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Factors affecting transduction efficiency ofpseudotyped viral vectors incorporating alphaviralglycoproteinsAditi KesariPurdue University

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Thesis/Dissertation Acceptance

This is to certify that the thesis/dissertation prepared

By

Entitled

For the degree of

Is approved by the final examining committee:

To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material.

Approved by Major Professor(s):

Approved by:Head of the Departmental Graduate Program Date

Aditi S. Kesari

Factors affecting Transduction Efficiency of Pseudotyped Viral Vectors Incorporating Alphaviral Glycoproteins.

Doctor of Philosophy

Peter J. HollenbeckChair

David A. Sanders

James F. Leary

Richard J. Kuhn

David A. Sanders

Jeffery Lucas 3/2/2016

FACTORS AFFECTING TRANSDUCTION EFFICIENCY OF PSEUDOTYPED

VIRAL VECTORS INCORPORATING ALPHAVIRAL GLYCOPROTEINS.

A Dissertation

Submitted to the faculty

of

Purdue University

by

Aditi Kesari

In Partial Fulfillment of the

Requirements for the Degree

of

Doctor of Philosophy

May 2016

Purdue University

West Lafayette, Indiana

ii

This Thesis is Dedicated to

P.P. Dr. Suchitdada Dattopadhye

And

My dear Son, Aariv Josyula

iii

ACKNOWLEDGEMENTS

Firstly, I would like to thank the Almighty for continuously showering blessings

on me. It is because of Your grace that I am able to write this today.

I would like express my sincere gratitude to my advisor, Dr. David Sanders for

giving me an opportunity to work in his lab and believing in me. I wouldn’t have been

able to fulfill my dream of pursuing a PhD, if it wasn’t for his support. His immense

knowledge, the thought provoking discussions we had, the freedom he gave me to

explore the field of science and the scientific insights he provided have always been a

source of inspiration. Above all, the friendly atmosphere that he maintained in the lab

made this journey thoroughly enjoyable.

The continuous support and guidance I received from my committee members,

Dr. Peter Hollenbeck, Dr. Richard Kuhn and Dr. James Leary, through all these years

was extremely valuable. Their suggestions and comments broadened the scope of my

research. I really appreciate the help they provided me at different stages of this journey.

I would like to thank all the current and past members of Dr. Sanders’ lab,

especially, Dr. Anita Ghosh, Jason Segura, Dr. C. Matthew Sharkey and Dr. Swapna

iv

Apte for their support and help. Also, it was great working with Dr. Oya Bulut, Sarah

Talamentez, Melanie Bumbalough and Tycho Jaquish. I would also like to thank all

graduate students, post-doctoral researchers and technicians in the Department of

Biological Sciences and the Structural Biology building, who gave me access to different

research facilities, helped me with different techniques, advised me on presentation skills,

etc. I am especially grateful to Dr. Rushika Perera, Dr. Joyce Jose, Dr. Thomas Edwards

and other members from Dr. Kuhn’s laboratory for their guidance and help. I would also

like to thank Dr. Ronald Hullinger, Dr. Laurie Jaeger and other faculty members from

department of Basic Medical Sciences for their support.

My sincere thanks to all my friends and well-wishers, who made these years of

my life so memorable. I sincerely thank Pravinsinh Wagh, Jalaja Padma, Prasanth

Tanikella, Sanmit Ambedkar, Sudhir Bangaru, Chaitra Chavali, Harish Suryanarayana

and Deenal Mannava for helping me during a challenging phase of my life.

Finally I would like to thank my entire family. My father, Shashikant Kesari who

taught me no dream is too big to achieve, my mother, Shubhada Kesari who imbibed in

me the strength and the confidence to achieve those dreams, my brother, Amit Kesari,

who showed me the right path to achieve those dreams, I am forever indebted to them.

Last but not the least, my husband, Aditya Teja Josyula, you have been God’s best gift to

me. Thank you for being my best friend, teacher, mentor, confidant, my audience, my

editor, my ‘tech-guy’, but beyond everything the best partner. Thank you for everything.

Looking forward to spending many more sweet and memorable moments with you.

v

TABLE OF CONTENTS

Page

LIST OF TABLES ........................................................................................................... viii

LIST OF FIGURES ........................................................................................................... ix

LIST OF ABBREVIATION .............................................................................................. xi

ABSTRACT ..................................................................................................................... xiv

CHAPTER 1. GENE THERAPY/ TRANSFER VECTORS AND RETROVIRUSES ..... 1

1.1 Introduction to Gene Therapy ................................................................................... 1

1.2 Gene delivery vectors ................................................................................................ 4

1.3 Retroviruses ............................................................................................................... 5

