Ulm University Medical Center Institute of Molecular ...

Ulm University Medical Center

Institute of Molecular Virology

Prof. Dr. Frank Kirchhoff

A small peptide derived from the HIV-1 gp120 glycoprotein

forms positively charged fibrils that enhance transduction

efficiencies of retro- and lentiviral vectors

Dissertation

to obtain the Doctoral Degree of Human Biology (Dr. biol. hum.)

at the Faculty of Medicine, Ulm University

presented by

Maral Yolamanova

Ashgabat, Turkmenistan

2016

2

Present Dean: Prof. Dr. Thomas Wirth

1st reviewer: Prof. Dr. Jan Münch

2nd reviewer: Prof. Dr. Barbara Spellerberg

Date of graduation: 15th April 2016

Parts of this dissertation have already been published in the following journal article:

Yolamanova M, Meier C, Shaytan AK, Vas V, Bertoncini CW, Arnold F,

Zirafi O, Usmani SM, Müller JA, Sauter D, Goffinet C, Palesch D, Walther P,

Roan NR, Geiger H, Lunov O, Simmet T, Bohne J, Schrezenmeier H,

Schwarz K, Ständker L, Forssmann WG, Salvatella X, Khalatur PG,

Khokhlov AR, Knowles TP, Weil T, Kirchhoff F and Münch J.

Peptide nanofibrils boost retroviral gene transfer and

provide a rapid means for concentrating viruses.

Nature Nanotechnology 2013, 8:130-136. DOI: 10.1038/nnano.2012.248

Copyright © by Macmillan Publishers Limited

TABLE OF CONTENTS I

TABLE OF CONTENTS

TABLE OF CONTENTS ....................................................................................................... I

LIST OF ABBREVIATION ................................................................................................ III

1 INTRODUCTION ............................................................................................................ 1

1.1 Retroviruses ............................................................................................................... 1

1.2 Retroviral vectors ...................................................................................................... 3

1.3 Pseudotyping ............................................................................................................. 5

1.4 Limitations for efficient retroviral gene transfer ....................................................... 5

1.5 Approaches to enhancing retroviral transduction efficiency ..................................... 6

1.6 Peptide derived from HIV-1 envelope glycoprotein boost HIV-1 infection ............. 8

1.7 Aim of present study ............................................................................................... 10

2 MATERIAL AND METHODS...................................................................................... 11

2.1 Material .................................................................................................................... 11

2.1.1 Eukaryotic cells ................................................................................................ 11

2.1.2 Primary cells ..................................................................................................... 12

2.1.3 Bacteria ............................................................................................................. 12

2.1.4 Nucleic acids..................................................................................................... 12

2.1.5 Proteins ............................................................................................................. 14

2.1.6 Peptides ............................................................................................................. 14

2.1.7 Chemical reagent .............................................................................................. 15

2.1.8 Consumables ..................................................................................................... 17

2.1.9 Technical equipment......................................................................................... 17

2.1.10 Kits ................................................................................................................. 18

2.1.11 Buffers and solutions ...................................................................................... 19

2.1.12 Media .............................................................................................................. 19

2.2 Methods ................................................................................................................... 20

2.2.1 DNA methods ................................................................................................... 20

2.2.2 Bacterial methods ............................................................................................. 21

2.2.3 Cell culture ....................................................................................................... 21

2.2.4 Viral methods ................................................................................................... 22

2.2.5 Imaging methods .............................................................................................. 28

2.2.6 Proteomic methods ........................................................................................... 29

3. RESULTS ...................................................................................................................... 30

3.1 Identification of gp120 fragments that promote HIV-1 infection ............................. 30

3.2 gp120 fragments are not cytotoxic. ........................................................................... 32

3.3 gp120 peptides that enhance HIV infection form amyloid fibrils ............................. 32

TABLE OF CONTENTS II

3.4 Formation of EF-C fibrils can be achieved in several solvents and their virus enhancing activity is stable .............................................................................................. 35

3.5 EF-C fibrils increase viral attachment and fusion ..................................................... 36

3.6 EF-C fibrils allow to concentrate viral particles ........................................................ 39

3.7 EF-C fibrils are potent enhancer of lenti- and retroviral transduction ...................... 41

3.8 Comparison between EF-C fibrils and other agents used to promote retroviral transduction ..................................................................................................................... 45

3.9 Immobilized EF-C nanofibrils capture virus and increase transduction. .................. 47

4. DISCUSSION ................................................................................................................ 52

4.1 Application of EF-C nanofibrils as enhancer of transduction efficiency. ............... 52

4.2. Analysis of the mechanism of fibril-mediated enhancement of viral infection. .... 55

5. SUMMARY .................................................................................................................. 59

6. REFERENCES .............................................................................................................. 60

LIST OF ABBREVIATION III

LIST OF ABBREVIATION

°C Degree Celsius

μg microgram

μl microliter

aa amino acid

β Beta

β-gal Beta-galactosidase

ATCC American Type Culture Collection

bp Basepair

CaCl2 Calcium chloride

CCR5 C-C chemokine receptor type 5

DEAE Diethylaminoethyl

DMEM Dulbecco’s Modified Eagle’s Medium

DMSO Dimethyl sulfoxide

DNA Desoxyribonucleic acid

DRK German: Deutsches Rotes Kreuz

ds Double strand

E. coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

EF-A Enhancing factor A

EF-C Enhancing factor C

env Envelope

FCS Fetal calf serum

Fig. Figure

g gravitational force

g Gram

gag Group specific antigen

Gal Galactosidase

GALV Gibbon ape Leukemia Virus

GFP Green Fluorescent Protein

G-CSF Granulocyte colony stimulating factor

gp Glycoprotein

gp120 Envelope glycoprotein of HIV, 120 kDa

gp41 Envelope glycoprotein of HIV, 41 kDa

HCl Hydrochloric acid

HEPES 2-(4-(2-Hydroxyethyl)- 1-piperazinyl)-ethansulfonsäure

LIST OF ABBREVIATION IV

h Hour(s)

HIV Human Immunodeficiency Virus

i.e. id est

IL-2 Interleukin-2

kb Kilo base pairs

kDa Kilodalton

KCl Potassium chloride

l Liter

LB Lysogeny Broth

LSM Laser Scanning Microscope

LTR Long Terminal Repeat

m Meter

mM Millimolar

MgCl2 Magnesiumchloride

min Minute(s)

ml Milliliter

MLV Murine Leukemia Virus

NaCl Sodium chloride

NEB New England Biolabs

nef Negative factor

ng Nanogram

NIH National Institutes of Health

nm Nanometer

p24 Capsid protein of HIV, 24 kDa

PAP Prostatic acid phosphatase

PBL Peripheral blood lymphocyte

PBS Phopsphate buffered saline

pH Potentia hydrogenii

PHA Phytohaemagglutinin

pol Polymerase

RD114 Envelope glycoprotein of Feline Leukemia Virus

rev Regulator of expression of virion proteins

rhIL-6 Recombinant human interleukin-6

rhSCF Recombinant human Stem Cell Factor

RLU Relative Light Units

RPMI Roswell Park Memorial Institute medium

rpm Revolutions per minute

LIST OF ABBREVIATION V

RRE Rev responsive element

RT Room temperature (21°C - 25°C)

sec Second(s)

SEVI Semen-derived Enhancer of Viral Infection

SIN Self-inactivating vector

S.O.C Super Optimal broth with Catabolite repression

ss Single strand

TAE Tris-acetate-EDTA

tat Transactivator of transcription

TEM Transmission electron microscopy

ThT Thioflavin T

Tris Tris(hydroxymethyl)aminomethane

U3 Unique 3

U5 Unique 5

V3 Variable loop 3

v/v Volume per volume

vif Viral infectivity factor

vpr Viral protein R

vpu Viral protein U

VSV Vesicular Stomatitis Virus

w/v Weight per volume

YFP Yellow Fluorescent Protein

Amino acid code

A Alanine N Asparagine C Cysteine P Proline D Aspartic acid Q Glutamine E Glutamic acid R Arginine F Phenylalanine S Serine G Glycine T Threonine H Histidine U Selenocysteine I Isoleucine V Valine K Lysine W Tryptophan L Leucine Y Tyrosine M Methionine

1 INTRODUCTION 1

1 INTRODUCTION

Introduction of genetic material into the cells offers many prospects for basic research, e.g.

for constitutive or regulated gene expression, cDNA library construction, siRNA delivery,

insertional mutagenesis or generation of transgenic animals (Matrai et al., 2010; Tiscornia

et al., 2002). Furthermore the opportunity to treat disease by either modifying the gene

expression or correction of abnormal genes has been used in approaches for therapeutic

application (Bunnell et al., 1995; McTaggart et al., 2002; Frecha et al., 2010; Matrai et al.,

2010). Gene therapy holds great promise for the treatment of inherited metabolic disorders

such as adenosine deaminase deficiency and cystic fibrosis as well as acquired diseases

such as cancer, cardiovascular and some infectious diseases (Candotti et al., 1012; Isner,

2002; Bunnell BA and Morgan RA, 1998). Current methods for transferring therapeutic

genes can be divided into two large groups: viral and non-viral methods. To the non-viral

delivery systems belong naked DNA, liposomes and DNA-protein complexes

(Ramamoorth M and Narvekar A, 2015). The main problems with non-viral delivery are (i)

relatively low transduction efficiency; (ii) transiency of gene expression; (iii) lack of

specific targeting. Therefore, the application of non-viral systems in gene therapy is low

(Journal of Gene Medicine, © 2015 John Wiley and Sons Ltd). Viral vectors are the most

efficient vehicles for gene transfer in vivo. The major advantages of viral vectors are high

transduction efficiency and specificity of transgene delivery using the cell’s own

biosynthetic machinery. Viral gene delivery can be classified into two general groups: the

viral vectors which integrate into the host cell genome, such as, retroviral, lentiviral and

adeno-associated viral vectors and non-integrating vectors like herpes simplex virus type 1

and adenoviral vectors. Moreover, some of viral vectors; e.g. lentiviruses, have the unique

ability to efficiently replicate in non-dividing cells as they target the nucleus without the

requirement of mitosis (von Schwedler et al., 1994). Nowadays viral vectors are employed

in more than 70% of all clinical gene therapy trials (Journal of Gene Medicine, © 2015

John Wiley and Sons Ltd).

1.1 Retroviruses

Retroviruses are classified into three genera: the oncoretroviruses (e.g. MLV, murine

leukemia virus), the lentiviruses (e.g. HIV, Human Immunodeficiency Virus), and the

spumaviruses including the Human Foamy Virus (HFV) (Coffin et al., 1997; Heneine et

al., 1998). Retroviruses are lipid-enveloped viruses carrying two copies of single-stranded

1 INTRODUCTION 2

positive RNA, which range between 7 to 13 kb in length. The virus enters the cell through

the specific interactions between viral envelope glycoproteins and the cellular receptors

leading to fussion between the viral envelope and the host membrane. The viral RNA

enters the cytoplasm and is reversely transcribed into double-stranded DNA. The viral

DNA is permanently integrated into the host cell chromosomal DNA and called a provirus

(Coffin et al., 1997; Turner and Summers, 1999).

Basic genomic features are common to all retroviruses. Two copies of the long terminal

repeat (LTR) flank the ends of the viral genome (Fig. 1) and contain all of the requisite

signals for gene expression: enhancer, promoter, transcription initiation (capping),

transcription terminator and polyadenylation signal. Each LTR can be divided into three

elements: U3, R and U5. While the promoter region with the regulatory sequences is

contained in the U3 region at the 5’-extremity of the LTR, the polyadenylation signal is

located in the U5 region at the 3’-extremity of the LTR. The sequence identity of the LTR

is important in the process of reverse transcription, because the polymerase jumps from

one end of the template to the other. A sequence named packaging signal (psi, Ψ) is found

near the 5’end of the viral genome (Fig. 1) and is required for specific packaging of the

viral RNA into newly forming virions (Coffin et al., 1997; Turner and Summers, 1999).

Retrovirus genomes contain three essential genes: gag (group-specific antigen), pol

(polymerase), and env (envelope glycoprotein) (Fig. 1). The gag sequence encodes the

three main structural proteins capsid, matrix and nucleocapsid, which are necessary for the

assembly and release of virus and virus-like particles. The pol sequence encodes the

enzymes protease, reverse transcriptase and integrase, which are responsible for the reverse

transcription of the viral genome from RNA to DNA during the infection process and for

Figure 1. Scheme of the genetic organization of MLV, a gammaretrovirus, and HIV-1, a lentivirus. The proviruses of all retroviruses are flanked by Long Terminal Repeats (LTR) and encode gag, pol and env genes. HIV-1 virus carries additionally six regulatory/accessory genes (tat, rev, vif, vpr, vpu and nef).

1 INTRODUCTION 3

the integration of the proviral DNA into the host cell genome. The env sequence encodes

the glycoproteins, necessary for interaction with host receptors and virus entry (Coffin et

al., 1997; Turner and Summers, 1999).

Moreover, lentiviruses possesses several additional genes, i.e. tat and rev encoding

regulatory proteins and vif, vpr, vpu and nef encoding accessory proteins (Turner and

Summers, 1999). The Tat (transactivator of transcription) protein stimulates viral gene

expression during transcription initiation and elongation (Mahlknecht et al., 2008). The

Rev (regulator of expression of viral proteins) protein is required for efficient cytoplasmic

accumulation of viral RNA (Malim and Emerman, 2008). The Vif (virion infectivity

factor) protein counteracts a cellular restriction factor, APOBEC3G, that inhibits HIV-1

(Sheehy et al., 2002). The Vpu (viral protein unknown) degrades CD4 and promotes virus

release by counteracting the cellular restriction factor tetherin (Neil et al., 2006). The Vpr

(viral protein regulatory) arrests cellular proliferation in the G2 phase of the cell cycle,

promotes cellular differentiation and interacts with cellular proteins involved in DNA

repair (Malim and Emerman, 2008). The Nef (negative factor) performs various activities

that promote viral immune evasion and replication (Kirchhoff et al., 2004; Münch et al.,

2005; Schindler et al., 2006).

1.2 Retroviral vectors

Retroviruses provide a well suited platform for the stable transfer of genetic information

into target cells (McTaggart et al., 2002). The 1-st generation of retroviral vectors contains

a single packaging plasmid encoding the all HIV-1 genes besides envelope protein which

was encoded on a separate plasmid (Wei et al., 1981). In the 2-nd generation of retroviral

vector, the HIV-derived packaging component was reduced to the gag, pol, tat and rev

genes. The transfer plasmid contains the viral LTRs and Ψ packaging signal and plasmid

encoded env gene were separated into additional plasmids (Naldini et al., 1996). For

reasons of biosafety, the 3-d generation plasmid systems were developed (Markowitz et

al., 1988). Currently used systems comprise a transfer construct, a packaging construct and

an envelope construct. In the case of lentiviral vectors a fourth plasmid which codes rev

gene is also necessary because it was shown that Rev facilitates export of the RNA from

the nucleus. The transfer construct carrying the transgene of interest contains the LTR

sequences necessary for the genomic integration of the virus into the target cells, the Ψ

signal for packaging the genomic viral RNA into the viral capsid and Rev Response

Element (RRE), a binding site for the Rev protein, needed for lentiviral vectors.

