubator with 5% CO2.

Antibodies used in this study include: Anti-Histone H3 (ab1791, Abcam), Anti-Histone H3 tri-

methyl K9 (ab8898, Abcam), Anti-Acetyl-Histone H3 (06-599, Millipore), anti-MLV NC

(National Cancer Institute serum 79S-), anti-MLV CA (National Cancer Institute serum 79S-804)

and rabbit IgG control antibody (sc-2027, Santa Cruz).

Plasmids

pCMV-intron expresses wild-type gag and pol from NB-tropic MLV (Soneoka et al, 1995).

pCMV-intron D184A-IN is an integrase dead mutant of pCMV-intron created by site directed

mutagenesis. pMD.G expresses the vesicular stomatitis virus (VSV) envelope glycoprotein.

pNCA-GFP is a replication-defective single-round MLV vector described previously (Ooi et al,

2010). pSARM-EGFP is a replication-defective single-round M-PMV vector in which the env

gene has been replaced with EGFP as described (Newman et al, 2006).

Retroviral transduction assay and flow cytometry analysis

63

NB-tropic MLV-GFP reporter viruses were produced by 293T cell transfection with 8 g of

pNCA-GFP, 4 g of pCMV-intron, and 4 g of pMD.G DNAs using Polyethylenimine (PEI).

Similarly, M-PMV-GFP reporter viruses were produced by transfection of 293T cells with 12 g

of pSARM-EGFP and 4 g of pMD.G. All reporter viruses were harvested 48 h later, filtered

(0.45 m), DNase treated for 1 h (Turbo DNase, Thermo Scientific), and used directly for

transduction assays as described previously (Wang & Goff, 2015). For some experiments, 10 M

of raltegravir (Santa Cruz Biotechnology) was added to the culture medium for the entire

duration of infection. Forty-eight hours post-infection, cells were trypsinized, diluted using flow

cytometry buffer (PBS with 1% BSA), and subjected to flow cytometry using automated cell

analyzer (LSRII, BD Bioscience).

Chromatin immunoprecipitation

5×106 cells were crosslinked with 1% formaldehyde at room temperature for 10 min, followed

by quenching with 0.125M glycine for 5 min. Cells were lysed in 0.5 mL of ChIP lysis buffer

(50mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA) containing Protease inhibitor cocktail (Roche)

and sonicated to produce an average fragment size of 200–800 base pairs. Each

immunoprecipitation was performed by incubating 30 g of sonicated chromatin together with 4

g of respective antibodies in ChIP dilution buffer (10 mM Tris-HCl pH 8.0, 1% Triton X-100,

0.1% SDS, 150 mM NaCl, 2 mM EDTA) overnight at 4°C. Next day, 25 μl of Protein A/G

Dynabeads (Life Technologies) were added for an additional 4 h. Captured antibody-antigen

complexes were washed 2 times each in ChIP low salt buffer (20 mM Tris-HCl pH 8.0, 1%

Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM EDTA), ChIP high salt buffer (20 mM Tris-HCl

pH 8.0, 1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 mM EDTA), ChIP LiCl buffer (10 mM

64

Tris-HCl pH 8.0, 1% NP-40, 250 mM LiCl, 1 mM EDTA), and TE buffer (10 mM Tris-HCl pH

8.0, 1 mM EDTA). DNA was eluted from beads in 200 μl elution buffer (TE buffer containing 1%

SDS, 100 mM NaCl, 5mM DTT), reverse crosslinked (65°C overnight), treated with RNase A

(37°C, 1 h) and Proteinase K (37°C, 2 h), and purified using QIAquick PCR purification kit

according to the manufacturer’s instructions (Qiagen). Quantitative real-time PCRs were

performed with indicated primers. The number of copies of each PCR product in both input and

immunoprecipitated DNA was first determined using plasmid derived standard curves. Relative

enrichment is shown as percent of input DNA calculated by dividing the number of copies from

immunoprecipitated DNA by input DNA and multiplying it by 100%.