1.4 Moloney Murine Leukemia Virus (MoMuLV) ......................................................... 6

1.5 Replication cycle of MoMuLV ................................................................................. 8

1.6 Limitations of retroviruses as gene therapy vectors ................................................ 10

CHAPTER 2. ALPHAVIRUSES AND PSEUDOTYPING ............................................ 12

2.1 Introduction ............................................................................................................. 12

2.2 Structure of alphaviruses ......................................................................................... 12

vi

Page

2.3 Replication cycle of Alphaviruses........................................................................... 15

2.4 Pseudotyping ........................................................................................................... 18

2.5 Generation of pseudotyped viruses ......................................................................... 19

CHAPTER 3. MATERIALS AND METHODS .............................................................. 22

3.1 Cell lines and cell culture ........................................................................................ 22

3.2 Plasmids .................................................................................................................. 23

3.3 Production of virus by transient expression of envelope glycoproteins .................. 24

3.4 Transduction assay .................................................................................................. 25

3.5 Immunoblot assay ................................................................................................... 25

3.6 Immunofluorescence assay ..................................................................................... 26

3.7 Addition of Heparinase I to Producer cells ............................................................. 27

3.8 Addition of Heparinase I to target cells .................................................................. 27

3.9 Normalization following quantitative analysis of the immunoblot assay ............... 27

3.10 Transduction assays in cells treated with lipid-lowering drugs ............................ 28

3.11 Production of baculovirus virus pseudotyped with alphavirus envelope

glycoproteins ................................................................................................................. 29

3.12 Transduction assay in C6/36 cells grown in sterol-free conditions ...................... 29

3.13 Transduction of ASMase-/- cells........................................................................... 29

vii

Page

3.14 Verification of cholesterol concentrations in ASMase-/- cells and ASMase+/+

cells................................................................................................................................ 30

CHAPTER 4. ROLE OF HEPARAN SULFATE IN ENTRY AND EXIT OF ROSS

RIVER VIRUS GLYCOPROTEIN-PSEUDOTYPED RETROVIRAL VECTORS ...... 31

4.1 Summary ................................................................................................................. 31

4.2 Introduction ............................................................................................................. 32

4.3 Results ..................................................................................................................... 38

4.4 Discussion ............................................................................................................... 53

CHAPTER 5. ROLE OF CHOLESTEROL IN ENTRY OF PSEUDOTYPES

INCORPORATING ALPHAVIRAL GLYCOPROTEINS AND NIEMANN PICK’S

DISEASE .......................................................................................................................... 57

5.1 Summary ................................................................................................................. 57

5.2 Introduction ............................................................................................................. 58

5.3 Results ..................................................................................................................... 62

5.4 Discussion ............................................................................................................... 70

REFERENCES ................................................................................................................. 72

VITA ................................................................................................................................. 85

viii

LIST OF TABLES

Table Page

1.1 Viral Vectors ................................................................................................................ 5

4.1 Paired T-test ............................................................................................................... 46

4.2 Pearson Coefficient BHK .......................................................................................... 49

4.3 Pearson Coefficient CHO22 ...................................................................................... 49

4.4 Pearson Coefficient CHO18. ..................................................................................... 49

ix

LIST OF FIGURES

Figure Page

Figure 1.1 Types of Gene Therapies ................................................................................... 2

Figure 1.2 MoMuLV particle .............................................................................................. 7

Figure 1.3 MoMuLV genome ............................................................................................. 7

Figure 1.4 Gag polyprotein ................................................................................................. 8

Figure 1.5 Replication cycle of MoMuLV ....................................................................... 10

Figure 2.1 Structure of Alphaviruses ................................................................................ 13

Figure 2.2 Alphaviral genome .......................................................................................... 14

Figure 2.3 Alphaviral Structural Proteins ......................................................................... 15

Figure 2.4 Replication cycle of alphaviruses .................................................................... 17

Figure 2.5 Pseudotyping ................................................................................................... 21

Figure 4.1 Structure of Heparan Sulfate ........................................................................... 35

Figure 4.2 RRV-E2 Envelope glycoprotein amino-acid sequence ................................... 36

Figure 4.3 RRV-E2 Envelope glycoprotein and HS ......................................................... 37

Figure 4.4 T216R-RRV and N218R-RRV pseudotypes have lower transduction titers .. 39

Figure 4.5 T216R-RRV and N218R-RRV envelope glycoproteins are inefficiently

incorporated in pseudotyped virus .................................................................................... 41

x

Figure Page

Figure 4.6 Intracellular retention of T216R-RRV and N218R-RRV in HS-expressing

cells ................................................................................................................................... 43

Figure 4.7 Addition of Heparinase I to Producer ØNXnlslacZ cells releases T216R-RRV

and N218R-RRV pseudotyped viruses ............................................................................. 46

Figure 4.8 Heparinase treatment of target cells reduces transduction by T216R-RRV and