1 INTRODUCTION 4

(Markowitz et al., 1988; Coffin et al., 1997). The gag and pol (gag/pol) genes, which are

required for particle production and transduction of target cells, env gene, that encodes the

envelope glycoproteins, and rev gene, are separated onto three different helper plasmids, in

which the Ψ signal and LTRs are removed (Fig. 2). The tat is eliminated from the 3-d

generation system through the addition of a chimeric 5’ LTR fused to a heterologous

promoter on the transfer plasmid. Expression of the transgene from this promoter is no

longer dependent on Tat transactivation.

Figure 2: Schematic representation of the retroviral vectors and workflow. The gene of interest is cloned in a plasmid (transfer vector) that contains the retroviral regulatory elements (LTR, long terminal repeats) as well as elements to regulate the expression of the transgene. For gammaretrovirus based packaging systems, the transfer vector is co-transfected in HEK293T cells with two additional vectors carrying gag/pol and env genes, respectively. For lentivirus based packaging systems, the transfer vector is co-transfected in HEK293T cells with three additional vectors carrying gag/pol, env and rev, respectively. In the HEK293T cells the viral particles are formed, bud from the cell surface and accumulate in the culture medium (supernatant).

These three or four plasmids are co-transfected into producer cells, usually the human

embryonic kidney HEK293 cell line. The helper plasmids include all the structural proteins

needed in order to package a new virus. Due to the Ψ deletion within the helper plasmid,

the transcribed helper RNA does not get incorporated into the new recombinant virus.

Thus, progeny virions are replicative incompetent. They are able to infect cells and

promote integration of the transgenes into the cellular genome, but no viral particles can be

further generated. This system avoids the possibility of homologous recombination

between the three plasmids, which could give rise to replication-competent retroviruses

1 INTRODUCTION 5

(Miller and Buttimore, 1986; Miller et al., 1986; Naldini et al., 1996; Dull et al., 1998).

The process by which DNA is transferred by a retroviral vector is termed transduction.

Furthermore, self-inactivating retroviral vectors were developed. These vectors are

constructed by deleting the enhancer and/or the promoter within the U3 region of the 3'

LTR. After reverse transcription, the deletion is transferred to the 5’ LTR of the proviral

DNA, resulting in an inactive provirus (Miyoshi et al., 1998; Yu et al., 1986).

1.3 Pseudotyping

One interesting property of retroviral vectors is their ability to be enveloped by different

glycoproteins (GP). The incorporation of heterologous GP into retroviral particles is

termed pseudotyping and is used to alter the target cell tropism or to increase the stability

and infectivity of retroviral vectors (Sanders, 2002). Nowadays the following GPs are

extensively used for pre-clinical and clinical studies due to their attractive cell tropism: the

envelope protein of Gibbon-ape Leukemia Virus (GaLV), Feline Endogenous Virus

(RD114), amphotropic Mirine Leukemia Virus (MLV) and Vesicular Stomatitis Virus

(VSV-G) (Akkina et al., 1998; Neff et al., 2004; Cronin et al., 2005). The most widely

used GPs for pseudotyping retroviral vectors is VSV-G GP (Burns et al., 1993), which

confers a broad tropism and generates stable particles that can be purified and

cryopreserved. GaLV and RD114 pseudotyping have shown excellent gene transfer

efficiencies for primary T lymphocytes and human hematopoietic cells (CD34+) (Bauer et

al., 1995; Bunnell et al., 1995; Uckert et al., 2000, Kelly et al., 2001). However,

pseudotyping of lentiviral vectors with RD114 and GaLV GP showed reduced infectivity.

To overcome this problem, chimeric GPs were developed by replacing the cytoplasmatic

tail of the RD114 and GaLV envelope proteins (designated TR) with that of MLV GP to

increase their incorporation into viral particles (Stitz et al., 2000; Sandrin et al., 2002;

Sandrin et al., 2004). Lentiviral vectors pseudotyped with GaLV/TR and RD114/TR GP

have shown high titer, stability of viral particle and resistance to inactivation in the

presence of human sera.

1.4 Limitations for efficient retroviral gene transfer

Major obstacles associated with retroviral vectors include the production of low-titer viral

stocks and inefficient transduction rates, particularly into specific primary human cells,

such as macrophages and CD34+ hematopoietic stem cells. In the best cases, between 106

1 INTRODUCTION 6

to 107 infective viral particles per mL of cell culture supernatant are produced by

commonly used producer systems (Pear et al., 1993; Dull et al., 1998). These

concentrations are enough for certain in vitro applications. However, high concentrations

of viral stocks are required for gene therapy applications in order to improve transduction

efficiencies (Andreadis et al., 1999). Several techniques to concentrate virus stocks like

optimizing transfection protocols, use of new designed retroviral vectors and/or

ultracentrifugation have been developed (Kotani et al., 1994; Reiser, 2000; Bajaj et al.,

2001; Gatlin et al., 2001; Zhang et al., 2001).

Usually, virus attachment requires the interaction of the viral envelope GP with specific

receptors on the cell surface. The densities of GP on the virions and the appropriate entry

receptors on the targets cells are often low and in the majority of encounters the viral

particle will be repelled by the repulsion between the negatively charged viral and cellular

membranes. Furthermore, the slow diffusion and rapid decay of retrovirus particles are

important reasons for the low transduction efficiencies (Andreadis et al., 2000; Chuck et

al., 1996; Le Doux et al., 1999). Various approaches have been developed to increase the

efficiency of virion attachment and thus gene delivery but they all have significant

limitations and improved methods are still urgently needed.

1.5 Approaches to enhancing retroviral transduction efficiency

Frequently used strategies to increase attachment rates include low speed centrifugation of

virions onto their target cells (Ho et al., 1993; Bahnson et al., 1995; Quintas-Cardama et

al., 2007) and/or treatment with cationic polymers (e.g., hexadimethrine bromide

(polybrene), diethylaminoethyl (DEAD)-dextran) (Hodgson and Solaiman, 1996; Vogt,

1967), and cationic peptides (e.g., protamine sulfate) (Cornetta and Anderson, 1989;

Hodgson and Solaiman, 1996). Positively charged compounds may enhance transduction

efficiency via charge shielding, in which the compounds reduce electrostatic repulsion by

neutralizing virus and cell surface charge (Davis et al., 2004). Moreover, cationic

compounds could allow the virus to overcome electrostatic repulsion by aggregation of the

virus, thereby enhancing the rate of sedimentation of the virus onto the cell surface (Davis

et al., 2002). These methods are often inconvenient, associated with cytotoxicity, cell type

dependent and/or poorly effective. Furthermore, most adjuvants and agents that facilitate

virion attachment are not suitable for transduction of highly sensitive primary human cells

because they affect their viability and functionality.

1 INTRODUCTION 7

The most commonly used and most effective technique to promote viral gene delivery is

coating of cell culture plates, flasks or bags with RetroNectin (Williams et al., 1991;

Hanenberg et al., 1996; Moritz et al., 1996; Hanenberg et al., 1997). RetroNectin is a

chimeric protein that comprises 574 amino acids and contains a cell-binding domain, a

heparin-interacting region and a CS-1 site (Fig. 3). The fibronectin cell-binding and CS-1

sites interact with the very late antigens (VLA) 4 and 5 on the cell surface and the heparin

domain binds the virions, thereby bringing them into close proximity and promoting

transduction (Williams et al., 1991; Hanenberg et al., 1996; Moritz et al., 1996). However,

RetroNectin has to be coated onto plates to capture virions and several washing steps have

to be applied. Thus, the use of this relatively expensive compound is time consuming and

laborious.

Figure 3. Mechanism of RetroNectin mediated increase of transduction efficiencies.

1 INTRODUCTION 8

1.6 Peptide derived from HIV-1 envelope glycoprotein boost HIV-1 infection

To better characterize the interaction of HIV-1 with its cellular receptors, we screened a

library of peptides derived from different regions of the surface envelope glycoprotein

gp120 of HIV-1 (group M, subtype B, strain HxB2) (Gurgo et al., 1988). HIV-1 group M

is the ‘major’ group and is responsible for the majority of the global HIV epidemic

(Hemelaar, 2012). This GP together with gp41 produces by the proteolytic cleavage of the

precursor protein gp160, which is encoded by the env gene of HIV-1. gp120 is responsible

for virus binding to the receptor (CD4) and co-receptors (CCR5 and CXCR4) expressed by

target cells and triggers the subsequent fusion between viral envelope and cell membrane

It has been previously shown, that semen-derived

peptides form amyloid fibrils, termed Semen-derived

Enhancer of Virus Infection (SEVI), promote HIV-1

infection (Münch et al., 2007) (Fig. 4). Under

conditions of limiting viral dilutions SEVI boost the

HIV infection by up to 105-fold (Münch et al., 2007). It

was hypothesized, that positively charged fibrils bind

to negatively charged surface of virions and target

cells. This reduces the charge-charge repulsions

between them and promotes virion attachment and

fusion. (Roan et al., 2009, Brender et al., 2009). Last

studies showed that SEVI improve the efficiency of

retroviral gene transfer (Wurm et al., 2010; Wurm et

al., 2011). However, SEVI has some drawbacks for

usage as an enhancer of viral gene transfer. For

example, the amyloidogenic peptides that form SEVI

are relatively large (38 amino acids) and thus relatively

expensive to produce. Furthermore, it takes several

hours to generate the active amyloid fibrils (Münch et

al, 2007). Finally, sometimes very large amyloid

aggregates are obtained particularly after longer storage

and repeated freeze-thaw cycles that are poorly

effective in boosting retroviral transduction.

Figure 4. Putative mechanism of SEVI action. Abundant semen protein prostatic acid phosphatase (PAP) form amyloid fibrils termed SEVI. The fibrils capture HIV virions and promote their attachment to target cells. (Source: N. Roan and W. Greene, 2007, Cell, 131(6):1044-1046).

1 INTRODUCTION 9

(Kwong et al., 1998). gp120 is composed of five regions called C1–C5 and conserved

among different HIV-1 strains, five highly glycosylated and hypervariable regions called

V1–V5 and contains 30 β-strands and 6 α–

helices (Fig. 5). One of peptides derived

from HIV-1 gp120 efficiently enhanced

HIV-1 infection. This peptide corresponds to

amino acids 413 to 431 of the HIV-1 gp120.

This peptide partially covers the β19 and

β20 strands which proposed to be involved

in co-receptor interaction (Kwong et al.,

1998). We hypothesised that this peptide

might be used as an effective means to

increase retroviral gene transfer in basic

research and clinical applications. Figure 5. Structure of the HIV-1 gp120. Strands ß19 and ß20 overlapping the amino acids 413 to 431. (Source: Kwong et al., 1998, Nature, 393:648-59).

1 INTRODUCTION 10

1.7 Aim of present study

Retroviral and lentiviral gene transfer is the method of choice for the stable introduction of

genetic material into cells and offers many prospects for basic research and for the

treatment of genetic disorders, malignancies and infection diseases. Application of retro-

and lentiviral gene transfer systems is often hampered by low transduction efficiencies,

particularly into specific primary human cell types, such as macrophages and CD34+

hematopoietic stem cells. Various approaches have been developed to increase the

efficiency of gene delivery but they all have significant limitations and improved methods

are urgently needed. Therefore, the aim of the present study was to develop convenient and

broadly applicable tools for effective and safe retroviral gene transfer, based on a peptide

derived from HIV-1 external gp120. To achieve this goal, the following points were

investigated:

• Identification of most effective peptide fragment required for infection

enhancement

• Determination of effect of peptide on cell viability

• Investigation of interaction between peptide and virus particles

• Investigation of interaction between peptide and cells surface

• Determination of effect of peptide on retroviral transduction efficacies and cell

viability in comparison to other enhancer in vitro

• Analysis of peptide in vivo studies using a mouse model

2 MATERIAL AND METHODS 11

2 MATERIAL AND METHODS

2.1 Material

2.1.1 Eukaryotic cells

HEK293T Human renal epithelial cell line which was transformed with adenovirus type 5 and expresses SV40 (simian virus 40) large T-antigen (Graham et al., 1977). The cells were obtained from the American Type Culture Collection (ATCC).

HeLa Human epithelial carcinoma cell line (Gey, 1952). The cells were obtained from ATCC.

TZM-bl CXCR4-positive HeLa cell clone that was engineered to express CD4 and CCR5. TZM-bl cells contain HIV LTR-driven β-galactosidase and luciferase reporter cassettes that are activated by HIV tat expression. (Wei, 2002). The cells were obtained from the NIH AIDS Reagent Program.

CEM-M7 Human T lymphoid cell line, which expresses luciferase and GFP under control of the LTR promoter (Hsu et al., 2003). The cells were kindly provided by N. Landau (New York, NY, USA).

KG-1 Human acute myeloid leukemia cell line which express transmembrane and soluble forms of CD34. (Koeffler, 1978; Fernández M, 2000). The cells were obtained from ATCC.

HFF Human foreskin fibroblasts cell line. The cells were kindly provided by J. von Einem (Institute of Virology, Ulm, Germany.).

THP-1 Human acute monocytic leukemia cell line (Tsuchiya et al., 1980). The cells were obtained from the American Type Culture Collection. The cells were obtained from ATCC.

K562 Human erythroleukemic cell line (Anderson LC et al., 1979). The cells were obtained from ATCC.

U-87 MG Human astroglioma cell line with epithelial-like morphology (Ponten and Macintyre, 1968). The cells were obtained from the NIH AIDS Reagent Program.

BON Human pancreatic carcinoid cell line (Evers, 1994). The cells were obtained from ATCC.


2.1.2 Primary cells

Human Peripheral Blood Lymphocytes (PBL)

Human Peripheral Blood Mononuclear Cells (PBMCs) were separated from buffy coat using a Ficoll gradient. PBL were enriched from the PBMC fraction by an overnight plastic adherence at 37°C to remove adherent monocytes. Lymphocytes were activated for 3 days with 20 U/ml Il2 and 2 µg/ml PHA.

Human Macrophages Monocytes were separated using adherence on plastic. After 45 min, non-adherent cells were removed by rinsing in PBS. Monocytes were differentiated to macrophages by incubation for 7 days with 10 ng/ml GM-CSF.

Human CD34+ cells Cells were received from the Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, and collected by apharesis of G-CSF (granulocyte colony stimulating factor) treated individuals. Cells were maintained in CellGro® SCGM medium (CellGenix Germany) supplemented with 10% FCS, 100 ng/ml rhSCF, 20 ng/ml rhIL-3, 25 ng/ml rhIL-6.

2.1.3 Bacteria

E.coli XL-2 blueTM rec A1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1,

lac [F ́ proAB laclqZΔM15 Tn10 (Tetr) Amy Camr]

Agilent Technologies, Santa Clara, CA, USA)

2.1.4 Nucleic acids

Plasmid DNAs

pBRHIV-1NL4-3_92Th014 The plasmid encodes the HIV-1 NL4-3 provirus. The V3-loop region has been exchanged by the V3-loop of the R5-tropic 92th014.12 isolate (Papkalla et al., 2002).

pBRNL4-3_92Th014.12(R5)nef+_ IRES_g-Luc

The plasmid encodes the pBRNL4-3_92Th014.12 provirus and a Gaussia-luciferase cassette in place of nef.

pSRS11 SF GFPpre Self-inactivating (SIN) retrovirus transfer vector, driving by the Rous sarcoma virus (RSV) promoter in the producing cell. Expression of the green fluorescent protein (GFP) is directed by an internal spleen-focus forming virus promotor (SF) (Schambach et al., 2006b). The plasmid was kindly provided by J. Bohne.