Quantitative real-time PCR analysis of viral replication intermediates

At various time points following infection, cells were washed with PBS, and total DNA was

isolated using Qiagen DNeasy kit according to the manufacturer’s instructions. For analysis of

viral replication intermediates, 75 ng of total DNA was mixed with SYBR Green PCR master

mix (Roche) containing 15 pmol of indicated primers (see Supplemental table 1). PCRs were

performed in 96-well plates using 7900 Fast Real-Time PCR system (Applied Biosystems) with

the following reaction conditions: 10 min at 95°C, followed by 45 cycles of 30 s at 95°C, 30 s at

60°C and 30 s at 72°C. For all reactions, the number of copies of each PCR product was

determined using plasmid derived standard curves.

Aphidicolin treatment and cell cycle analysis of DNA content

NIH-3T3 cells were treated with aphidicolin (Sigma, 2 g/mL) for 24 h prior to infection.

Efficiency of aphidicolin treatment was confirmed by analyzing DNA content. Cells were

65

stained with Hoechst 33342 (Invitrogen) at 1 g/mL in cell culture medium for 30 min at 37°C,

followed by flow cytometry using automated cell analyzer (LSRII, BD Bioscience).

66

ACKNOWLEDGEMENTS

This work was supported by NCI grant R01 CA 30488 from the National Cancer Institute. S.P.G.

is an investigator of the Howard Hughes Medical Institute.

AUTHOR CONTRIBUTIONS

GZW and SPG designed and supervised the study. GZW and YW conducted all the experiments.

GZW and SPG analyzed the data and wrote the manuscript.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

67

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FIGURE LEGENDS

FIGURE 1. Core histone loading onto retroviral DNAs occur rapidly following infection

(A) MEF cells were infected with VSV-G pseudotyped MLV-GFP reporter virus. To control for

potential plasmid DNA carry over in the viral supernatant, heat inactivated (HI) virus were used

in parallel. Total DNA from infected cells was isolated at indicated time points, followed by real

time quantitative PCR using primers targeting GFP (total viral DNA) and 2-LTR circles.

Absolute copy number is calculated based on standard curves generated using plasmid DNA.

Results shown are means ± SDs from two independent experiments performed in duplicates.

(B) Histone H3 chromatin immunoprecipitation (ChIP) analysis of chromatin harvested at

indicated time points following MLV-GFP infection of MEF cells. ChIP data is presented as %

of input DNA, calculated by dividing the ChIP copy number for each gene target by the copy

number from input DNA and multiplying by 100%. Results shown are means ± SDs from two

independent experiments performed in duplicates. ND denotes not determined.

(C) ChIP analysis as outlined in (B) using rabbit IgG control antibody.

(D) Same experiment as outlined in (A) using F9 embryonic carcinoma cells.

(E) Same experiment as outlined in (B) using F9 embryonic carcinoma cells.

(F) Same experiment as outlined in (C) using F9 embryonic carcinoma cells.

FIGURE 2. Histone loading of retroviral DNAs occurs independently of viral integration

(A) Schematic of experimental setup.

(B) Flow cytometry analysis of NIH-3T3 cells infected with VSV-G pseudotyped wild-type (WT)

or integrase dead mutant (D184A) MLV-GFP reporter viruses. This is 2 days post infection. Y-

axis shows side scatter (SSC), X-axis shows GFP intensity. One representative experiment of

three independent experiments is shown.

(C) NIH-3T3 cells were infected with VSV-G pseudotyped wild-type (WT) or integrase dead

mutant (D184A) MLV-GFP reporter viruses. Total DNA from infected cells was isolated at

indicated time points post infection and number of copies of viral replication intermediates was

determined as described above. Results shown are means ± SDs from two independent

experiments performed in duplicates.

(D) Histone H3 ChIP analysis of chromatin harvested at indicated time points following MLV-

GFP infection of NIH-3T3 cells. ChIP data is presented as % of input DNA as described above.

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Results shown are means ± SDs from two independent experiments performed in duplicates. ND

denotes not determined.

(E) Schematic of experimental setup.

(F) Flow cytometry analysis of MEF cells infected with VSV-G pseudotyped WT MLV-GFP

reporter viruses in the presence or absence of raltegravir (Ral). This is 2 days post infection. Y-

axis shows side scatter (SSC), X-axis shows GFP intensity. One representative experiment of

three independent experiments is shown.

(G) MEF cells were infected with VSV-G pseudotyped wild-type (WT) MLV-GFP reporter

viruses in the presence or absence of raltegravir (Ral). Total DNA from infected cells was

isolated at indicated time points post infection and the number of copies of viral replication

intermediates was determined as described above. Results shown are means ± SDs from two

independent experiments performed in duplicates.