N218R-RRV pseudotyped viruses .................................................................................... 48

Figure 4.9 T216R-RRV and N218R-RRV pseudotyped viruses possess higher

transduction efficiencies when transductions by viruses are normalized by glycoprotein

content ............................................................................................................................... 51

Figure 5.1 Cholesterol and Sphingolipid rich lipid rafts .................................................. 61

Figure 5.2 Alphavirus-MoMuLV pseudotypes require cholesterol for entry in mammalian

cells ................................................................................................................................... 64

Figure 5.3 Alphavirus-baculovirus pseudotypes require cholesterol for entry in insect

cells ................................................................................................................................... 66

Figure 5.4 Transduction efficiency of alphaviral pseudotypes is higher in ASMase-/- cells

........................................................................................................................................... 69

xi

LIST OF ABBREVIATION

SCID Severe Combined Immuno-deficiency syndrome

ADA

Adenosine deaminase

OTC Ornithine transcarbamylase

MoMuLV Moloney Murine Leukemia Virus

RRV Ross River Virus

HIV Human immunodeficiency virus

DNA Deoxyribonucleic acid

RNA Ribonucleic acid

RA Reverse transcriptase

IN Integrase

PR Aspartic protease

SU Surface unit

TM Transmembrane domain

PBS Primer binding site

PPT Polypurine tract

MA Matrix domain

CA Capsid

xii

NC Nucleocapsid

ER Endoplasmic reticulum

ESCRT Endosomal sorting complexes required for transport

SFV Semliki forest virus

SINV Sindbis virus

VEEV Venezuelan equine encephalitis virus

EEEV Eastern equine encephalitis virus

ORF Open reading frames

CMV Cytomegalovirus

LTR Long terminal repeats

BHK Baby hamster kidney cells

CHO Chinese hamster ovary cells

DMEM Dulbecco’s Modified Eagle’s Medium

FBS Fetal bovine serum

ASMase Acid sphingomyelinase

ASMase-/- cells Acid sphingomyelinase deficient cells

PBS Phoshate buffered saline

VSV Vesicular stomatitis virus

TU/mL Transduction Units/ml

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

ß-mCD Methyl-beta-cyclodextrin

HS Heparan sulfate

xiii

FIV Feline immunodeficiency virus

SD Standard deviation

HSV Herpes Simplex Virus

NPD Niemann Pick’s disease

xiv

ABSTRACT

Kesari, Aditi PhD. Purdue University, May 2016. Factors Affecting Transduction Efficiency of Pseudotyped Viral Vectors Incorporating Alphaviral Glycoproteins. Major PI: David A. Sanders

The genome of an organism has the complete set of biochemical instructions

required for sustenance of life. Mutations or abnormalities in this genome lead to genetic

disorders. Currently available therapeutic options mostly focus on treating the symptoms,

but not curing them. Gene therapy promises to be a curative form of medicine. In gene

therapy cells carrying a defective gene are targeted and replaced with a healthy copy of

that gene. The vehicles used for delivering this gene are known as vectors. Retroviruses

are popularly used gene therapy/transfer vectors. However, retroviruses are limited in the

range of cells they can enter and infect. The range of cells targeted by the viral vector can

be either expanded or narrowed by replacing the envelope of the virus with the envelope

of another virus that has the desired range of tissue tropism. Such hybrid viruses are

called pseudotyped viruses. Previously, we have pseudotyped Moloney Murine Leukemia

Virus (MoMuLV), a retrovirus with the envelope of Ross River Virus (RRV), an

alphavirus. Here, we substituted amino-acid residues of RR-envelope glycoprotein with

xv

basic amino-acid residues. We show that this makes the pseudotyped virus utilize

heparan sulfate, a ubiquitous molecule present on the surfaces of most cells, as an

attachment factor. Attachment to cellular heparan sulfate helps in concentration of virus

particles on the cell surface and thus enhancing their chances of cell entry, thereby

increasing their transduction efficiency of this viral vector. The same affinity towards

heparan sulfate, however, poses a challenge in terms of release of this pseudotyped virus

from the producer cells. General principles concerning viral adaptation to the use of

attachment factors and improving pseudotyped virus titers through modifying cell

membrane components can be derived from these results. We also show that alphavirus-

glycoprotein retroviral pseudotypes require cholesterol for entry into the cells.

Furthermore, excess cholesterol in cells facilitates the entry of alphavirus-glycoprotein

retroviral pseudotypes. We show that alphavirus-glycoprotein pseudotypes transduce the

acid sphingomyelinase deficient (ASMase-/-) cells derived from Niemann Pick’s disease-

model mice more efficiently than the cells from healthy mice (ASMase+/+). Thus

alphavirus-glycoprotein pseudotypes hold a potential as suitable vectors for gene therapy/

transfer in Niemann Pick’s Disease-Type A, a genetic lipid-storage disorder.