M57-DAW MLV gag/pol expression plasmid utilizing an SV promotor (Schambach et al., 2006b). The plasmid was kindly provided by J. Bohne.

phCMV-RD114 Construct expressing endogenous feline virus (RD114) glycoprotein. The plasmid was kindly provided by F.L. Cosset.

phCMV-GaLV Construct expressing Gibbon Ape Leukemia Virus (GaLV) glycoprotein. The plasmid was kindly provided by F.L. Cosset.

phCMV-MLV Construct expressing amphotropic Murine Leukemia Virus (MLV4070A) glycoprotein. The plasmid was kindly provided by F.L. Cosset.

pRRL.cPPT.SFeGFP.pre Self-inactivating (SIN) lentiviral vector, contains the spleen focus-forming virus LTR (SF), the central polypurine tract (PPT), which increase the efficiency of reverse transcription and the posttranscriptional regulatory element (PRE) of woodchuck hepatitis virus (increase infectious titer). (Zufferey et al., 1999; Schambach et al., 2006a). The plasmid was kindly provided by L. Naldini

pMDLg/pRRE CMV-driven expression plasmid that contains gag, pol and Rev-responsible element (RRE) from HIV-1, a binding site for the Rev protein which facilitates export of the TNA from the nucleus (Dull et al., 1998). The plasmid was kindly provided by J. Bohne.

pRSV-Rev Rev cDNA-expressing plasmid in which the joined second and third exons of HIV-1 rev under the transcriptional control of RSV U3 (Dull et al., 1998). The plasmid was kindly provided by D. Trono.

phCMV-RD/TR

Chimeric envelope gp carrying the MLV gp cytoplasmic tail. Relatively short cytoplasmic tail of MLV increases incorporation of RD gp on lentiviral core (Sandrin et al., 2002). The plasmid was kindly provided by F.L. Cosset.

phCMV-GaLV/TR Chimeric envelope gp carrying the MLV gp cytoplasmic tail. Relatively short cytoplasmic tail of MLV increases incorporation of GaLV gp on lentiviral core (Sandrin et al., 2002). The plasmid was kindly provided by F.L. Cosset.

VSV-G Vector which expresses the envelope protein of the vesicular stromatitis virus (VSV-G) (Schindler et al., 2003)

MLV GAG YFP The plasmid encodes MLV gag proteins fused to YFP. The particles are non-infectious (kindly provided by W. Mothes).


Molecular weight size marker

1 kb Plus DNA Ladder Life Technologies GmbH, Darmstadt

2.1.5 Proteins

Enzymes

Restriction endonucleases New England Biolab, Ipswich, MA, USA

EDTA-Trypsin Invitrogen/Gibco (Karlsruhe)

RetroNectin

RetroNectin was obtained from TAKARA (TAK T100B) and coated on “Non-Tissue

Culture Treated 96 well plates” (BD 351172) as recommended by the manufacturer.

2.1.6 Peptides

HIV-1 p12 Abcam, Cambridge, UK

PAP248-286 GIHKQKEKSRLQGGVLVNEILNHMKRATQIPSYKKLIMY

EF-A NITLQCKIKQIINMWQEVG

EF-B NITLQCKIK

EF-C QCKIKQIINMWQ

EF-D KIKQIINMWQ

EF-E QCKIKQ

EF-F QCKIKQIINMW

EF-G KIK

EF-H QIINMWQEVG

EFcon TITLPCRIKQIINMWQGVG

The peptides were synthesized by Celtek Bioscience (Nashville, TN, USA) and

ViroPharmaceutical GmbH & Co KG (Hannover, Germany) using standard Fmoc solid-


phase peptide synthesis, purified by preparative RP HPLC and analyzed by HPLC and MS.

Peptides derived from HIV-1 gp120 were dissolved in DMSO (Merck, 1029521000) to 10

mM or 10 mg/ml stocks and stored at -20°C. PAP248-286 was dissolved in PBS at a

concentration of 10 mg/ml and agitated overnight at 37°C and 179 g in a thermomixer to

promote amyloid fibril formation.

2.1.7 Chemical reagent

Agarose Carl Roth GmbH + Co. KG, Karlsruhe

Ampicillin Ratiopharm GmbH, Ulm

Bacto tryptone Becton Dickinson, Franklin Lakes, NJ, USA

Bacto yeast extract Becton Dickinson, Franklin Lakes, NJ, USA

Buffers for restriction digestion New England Biolabs, Ipswich, MA, USA

Calcium chloride dihydrate (CaCl2 x 2H2O) AppliChem GmbH, Darmstadt

CellGro® SCGM medium CellGenix, Germany

CellMask™ Deep Red Thermo Fisher Scientific, Marietta, USA

Chloroquine diphosphate salt Sigma Aldrich, St. Louis, MO, USA

Congo Red Sigma Aldrich, St. Louis, MO, USA

DEAE-dextran Sigma Aldrich, St. Louis, MO, USA

Dimethyl sulfoxide (DMSO) Sigma Aldrich, St. Louis, MO, USA

Disodium hydrogen phosphate dihydrate (Na2HPO4 x 2H2O)

AppliChem GmbH, Darmstadt

DMEM Life Technologies GmbH, Darmstadt

Ethanol Sigma Aldrich, St. Louis, MO, USA

Ethidium bromide AppliChem GmbH, Darmstadt

Fetal calf serum (FCS) Life Technologies GmbH, Darmstadt

Ficoll separation solution Merck KGaA, Darmstadt

Gentamicin Life Technologies GmbH, Darmstadt

Glucose Sigma Aldrich, St. Louis, MO, USA

Granulocyte-macrophage colony-stimulating factor (GM-CSF)

R&D Systems, Wiesbaden-Nordenstadt


L-Glutamine Life Technologies GmbH, Darmstadt

Hepes (C8H18N2O4S) Sigma Aldrich, St. Louis, MO, USA

HPLC water VWR International GmbH, Darmstadt

Hydrogen chloride (HCl) VWR International GmbH, Darmstadt

Isopropanol Merck KGaA, Darmstadt

Methanol Merck KGaA, Darmstadt

MTT (3-(4,5-dimethylthiazole-2-yl)-2,5 diphenyltetrazolium bromide

Sigma Aldrich, St. Louis, MO, USA

Penicillin/Streptomycin (Pen/S) Life Technologies GmbH, Darmstadt

Phosphate buffered saline (PBS) Life Technologies GmbH, Darmstadt

PHA (Phytohaemagglutinin) Thermo Fisher Scientific, Marietta, OH, USA

Protamine sulfate Sigma Aldrich, St. Louis, MO, USA

Potassium chloride (KCl) Merck KGaA, Darmstadt

Roti®-Load DNA Carl Roth GmbH + Co. KG, Karlsruhe

RPMI-1640 medium Life Technologies GmbH, Darmstadt

Recombinant Human Interleukin-2 (rhIL-2) Miltenyi Biotec GmbH, Bergisch Gladbach

Recombinant Human Interleukin-3 (rhIL-3) R&D Systems, Wiesbaden-Nordenstadt

Recombinant Human Interleukin-6 (rhIL-6) R&D Systems, Wiesbaden-Nordenstadt

Recombinant Human Stem Cell Factor (rhSCF)

R&D Systems, Wiesbaden-Nordenstadt

SOC medium Life Technologies GmbH, Darmstadt

Sodium ascorbate (C6H7NaO6) Sigma Aldrich, St. Louis, MO, USA

Sodium chloride (NaCl) Merck KGaA, Darmstadt

Sodium hydroxide (NaOH) Merck KGaA, Darmstadt

Sulfuric acid (H2SO4) Sigma Aldrich, St. Louis, MO, USA

SureBlue™ TMB Microwell Peroxidase Substrate

KPL, Gaithersburg, MD, USA

TAE buffer (50x) 5 PRIME, Gaithersburg, MD, USA

Thioflavin T (ThT) stock solution Sigma Aldrich, St. Louis, MO, USA


Tris VWR International GmbH, Darmstadt

Tryptone Becton Dickinson, Franklin Lakes, NJ, USA

Trypsin / EDTA, 0.05% Life Technologies GmbH, Darmstadt

Triton X-100 Sigma Aldrich, St. Louis, MO, USA

Tween 20 Sigma Aldrich, St. Louis, MO, USA

2.1.8 Consumables

8-channel manifold for QuikSip™ BT-Aspirator

BRAND GMBH + CO KG, Wertheim

Cell culture flasks Sarstedt, Nümbrecht

Cell culture well plates Greiner Bio-One GmbH, Frickenhausen

Falcons (15 ml and 50 ml) Sarstedt, Nümbrecht

Micro tubes, 1.5 and 2.0 ml, PP, with attached PP cap

Sarstedt, Nümbrecht

Millex-HA Filter Unit 0.45 μm Merck KGaA, Darmstadt

Polystyrene microplates Corning, Corning, NY, USA

MaxiSorp™ Thermo Scientific, Waltham, MA, USA

White plates Thermo Scientific, Waltham, MA, USA

Serological pipettes (5 ml, 10 ml and 25 ml)


µ-Slide VI0.4 Ibidi, Planegg / Martinsried

Tips without filter, Type A/B/E (10, 200, 1,000 μl)


2.1.9 Technical equipment

Centrifuge 5417 R (Rotor: F-45-30-11) Eppendorf AG, Hamburg

Centrifuge 5810 R (Rotor: A-4-81) Eppendorf AG, Hamburg

Multipette plus Eppendorf AG, Hamburg

Thermomixer comfort Eppendorf AG, Hamburg


MR Hei-Standard (stirrer) Heidolph Instruments GmbH & Co. KG, Schwabach

Titramax 100 (plate shaker) Heidolph Instruments GmbH & Co. KG, Schwabach

Pipetus® Hirschmann Laborgeräte GmbH & Co. KG, Eberstadt

DISCOVERY Comfort Multichannel Pipette

HTL Lab Solutions Warsaw, Poland

FE20–FiveEasy™ pH-meter Mettler-Toledo, Columbus, OH, USA

VMax Kinetic ELISA Microplate Reader SoftMax Pro 5.3

Molecular Devices, Sunnyvale, CA, USA

HERAsafe® KSP 18 Thermo Fisher Scientific, Marietta, OH, USA

Steri-Cult CO2-Inkubator Modell 3311 Thermo Fisher Scientific, Marietta, OH, USA

Vortex-Genie® VWR International GmbH, Darmstadt

Orion II Microplate Luminometer Software Simplicity

Zylux Corporation, Pforzheim

Thermomax microplate reader Molecular devices, UK

2.1.10 Kits

Tropix® Gal-Screen® assay Applied Biosystems, Foster City, CA, USA

CellTiter-Glo®Luminescent Cell Viability Assay

Promega, Madison, WI, USA

Luciferase Assay System (E1501) Promega, Madison, WI, USA

Wizard® Plus Midipreps DNA Purification System

Promega, Madison, WI, USA


2.1.11 Buffers and solutions

2 M CaCl2

Gaussia substrate dilution buffer: 0.1 M Tris, 0.3 M sodium ascorbate,

adjust pH to 7.4

10x HBS

8.18 g NaCl

5.94 g Hepes

0.25 g Na2HPO4 x 2 H2O

dissolved in 100 ml H2O, adjusted to pH 7.12

2.1.12 Media

Bacteria culture media

LB medium

10 g/l bacto tryptone, 5 g/l bacto yeast extract, 8 g/l NaCl, 1 g/l glucose, add 100 mg/l ampicillin prior to use

LB/AMP agar

add 15 g/l agar and 100 mg/l ampicillin to LB medium

S.O.C. medium LifeTechnologies/Invitrogen (Carlsbad)

Cell culture media

DMEM supplemented with

350 μg/ml L-glutamine 120 μg/ml penicillin 120 μg/ml streptomycin sulfate 10% (v/v) heat-inactivated FCS

used for TZM-bl, HEK293T, HeLa, HFF cells

RPMI 1640 supplemented with

350 μg/ml L-Glutamine 120 μg/ml penicillin 120 μg/ml streptomycin sulfate 10% (v/v) heat-inactivated FCS (add 10 ng/ml IL-2, 1 μg/ml PHA to stimulate PBMCs)

used for CEMx-M7, KG-1, K562, Jurkat T, BON and PBLs


EMEM supplemented with 350 μg/ml Glutamine 120 μg/ml penicillin 120 μg/ml streptomycin sulfate 1% Non-Essential Amino Acids (NEAA) 1mM Sodium Pyruvate (NaP) 10% (v/v) heat-inactivated FCS used for U-87 MG cells

CellGro® SCGM medium

supplemented with

100 ng/ml rhSCF 20 ng/ml rhIL-3 25 ng/ml rhIL-6 10% (v/v)heat-inactivated FCS used for human CD34+ cells which were obtained from the Institut für Klinische Transfusionsmedizin und Immungenetik, Ulm

2.2 Methods

2.2.1 DNA methods

2.2.1.1 Plasmid DNA preparation

Plasmid DNA for transfection was prepared using the WizardTM

Plus Midiprep Kit

according to the manufacturer´s protocol. The DNA concentration and quality was

determined using a Nano Drop 2000 spectrophotometer.

2.2.1.2 Restriction digest

Plasmid DNA was checked by incubating 1 µg of DNA with 0.5 μl of appropriate

restriction enzymes and 2 μl of the recommended 10 x NEB restriction buffer at 37 °C for

1 h on a thermomixer. Sterile water was added to a final volume of 20 μl.

2.2.1.3 Agarose gel electrophoresis

The restricted plasmid DNA was analysed by agarose gel electrophoresis. 20 µl of the

digested DNA was mixed with 5 µl loading dye and separated on 0.7% agarose gels

(containing 0.05 μg/ml ethidium bromide) in 1 x TAE buffer at 120 V for 30 min using the

Voltage PowerPAC Basic Power Supply. The 1kb+ ladder (Life Technologies GmbH,

Darmstadt) was used as a standard. DNA fragments were visualized with the Gel Doc™

XR system (Bio-Rad).


2.2.2 Bacterial methods

2.2.2.1 Bacterial culture

The used plasmids contained a gene coding for ampicillin resistance. Therefore,

transformed bacteria were grown in LB medium or on LB agar plates containing 50 µg/ml

ampicillin. The bacteria were grown in LB medium for 14-16 h at 37 °C on a shaker and

the inoculated LB agar plates were incubated at 37 °C.

2.2.2.2 Bacterial transformation

1 µg of plasmid DNA was incubated with 5 μl of Escherichia coli XL2 BlueTM cells on ice

for 20 min. After the cells were heat-shocked for 30 sec at 42 °C they were incubated on

ice for 2 min, followed by the addition of 200 μl SOC medium. The transformed cells were

incubated at 37 °C on a shaker for 30 min and plated on LB agar plates containing 50µg/ml

ampicillin. The plates were incubated at 37 °C in a compartment drier overnight.

2.2.3 Cell culture

2.2.3.1 Adherent and suspension cell culture

The adherent and suspension cells were maintained at 37 °C in an incubator in 5 % CO2

and 95 % air humidity. The cells were grown in 25 cm2 or 75 cm2 cell culture flasks

(Sarstedt, Nümbrecht) depending on the requirement of cells, in DMEM or RPMI-1640,

respectively. The adherent cells were split by trypsinization twice a week. Trypsin

treatment of cells was performed by removal of the culture medium from the cells. The

cells were washed with PBS and 1-3 ml trypsin was applied to the cells. The cells were

incubated at 37 ºC until they became detached. The cells were collected, centrifuged at

1,300 rpm for 3 minutes, resuspended in the fresh medium and re-seeded at a lower density

in new flasks. Suspension cells were collected and afterwards centrifuged at 1,300 rpm for

3 min and passaged 1:5 in fresh culture medium.