(H) Histone H3 ChIP analysis of chromatin harvested at indicated time points following MLV-

GFP infection of MEF cells treated with or without raltegravir (Ral). ChIP data is presented as %

of input DNA as described above. Results shown are means ± SDs from two independent

experiments performed in duplicates. ND denotes not determined.

FIGURE 3. Histone loading of retroviral DNAs requires viral nuclear entry

(A) Schematic of experimental setup.

(B) Cell cycle analysis of NIH-3T3 cells treated with or without DNA polymerase inhibitor

aphidicolin for 24 h prior to infection. Y-axis shows cell count, X-axis shows DNA content

measured by Hoechst staining. One representative experiment of three independent experiments

is shown.

(C) NIH-3T3 cells pretreated with or without aphidicolin (Aph) to induce cell cycle arrest were

infected with VSV-G pseudotyped WT MLV-GFP reporter virus. Total DNA from infected cells

was isolated 24 h post infection and the levels of viral replication intermediates were determined

as described above. Results shown are means ± SDs from two independent experiments

performed in duplicates.

(D) Histone H3 ChIP analysis of chromatin harvested from NIH-3T3 cells outlined in (C) 24 h

post infection. ChIP data is presented as % of input DNA as described above. Results shown are

means ± SDs from two independent experiments performed in duplicates. ND denotes not

determined.

(E) Schematic of experimental setup.

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(F) Flow cytometry analysis of Ba/F3 cells infected with VSV-G pseudotyped single-round

MLV or M-PMV-GFP reporter viruses. This is 2 days post infection. Y-axis shows side scatter

(SSC), X-axis shows GFP intensity. One representative experiment of three independent

experiments is shown.

(G) Level of viral replication intermediates from Ba/F3 cells infected with MLV or M-PMV-

GFP reporter viruses. Total DNA from infected cells was isolated at indicated time points post

infection and number of copies of viral replication intermediates was determined as described

above. Results shown are means ± SDs from two independent experiments performed in

duplicates.

(H) Histone H3 ChIP analysis of chromatin harvested from infected Ba/F3 cells outlined in (G).

ChIP data is presented as % of input DNA as described above. Results shown are means ± SDs

from two independent experiments performed in duplicates. ND denotes not determined.

FIGURE 4. Loading of epigenetically modified histones onto viral DNAs is delayed compared

to core histones

(A) Flow cytometry analysis of F9 cells infected with VSV-G pseudotyped WT MLV-GFP

reporter viruses at the indicated time points. Y-axis shows side scatter (SSC), X-axis shows GFP

intensity. One representative experiment of three independent experiments is shown.

(B) H3K9 trimethylation (H3K9me3) ChIP analysis of infected F9 cells at the indicated time

points. ChIP data is presented as % of input DNA as described above. Results shown are means

± SDs from two independent experiments performed in duplicates. Mitochondrial DNA

polymerase (positive control gene), GAPDH (negative control gene). ND denotes not determined.

(C) Acetyl-histone H3 (H3Ac) ChIP analysis of infected F9 cells at the indicated time points.

ChIP data is presented as % of input DNA as described above. Results shown are means ± SDs

from two independent experiments performed in duplicates. Mitochondrial DNA polymerase

(negative control gene), GAPDH (positive control gene). ND denotes not determined.

(D) Similar experiment as (B) performed in MEF cells.

(E) Similar experiment as (C) performed in MEF cells.

FIGURE 5. Characterization of the expression of unintegrated MLV DNA in NIH-3T3 cells

(A) Schematic of experimental setup.

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(B) Flow cytometry analysis of NIH-3T3 cells infected with VSV-G pseudotyped wild-type (WT)

or integrase dead mutant (D184A) MLV-GFP reporter viruses in the presence or absence of TSA

treatment. This is 2 days post TSA treatment. Y-axis shows side scatter (SSC), X-axis shows

GFP intensity. One representative experiment of three independent experiments is shown.

(C) NIH-3T3 cells infected with VSV-G pseudotyped WT or integrase dead mutant (D184A)

MLV-GFP reporter virus and each treated with or without TSA. Total DNA from infected cells

was isolated at indicated time points and levels of viral replication intermediates were

determined as described above. Results shown are means ± SDs from two independent

experiments performed in duplicates.