1

CHAPTER 1. GENE THERAPY/ TRANSFER VECTORS AND RETROVIRUSES

1.1 Introduction to Gene Therapy

The genome of an organism has the complete set of biochemical instructions

required for sustenance of life. If this genome carries any abnormality or mutation, it

leads to genetic disorders. Currently available therapeutic options mostly focus on

treating the symptoms but not curing them. Gene therapy promises to be a curative form

of medicine.

In gene therapy, cells carrying a defective gene are targeted and replaced with a

healthy copy of that gene. The gene delivery can be achieved by administering genetic

material into the body. This type of gene therapy is referred to as in vivo gene therapy

(Figure 1.1). Gene delivery can also take place outside the body, where affected cells are

removed from the body, delivered with genetic material followed by re-administering the

cells back in the body. This type of gene therapy is termed as ex vivo gene therapy

(Figure1.1).

2

Figure 1.1 Types of Gene Therapies

In ex vivo gene therapy, defective cells are removed from the body and cultured. The genetic material is introduced into the cells outside the body, before re-injecting the cells back into the body. In in vivo gene therapy the corrective gene is directly administered into the body.

Genetic material can be delivered either to the somatic (non-reproductive) cells or

to the germline cells. The effect of delivery of genetic material to the somatic cells

doesn’t pass on to the next generation, whereas the effect of gene delivery in germline

cells is carried on to the next generation. Thus, the therapeutic effect of germline gene

therapy, unlike somatic gene therapy, is permanent. However one potential hazard of the

former method is that the negative effects can also be permanently passed down to

subsequent generations (www.genetherapynet.com/viral-vectors.html n.d.).

Gene therapy to cure human diseases has been proposed since a long time. In

spite of advancement in this field, currently there is no approved treatment available. The

first attempt at gene therapy to treat diseases in humans was done in 1990. A 4 year old

3

girl suffering from Severe Combined Immuno-deficiency syndrome (SCID), a

monogenetic disease, which is caused due to the deficiency of adenosine deaminase

(ADA), was treated at NIH Clinical Center through an ex vivo gene therapy approach.

Her white blood cells were removed from her body and a healthy gene expressing ADA

was introduced into them. These cells were re-administered into her body (Blaese R

1995).

Multiple gene therapy/transfer studies were performed until late nineties. In 1999,

this field suffered a major set-back when a patient named Jesse Gelsinger, who had

participated in a clinical trial, died within a week of receiving the treatment. He had

deficiency of ornithine transcarbamylase (OTC), one of the enzymes required for

converting excess nitrogen in blood into urea. The gene expressing OTC was

administered using adenovirus vector. His body invoked a major immune reaction in

response to the high dosage of the virus (P. N.-H. Wirth T 2013). The failure of this trial

resulted in withdrawal of many studies.

Despite this, there have been many studies that have shown encouraging results

such as SERCA gene therapy for cardiac failure (Periasamy M 2008), gene therapy for

X-linked Severe Combined immunodeficiency syndrome (Hacein-Bey-Abina S 2010),

etc. China was the first country to approve a gene therapy product for therapeutic use (P.

N.-H. Wirth T 2013). However, this approval wasn’t backed up by clinical trial data.

Cerepro, an adenoviral vector carrying thymidine kinase gene, is the first gene therapy

product used for treating malignant brain tumors to have passed phase III clinical trial (S.

H.-H. Wirth T 2009). Glybera, an adeno-associated viral vector delivering lipoprotein

4

lipase gene, is the first gene therapy product to be recommend by European Medicines

Agency for approval in Europe (P. N.-H. Wirth T 2013). Given the recent advancements

in this field, gene therapy definitely seems to be an attainable therapeutic option in the

near future.

1.2 Gene delivery vectors

The vehicles used for delivering the genes are known as vectors. The gene

therapy/transfer vectors are broadly classified into viral and non-viral vectors. Liposomes

and naked DNA delivery have been used for non-viral gene delivery. Even though these

physical methods tend to be safer and less immunogenic than viral vectors, viral vectors

have proven to be efficient gene delivery vectors as compared to the non-viral ones (Li

SD 2006).

There are many viruses that can be used as gene delivery vectors. Table 1.1

discusses the properties of a few such viruses (www.genetherapynet.com/viral-

vectors.html n.d.).