2.2.3.2 Isolation of primary blood cells

Buffy-coat (lymphocyte concentrate from 500 ml whole blood), obtained from the

Transfusion Center of the Ulm University Hospital, was diluted 1:3 with PBS containing

2% FCS. Ficoll separating solution was overlayed with the diluted blood and centrifuged at

515 g for 20 min without brakes. The white interphase layer formed by peripheral blood


mononuclear cells (PBMCs) was transferred in a fresh tube and washed twice with PBS

containing 2 % FCS.

Peripheral blood lymphocytes (PBL) were enriched from the PBMC fraction by

overnight plastic adherence at 37 °C to remove adherent monocytes. After separation

lymphocytes were cultured in supplemented RPMI culture medium and activated for 3

days with 20 ng/ml IL-2 and 2 µg/ml PHA prior to transduction.

Monocytes-derived macrophages: Monocytes were separated from lymphocytes by

adherence to tissue culture plastic (Nunc™ Cell Culture Treated Flasks, Thermo Scientific,

#136196) for 45 min. Non-adherent cells were removed by rinsing with RPMI medium.

Monocytes were stimulated with 10 ng/ml GM-CSF in RPMI cell culture medium. At day

3 the medium with growth factor was replaced and the cells were used for experiments at

day 7.

2.2.4 Viral methods

2.2.4.1 Virus stocks generation via calcium phosphate transfection of HEK293T cells

R5-tropic HIV-1 was produced by transient transfection of HEK293T cells with the

pBRHIV-1NL4-3_92Th014 construct using calcium phosphate precipitation. The calcium

phosphate transfection is a method for introduction of DNA into mammalian cells based on

the formation of a calcium phosphate-DNA precipitate (Graham and van der Eb, 1973;

Wigler et al., 1977). One day before transfection, 293T cells were seeded in 6-well plates.

At a confluence of 60-70 % the cells were used for transfection. For this 5 μg DNA per

well was mixed with 13 μl 2 M CaCl2 and the total volume was made up to 100 μl with

water. This solution was added dropwise to 100 μl of 2xHBS. The transfection cocktail

was vortexed for 5 sec and added dropwise to the cells. The transfected cells were

incubated for 8-16 h before the medium was replaced by fresh supplemented DMEM. 48 h

post transfection, virus stocks were prepared by collecting the supernatant and centrifuging

it at 805 g for 3 min. Virus stocks were quantified by p24 Elisa (AIDS Reagent and

Reference Programme, SAIC Frederick, USA) and stored at -80 °C.

Retroviral vectors were produced by transient transfection of HEK293T cells with calcium

phosphate using GFP encoding pSRS11 SF GFPpre, M57-DAW (gag/pol plasmid) and

glycoprotein expression plasmid. In the present study retroviral particles were pseudotyped

with Gibbon Ape Leukemia virus (GaLV), endogenous feline leukemia virus (RD114),

Vesicular Stomatitis Virus (VSV-G) or amphotropic Murine Leukemia Virus (MLV)

envelope glycoproteins. 24 hours prior to transfection, HEK293T cells were seed at 2 x 106


cells per 100 mm cell culture dish in DMEM 10 % FCS to achieve 70-80 % confluence at

the time of transfection. At the day of transfection DMEM medium was replaced with 10

ml medium containing chloroquine (final concentration of 25 μM). Chloroquine decreases

the intracellular degradation of plasmid DNA in the lysosomes, thus increasing the amount

of plasmid DNA that reaches the nucleus (Walker et al., 1996). Thereafter, 5 μg transfer

vector, 13 μg M57-DAW and 2 μg envelope plasmid were mixed directly with 0.25 mM

CaCl2 in dH2O (total volume - 500 µl). This is then added dropwise to the same volume of

2xHBS buffer where a precipitation of calcium phosphate occurs. The transfection cocktail

was vortexed for 5 sec and added dropwise to the cells. The medium was changed after 16

hours to avoid cell toxic effects of the chloroquine treatment. Supernatant containing the

viral particles was collected 48 hours after transfection, centrifuged at 805 g for 3 min and

used immediately for transduction or stored at -80ºC until required.

Luciferases expressing retroviral vectors were generated by co-transfecting a luciferase

encoding mouse leukaemia virus vector with M57-DAW and GaLV, RD114, VSV-G or

MLV glycoprotein expression plasmids using calcium phosphate precipitation as described

above.

Lentiviral vectors encoding GFP were generated by transient transfection of 293T cells as

described above. 4 µg pRRL.cPPT.SFeGFP.pre, 8 µg pRSV-Rev, 4 µg pMDLg/pRRE and

2-4 µg appropriate chimeric envelope glycoprotein with the cytoplasmic tail derived from

the MLV gp: phCMV-RD114/TR, phCMV-GaLV/TR, Ampho MLV (kindly provided by

F.L. Cosset) or VSV-G were used.

Luciferase encoding lentiviral particles were generated by cotransfection of an env-deleted

pBRHIV-1 NL4_3 derivative encoding luciferase instead of nef and constructs expressing

VSV-G, MLV or chimeric envelope glycoprotein derived from GaLV and RD114.

2.2.4.2 Determination of titer of retro- and lentiviral supernatants

Titers (transducing units/ml) of the viral supernatants were determined by transducing 105

HeLa cells with serial dilutions of viral supernatant.After 3 days post-transduction the

percentage of GFP positive cells was determined by FACS analyses.


2.2.4.3 Effect of EF-C on infection of TZM-bl reporter cell line

One day before infection, TZM-bl cells were seeded (10.000 cells/well) in F-96-well plates

(Greiner Bio-one, Frickenhausen). Virus stocks were normalized based on their amounts of

HIV-1 p24 capsid antigen. Infections were performed with 0.1 or 1 ng p24. 40 µl viral

stocks were treated with the 40 µl various dilutions of peptides, vortexed and incubated for

5 min room temperature. Concentrations of peptide during preincubation with virus were

100, 10, 1, 0.1 and 0.0 µg/ml. Subsequently, 20 µl of these virus stocks were used to infect

180 µl cell cultures. Infection rates were determined 2 or 3 days post infection by

quantifying β-galactosidase in cellular lysates.

2.2.4.4 Effect of EF-C on transduction efficiencies of different cell line

One day before transduction, adherent cells were seeded (10,000 cells/well) in F-96-well

plates (Greiner Bio-one, Frickenhausen). 200,000 suspension cells were seeded on the day

of experiment. Virus stocks were normalized based on their HeLa-transducing units

content. Transduction experiments were performed with viral vector stocks at a MOI of 1.

40 µl viral stocks were treated with the 40 µl various dilutions of peptides, mixed and

incubated for 5 min at room temperature. Concentrations of peptide during pre-incubation

with virus were 100, 10, 1, 0.1 and 0.0 µg/ml. Subsequently, 20µl of these virus stocks

were used to infect 180 µl cell cultures. After overnight incubation, supernatants were

removed and cells were further cultivated in 200 µl fresh media. Infection rates were

determined 2 or 3 days post infection by quantifying β-galactosidase or luciferase activities

in cellular lysates. After 3 days post transduction, cells were used for FACS analyses or

analysed by detecting luciferase activity in the cell supernatant.

2.2.4.5 Effect of EF-C on transduction efficiencies of primary cells

Transduction experiments with primary cells were performed with viral vector stocks at a

MOI of 1 and 10. Primary cells (PBL, macrophages, CD34+) were resuspended in virus

stocks containing 50, 25 and 0 µg of EF-C. After 4 hours incubation at 37 °C, cells were

washed and further cultivated in their own specific growth media. GFP positive cells were

determined 3 or 4 days later by using flow cytometry analysis.

2.2.4.6 EF-C mediated pull down assay

900 µl of R5-tropic HIV were mixed with 100 µl of EF-C (final concentration: 50 µg/ml)

or PBS. After 10 min incubation at the room temperature, stocks were centrifuged for 5


min at 10,000 g. Fig. 16a, b: The supernatants were collected and the pellets resuspended

to the original volume in PBS and cell culture medium DMEM. Both, pellets and

supernatants were used to infect TZM-bl cells. Infection rates were measured at day 2 post-

infection using β-galactosidase screen assay. Fig. 16c: The supernatant was collected and

the resulting pellet resuspended in 100 µl DMEM. The amount of HIV-1 p24 capsid

antigen in EF-C precipitated pellet and in the original virus stock was determined by a

HIV-1 p24 ELISA.

2.2.4.7 Comparison of infection enhancing activity of EF-C and others enhancers

Fig. 21a: 40 µl of freshly prepared EF-C, polybrene, protamine sulphate, DEAD dextran

and SEVI fibrils (obtained after overnight agitation) were serially diluted in PBS and

subsequently incubated with equal volumes of HIV-1 containing 0.1 ng p24 antigen.

Peptide concentrations during preincubation with virus were 50, 25, 12.5, 6.25, 3.1, 1.56,

0.78, 0.0 µg/ml. After 5 min incubation at room temperature 20 µl of these mixtures were

added to 180 µl TZM-bl cells (1 x 104/well) seeded the day before. After overnight

incubation, supernatants were removed and cell were further cultivated in fresh media.

Infection rates were determined 2 days later by quantifying β-galactosidase activities in

cellular lysates.

Fig. 22: 120.000 KG-1 cells were plated in 96-well plate in 100µl RPMI. Transduction

experiments were performed with viral vector stocks at a MOI of 1. 10µl of different

dilution of EF-C (1,000; 500; 250 µg/ml) or polybrene/protamine sulphate (320 µg/ml; 160

µg/ml) were added to 90µl VS, mixed and incubated for 5 min at room temperature. 100 µl

KG-1 cells were transduced with 100 µl of these mixtures. After overnight incubation,

supernatants were removed and cell were further cultivated in fresh RPMI media.

Transduction rates were measured 5 day later by FACS.

2.2.4.8 Coating experiments using EF-C and RetroNectin

RetroNectin was obtained from TAKARA (TAK T100B) and coated on “Non-Tissue

Culture Treated 96 well plates" (BD Biosciences #351172) as recommended by the

manufacturer.

Fig. 23b: 50, 10, 1 and 0 µg of EF-C per well were used to coat Costar 96 well plates over

night at 4°C. The next day, peptide was removed and plates were incubated with HIV-1 for

1 or 4 hrs. Wells were then washed once in PBS and bound virus was determined by p24

ELISA.


Fig. 24a: Four different microtiter plates (A: Thermo Scientific NuncTM Nunc F96

MicroWell™ Plates Polystyrene Clear No.:167008; B: Thermo Scientific NuncTM Nunc

F96 MicroWell™ Plates Polystyrene Clear No.: 442404; C: CORNING (96 well)

EIA/RIA Flat Bottom, High Bind, Nonsterile No.: 3590; D: CORNING (96-Well) Multiple

Well Cluster Plate TC Treated No.: 3596) were coated overnight at 4 °C with 10, 1, 0.1,

and 0.0 µg freshly diluted EF-C (50 µl each). Thereafter, the peptide solutions were

removed, wells washed and 50 µl HIV-1 R5 (0.1 ng p24 antigen) was added. After 4 hrs

incubation at 37 °C, virus was removed and 1 x 105 TZM-bl cells (200 µl) per well were

added, and 2 days later infection rates were determined by Gaussia-luciferase assay.

Fig. 24b: 50, 10, 1 and 0 µg of EF-C per well were added to Costar 96 well plates and

incubated overnight at 4 °C. Peptide was then removed and 50 µl of a lentiviral vector

containing the GaLV env was added to each well. 4 hrs later, the inoculum was removed

and wells were either washed once in PBS or left untreated. Thereafter, 1 x 106 KG-1 cells

were added. After 5 days, transduction rates were determined using FACS analysis.

2.2.4.9 Fusion assay.

This sensitive flow cytometry-based HIV-1 virion-fusion assay was conducted essentially

as described (Cavrois et al., 2002; Cavrois et al., 2004). VSV-G HIV-1 NL4.3 Env- GFP

containing BlaM-Vpr chimeric proteins were produced by triple-transfection of 293T cells.

Medium only and VSV-G HIV-1 NL4.3 Env- GFP BlaM-Vpr virions were incubated with

EF-C at the indicated concentrations and then added to TZM-bl cells. After 4 hours, cells

were washed and loaded with CCF2/AM dye overnight at room temperature. Fusion was

monitored by BD FACSCanto II Flow Cytometer and the corresponding software BD

FACS Diva. Since EF-C caused some auto-fluorescence in this assay, samples containing

EF-C in the absence of virus were used as negative control and subtracted from all values.

2.2.4.10 ß-galactosidase assay

HIV-1 infection rates in TZM-bl cells were quantified by removing the cell culture

medium 2 or 3 days post infection, adding 50 µl of 1:1 dilution of Gal-sceen® substrate

(Applied Biosystems, T1027) in PBS to each well. After 30 min incubation at room

temperature, 40µl of the cell lysates were transferred into a F-96-Nunclon-delta white

micro-well plate (NuncTM, Langenselbold) and the light emission was monitored with an

Orion Microplate Luminometer (Berthold Detection systems, Pforzheim). The enzyme


activity was measured as relative light units/second (RLU/sec) using the computer program

Simplicity 4.02 (Berthold Detection systems, Pforzheim).

2.2.4.11 Luciferase Assay

To detect transduction rates of luciferase encoding lentiviral and retroviral vectors,

suspension cells were pelleted by centrifugation at 1,300 rpm for 3 min. The supernatant

was then removed and cells were lysed in 40 µl 5-fold diluted Luciferase Cell Culture

Lysis Reagent (Promega, E1531). When adherent cells were used, the supernatant was

removed, and 40 µl Luciferase Lysis Buffer added. After 5 min, cells were resuspended

and 30 µl of the lysates were transferred into white microtiter plates. Thereafter, 70µl

luciferase substrate reconstituted in luciferase buffer (Luciferase Assay system, Promega,

E1501) was added, and luminescence was measured using a luminometer (Orion

microplate luminometer).

2.2.4.12 Cell Viability

To determine the effect of the peptides on cellular metabolic activity, different cell lines

were incubated with various concentrations of peptides under experimental conditions

corresponding to those used in the respective infection and transduction assays in the

absence of virus. After 2 or 3 days of incubation, CellTiter-Glo® Luminescent Cell

Viability Assay (Promega, G7571) was used as recommended by the manufacturer. Cells

were treated for three days with the indicated final concentrations of fibrils. Thereafter,

CellTiter-Glo® Buffer was thawed, equilibrated to room temperature, and mixed with

CellTiter-Glo® Substrate. The cell culture medium was removed, 100 µl fresh medium and

100 µl CellTiter-Glo® Reagent were added, agitated for 2 minutes to induce cell lysis,

pipetted into white lumiplates, and incubated for 10 min to stabilize the luminescence

signal. Luminescence intensities were the recorded in a luminometer (Orion microplate

luminometer).