(D) H3Ac ChIP analysis of infected NIH-3T3 cells treated with or without TSA. ChIP data is

presented as % of input DNA as described above. Results shown are means ± SEMs from three

independent experiments performed in duplicates. Mitochondrial DNA polymerase (negative

control gene), GAPDH (positive control gene).

FIGURE 6. Characterization of the kinetics of nucleocapsid (NC) and capsid (CA) association

with viral DNA following reverse transcription

(A) MLV-NC ChIP analysis of chromatin harvested at indicated time points following MLV-

GFP infection of NIH-3T3 cells. ChIP data is presented as % of input DNA as described above.

Results shown are means ± SDs from two independent experiments performed in duplicates.

(B) Similar experiment as (A) using MLV-CA antibodies for ChIP.

(C) MLV-NC and CA ChIP analysis of chromatin harvested from infected, aphidicolin (Aph)

treated NIH-3T3 cells. ChIP data is presented as % of input DNA as described above. Results

shown are means ± SDs from two independent experiments performed in duplicates.

FIGURE 7. Model of the states of retroviral DNA following infection

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81

CHAPTER 6 - DISCUSSION

The data presented in the previous chapters encompasses various aspects of the retroviral life

cycle during early phases of infection. In the following section, I will individually discuss the

implications of our findings and suggest some future investigations.

EBP1, a novel host factor involved in primer binding site-dependent restriction of moloney

murine leukemia virus in embryonic cells

I began my graduate work studying the phenomenon of MLV silencing in mouse embryonic cells

(Chapter 2). The ability of mouse embryonic cells to silence the MLV promoter lies in its

expression of the sequence specific KRAB-ZFP ZFP809. ZFP809, through its interaction with

the PBSpro of MLV, serves to coordinate the assembly of macromolecular silencing complexes

containing TRIM28, ESET, HP1 and others, thereby establishing of a transcriptionally inactive

provirus. As a continuation of work done by Dr. Daniel Wolf in the lab, we identified a novel

ZFP809 interacting protein, EBP1, and showed its involvement in facilitating ZFP809/TRIM28

dependent MLV silencing in mouse embryonic cells. There was no previous history of an

involvement of EBP1 in ES-cell specific silencing. EBP1 is a known transcriptional repressor of

E2F1 and androgen receptor regulated promoters (Ghosh et al, 2013; Zhang et al, 2005a; Zhang

et al, 2002; Zhang et al, 2005b; Zhang et al, 2003) in breast and prostate cancer cell lines.

Interestingly, our observation of the interaction between EBP1 and TRIM28 coincides with

previous findings showing the involvement of both proteins at the E2F target promoter (Wang et

al, 2007). Therefore, it is conceivable that for other cellular promoters, specific DNA binding

proteins, acting in a manner similar ZFP809, may recruit both EBP1 and TRIM28 to the target

promoter and modulate gene expression epigenetically. Given that both EBP1 and TRIM28 are

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ubiquitously expressed and could regulate target genes in many cell types, we postulate that cell

specific promoter regulation will be determined by cell specific zinc finger proteins, which in the

case of embryonic cells, is ZFP809.

Of note, EBP1 has been implicated in the biology of RNA viruses in other settings.

Honda et al identified EBP1 as a novel binding partner for the PB1 subunit of influenza virus

RNA polymerase and showed that overexpression of EBP1 blocked the transcription of influenza

virus and led to reduced viral titers (Honda et al, 2007). In another study, it was reported that

infection of Vero cells by rinderpest virus led to the downregulation of EBP1 expression, and

that overexpression of EBP1 led to the inhibition of rinderpest virus transcription (Gopinath et al,

2010). EBP1’s role in antagonizing retroviral infection in embryonic cells expands the repertoire

of RNA viruses sensitive to EBP1 regulation. Preliminary tests suggest that cellular EBP1

expression is not altered by retroviral infection (data not shown), and that the course of viral

DNA synthesis by reverse transcriptase was not affected.

Mouse embryonic cells suppress the expression of both incoming exogenous retroviruses

as well as endogenous retroelements. The loss of either ZFP809 (Wolf et al, 2015), TRIM28

(Rowe et al, 2010) or SETDB1 (Matsui et al, 2010) in mouse ES cells led to robust activation of

endogenous retroviruses, as well as failure to block new retroviral infections. In F9 EC cells,

depletion of EBP1 did not result in the upregulation of various endogenous retroelements (data

not shown). Therefore, we suggest that although EBP1 is required for the silencing of newly

integrated provirus, it may be dispensable for the silencing of endogenous retroviral elements.