5

Table 1.1 Viral Vectors

Virus Retrovirus/ lentivirus

Adenoviruses Adeno-associated

Herpesvirus

Family Retroviridae Adenoviredae Parvoviridae Herpesviridae

Types of cells entered

Dividing and non-dividing (only lentivirus)

Dividing and non-dividing



Integration with the host genome

Yes No Yes (lower frequency)

No

Expression of transgene

Long lasting Transient Long lasting Transient

Packaging capacity

8kb 7.5kb 4.5kb 30kb

Immune response

Low (moderate for lentivirus)

High Low Low

1.3 Retroviruses

Retroviruses are a large group of viruses responsible for causing

immunodeficiency syndromes and leukemias in vertebrates. These viruses contain a

single-stranded positive-sense RNA genome, which through the process of reverse

transcription forms a DNA intermediate. This DNA intermediate can integrate with the

host genome leading to formation of a provirus. Retroviruses are transmitted by coming

in close contact with infected animal, exposure to body fluids of infected animals,

6

maternofetal transmission, accidental inoculation and experimental infection (Coffin JM

1997). Retroviruses are increasingly being used as research tools in cell and molecular

biology.

Retroviruses like Human immunodeficiency virus (HIV), Moloney murine

leukemia virus (MoMuLV) etc. are used as vectors of gene therapy because they allow

fast and stable transfer of genetic material to cells, and have strong promoter system that

results in efficient and long lasting expression of genetic material. However, the host

range and tissue tropism of this viral vector are limited.

1.4 Moloney Murine Leukemia Virus (MoMuLV)

MoMuLV (gammaretrovirus genus) is one of the simplest retroviruses and hence is used

as a model for studying retroviruses. The MoMuLV is a polymorphic virion around 100-

120 A˚ in size (Figure 1.2) (Yeager M 1998). Two strands of single-stranded RNA

genome are incorporated into the virus particle. The viral genome has a coding region

and a non-coding region (Figure 1.3). Coding region has genes gag-pol-env that code for

the three polyproteins, Gag (Figure 1.4), Pol and Env respectively. These are involved in

assembly of the virion. The capsid of the virion is formed from Gag while the envelope is

developed from the Env polyprotein and the lipid bilayer of the host. The three enzymes-

reverse transcriptase (RA), integrase (IN) and aspartic protease (PR) are derived from the

Pol polyprotein (Rein 2011).

7

Figure 1.2 MoMuLV particle

The outermost layer is formed from Env glycoproteins and host-derived lipid bilayer. The Env glycoprotein is made up of the surface unit (SU), which contais the receptor binding domain and transmembrane (TM) unit, which has the fusion peptide. The lipid bilayer and the embedded glycoproteins constitute the envelope of the virus particle. Inside, lies the core made up of the Gag polyprotein. Within the core there are two strands of single-stranded RNA genome. Along with that, there is Pol, the polyprotein which comprises of three enzymes: (i) reverse transcriptase, which converts the viral RNA genome into a DNA copy, (ii) viral aspartic protease, which leads to maturation of the virion by cleaving the polyproteins into their respective domains and (iii) integrase, which processes the DNA copy of viral genome and inserts it into the host genome to form the provirus. (Figures from Rein A, Advances in Virology, 2011 and Silverman RH et al., Nature Reviews urology, 2010) (Rein 2011) (Silverman RH 2010)

Figure 1.3 MoMuLV genome

The coding region of the viral genome includes the gag, pol and env regions that encode Gag, Pol and Env polyproteins respectively. The non-coding region has a LTR sequence, which has all the promoters and enhancers, the psi (Ψ) packaging sequence, a primer binding site (PBS) and the polypurine tract (PPT), which acts a primer for synthesis of second strand of DNA (Shinnick TM 1981)

8

Figure 1.4 Gag polyprotein

Gag polyprotein is made up of a matrix (MA) domain, which lies on the outer side of the core and associates with the lipid bilayer through myristylation, a small proline rich p12 region, which is thought to promote the release of virus particles, a capsid (CA) domain and a nucleocapsid (NC) domain which lies on the inner side and is in association with the RNA genome (Rein 2011, Shinnick TM 1981)

1.5 Replication cycle of MoMuLV

MoMuLV infects the cell by receptor-mediated membrane fusion (Figure 1.5). The SU

subunit of the envelope glycoprotein of the virus binds to the cell surface receptor. This

causes structural change in the SU subunit which leads to disruption of the disulfide bond

between the SU and TM subunits (Freed EO 1987, Pinter A 1997), exposing the fusion

peptide of the TM subunit. Fusion between the viral and cell membranes releases the

capsid into the cytoplasm which later disintegrates and the viral RNA escapes into the

cytoplasm. The RNA genome is copied into DNA by reverse transcriptase. This DNA

enters the nucleus and gets inserted into the host genome with the help of viral integrase

to form a provirus. The integrated DNA is transcribed into mRNA which then travels out

of the nucleus. In the cytoplasm it is translated into the assembly proteins. Envelope

glycoproteins are synthesized from spliced mRNA and translocated to ER. Glycosylation

takes place in the Golgi along with the furin cleavage of Env polyprotein into the SU and