2.2.4.13 Flow cytometric analysis

Adherent cells were washed once in PBS, trypsinized, resuspended in DMEM + 10% FCS,

transferred into FACS tubes, and pelleted for 3 min at 1,300 rpm. The supernatant was

removed, and after an additional washing step in PBS and subsequent centrifugation, cells

were fixed with 200 μl FACS buffer containing 2% paraformaldehyde and incubated for 30

min at 4 °C. Suspension cells were resuspended, transferred into FACS tubes, washed one


time in PBS and fixed as described above. FACS analysis was performed using the BD

FACSCanto II Flow Cytometer and the corresponding software BD FACS Diva.

2.2.5 Imaging methods

2.2.5.1 Fluorescence microscopy

To detect the interaction of EF-C fibrils with virion, fibrils were labelled with a

Rhodamine B dye (Rho-EF-C). 10 µl of Rho-EF-C (250 µg/ml) were mixed with 90 µl of a

MLV GAG YFP virus-like particles and 30 μl of this mixture were transferred to an IBIDI

μ-slide VI0.4 chamber (Ibidi) for visualization. To investigate the interaction of EF-C with

cells, 5 x 104 HeLa cells were seeded in 8 well slides and incubated overnight. Prior to

imaging, cell membranes were stained with CellMask™ Deep Red according to the

manufacturer's instruction (Invitrogen) and virus or Rho-EF-C/virus mixtures were added

to cells. For EF-C coating experiments on plastic slides and 3D reconstitution, EF-C (50

µg/ml) was stained with ProteoStat® Amyloid Plaque Detection Kit (Lörrach, Germany)

as per manufacturer’s instructions. EF-C was incubated for 10-12 hours on IBIDI slide.

Afterwards, EF-C was thoroughly washed with PBS and further incubated with YFP

labelled MLV-Gag virions for 1 hour. EF-C/virion complex was washed with PBS to

remove unattached virions. Fluorescence images were taken with the AxioVision software

ZEN 2009 using Zeiss LSM 710 confocal scanning equipment with a 63 × 1.4 NA

immersion oil objective.

2.2.5.2 Atomic force microscopy (AFM)

A 10 µg/ml infection-enhancing EF-C stock solution, prepared by diluting the peptide

dissolved in DMSO into Mili-Q water was further diluted to give a final concentration of 5

µg/ml. Next, 10 µL were added to a freshly cleaved mica surface and dried at 65 °C

overnight to remove DMSO. AFM measurements were performed on a Multimode III

AFM (Bruker) Images were analysed with the SPIP software version 5.1.5 (Image

Metrology A/S, DK). More than 50 individual fibrils were extracted and their geometrical

parameters were then analyzed to calculate the corresponding fibril diameters. Dr.

Christoph Meier (Institute for Organic Chemistry III/Macromolecular Chemistry,

University Ulm, Germany) performed the AFM analysis of the samples.


2.2.6 Proteomic methods

2.2.6.1 Thioflavin T (ThT) binding.

ThT stock solution (Sigma) was prepared by adding 8mg ThT to 10ml phosphate buffer

(10mM phosphate, 150mM NaCl, pH 7.0). This solution was filtered through a 0.2 µm

syringe filter, and further diluted 50-fold in PBS. 1.5 µl of a 1 mg/ml peptide stock

solutions were added to 148.5 µl Thioflavin T solution. Mixtures were vortexed and

transferred into Corning® CellBIND® 96 Well Flat Clear Bottom Black Polystyrene

Microplates (Corning). Fluorescence emission intensities were measured from 470 to 590

nm (band-pass 10 nm) using a spectrofluorometer (SAFAS flx Xenius n°5473, SAFAS).

2.2.6.2 CongoRed interaction.

Peptide solutions (10 µl) were placed into 1.5 ml tubes and 200 µl CongoRed (CR, Sigma

Aldrich, HT60-1KT) was added. The solutions were vortexed and incubated for 2 min at

room temperature. Stained amyloid was pelleted by centrifugation at 14,000 rpm for 5 min.

The supernatant was removed and the red pellet dissolved in DMSO (100 µl). This solution

was transferred into a 96-well plate and the optical density was measured at 490/650 nm

using Thermomax microplate reader (Molecular Devices Vmax).

2.2.6.3 ζ-potential measurement

For analyzing ζ-potentials of the fibrils, 50 µl of freshly prepared EF-C (1 mg/ml) were

diluted to 1 ml of an aqueous solution of KCl (10-3 M). The ζ -potential was derived from

the electrophoretic mobility of the peptides and measured using a Zeta Nanosizer (Malvern

Instruments, UK) and the corresponding DLS Nano software. Each measurement was

performed in triplicate. ζ-potentials of the buffers were measured and the values of each

buffer were subtracted from all corresponding samples.

3. RESULTS 30

3. RESULTS

3.1 Identification of gp120 fragments that promote HIV-1 infection

To analyse the interaction of HIV-1 with its cellular receptors, synthetic peptides

corresponding to different regions of the external viral glycoprotein gp120 of HIV-1 strain

HxB2 were tested. For this purpose, a CCR5 tropic HIV-1 (R5 HIV-1) virus stock was

generated by transient transfection of HEK293T cells with proviral DNA. The viral titer

was determined by p24 ELISA and virus dilution containing 1 ng p24 antigen was treated

with different concentrations of freshly prepared gp120 fragments. After 5 minutes of

incubation, these mixtures were then used to infect the TZM-bl reporter cell line. Infection

rates were determined 3 days later by β-galactosidase assay. Surprisingly these rates

showed that one of the tested gp120 fragments (Enhancing Factor A; EF-A) efficiently

enhanced HIV-1 infection. In the presence of 12.5 µg/ml of EF-A infection rates of R5

HIV-1 were increased by 34 fold relative to control infections containing no peptide (0

µg/ml) (Fig. 6a). This peptide with the sequence NITLQCKIKQIINMWQEVG

corresponds to amino acids (aa) residues 413 to 431 of the HIV-1 gp120.

Figure 6. Identifikation of gp120 fragments that enhance HIV-1 infection. (a) The EF-A gp120 fragment enhances HIV-1 infection. Virus was treated with indicated concentrations of the peptide and mixtures were then used to infect TZM-bl reporter cells. Infection rates were determined 3 days after infection using β-galactasidase assay. The numbers above the bars give the n-fold enhancement of infection relative to that measured in the absence of EF-A. RLU/s, relative light units per second; the asterisk indicates over-infection. Data represent mean values ±SD obtained from triplicate infection. (b) Overview of analyzed gp120 fragments. The localization in the HIV-1 Env precursor is indicated schematically. Segments of gp120 are designated as follows: black boxes, conserved regions C1–C5; white boxes, variable regions V1–V5. Numbers correspond to the amino acid position in the HIV-1 strain HxB2 gp120 sequence. Active peptides are indicated in red. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 31

Treatment of virus with 25 µg/ml of EF-A and subsequent infection of TZM-bl cells

caused massive virus induced cytopathic effect (CPE) which was observed by light

microscopy (data not shown). The reason for this phenomenon is overinfection of cells

(Fig. 6a). The cells became rounded, fused with adjacent cells and form a giant,

multinucleate cells called syncytia.

To confirm this effect of EF-A and to determine the minimal peptide fragment required for

infection enhancement, next seven length variants (B to H) of EF-A and another peptide

(EFcon) representing the consensus sequence of all strains of HIV-1 group M subtype were

synthesized (Fig. 6b). These peptides were analysed using the infectivity assay described

above. The peptides corresponding to C- (EF-B) and N-terminal (EF-H) halves of EF-A

did not enhance HIV-1 infection (Fig. 7), whereas peptides that contain the core region of

EF-A (aa residues 419 to 427 of gp120) promoted HIV-1 infection (Fig. 7). Moreover, the

size of peptide has played a decisive role, because too small peptides (EF-E and EF-G) did

not augment infection. In detail, treatment of 0.1 ng virus with 100 µg/ml of EF-C, EF-D,

EF-F and EFcon boosted HIV infection between 17 to 27 fold, while EF-B and EF-H

displayed no or slight effects (Fig. 7). The small 12-aa peptide (EF-C) with the sequence

QCKIKQIINMWQ was identified as the most effective enhancer (Fig. 7). This peptide has

been named Enhancing Factor C (EF-C).

Figure 7. Activity of various synthetic gp120 fragments in promoting HIV-1 infection. 0.1 ng R5 HIV-1 virions were treated with equal amount of different concentrations of freshly prepared gp120 fragments. After 5 min incubation, these mixtures were used to infect TZM-bl cells. Infection rates determined 2 days later by β-galactosidase assay. Shown are average values derived from triplicate measurements +/- SD. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 32

3.2 gp120 fragments are not cytotoxic.

To analyse possible cytotoxic effects, freshly dissolved peptides, which enhanced HIV-1

infection, were incubated with TZM-bl cells for three days and then the metabolic activity

in these samples was assessed by CellTiter-Glo® Luminescent Cell Viability Assay

(Promega). This method is based on the use of the luciferase reaction to measure the

amount of ATP from metabolically active (viable) cells. Quantification of intracellular

ATP levels showed that none of the tested peptides affected cell growth and viability (Fig.

8a). Next, the effect of the most potent HIV enhancing peptide, EF-C, on the viability of

different cell lines and primary cells was tested using the same bioluminescence assay. The

TZM-bl cell line, a Jurkat leukaemic T-cell line, a progenitor like cell line (KG-1), a B/T

cell hybrid cell line (CEM-M7) and primary peripheral blood mononuclear cells (PBMC)

were incubated with various concentrations of EF-C peptide for 3 days. The cell viability

assay showed that EF-C has no toxic effects for the indicated cells (Fig. 8b), a high

metabolic activity was determined for all tested cells even in the presence of 100µg/ml of

EF-C.

3.3 gp120 peptides that enhance HIV infection form amyloid fibrils

Dissolving the peptide in Dimethylsulfoxid (DMSO) (10 mg/ml) prior to introduction into

the aqueous solution (1 mg/ml) promoted the formation of a turbid solution. This property

of EF-C seemed similar to earlier observations with SEVI. Freshly dissolved solution of

Figure 8. gp120 derived fragments are not cytotoxic. (a) Indicated peptide solutions were incubated with 1 x 105 TZM-bl cells for 3 days at 37°C. Metabolic activity was determined by CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Shown are average values derived from triplicate measurements +/- SD. Concentrations shown are final cell culture concentrations. (b) Indicated cells were incubated with EF-C peptide for three days and cellular ATP concentrations were measured by the Cell Viability Assay. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 33

chemically synthesized PAP248-286 is unable to increase infection. Upon overnight

agitation of PAP248-286 at 37 °C the solution became turbid and the enhancement activity

was restored (Münch et al., 2007). Biochemical and biophysical analyses showed that

positively charged amyloid fibrils were thereby formed. It was hypothesized that these

fibrils decrease the electrostatic repulsion between negatively charged viral and cell

membranes (Münch et al., 2007; Roan et al., 2007; Ye et al., 2009).

To determine whether the gp120 fragments may also form amyloid aggregates, the

reactivity of a freshly prepared peptide solutions to the amyloid specific dyes Thioflavin T

(ThT) (Biancalana et al., 2009) and Congo Red (Puchtler and Sweat, 1965; Elghetany and

Saleem, 1988) was analysed as described for SEVI (Münch et al., 2007). ThT is a

benzothiazole dye that exhibit enhanced fluorescence upon binding to β-sheet structure of

amyloid proteins and is commonly used to diagnose amyloid fibrils. ThT fluorescence

scans showed that solutions containing the peptides EF-C, EF-A, EFcon, EF-D and EF-F

displayed increased fluorescence intensities (Fig. 9a). Further evidence that gp120 derived

peptides form amyloid aggregates was derived by a Congo Red (CR) staining assay. Congo

red is a hydrophilic chemical agent that binds to amyloid fibrils and causes an apple green

birefringence under polarized light. In contrast to the PBS control, the EF-C, EF-A, EFcon,

EF-D and EF-F solutions formed a red coloured pellet after centrifugation, indicating the

dye specifically intercalated into the amyloid (Fig. 9b).

Figure 9. Formation of amyloid material by the indicated gp120 fragments monitored by (a) Thioflavin T fluorescence and (b) Congo Red staining. OD, optical density; CPS, counts per second. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 34

Notably, ThT fluorescence intensities and results from Congo Red staining correlated with

the activity of gp120 fragments to increase virus infection and EF-C showed the highest

values (Fig. 10). In addition, EF-C, which was direct dissolved in PBS, contained only

minor amounts of Congo Red stainable material and was largely inactive. This indicates

that the activity of the gp120 fragments to enhance infection emerges from the presence of

amyloid aggregates.

For further clarification of the mechanism underlying infectivity enhancement, structure

and morphology of EF-C was analysed using atomic force microscopy (AFM) and ζ (Zeta)

potential measurement. AFM is a powerful technique that enables to visualize nanoscale

structures (e.g., amyloid fibrils) in an aqueous environment and measure their properties

(e.g., fibril size) (Stine et al., 1996; Mostaert et el., 2006). AFM analyses of an aqueous

EF-C peptide solution obtained immediately after diluting the peptide DMSO stock (10

mg/ml) tenfold in water (1 mg/ml) revealed the formation of fibrillary aggregates with a

diameter of 3.4 ± 0.1 nm (Fig. 11). This is significantly smaller than the diameter of SEVI

fibrils derived after overnight agitation (5.3 ± 0.1 nm) (Fig. 11), therefor I referred to EF-C

fibrils also as nanofibrils herein after.

Figure 10. Amyloid characteristics correlate with enhancement of HIV-1 infection. Values were derived from experiments shown in Fig. 7 and 9 a, b. Correlation analyses were performed with GraphPad Prism 5 software. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 35

To determine the surface charge of the fibrils, the ζ-potential of the EF-C fibrils was

analysed. ζ-potential measurements showed that EF-C fibrils exhibit positive charges of

+17.7 ± 1.7 mV, similar to that of SEVI (+17.8 ± 1.1 mV). Summarized, this results show

that the EF-C peptide adopts a fibrillary structure with a β-sheet arrangement and a positive

surface charge.

3.4 Formation of EF-C fibrils can be achieved in several solvents and their virus enhancing activity is stable

For certain experimental setups it might be beneficial to dilute EF-C stocks (10 mg/ml in

DMSO) in other buffers than PBS to form fibrils. To test this, I diluted a 10 mg/ml DMSO

stock of EF-C in PBS, H2O or the cell culture media RPMI 1640 (Gibco) and DMEM

(Gibco). The resulting 1 mg/ml solutions were further diluted 3-fold and mixed with 1 ng

p24 antigen of R5 HIV-1. 105 TZM-bl cells in 180 µl were then inoculated with 20 µl of

these mixtures and infection rates were determined by luciferase assays 2 days later. EF-C

preparation in all tested solvents showed similar results and enhanced HIV-1 infection

from 12 x 104 RLU/s measured in the absence of peptide to 9.5-10 x 106 RLU/s if virions

were exposed to 100 µg/ml EF-C (13-fold enhancement) (Fig. 12a). Thus, amyloid fibrils

can be formed in different solvents that might be advantageous for several approaches, e.g.

in experiments with cells depending on specific media.

To find out how long the dilutions peptide in PBS retain their virus enhancing activity, a

10 mg/ml DMSO stock of EF-C was diluted twice in PBS (final concentration: 100 µg/ml).

Dilutions were then stored at 4°C or -20°C. After 2 weeks, both samples and a freshly

prepared EF-C solution were further diluted with 0.1 ng p24 antigen of HIV-1 and

analysed for their HIV enhancing activity 2 days later. Infection assays revealed that the

stored peptide solutions increased virus infection with almost the same activity as the

Figure 11. Atomic force microscopy image of SEVI and EF-C fibrils.