Such division of labor has also been observed in the case of the histone methyltransferase G9a

(Leung et al, 2011).

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In summary, our study revealed EBP1 to be a novel component of the PBS-dependent

silencing machinery in mouse embryonic cells. It should be noted that retroviral silencing is

mediated through both PBS-dependent, as well as PBS-independent pathways involving the host

transcription factor Yin Yang 1 (Schlesinger et al, 2013), and further studies will be necessary to

address the potential crosstalk between these two arms. It will also be of interest to determine

whether TRIM28 in concert with other KRAB-ZFPs also requires EBP1 to mediate silencing in

other settings.

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Regulation of Yin Yang 1 by Tyrosine Phosphorylation

As an extension of YY1’s role in MLV silencing, I became interested in understanding

how YY1 activity is regulated through post-translational modifications. In chapter 3, I showed

that YY1 is a target of tyrosine phosphorylation across many cell types. Using a combination of

kinase inhibitors, kinase overexpression and kinase knockout studies, we arrived at the

conclusion that YY1 phosphorylation is likely mediated by multiple Src family kinases. The nine

Src family kinases exhibit different tissue expression patterns. For example, c-Src, Fyn, and Yes

are ubiquitously expressed (Stein et al, 1994), while Lyn shows preferential expression in

hematopoietic cells (Hibbs et al, 1995). Given the conservation of YY1 phosphorylation across

different cell types, we postulate that depending on the Src kinase signature within a cell,

different cell types may utilize different members of the kinase family for phosphorylating YY1.

Thus, this mechanism of YY1 regulation may be functioning in a wide range of tissues and

settings.

In Lyn-overexpressing cells, overall YY1 phosphorylation was greatly reduced by single

mutations at either tyrosine 8, 254 or 383, suggesting that these three sites are preferentially

phosphorylated by Lyn, and that phosphorylation of these sites may occur in a cooperative

manner, since mutations at each site greatly reduced the likelihood that the other sites become

phosphorylated in vivo. Phosphorylation of bacterially purified Y383F YY1 using recombinant

Lyn was also significantly reduced in vitro. However, in both Src and Yes overexpressing cells,

YY1 was preferentially phosphorylated on different residues, suggesting that the sites of YY1

phosphorylation in a given cell type may depend on the expression pattern of Src family kinases

within that cell. Importantly, it appears that tyrosine 383 is a target of all three kinases.

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To examine the functional importance of YY1 phosphorylation sites uncovered by our

mutagenesis analysis, we tested the effect of introducing phosphomimetic and

nonphosphorylatable mutations on the ability of YY1 to regulate gene expression driven by both

retroviral and cellular promoters. Our observation that overexpression of Y383E, but not Y383F,

affected the ability of YY1 to either transactivate the HTLV-1 promoter, or repress the MLV or

endogenous c-fos promoter highlighted the functional importance of this tyrosine residue.

YY1 contains four C2H2 zinc finger domains, each of which is comprised of conserved

cysteines, histidines and hydrophobic residues folding to form a structure surrounding a

central zinc ion (Houbaviy et al, 1996). Y383 is located at the very start of the fourth zinc finger,

before the first cysteine of the CCHH cluster chelating the zinc, and is not the highly conserved

bulky hydrophobic residue in the center of the zinc finger. A closer inspection of the co-crystal

structure of YY1 bound to the AAV P5 promoter initiator element (Houbaviy et al, 1996) reveals

that tyrosine 383, together with valine 384 and critical cysteine 385 participate in the formation

of the first -sheet of the fourth zinc finger. Hence, one would predict that the addition of a

phosphate at this position may perturb the first -sheet, thus interfering with zinc and/or nucleic

acid binding. We cannot rule out the possibility that phosphorylation induced changes in protein

folding may also affect YY1’s affinity for other transcriptional cofactors such as CTCF

(Donohoe et al, 2007) and Smads (Kurisaki et al, 2003).