TM subunits. The SU-TM heterodimer is linked with a disulfide bond. Three

heterodimers come together to form trimers and these are then transported to the

membrane. Gag-Pol polyproteins, which are synthesized together as fusion protein co-

9

assemble at the membrane. Myristylation of the N terminal of Gag targets it to the plasma

membrane (Rein A 1986). An alternative glyco-Gag is also synthesized and is thought to

target the virus assembly towards lipid rafts (Nitta T 2011). The psi packaging sequence

helps the viral RNA to compete against the cellular RNA to be incorporated in the

budding virus. The late motif (p12) of Gag interacts with the ESCRT machinery, which is

involved in the budding process of the assembled virus (Rein 2011, Pornillos O 2002).

One of the cleavage products of the Pol polyprotein, the viral aspartic protease (PR),

plays a role in maturation of the virus particle as it cleaves the Gag molecule into its

respective subunits. The maturation step is crucial for the infectivity of the virus (Coffin

JM 1997).

10

Figure 1.5 Replication cycle of MoMuLV

Retrovirus entry is initiated when the virus particle binds to the cell surface receptor. Fusion between the viral and cell membrane releases the capsid into the cytoplasm. The viral RNA escapes into the cytoplasm upon disintegration of the capsid. RNA is copied by into DNA by reverse transcriptase. This DNA enters the nucleus and gets inserted in the host genome with help of viral integrase to form a provirus. The integrated DNA is transcribed into mRNA. mRNA is gets transported to the cytoplasm, where it is translated into the assembly proteins. Envelope proteins are synthesized from spliced mRNA and translocated to ER. Gag-Pol polyprotein which are synthesized together as fusion proteins co-assemble at the membrane. The psi sequence helps in packaging of the viral RNA into compete the budding virus. Viral aspartic protease cleaves the Gag molecule into its respective subunits. This step of maturation of the virus is crucial for its infectivity.

1.6 Limitations of retroviruses as gene therapy vectors

The retroviruses have three major limitations as gene therapy/transfer vectors.

Firstly, the host range and tissue tropism are not very extensive. This limits the types of

cells they can enter into. Secondly, there is a chance of recombination and formation of

11

replicating and competitive virus particles capable of causing active infection in

individuals receiving the treatment (Anson 2004). And finally, clinical trials in the past

have shown instances of insertional mutagenesis leading to oncogenesis (Anson 2004).

Pseudotyping of the virus can be used to overcome the first two limitations. It is a

process in which the envelope protein of the virus is replaced with an envelope protein of

other virus. Previously, we have pseudotyped retroviruses by replacing its envelope with

the envelope of alphaviruses in our lab. The details about alphaviruses and process of

pseudotyping are discussed in the subsequent chapter.

12

CHAPTER 2. ALPHAVIRUSES AND PSEUDOTYPING

2.1 Introduction

Alphaviruses are arthropod-borne viruses that cause severe infections in a wide

range of vertebrates. The clinical manifestations in infected humans include fever, rash,

arthralgia, myalgia, headaches and polyarthritis (Strauss J H 1994). Alphaviruses belong

to the Togaviridae Family and are classified into two categories: (i) Old World viruses

which include Semliki forest virus (SFV), Sindbis virus (SINV) and Ross River virus

(RRV) (ii) New World viruses which include Venezuelan equine encephalitis virus

(VEEV), Eastern equine encephalitis virus (EEEV).

2.2 Structure of alphaviruses

An alphavirus is an enveloped, single-stranded, positive-sense, RNA virus

(11.5kb) (Figure 2.1) (Strauss J H 1994). The RNA genome (Figure 2.2) is surrounded by

the capsid, which is made up of 240 units of capsid protein. This is in turn is surrounded

by an envelope, which is a lipid bilayer membrane with 80 spikes of envelope

glycoprotein embedded into it (Figure 2.3). E1, E2 and E3 are the three envelope

glycoproteins. Each spike is a trimer of an E1/E2 heterodimer (Cheng RH 1995).

Whereas E2 is required for binding to cell-surface receptors, E1 is required for fusion of

the viral and cellular membrane. The envelope and the inner nucleocapsid core have

13

icosahedral symmetry with triangulation number T=4 (Strauss J H 1994, Cheng RH

1995). The E2/E1 heterodimer has an one-one association with each nucleocapsid

monomer via the C-terminal of E2 (Cheng RH 1995).

Figure 2.1 Structure of Alphaviruses

Alphaviruses have 80 spikes of envelope glycoproteins (blue) embedded in the lipid bilayer (green), which forms its envelope. Inside this, lies the nucleocapsid core (yellow and red) made up of 240 units of Capsid proteins and a single strand of a positive-sense RNA genome. Figure adapted from Cheng et al., Cell, 1995 (Cheng RH 1995).