3. RESULTS 36

freshly prepared peptide (Fig. 12b). Thus, EF-C peptide could be stored for at least two

weeks without losing its enhancing activity.

3.5 EF-C fibrils increase viral attachment and fusion

Like SEVI, EF-C fibrils are derived from a peptide with a net positive charge, suggesting

that the respective fibrils enhance infection by neutralizing the repulsion between the

negatively charged viral and cellular membranes (Münch et al., 2007). To directly

visualize the interaction of EF-C nanofibrils with virions, I labelled the nanofibrils with a

Rhodamine B dye (Rho-EF-C) and controlled their enhancing activity. Rho-EF-C fibrils

increase virus infection as efficiently as the unlabelled fibrils (Fig. 13a). After that, I

mixed labelled fibrils with CFP-labelled retroviral particles (MLV-CFP) and monitored

their interaction using fluorescence confocal microscopy (Fig. 13b). All virions (green)

associated with the fibrils (red) within seconds. In the presence of EF-C, no individual viral

particles could be detected, highlighting efficient and stable complex formation between

virions and fibrils (Fig. 13b).

Figure 12. The virus enhancing activity in EF-C dilutions is active in various solvents and media and is stable. (a) A 10 mg/ml DMSO stock of EF-C was diluted in either PBS, H20 or cell culture media RPMI1640 (Gibco) and DMEM (Gibco). Indicated concentrations of solutions were mixed with 1 ng R5 HIV-1. These mixtures were added to TZM-bl cells. Infection rates were determined 2 days later by luciferase assay. Shown are average values derived from triplicate measurements +/- SD. Concentrations given on the x-axes are EF-C concentrations during virion treatment. (b) Freshly prepared EF-C solution and samples, which were diluted and stored at 4°C or -20°C for two weeks, were analyzed for their HIV enhancing activity in TZM-bl cells as described above. Shown are average values derived from triplicate measurements +/-SD. Concentrations given on the x-axes are EF-C concentrations during virion treatment. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 37

To further clarify the mechanism underlying EF-C-mediated infectivity enhancement, I

studied whether the fibrils increase virion attachment to the cell surface and subsequent

fusion into the target cells. 10µl of Rho-EF-C (250µg/ml) were added to 90µl CFP labelled

MLV virus, and the mixtures were then added to HeLa cells. Fluorescence microscopy

after 2 and 4 hours of incubation revealed that the amount of viral particles attached to the

cell surface in the presence of fibrils is drastically increased (Fig. 14a). The viral uptake

and internalization of fibrils was evident from the loss of fluorescence intensities after 24

hours (Fig. 14a). In parallel, the HeLa cells were inoculated with MLV-CFP alone. Images

obtained after 24 hours of incubation showed that only few viral particles were entry into

the cells (Fig. 14b).

Figure 13. EF-C fibrils bind viral particles. (a) Rho-labelled peptide boosts HIV infection as efficiently as unlabelled EF-C. Both peptides were dissolved in DMSO, diluted in PBS and used to treat HIV-1 particles that were subsequently used to inoculate TZM-bl cells. Infection rates were determined 2 days later by ß-galactosidase assay. Shown are average values from triplicate measurements. Concentrations given on the x-axes are EF-C concentrations during virion treatment, cell culture concentrations were 10-fold lower. (b) Confocal microscopy images of MLV-CFP virions alone, Rho-Envitra alone, or mixtures thereof including a 5-fold magnification of the latter. Scale bars, 15 µm. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 38

To confirm the idea that EF-C efficiently delivers virions to the cell membrane and

enhances viral fusion, a flow cytometry-based HIV-1 virion-fusion assay was performed

(Cavrois et al., 2002). Therefore I produced VSV-G pseudotyped lentivirus particles

carrying a β-lactamase reporter protein (BlaM) which fused to the amino terminus of the

virion protein Vpr (BlaM-Vpr). During the fusion, BlaM-Vpr protein transfer into the

target cell and this transmission can be monitored by the enzymatic cleavage of CCF2, a

fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-

lactam ring changes the fluorescence emission spectrum and this change can be detected

by flow cytometry. I inoculated TZM-bl cells with either PBS-treated or EF-C-treated

virus. After 4 hours, cells were washed and loaded with CCF2/AM dye overnight. Next

Figure 14. Kinetics of fibril/virion interaction wi th cells. (a) HeLa cells were inoculated with MLV-CFP in the presence of Rho-EF-C and images were taken at indicated time points. Final concentration of Rho-EF-C is 25µg/ml. Upper panel: all three channels are shown (red –fibrils, green – virus, blue – cell membrane. Lower panel: only the green (virus) channel is shown for the same pictures as in the upper panel. Scale bars, 15 µm. (b) HeLa cells (blue) were inoculated with either MLV-CFP (green) alone or in the presence of Rho-EF-C (red) and analysed by confocal microscopy after 24 hours. Scale bars, 15 µm. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 39

day, the cells were washed and fusion was monitored with a BD FACSCanto II Flow

Cytometer (Becton Dickinson, San Jose, CA) (Fig. 15). Since EF-C caused some auto-

fluorescence in this assay, samples containing EF-C in the absence of virus were used as

negative control and subtracted from all values. Treatment of BlaM-Vpr with 50 µg/ml EF-

C resulted in 47% fused cells, whereas only 2.8% were fused in the absence of the

enhancer (Fig. 15). Thus, addition of EF-C fibrils increased attachment and subsequent

fusion of the viral particle to the target cells.

3.6 EF-C fibrils allow to concentrate viral particles

Nowadays, time-consuming ultracentrifugation is the most commonly used method to

concentrate retroviral stock solutions (Reiser, 2000; Bajaj et al., 2001; Gatlin et al., 2001;

Zhang et al., 2001). The efficient formation of macroscopic complexes between virions

and EF-C fibrils (Fig. 13b) prompted me to test whether this allows precipitation and

concentration of virions in a bench-top centrifuge. For this purpose R5 tropic HIV was

mixed with EF-C or PBS and centrifuged for 5 min at 10,000 g. The supernatants (used-up

medium) were taken and the pellets resuspended to the original volume in PBS and cell

culture medium DMEM (Fig. 16a). Both, pellets and supernatants were used to infect

TZM-bl cells. Βeta-galactosidase screen assay performed 2 days later showed that only the

pelleted fraction of EF-C increased reporter gene expression whereas the respective

supernatants lacked any virus enhancing activity demonstrating that the infectivity

Figure 15. EF-C fibrils enhance virion fusion. TZM-bl cells were transduced with a VSV-G pseudotyped BlaM-Vpr lentiviral vector in the presence of the indicated concentrations of EF-C and virion fusion was determined by FACS. The numbers in panels indicate the percentages of fused cells. Percentages from lower panel were subtracted from upper panel.

3. RESULTS 40

enhancing fibrils of EF-C are pelletable (Fig. 16a, b). The PBS treated virus sample much

less infectious than fibril-treated virus. Thus, centrifugation of HIV-1 with EF-C fibrils

allowed precipitation of essentially all virions. One advantage of this approach is that

metabolized old medium of the virus stock can be easily exchanged by fresh medium of

choice. A second advantage is the possibility to easily concentrate virus by reconstituting

pelleted virus in a lower volume than the original one. To test this, 1,000 µl of EF-C

treated HIV stock was centrifuged at 10,000 g for 5 min. Afterwards, the supernatant was

collected and the resulting pellet resuspended in 100 µl DMEM. The amount of HIV-1 in

each fraction was determined by an HIV-1 p24 ELISA. The EF-C precipitated pellet

contained almost 1.2 µg capsid antigen whereas the original virus stock contained less than

100 ng (Fig. 16c). Thus, EF-C fibrils do not only allow to precipitate virus from a virus

solution but also to increase virus concentration by one single centrifugation step.

Figure 16. Nanofibrils bind viral particles and allow their precipitation and concentration. (a) Schematic drawing of the experimental outline to pellet virions. 10 ml of a virus stock preparation containing used-up medium was treated with PBS (1) or 50 µg/ml EF-C (2) and subjected to low speed centrifugation (5 min; 10,000 g; RT) After removal of used-up medium (3), the pelleted virions were resuspended in fresh medium (4) or buffer (5). Reducing the volume of resuspension medium allows the virus to be concentrated (6). (b) Infectivity of the resulting solutions. (c) p24 ELISA of the original virus stock (1) and EF-C-treated virus that was pelleted and resuspended in 1/10th of the volume of the medium used as the original stock (6). (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 41

These results are of great importance because this fast and convenient fibril-based

approach allows to replace time-consuming (60-120 min) ultra-centifugation (50,000 g at

4°C) that is normally used to enrich virus. The convenient exchange of old and perhaps

toxic medium by EF-C along with the simultaneous increase of transduction efficiencies

could be particularly attractive for effective gene delivery into primary cells that require

special media. In sum, EF-C allows to precipitate and to concentrate virions by brief

centrifugation and simultaneously increases infection/transduction efficiency.

3.7 EF-C fibrils are potent enhancer of lenti- and retroviral transduction

Since low transduction rates represent a significant problem in retroviral gene transfer, the

effect of EF-C on transduction rates of relevant target cells by retro- and lentiviral vectors

was tested. I generated gamma-retroviral (MLV-derived) and lentiviral (HIV-derived)

particles expressing luciferase or GFP as transgene. These vectors were pseudotyped with

the Gibbon Ape Leukemia Virus (GaLV), the endogenous feline leukemia virus (RD114),

the Vesicular Stomatitis Virus (VSV-G) or the amphotrofic Murine Leukemia Virus

(MLV) envelope proteins. For the lentiviral vector truncated variants of GaLV and RD114

containing mutation in the cytoplasmic domain of Env were used. These Env proteins were

selected because they are commonly used for gene transfer and have already been tested in

animal models and/or in clinical trials (Burns et al., 1993; Dybing et al., 1997; Lee et al.,

2001; Relander et al., 2005).

At first, I analyzed the impact of EF-C on transduction of HEK 293T cells by luciferase

encoding lenti- and retroviral vectors pseudotyped with different glycoproteins. Luciferase

assays performed 2 days after transduction demonstrated that brief exposure to EF-C

enhanced transduction rates of all analysed vectors (Fig. 17a, b). For example luciferase

activities in cellular lysates transduced with untreated retrovirus particles resulted in values

below 5.0 x 105 RLU/s, whereas infection rates of nanofibril-treated virus were increased

up to 6.5 x 106 (RD114) or even 1.4 x 107 RLU/s (MLV) (Fig. 17b). Similar results were

obtained for lentiviral particles with the exception of the VSV-G pseudotype. Reporter

gene activities after infection with untreated vectors were below 3 x 104 RLU/s, but were

markedly increased after transduction with nanofibril-treated virions (1.6 x 105) (Fig. 17a).

The enhancing effect was dose-dependent and largely independent of the viral vector or

envelope. Notably, EF-C did not allow virus transduction in the absence of a functional

3. RESULTS 42

Env protein (Fig. 17a, b). This indicates that EF-C fibrils do not allow unspecific entry

into target cells.

To further examine whether fibrils are suitable for therapeutic application, I examined their

capability to promote retro- and lentiviral transduction in a variety of cell types. GFP

encoding viral vectors commonly used in gene transfer settings (Naldini, 1997; Dull et al.,

1998; Zufferey et al., 1998; Schambach et al., 2006) were produced by transient

transfection of HEK 293T cells. Cell culture supernatants containing retro- or lentiviral

vectors harvested two days later were used for transduction. Treatment of GaLV

pseudotyped retroviral vector (RV-GaLV) with EF-C efficiently enhanced transduction of

the human epithelial cervical cancer HeLa cell line from 5% in the control to 98% after

100 µg/ml EF-C treatment (Fig. 18a). Gene transfer into the skin fibroblast is a promising

approach to treat inherited or acquired dermatological diseases (Wei et al., 1999).

Therefore, I tested whether EF-C nanofibrils enhance gene transfer into foreskin fibroblasts

(HFF) cell line. Treatment of RV-GaLV with EF-C resulted in 40.8% GFP positive cells 3

days post transduction whereas only 1.8% were transduced in the absence of the enhancer

(Fig. 18b), corresponding to a 23-fold enhancement of transduction rates.

Figure 17. EF-C fibrils boost gene transfer by various retro- and lentiviral particles. (a) Lentiviral particles were generated by co-transfection of 293T cells with an env-defective HIV-1 proviral construct containing the luciferase reporter gene and expression plasmids encoding the indicated viral glycoproteins. Freshly harvested virions were treated with the indicated concentrations of EF-C fibrils and used to transduce 293T cells. (b) Retroviral particles were generated by transfection of 293T cells using an MLV-based, luciferase encoding vector, the M57-DAW packaging plasmid and expression plasmids encoding the indicated viral Env proteins. Retroviral particles were treated with EF-C fibrils and used to infect 293T cells. Both panels give average values obtained from triplicate infections +/-SD. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 43

I also performed experiments in non-adherent KG-1 cells, a human acute myeloid

leukemia cell line that represents an early stage of hematopoietic differentiation and that is

often used in colony formation assays instead of human bone marrow cells (Koeffler et al.,

1978). The rate of GFP expressing cells was 1.5 % in the control whereas treatment with

50 µg/ml EF-C led to GFP expression in 65.8 % of the cells (Fig. 19a). Thus, EF-C does

not only favour transduction of adherent cells but also of those growing non-adherent.

Furthermore, I transduced BON, a human pancreatic carcinoid cell line, and U87MG cell

line, a glioblastoma, astrocytoma cell line with RD114 pseudotyped lentiviral vector (LV-

RD114). Those cell lines have been widely used as models for translational study against

dangerous types of cancers, human pancreatic tumors and human malignant glioma (Yao et

al., 2007; Schmidt et al., 2013). In the presence of 50 µg/ml of EF-C, 83.5 % of BON cells

and 90.7 % of U87MG cells were GFP positive, whereas inoculation with untreated virus

showed transduction rates of 5% and 4.4 % respectively (Fig. 19b). In sum these results

show that EF-C nanofibrils generally facilitate transduction of different cell lines.

Figure 18. The enhancing effect of EF-C fibrils on viral transduction are cell-type independent. (a) FACS analysis of HeLa cells transduced with retroviral particles carrying the GaLV Env that were untreated or treated with the indicated concentrations of EF-C fibrils. (b) Analysis of HFF cells transduced with retroviral particles carrying the GaLV Env by fluorescence microscopy. The numbers in panels indicate the percentages of transduced (GFP+) cells. Scale bars, 50 µm. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 44

To analyse the transduction enhancing activity of EF-C fibrils in primary cells, I

transduced PHA/IL-2 activated human peripheral blood lymphocytes (PBL) with LV-

RD114 in the presence or absence of nanofibrils. FACS analyses revealed that EF-C

nanofibrils increased the percentage of GFP positive cells after infection with a multiplicity

of infection (MOI) of 1 from 2.3 % to more than 24 % and after infection with a MOI of 10

to a maximum of 44% (Fig. 20a). To test, whether EF-C might also favour transduction of

macrophages that are considered as hard to transduce cells (Leyva et al., 2011),

monocytes-derived macrophages were transduced with a LV-RD114 stocks that were

either treated with EF-C fibrils or PBS. Flow cytometry and fluorescence microscopy 3

days after transduction revealed that 50 µg/ml of EF-C fibrils enhanced lentiviral gene

delivery into macrophages about 13-fold and allowed viral gene expression in almost half

of them (Fig. 20b).