The importance of Y383 is supported by biochemical assays showing reduced DNA and

RNA binding by the Y383E mutant. Apart from tyrosine 383, it is surprising that

phosphomimetic mutations at the other five tyrosines failed to affect YY1 function in the context

of HTLV-1 transactivation. For example, tyrosine 185 lies in close proximity to serine 180 and

serine 184, sites previously shown to be a target of Aurora B kinase, leading to changes in DNA

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binding and YY1 acetylation (Kassardjian et al, 2012). In spite of this proximity, the Y185E and

Y185F mutants maintained full transactivation activity on the HTLV-1 promoter. Therefore, the

simplest explanation is that phosphorylation of the other five tyrosines is not important for

YY1’s activation function.

Inactivation of gene-specific transcription factors often affects control of cell division and

mitosis. One mechanism of transcription factor inactivation during the cell cycle involves

serine/threonine phosphorylation of the consensus TGEKP zinc finger linker sequence in C2H2

transcription factors such as Sp1, Ikaros (Dovat et al, 2002) and YY1 (Rizkallah et al, 2011a;

Rizkallah & Hurt, 2009), leading to reduced DNA binding., YY1 activity may also be similarly

controlled during the cell cycle by phosphorylation of tyrosine 383. To our knowledge,

regulation of a C2H2 transcription factor through tyrosine phosphorylation of its zinc finger has

not been documented. In YY1, tyrosine 383 makes up part of the consensus Y-X-C motif that is

also found at the start of many other C2H2 transcription factors (Brayer & Segal, 2008), thereby

raising that possibility that other C2H2 family members may be regulated in a similar manner.

YY1 is a stable, constitutive and widely expressed protein across many cell types (Austen

et al, 1997). This makes regulation of YY1 through post-translational modifications an attractive

mean of controlling its activity. The levels of tyrosine phosphorylated YY1 is low under basal

conditions, but becomes readily detectable upon the inhibition of endogenous tyrosine

phosphatases or the activation of cytosolic tyrosine kinases via EGFR signaling. Given that

protein phosphorylation is dictated by the balance between kinase and phosphatase activities, our

data suggests that the majority of YY1 is unphosphorylated and available for DNA binding under

steady-state conditions, leading to persistent activation or repression of YY1 responsive genes.

However, the presence of extracellular stimuli such as EGF allows for dynamic changes in the

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DNA binding capacity of YY1 through tyrosine phosphorylation, thereby permitting temporal

fine-tuning of YY1-responsive promoters.

Under steady-state conditions, YY1 is predominantly a nuclear protein with a minor

cytoplasmic presence (Austen et al, 1997). Cellular fractionation experiments using pervanadate-

treated Jurkat cells showed high levels of tyrosine phosphorylated YY1 in the nucleus and trace

amounts in the cytoplasm. This is consistent with emerging evidence showing that beside

cytoplasmic localization, a fraction of the Lyn (Radha et al, 1996), Src, Fyn, and Yes kinases

(Takahashi et al, 2009) is present in the nucleus. Nevertheless, the interplay between YY1

phosphorylation and cellular localization is a topic of future studies.

In summary, our work has revealed a novel means of regulating YY1 function through

tyrosine phosphorylation. Further studies will be necessary to address the specific tyrosine

phosphatase(s) responsible for YY1 dephosphorylation, as well as the localization and dynamics

of tyrosine phosphorylated YY1 in vivo. These studies will provide us with a better

understanding of the complex interplay between tyrosine kinases and phosphatases in regulating

YY1 function.

88

Postentry restriction of Mason-Pfizer monkey virus in mouse cells

My next project revolved around an interesting observation that mouse cells are resistant

to infection by M-PMV, a prototypical simian betaretrovirus. As shown in chapter 4, the block is

very potent (50-100 fold) and resistance to M-PMV appears to be widespread among many cell

types. Moreover, the observation that mouse cells blocked M-PMV reporter viruses pseudotyped

with various envelope proteins suggests that the block occurs post-entry. Moreover, the process

of reverse transcription, as measured by the levels of minus-strand strong-stop DNA and the

reporter GFP DNA, occurred normally in all the mouse cell lines tested. The levels of 2-LTR

circles, however, were drastically reduced, suggesting a significant block at the time of nuclear

entry. This conclusion is further supported by diminished levels of integrated proviral DNA

remaining in mouse cells after long-term passage. It is not known if infection of permissive cells

by M-PMV requires cell division as is the case for MLVs, but the cells used in all our

experiments were dividing, ensuring that this is not the basis for the block. Given that the defect

in viral circular DNA formation partially coincides with the timing of Fv1 restriction, we

hypothesized that the Fv1 alleles present in the various mouse lines might account for the block

in M-PMV infection. But overexpression of the Fv1n or Fv1

b alleles in human cells failed to

induce any resistance to M-PMV. This observation was consistent with the inability of M-PMV

to transduce mouse cells harboring different Fv1 alleles, making Fv1 an unlikely candidate for