14

Figure 2.2 Alphaviral genome

An alphavirus has a single-stranded, positive-sense RNA genome. The nonstructural proteins are translated from the genomic RNA and are involved in replication of the genome, whereas the structural proteins are translated from the subgenomic RNA, with a promoter as shown in the figure.

15

Figure 2.3 Alphaviral Structural Proteins

Alphaviral structural proteins are expressed as polyproteins. The Capsid protein © cleaves itself after getting translated (Arrow 1). The N terminus of the remaining portion of polyprotein (envelope glycoproteins) has a signal sequence due to which it translocates into the endoplasmic reticulum, while another signal sequence anchors it in the membrane. A proteolytic cleavage after this signal peptide releases E3-E2 (pE2) (Arrow 3) (Garoff H 1990). Another proteolytic cleavage at C terminal of heavily palmitoylated 6kD segment (Arrow 4), releases E1 which is then anchored by stop-transfer signal. Cleavage between E3 and E2 takes place in trans-golgi and is mediated by furin-like protease (Arrow 2)

2.3 Replication cycle of Alphaviruses

The virus enters the cell via receptor-mediated endocytosis (Figure 2.4) (DeTulleo L

1998). The presence of a cholesterol and sphingolipid rich bilayer has been shown to be

required for entry of SINV and SFV (H. A. Kielian MC 1984, Phalen T 1991, Lu YE

1999). Once inside the endosome, the acidic pH leads to dissociation of the E1-E2

heterodimer complex exposing the hidden fusion loop of the E1 glycoprotein. Insertion of

the fusion loop into the endosomal membrane is followed by formation of E1-homotrimer

complex (H. A. Kielian MC 1985, Boggs WM 1989, Justman J 1993, Li L 2010). This

16

fusion process releases nucleocapsid core into the cytoplasm. Disassembly of the capsid

releases he RNA genome, which is translated from two ORFs to form (I) the non-

structural polyprotein (P1234), which is required for the replication of the genome (early

phase) and (II) the structural polyprotein (late phase). The positive strand is first

converted to a negative strand of RNA, which is predominant during early phase. This

negative strand, during the late phase of replication cycle is either replicated into full-

length positive genomes, which are incorporated into new virion or it is spliced into

subgenomic positive-sense RNA. The subgenomic RNA is translated into structural

polyprotein (C-pE2-6K-E1) (Jose J 2009) (Leung JY 2011). Upon cleavage, Capsid

protein starts oligomerizing and then associates with the single-stranded RNA genome to

form the nucleocapsid (Tellinghuisen TL 1999). The cleavage of Capsid protein is

followed by the processing of the remaining polypeptide, which now consists of envelope

glycoproteins. The N-terminus of this polypeptide has signal sequence which helps in

translocation of the polypetide into the ER. A 30 residue-long signal sequence anchors C-

terminus of the polypeptide. A proteolytic cleavage after this signal peptide releases E3-

E2 (pE2) (Garoff H 1990). Another proteolytic cleavage takes place in remaining the

polypeptide between the hydrophobic 6K and E1 protein (Liljeström P 1991). The E1 and

pE2 undergo complex folding with the help of chaperones. Disulfide bond formation

takes place to form the pE2-E1 heterodimer in the ER (Anthony RP 1992). Post-

translational modifications like glycosylation, palmitoylation in case of E2 follow this

(Sefton 1977). Once the heterodimer reaches trans-golgi, furin-like protease cleaves E3

from E2 (Gaedigk-Nitschko K 1990). This cleavage is essential for entry of virus

(Salminen A 1992 ). The glycoproteins are transported to the plasma membrane where

17

the virions are assembled. Interaction between nucleocapsid unit and E2 takes place and

budding occurs (Cheng RH 1995).

Figure 2.4 Replication cycle of alphaviruses

Alphaviruses enter the cell via receptor mediated endocytosis. The process of fusion is triggered by the acidic pH in the endosomes. The RNA gnome is released in the cytoplasm and translated into non-structural polyproteins, which are required for replication of the genomic RNA, and structural polyproteins. The capsid proteins, which are derived from the structural polyproteins and the genomic RNA form nucleocapsid cores, which are transported to the plasma membrane. Envelope glycoproteins, which are derived from the structural polyprotein, reach the plasma membrane through secretory pathway. The assembly of the virus particle takes place at the plasma membrane, which is followed by budding. Figure adapted from Schwartz et al., Nature Reviews Microbiology, 2010 (Schwartz O 2010).

18

2.4 Pseudotyping

Viruses are commonly used as vectors to deliver a desired gene to target cells for

the purpose of gene therapy or gene transfer. For gene therapy/transfer, one either needs

to expand the host range of the gene delivery vector or narrow it down to a specific

target. The recognition and infection of host cells by viruses depends on their envelope

proteins. Hence by incorporating the envelope proteins of other viruses, the ability to

enter cells other than those a virus normally infects can be conveyed to this parent virus.