Figure 19. EF-C fibrils potently enhance lentiviral transduction in different cell types. (a) FACS analysis of KG-1 cell line transduced with LV-RD114 that were untreated or treated with the indicated concentrations of EF-C fibrils. The numbers in panel indicate the percentages of transduced (GFP+) cells. (b) Effect of nanofibrils on lentiviral gene transfer into human glioblastoma U87MG cells and human endocrine pancreatic tumour cell line BON. The numbers above the bars indicate n-fold enhancement compared to the titer measured in the absence of peptide. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 45

Next, I analysed whether EF-C enhances transduction of stem cells. Human CD34+

hematopoietic stem cells (HSC), which represent a major target in gene therapy studies,

were incubated with a LV-RD114 that has been treated with EF-C or PBS prior to

transduction. FACS analyses performed 4 days later showed that transduction of fibrils-

treated vector resulted in more than 25.6% of transduced and GFP positive cells, whereas

inoculation with untreated virus showed transduction rates of only 0.2% (Fig. 20c). These

results demonstrate that EF-C treatment of a vector preparation markedly enhanced gene

transfer into primary cells including HSCs.

3.8 Comparison between EF-C fibrils and other agents used to promote retroviral transduction

Next, I compared the efficiency of EF-C mediated enhancement with commercially

available enhancers like cationic compounds. Positively charged polymers such as

polybrene and (DEAE)-dextran enhance the attachment of the virus and subsequent

infection by bridging interactions between negatively charged viral and cell membranes.

Protamine sulphate is a highly cationic polypeptide derived from the sperm of salmon that

enhances retroviral gene transfer in a similar manner as cationic polymers. HIV enhancing

Figure 20. The enhancing effect of EF-C fibrils on viral transduction in primary cells. (a) Effect of nanofibrils on lentiviral gene transfer into human peripheral lymphocytes (PBL). The numbers above the bars indicate n-fold enhancement compared to the titer measured in the absence of peptide. (b) Analysis of monocytes-derived macrophages transduced with lentiviral particles carrying the RD114 Env by fluorescence microscopy. The numbers in panels indicate the percentages of transduced (GFP+) cells. Scale bars, 50 µm. (c) FACS analysis of CD34+ stem cells transduced with LV-RD114 that were untreated or treated with the indicated concentrations of EF-C fibrils. The numbers in panel indicate the percentages of transduced cells. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 46

activity of EF-C was also compared with SEVI (Semen-derived Enhancer of Viral

Infection), amyloid fibrils that are generated from the fragments of prostatic acidic

phosphatase (PAP248-286) in semen (Münch et al., 2007). In detail, 0.1 ng R5 HIV-1 viral

stocks were treated with equal concentrations of freshly prepared EF-C, polybrene,

protamine sulfate, DEAE dextran and SEVI fibrils obtained after overnight agitation. After

5 min incubation, these mixtures were used to infect TZM-bl cells. Infection rates

determined 2 days later showed that EF-C fibrils enhanced HIV-1 infection more

efficiently than polybrene, protamine sulfate and DEAE dextran and was also slightly more

effective than amyloid SEVI fibrils obtained after overnight agitation (Fig. 21a).

Next, I tested the effect of EF-C, protamine sulfate, polybrene, DEAD dextran on the

metabolic activity of TZM-bl cells using the CellTiter-Glo® Luminescent Cell Viability

Assay (Promega). The cells were incubated with various concentrations of these

compounds for 3 days. Measurement of intracellular ATP levels showed that DEAD

dextran decrease metabolic activity of TZM-bl cells at the concentration 25 - 50 µg/ml

(Fig. 21b), whereas treatment the cells with EF-C nanofibrils, polybrene and protamine

sulphate has no toxic effect for the cells (Fig. 21b).

Figure 21. Comparison of infection enhancing properties of EF-C fibrils and others enhancers. (a) Indicated compounds were serially diluted and subsequently incubated with HIV particles. 20 µl of these mixtures were used to infect 180 µl TZM-bl cells. Infection rates were determined 2 days later by quantifying ß-galactosidase activities in cellular lysates. Concentrations shown are those during virion treatment. (b) Indicated compounds were incubated with TZM-bl for 3 days and cellular ATP concentrations were measured by the Cell Viability Assay. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 47

In addition, the effect of EF-C, polybrene and protamine sulphate on transduction rates of

KG-1 cells was tested by retroviral vector carrying the GaLV GP (Fig. 22). For this

experiment, concentrations of polybrene and protamine sulphate were reduced to 8 and 16

µg/ml as recommended by literature (Davis et al., 2002; Cornetta et al., 1989). EF-C

nanofibrils increased the percentage of GFP expressing cells from 1.5 % in the control to

almost 56.2 % after treatment with 25 µg/ml EF-C, whereas only 2.6 % and 6.6 % cells

were transduced after addition of 8 µg/ml of protamine sulphate or polybrene respectively

(Fig. 22). In sum, compared to polybrene, protamine sulphate and DEAE dextran, EF-C

nanofibrils are more effective and less toxic.

3.9 Immobilized EF-C nanofibrils capture virus and increase transduction.

Currently, the most effective method to boost retroviral gene transfer is based on the use of

the fibronectin derivative RetroNectin (Williams et al., 1991; Hanenberg et al., 1996;

Moritz et al., 1996; Hanenberg et al., 1997). RetroNectin needs to first be pre-coated onto

plates. The heparin domain of RetroNectin binds the viral particles this allows to remove

the utilize virus medium. Finally the target cells attach to the cell-binding domain of

RetroNectin. The co-localization of virions and cells in close proximity increased cellular

uptake of the virus. I analysed whether EF-C nanofibrils can be applied in an analogue

Figure 22. Comparison of transduction efficiency of EF-C fibrils, polybrene (PB) and protamine sulphate (PS). Indicated concentrations of enhancers were incubated with GaLV pseudotyped retroviral particles and subsequently used to infect 2 x 105 KG-1 cells. Transduction rates were determined 5 days later by FACS. Concentrations shown are those during cells treatment.

3. RESULTS 48

manner. For this, plastic slides were coated with Rho-EF-C and incubated with YFP-

tagged retroviral particles. Confocal microscopy analyses showed that the fibrils were

immobilised on the surface and efficiently captured virions (Fig. 23a). The binding of

lentiviral particles to the dishes was demonstrated in a p24 ELISA (Fig. 23b). For that,

various concentrations of EF-C were coated on routinely used microtiter plates (Costar,

#3596) over night at 4°C. The next day, peptide was removed and plates were incubated

with HIV-1 for 1 or 4 hrs. Then, the wells were washed once in PBS and bound virus was

determined by p24 ELISA. The results showed that immobilised fibrils capture virions and

this occurred in a time- and dose-dependent manner (Fig. 23b).

To test if immobilized EF-C fibrils enhance viral infection, four microtiter plates obtained

from different providers (A: Thermo Scientific NuncTM Nunc F96 MicroWellTM Plates

Polystyrene Clear, #167008; B: Thermo Scientific NuncTM Nunc F96 MicroWellTM Plates

Polystyrene Clear, #442404; C: CORNING (96 well) EIA/RIA Flat Bottom, #3590; D:

CORNING (96 well) Multiple Well Cluster Plate TC Treated, #3596) were incubated with

freshly diluted EF-C. The next day, the solutions were removed and HIV-1 added. After 4

hours of incubation, the virus inoculum was removed and TZM-bl reporter cells were

plated. In some control samples, the virus inoculum has not been removed and cells were

directly added to the virus (ctrl). Beta-galactosidase assays performed 2 days later revealed

that infection rates of cells added to EF-C coated plates were strongly increased (Fig. 24a).

Figure 23. Coated EF-C fibrils can capture viruses. (a) Z-stack images of immobilised EF-C (top left), virus only (bottom left), immobilized EF-C exposed to virus (right); the upper image shows both channels (merged), the inlets below separately. (n) – number of capture viruses. (b) Indicated amounts of EF-C were used to coat standard cell culture plates over night at 4°C. The next day, peptide was removed and plates were incubated with HIV-1 for 1 or 4 hrs. Wells were then washed once in PBS and bound virus was determined by p24 ELISA. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 49

Maximum enhancement was observed for all 4 dishes between 1-10 µg EF-C per well

(Fig. 24a). Importantly, infection rates observed in control samples were even lower than

those observed in wells that were coated with nanofibrils and where the whole inoculum

has been removed (Fig. 24a). Similar experiment was performed with GaLV pseudotyped

retroviral vector and KG-1 cells. Analyse of flow cytometric data showed that EF-C

nanofibrils coated onto plates increased the transduction rates of suspension cells by more

than 5-fold (Fig. 24b). Thus, EF-C can be coated onto the surfaces of cell culture dishes

allowing to increases viral infection and transduction rates.

To compare the ability of immobilized RN and immobilized EF-C nanofibrils to promote

retrovirus transduction, both compounds were tested in a site by site approach. Microtiter

plate from BD Bioscience (#351147) that was recommended by TAKARA BIO INC was

coated with RetroNectin. Routinely used microtiter plate from Costar (#3596) was coated

with EF-C. Both plates were then incubated with similar amounts of a RV-GaLV for 4

hours. RN coated microplate was washed as recommended by the manufacturer and EF-C

fibrils coated microplate was washed with 100 µl PBS. Thereafter, KG-1 cells were plated

and the transduction rates were measured by FACS analysis. The results showed that both

Figure 24. Immobilized EF-C nanofibrils capture virus. (a) EF-C fibrils that have been coated on different microtiter plates facilitate viral infection. See text for detail information. (b) Immobilized EF-C fibrils boost lentiviral vector transduction into KG-1 cells. Freshly diluted EF-C was added to Costar 96 well plates and was incubated over night at 4°C. Peptide was then removed and 50 µl of a lentiviral vector containing the GaLV GP was added to each well. 4 hrs later, the inoculum was removed and wells were either washed once in PBS or left untreated. Thereafter, 1 x 106 KG-1 cells were added. After 5 days, transduction rates were determined using FACS analysis. ctrl, control in which the inoculum was not removed. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 50

compounds dose dependently increased the rate of GFP expressing cells to a maximum of

~ 11% (Fig. 25a). This value was reached in the presence of 1.2 µg of RetroNectin or 10

µg of EF-C. Interestingly, transduction rates in the absence of RetroNectin in BD

Bioscience microtiter plates were lower than those in the absence of EF-C in tissue culture

treated plates from Costar (Fig. 25a), suggesting that cell growth in non-tissue BD

Bioscience plates might be compromised.

To compare the effects of soluble RetroNectin and EF-C, HIV-1 virus stocks containing 1

ng p24 antigen were incubated with 0, 0.8, 4, 20 µg/ml enhancer and used to infect TZM-

bl cells. EF-C nanofibrils strongly enhanced infection whereas treatment of virus stocks

with RetroNectin had no effect (Fig. 25b). Additionally, the activities of EF-C fibrils and

RetroNectin were compared under conditions in which the target cells were incubated first

with compounds. TZM-bl cells were incubated with 0, 0.8, 4 or 20 µg/ml enhancer and

subsequently infected with HIV-1. Also under these conditions, EF-C nanofibrils potently

favoured infection, whereas levels of infection in cells treated with RetroNectin were

comparable to those not treated by any enhancer (Fig. 25b). In sum these results

demonstrate that in contrast to RetroNectin that is active only if bound on surfaces, EF-C

potently enhanced viral infection when added to virions directly, or when used for

treatment target cells before infection.

Figure 25. Comparison of infection enhancing properties EF-C fibrils and RetroNectin. (a) Coated EF-C fibrils (EN) enhance viral transduction with similar efficiency as RetroNectin (RN). Retroviral particles were added to RN-coated or EN-coated plates and incubated with KG-1 cells. (b) In contrast to RN, EF-C fibrils enhance infection by directly treating virions or cells. Nanofibrils or RN were mixed with cells (left) that were subsequently infected with HIV-1, or mixed with HIV-1 virions (right) that were then used to infect cells. Infection rates were determined 2 days later. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

3. RESULTS 51

Figure 26. Additive effects of RetroNectin and EF-C nanofibrils. Virions were added to RN-coated plates and upon removal of the inoculum, KG-1 cells in the presence or absence of fibrils-treated virus were added. (Reprinted with friendly permission from Yolamanova, Meier et al., 2013, Nature Nanotechnology, 8(2):130-136. Copyright © by Macmillan Publishers Limited)

Finally, it was examined whether EF-C fibrils and

RetroNectin can be used in combination. The plate from

BD Bioscience was coated with 6 µg of RetroNectin and

incubated overnight at 4 °C. The microplate was washed

as recommended by the manufacturer and retroviral

particles pseudotyped with GaLV Env were inoculated.

After 1 hour virus stock was removed and 106 KG-1 cells

were plated in each well. Further RV-GaLV stocks were

treated with EF-C nanofibrils or PBS and after 5 min

incubation these mixture were added to the KG-1 cells.

The results showed that fibrils-treatment increases the

strong enhancement of lentiviral transduction of KG-1

cells by RetroNectin treatment even further by up to 5-

fold (Fig. 26). Hence, both methodologies can be

combined to achieve maximal gene transfer efficiencies.

4. DISCUSSION 52

4. DISCUSSION

4.1 Application of EF-C nanofibrils as enhancer of transduction efficiency.

Retroviral gene transfer is the method of choice for the stable introduction of genetic

material into cells and offers many prospects for basic research and for the treatment of

genetic disorders, malignancies and infectious diseases. The low titer of infectious virions

in the stocks is one of the major limitations of retroviral gene transfer. To produce high-

titered virus stocks various approaches like the development of new viral vectors systems,

the optimization of transfection protocols and ultracentrifugation were established (Kotani

et al., 1994; Reiser, 2000; Bajaj et al., 2001; Gatlin et al., 2001; Zhang et al., 2001).

Furthermore, work with retrovirus vectors is often hampered by low transduction rates,

particularly into primary cells. Transduction efficiency is determined by rate of virus

attachment to the target cell. Viral adsorption can be divided into three steps: (i) passive

diffusion of virions to the proximity of the cell, (ii) nonspecific binding of the virus to the

cell surface and (iii) binding of the viral glycoproteins to a specific cell surface receptor

that triggers the subsequent fusion (Davis et al., 2002). Passive diffusion can hamper by

electrostatic repulsion forces from the negatively charged phospholipid bilayers of

retrovirus and cell. Frequently used strategies to overcome this obstacle are low speed

centrifugation of virions together with their target cells and/or treatment with cationic

compounds, such as the hexadimethrine bromide (polybrene), the diethylaminoethyl

(DEAE)-dextran, protamine sulfate, poly-L-lysine, or cationic liposomes (Hodgson and

Solaiman, 1996; Vogt, PK, 1967; Cornetta et al., 1989; Hennemann et al., 2000). These

compounds are thought to reduce the repulsion forces between the cell and the virus and

mediate the binding of retroviral particle to the cell surface resulting in a higher efficiency

of transduction. The big disadvantage of some cationic polymers, like polybrene and

DEAE-dextran, is their cytotoxicity. Therefore, these compounds are used at only low

concentrations (4–20 μg/ml) (Toyoshima and Vogt, 1969; Manning et al., 1971; Cornetta

and Anderson, 1989). Furthermore, application of polybrene has a negative impact on cell

proliferation in primary cells, such as keratinocytes or human mesenchymal stem cells,

even when it is used at low concentrations (Seitz et al., 1998; Lin et al., 2010). Currently,

the most common method to increase the efficiency of retroviral transduction is coating of

the cell cultures dishes with RetroNectin, a fibronectin derivative which has been

discovered almost 20 years ago (Hanenberg et al., 1996; Moritz et al., 1996; Hanenberg et

al., 1997). When RetroNectin is coated onto cell culture dishes it induces the colocalization

4. DISCUSSION 53

of virus particles and target cells in close proximity resulting in increased transduction

efficiencies. However, utilization of RetroNectin is relatively time consuming and

expensive. Thus, improved convenient, flexible and effective transduction enhancers are

still needed. Novel classes of transduction enhancers are peptide-derived amyloidogenic

fibrils. It has been found that fibrils derived from human semen strongly enhance HIV-1

infection (Münch et al., 2007) and allow to improve viral gene transfer (Wurm et al., 2010;

Wurm et al., 2011). These fibrils, termed semen-derived enhancer of virus infection

(SEVI), bind virions and increase their adsorption to cells and subsequent fusion.