M-PMV restriction. Interestingly, it has been reported that Fv1 also failed to block primate

lentiviruses (Hatziioannou et al, 2004). Of note, although our data suggest a major defect at the

approximate time of nuclear entry, we cannot exclude the possibility that additional blocks at the

level of integration per se may also exist in mouse cells.

89

The inability of M-PMV to replicate in mouse cells may be due to either the absence or

incompatibility of critical host factors, or to the presence of active viral restriction factors

exemplified by Fv1. Infection of heterokaryons formed by the fusion between permissive and

non-permissive cells can be used to distinguish between the two possibilities. In preliminary tests,

we found that heterokaryons formed by the fusion of HeLa and NIH-3T3 cells remained resistant

to M-PMV infection (data not shown), implying that mouse cells may contain a novel dominant

restriction factor with Fv1-like activity. We cannot rule out the possibility, however, that the

fusion process itself is causing virus resistance and that the heterokaryons are resistant for other

reasons. Recently, the effects of human and mouse TRIM proteins on the early and late events of

HIV and MLV life cycle identified numerous family members with antiviral activities (Uchil et

al, 2008). With that in mind, it is possible that a ubiquitously expressed mouse-specific TRIM

protein is responsible for the M-PMV resistance. Preliminary tests of a panel of mouse TRIMs

expressed in HeLa cells did not reveal any TRIM capable of blocking M-PMV. Identification of

the putative dominant mouse restriction factor, if present, remains a subject for future

investigations. Finally, although mouse cells are non-permissive to M-PMV transduction, we

note that they are susceptible to infection by other betaretroviruses such as mouse mammary

tumor virus (MMTV). It remains to be determined whether this is due to MMTV’s ability to

escape host restriction, or the presence of compatible co-factors necessary for infection in mouse

cells.

90

Histones are rapidly loaded onto unintegrated retroviral DNAs soon after nuclear entry

Finally, the last project examines the question of what is the “state” of the viral DNAs

following infection. Given that reverse transcription generates de novo foreign DNAs in the host

cell, this raises the question of what is the histone state of these retroviral DNAs following

infection. Are retroviral DNAs a target of histone loading? If so, when, where and on what forms

of viral DNA does this occur? Following reverse transcription, do retroviral DNAs remain

associated with their cognate structural proteins such as NC and CA? When and where does NC

and CA dissociate from the viral DNA? Finally, given that unintegrated viral DNAs contribute

very little to viral gene expression in the infected cell, why is this so?

By performing histone H3 ChIP analysis on murine cells infected with MLV, we show

that core histone H3 rapidly associates with total viral DNAs following infection. Moreover,

experiments using either integrase mutant MLV or raltegravir treated cells demonstrate that

histone loading of total viral DNAs occurs independently of viral integration. Total viral DNA is

composed of linear DNA, a direct product of reverse transcription and a substrate for integration,

circular DNA, a marker of nuclear entry, and fully integrated proviral DNA. 2-LTR circles

constitute 10-15% of total viral DNAs, while the remaining is made up of mostly linear DNA,

other forms of circular DNA, and fully integrated proviral DNA. From this, we deduce that

during early infection, histone loading of linear DNA must account for a fraction of the total

ChIP signal. Since linear DNA also serve as precursors to 2-LTR circles, one can envision

several potential mechanisms of histone loading onto 2-LTR circles, including (1) histone

loading onto linear DNA followed by circularization, (2) circularization followed by histone

loading, or (3) histone loading and DNA circularization occurring simultaneously. Our current

data cannot distinguish between these possible modes of histone loading.

91

The majority, if not all of the histone loading onto viral DNAs occurs in the nucleus. This

is supported by our observation that perturbation of viral nuclear entry, either by infecting

aphidicolin arrested cells with MLV, or by infecting mouse cells with M-PMV, a virus

previously shown to be defective in nuclear entry, all caused a marked reduction in the extent of

histone H3 association with total viral DNAs.

Using a previously characterized model of MLV promoter silencing in mouse embryonic

cells, we were able to directly measure the timing of epigenetically modified histone association

with viral DNAs. In F9 embryonic carcinoma cells, significant core histone loading occurred as

early as 12 h post infection. In contrast, modified histone loading, as measured by the presence

of H3K9 trimethylation (H3K9me3), became evident much later at 6 days post infection. The

delay in the appearance of modified histones on the viral DNAs correlates with the delay in the

complete transcriptional silencing of the MLV promoter. In the case of differentiated cells,

where the MLV promoter is fully active, acetylated histone H3 loading was also found to occur

much later at 6 days compared to core histone loading as early as 12 h. Together, these data

suggest that the appearance of both repressive and active histone marks occur after core histone

loading, thereby causing a transient delay in the host cell modulation of viral gene expression. In

host cells, DNA replication during S-phase is associated with rapid reassembly of nucleosomes

and inheritance of parental histone modifications (Zentner & Henikoff, 2013). This is achieved

by histones around the parental DNA strand acting as a template to direct histone modifications

of the daughter DNA strand (Zhu & Reinberg, 2011). Retroviral infection generates de novo

foreign DNA that the cell has never seen before, hence the delay in the appearance of histone

marks may reflect the absence of previously established epigenetic memory, and the timing

required to establish it in the first place.

92

Using a catalytically inactive IN mutant (D184A), we were able to study gene expression

derived from unintegrated viral DNAs. Under basal conditions, the expression level from

unintegrated viral DNAs was very low compared to integrated proviral DNAs, consistent with

previous reports (Nakajima et al, 2001; Poon & Chen, 2003). However, the addition of HDAC

inhibitor TSA following infection caused a marked increase in the expression of unintegrated,

but not integrated viral DNAs. The increase in expression from unintegrated DNAs appears to be

a product of two mechanisms. First, the increase in gene expression from unintegrated viral DNA

was accompanied by an increase in the association of acetylated histone H3, a marker of actively

transcribed genes, with the viral promoter, thus favoring the recruitment of the host

transcriptional machinery. Interestingly, such H3Ac enrichment was only observed on total viral

DNA, but not 2-LTR circles, implying that the enhanced gene expression may be a product of

increased transcription from only linear DNA templates and not circular forms. Secondly, TSA

appears to increase the stability of unintegrated, but not integrated DNA forms, thereby

increasing the amount of viral template available for transcription. Why this is the case is a topic

for future investigations, one possibility is that TSA treatment may prolong the half-life of

unintegrated DNA forms by reducing the activity of the DNA degradation machinery in the

nucleus.

Using antibodies specific for the NC and CA proteins of MLV, we were able to follow

the kinetics of NC and CA dissociation from the viral DNA following infection. High degree of

NC and CA association with the viral DNA early during infection followed by rapid decay

coincides with the timing of histone loading, suggesting that the two events may be mutually

exclusive. Indeed, by blocking nuclear entry and preventing histone loading, we were able to

prolong the association of NC and CA with the viral DNAs. One possible model is that following

93

entry of viral DNAs into the nucleus, core histones may compete with NC and CA for binding to

the viral DNAs, leading to their disassembly.

The precise mechanism by which core histones are loaded onto retroviral DNAs remains

a mystery, and likely requires the activity of cellular histone chaperones. Since retroviral

infection generates newly synthesized DNA through reverse transcription, one may predict that

histone loading of viral DNA is analogous to histone loading of newly synthesized cellular DNA

during DNA replication. For cellular DNA, the Caf1 complex is thought to mediated de novo

histone H3/H4 assembly of newly synthesized DNA during replication (Gaillard et al, 1996;

Kaufman et al, 1995). However, preliminary experiments using Caf1 knockdown cells showed

no impairment in the extent of histone loading onto viral DNAs (data not shown), highlighting

the likely involvement of other chaperones, a topic of future studies. Equally interesting is the

question of whether viral encoded proteins play a role in facilitating histone loading onto its own

DNA.

Future studies will be aimed at elucidating the effect of histone loading on the retroviral

replication potential. For instance, does histone loading protects linear DNA from degradation?

Does histone loading of linear DNA promote efficient integration or integration site selection?

Answering these questions will require an understanding of the cellular machinery responsible

for histone loading onto viral DNAs.

94

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