However, SEVI also has some drawbacks for a use as transduction enhancer. Firstly, the

amyloidogenic peptides that form SEVI are relatively large (38 amino acids) and thus

expensive to produce. Secondly, it takes hours to generate the active fibrils (Münch et al.,

2007). Finally, very large aggregates that are less effective in boosting retroviral

transduction may form after long-term storage (Münch J, personal communication).

Newly, Fenard et al. identified the new viral entry enhancer, Vectofusin-1. This histidine-

rich cationic amphipathic peptide efficiently enhance gene transfer of lentiviral vectors into

human CD34+ cells by promoting the adhesion and the fusion between viral and cellular

membranes, without inducing toxicity (Fenard et al., 2013). Vectofusin-1 shows similar

efficiencies as RetroNectin with the advantage that it can be easily synthetized and

purified.

In this study a small HIV-1 gp120-derived peptide named EF-C that instantaneously

formed nanofibrils which greatly increased HIV-1 infection was investigated. This peptide

effectively boosted the efficiency of lenti- and retroviral gene delivery into various cell

types. EF-C nanofibrils increased the efficacy without affecting the specificity of retro- or

lentiviral gene delivery suggesting that the fibrils might also boost recently developed

lentiviral vectors that are based on specific single chain antibodies recognizing their

cognate cell surface antigens (Anliker et al., 2010). This is noteworthy, because many gene

therapy approaches aim to target specific cell types to avoid possible avoid side effect.

Moreover, EF-C allowed to cross-link viral particles and to precipitate them by low speed

centrifugation. The precipitated virions remained fully infectious and could be dissolved in

the medium and volume of choice prior to transduction. Thus, EF-C represents a

convenient and effective tool to concentrate virions, and to remove toxic compounds that

may affect the functionality of the target cells. EF-C facilitated higher efficiency of viral

transduction compared to polybrene, protamine sulphate, DEAE dextran and SEVI. EF-C

was tested as non-cytotoxic for several cell lines and primary cells. Additionally, to

4. DISCUSSION 54

exclude negative effect of EF-C on hematopoietic stem cell differentiation capacity, our

colleagues from Institute for Clinical Transfusion Medicine and Immunogenetics (DRK

Blood Service Baden-Württemberg, Ulm, Germany) examined the CD34+ cells in Colony

Forming Cell Assays. The results showed that EF-C did not affect differentiation capacity

of human stem cells (Yolamanova, Meier et al., 2013). Thus, in contrast to the former

enhancers, it can be used for gene transfer into sensitive primary human cells, such as

macrophages or hematopoietic stem cells. Lack of cytotoxicity in these cell types has also

been reported for amyloid SEVI fibrils (Wurm et al., 2011). When compared to

RetroNectin EF-C fibrils can be used to promote viral gene transfer in a more flexible and

simple manner, because it is/was added to the virions or to the target cell culture prior to

transduction. Usage of EF-C nanofibrils instead of RetroNectin reduces the workflow from

100 minutes (not including the overnight incubation after coating and the second spin-

infection step) to 10 minutes. Notably the highest transduction efficiencies were observed

when RetroNectin and EF-C nanofibrils were combined. Thus, treatment with EF-C has

significant advantages over other methods that are currently employed to increase lenti- or

retroviral transduction efficiencies, EF-C fibrils have no effect on cell viability of the

transduced cells and differentiation capacity of stem cells and might be suitable tool for

gene therapy.

In mice studies, which were carried out in the Department of Experimental Dermatology

and Allergic Diseases (Ulm University, Ulm, Germany), the effects of EF-C nanofibrlis

and RetroNectin on viral gene delivery were compared. For this, lineage depleted bone

marrow cells were transduced ex vivo (i) in the absence of enhancers; (ii) using a

commonly used multi-step RetroNectin protocol including a spin transduction (Zhou et al.,

2001); (iii) or a one-step approach using EF-C-treated virus. The results showed that

RetroNectin and EF-C fibrils increased transduction rates from 3.1 % in the control to 23.9

% and 18.9 %, respectively. Transplantation of these cells into recipient mice showed no

obvious side effects, and engraftment was determined by blood cell analysis 4 and 12

weeks later. The results showed a significantly higher proportion of transduced cells in the

RetroNectin (19.7 % and 12.3 %) and EF-C (13.5 % and 6.1 %) groups compared to the

control mice (0.1 %) (Yolamanova, Meier et al., 2013). Notably, brief EF-C treatment

resulted in almost similar transduction and engraftment rates as the multi-step RetroNectin

protocol, which involves an additional spin-transduction step and thus exposure of the cells

to twice as much virus. Taken together, EF-C represents a convenient and effective tool to

boost lenti- and retroviral gene delivery, to concentrate virions, and to remove toxic

4. DISCUSSION 55

compounds that may affect the functionality of the target cells, and is suitable for ex vivo

gene therapy applications.

A possible drawback of EF-C for clinical application is that it attached to and was taken up

by the cells and would thus be transferred to the patients. However, in the mice studies we

did not observe any side effects after transplantation of EF-C treated cells over 12 weeks of

follow-up. The lack of toxicity in cell culture suggests that the amyloid fibrils may just be

degraded without causing undesired effects. The possible induction of antibodies to EF-C

cannot be excluded but would not represent a major caveat because most clinical gene

transfer studies aim for a single treatment. However, follow-up studies on the fate of

nanofibrils in the cells or tissues seem highly necessary.

4.2. Analysis of the mechanism of fibril-mediated enhancement of viral infection.

Beside the application of amyloidogenic fibrils as efficient transduction enhancer, the

mechanism of fibril-mediated enhancement of viral infection is of great interest. It is

highly likely that EF-C fibrils promote virion attachment by serving as a bridge between

the virions and the cells allowing them to overcome the repulsion between the negatively

charged membranes as it has been proposed for cationic compounds like polybrene and

protamine sulphate (Davis et al., 2002). Indeed, it has been previously established that the

cationic properties of SEVI and other semen-derived amyloidogenic peptides are necessary

for efficiently promoting virus infection (Münch, et al, 2007; Roan et al., 2009; Arnold et

al., 2012). Roan et al. showed that a negatively charged SEVI variant, in which lysines and

arginines were replaced by alanines, was capable to form amyloid fibrils, but does not

efficiently enhance HIV-1 infection (Roan et al., 2009). Moreover, addition of polyanionic

compounds like heparin to fibrils or to the targets cells abrogate the enhancing activity of

SEVI and other semen-derived fibrils as heparin shields the charged surface of the fibrils

(Roan et al., 2009; Arnold et al., 2012). Recently, Zhang et al. identified another 13-

residue peptide in the HIV-1 gp120 glycoprotein which also form amyloid fibrils and

augment HIV-1 infection. They termed this peptide P13 and extend them with three lysine

amino acids at its C-terminus to increase its cationic charge (Zhang et al., 2013). The

modified peptide was designated as P16 and exhibited a pI of 10.48 and a net charge of +3.

It has been also observed that mutation form of P16, which contained alanine residues

instead of tryptophan, formed fibrils but much less enhanced infection. Furthermore,

anionic polymer completely blocked the activity of peptide to boost virus infection. These

4. DISCUSSION 56

interesting observations also show that electrostatic interaction is driving in fibril mediated

HIV infectivity enhancement.

Indeed the EF-C sequence contains positively charged basic residues and has a net charge

of +2 and an isoelectric point of 9.9. Zeta potential measurements revealed that EF-C

nanofibrils display a positively charged surface (+17.7±1.7 mV). Furthermore, our

colleagues from Institute of polymer science (University of Ulm, Germany) constructed a

structural model of the peptide nanofibrils. The resulting fibril model suggests that the

lysine side chain forms a hydrophilic surface with a high density of cationic charges at

physiological pH (Yolamanova, Meyer et al., 2013). Besides, it is conceivable, that the

magnitude of the enhancing effect will depend on the intrinsic capability of both virions

and cells to interact. The results derived from pseudotyped experiment with lentivirus

based virion showed that EF-C fibrils had only a moderate effect on transduction rates of

VSV-G carrying particles (Fig. 17a). The similar results were observed in the same

experiments with SEVI (Münch J, personal communication). I hypothesised that amount of

glycoproteins on the viral envelope can play substantial role in the mechanism of

enhancing activity of amyloid fibrils. HIV-1 virions carry usually only 8 to 10 Env trimers

per virus particle (Zhu et al., 2003), whereas about 400 envelope glycoprotein incorporated

in the VSV-G particle (Brown et al., 2010). The high density of glycoproteins on the

surface of VSV-G particle probably shields the negatively charge of phospholipid bilayer

of virus and makes the amyloid fibrils less effective for enhance infection. It would be

interesting to control in additional experiments whether the degree of virus infection

enhancement indeed correlates with amount of envelope glycoproteins. For this purpose

pseudoparticles with low, median and high amounts of VSV-G glycoprotein should be

generated and used for comparative analyses with amyloidogenic peptides.

Noteworthy, cationic properties do not seem to be an absolute requirement for the

enhancement of HIV infection by amyloid fibrils. Another interesting aspect in the

mechanism of enhancement activity is the self-assemble of peptide into fibrils. It has

previously been shown that amyloid fibrils in semen promote HIV-1 infection, whereas the

monomeric peptide has no effect (Münch et al., 2007; Roan et al., 2011; Arnold et al.,

2012). Albeit the EF-C peptide possesses a positive net charge, H2O or PBS dissolved EF-

C had no effect on infection. However dilution of DMSO-dissolved peptide in H2O or PBS

potently enhanced infection and transduction rates, this solution became turbid and

formation of fibrils could be observed by Thioflavin T binding and Congo red staining.

Existence of amyloid fibrils was further confirmed by atomic force microscopy, X-Ray

4. DISCUSSION 57

powder diffraction and circular dichroism spectroscopy (Yolamanova, Meyer et al., 2013).

Like SEVI, EF-C adopts a classic cross β amyloid structure in which β-sheets run

perpendicular to the axis of the fibrils. This indicates that in addition to cationic properties

of EF-C, fibril formation is a prerequisite for EF-C enhancement activity Moreover, EF-C

is usually even more effective than SEVI in boosting retro- and lentiviral gene transfer.

The reason for this is most likely that EF-C consists of small amyloid nanofibrils, whereas

SEVI forms large amyloid fibrils that may be too immotile to make efficient contact with

the target cells. Interestingly, negatively charged SEVI was capable to slightly enhance

virus infection (Roan et al., 2009). This can be explained by formation of a fibril network,

which captures the virus via hydrophobic interaction. Fibrils-virus complexes, which due

to their large size rapidly sediment onto the target cells, increase the rate of infection.

Since the cross β-sheet structure is common to all amyloid proteins but the ability to

enhance viral infection possess not all amyloid fibers even with positively charge surface.

For example, bacterial amyloid curly protein which also formed positively charged fibrils

(net charge of +5, pI of 9.9) do not enhance viral infection (Hartmann et al., 2012). Hence,

enhancement of virus infection rates is an intrinsic property of amyloid structure.

An interesting question is whether it is just coincidence that gp120 fragments form fibrilar

virion attachment factors or whether HIV-1 generates its own enhancer to facilitate viral

spread. It has been estimated that the sum of soluble, cell- and virion-associated gp120 in

HIV-infected individuals reaches up to 5 µg per ml blood (Cummins et al., 2010). Even if

all gp120 molecules are degraded to EF-C fragments, this would only result in

concentrations of approximately 50 ng/ml of the fibrils, which is far below those required

to enhance HIV infection in vitro. As mentioned above fibril formation is a prerequisite for

EF-C mediated transduction enhancement. For fibril formation EF-C has to be dissolved

first in organic solvent, conditions that are not found in vivo. Altogether, it seems unlikely

that gp120 fragments play a significant role for HIV-1 replication in vivo. Other amyloids,

however, may well play a relevant role in virus spread and the pathogenesis of AIDS

because amyloid deposits are fairly common (Green et al., 2005) and ß-amyloid fibrils

associated with Alzheimer`s or other diseases also promote HIV-1 infection (Wojtowicz et

al., 2002).

In sum, EF-C is an effective, simple, and inexpensive tool to boost viral gene delivery into

various cell types. Moreover, this peptide has great potential as enhancer of viral

transduction because it can be easily modified with various functional groups that raise its

natural ability to self-assemble into nanofibrils and increase their stability in vivo models.

4. DISCUSSION 58

Therefore application of EF-C and its derivatives will significantly facilitate the utilization

of retro- and lentiviral vectors in basic research and potentially in the treatment of human

diseases.

5. SUMMARY 59

5. SUMMARY

Low transduction efficiencies are a common problem in retro- and lentiviral gene transfer.

Here, I have presented a new approach to boost retroviral gene transfer using self-

assembled nanofibrils formed by the 12-amino acid peptide termed EF-C (Enhancement

Fragment C). This peptide was derived from the HIV-1 external envelope glycoprotein

gp120. The fibrils form instantaneously upon dilution of peptide in aqueous media and

adopt a typical amyloid structure that was observed due to Thioflavin T and Congo Red

staining and atomic force microscopy. Measurement of ζ-potential charge of aqueous

solution of EF-C revealed that the nanofibrils have positively charged surface, supposed

that their enhance infection by neutralizing the repulsion between the negatively charged

viral and cellular membranes. My data obtained using confocal fluorescence microscopy

shown that fibrils bind virions and increase their adsorption to cells and subsequent

infection. Addition of EF-C to virus stocks or target cells boosted gene delivery into

multiple cell types, including primary T cells, macrophages and CD34+ stem cells, and

was substantially more effective than other reagents commonly used to increase

transduction. Moreover, like RetroNection, the most commonly used reagent to increase

retroviral transduction, EF-C potently boosts transduction also when immobilized on cell

culture dishes. This effect was confirmed with retro- and lentiviral particles carrying

various viral envelope proteins frequently used for gene transfer and therapy. The

enhancing effect was independent of the viral glycoprotein but still required the respective

cellular receptor(s). Furthermore, the fibrils allow a fast and convenient concentration of

virions without the need of ultracentrifugation. The result presented in this thesis

demonstrate that EF-C nanofibrils provide a convenient, flexible and effective means to

increase retro- and lentiviral gene transfer in basic research and clinical applications.

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