The chimeric virus having the desired host range/tissue tropism thus formed is called a

pseudotyped virus (Sanders 2002).

Pseudotyped viruses have many experimental and clinical applications. Gene

therapy/transfer is one of the important applications of these pseudotyped viruses. Based

on the therapeutic need, the range of cells targeted by the viral vector for gene

therapy/transfer can be modified by pseudotyping the virus with the envelope of another

virus (Sanders 2002). Apart from their utility as gene-delivery vectors, pseudotyped

viruses can also be used as tools to study the entry of enveloped viruses.

Retroviruses, such as human immunodeficiency virus (HIV), feline

immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMuLV) are used

as gene-delivery vectors, because they allow fast and stable transfer of genes under the

influence of strong promoters to cells (Naldini L 1996, Kafri T 1997, Poeschla EM 1998,

Noh MJ 2010). However, they have limited host range and tissue tropism. For the

treatment of systemic genetic disorders a viral vector needs to be pseudotyped with an

envelope of a virus that has a broad tissue tropism. Since RRV, an alphavirus, has wide

host range and tissue tropism, it was incorporated in ØNX pseudotyped system to

19

produce RRV-MoMuLV pseudotyped virus (Sharkey CM 2001). Recombinant

retroviruses and lentiviruses bearing alphavirus glycoproteins have been shown to be

efficient gene transfer/therapy vectors (Sharkey CM 2001, Kang Y 2002). Previous

studies showed that pseudotyping with RRV was far less cytotoxic but equally efficient

as pseudotypes incorporating envelope glycoproteins of Vesicular Stomatitis virus (VSV-

G), a prototype pseudotyped virus (Kang Y 2002). Retroviruses pseudotyped with RRV

envelope glycoproteins were successfully used for in vivo gene-transfer studies in

hepatocytes, glial cells, muscle tissue and airway epithelial tissue (Kang Y 2002).

2.5 Generation of pseudotyped viruses

Producer ØNXnlslacZ cells (a second generation 293-T based retroviral packaging cell

line that is stably transfected with a MoMuLV gag-pol plasmid and the MFG.S-nlslacZ

plasmid, which produces envelope-protein-deficient, replication-incompetent MoMuLV

particles and encodes nuclear-localized β-galactosidase) are transfected with plasmid

encoding for alphaviral glycoproteins to generate alphaviral-MoMuLV pseudotypes

(Figure 2.5) (Sharkey CM 2001). The gag-pol plasmid expresses the core proteins of

MoMuLV under the influence of RSV promoter. The MFG.S-nlslacZ plasmid has the

gene of choice, which for experimental purposes is lacZ, along with the nuclear

localizing sequence (nls), LTR and psi (Ψ) sequence. The Ψ sequence helps in packaging

of LacZ into the budding pseudotyped virus. These naked nucleocapsid cores however,

are incapable of transducing the cells and require an envelope to do so. Hence these

producer ØNXnlslacZ cells are transfected with plasmid encoding the envelope protein

of other viruses, such as alphaviruses. The plasmid encoding for alphaviral glycoproteins

20

is expressed under the influence of CMV promoter. The retroviral promoter in the LTR

region is required for in expression of LacZ gene into β-galactosidase. β-galactosidase

then gets translocated to the nucleus under the effect of nuclear localizing sequence.

The pseudotyped viruses generated using this method are non-infectious and

replication incompetent. By replacing the MFG.S-nlslacZ gene with a vector-expressing

gene they can be used as efficient gene-delivery agents. Since the three plasmids involved

in formation of these pseudotypes have three different promoters, the chances of

recombination are very minimal. This makes the retroviral pseudotypes incorporating

alphaviral glycoproteins ideal candidates as gene-therapy/ gene-transfer vectors.

21

Figure 2.5 Pseudotyping

Alphaviral-MoMuLV pseudotypes are generated by transfecting producer ØNXnlslacZ cells with a plasmid encoding alphaviral glycoproteins (figure shows RRV-envelope plasmid). The ØNXnlslacZ cell-line is a second generation 293-T based retroviral packaging cell line that is stably transfected with the MoMuLV gag-pol and MFG.S-nlslacZ plasmids. The pseudotyped viruses generated using this method are replication incompetent and function as gene-delivery agents.

22

CHAPTER 3. MATERIALS AND METHODS

3.1 Cell lines and cell culture

Producer ØNXnlslacZ cells (a second generation 293-T based retroviral packaging cell

line that is stably transfected with MoMuLV gag-pol plasmid and MFG.S-nlslacZ

plasmid, which produces envelope-protein-deficient, replication-incompetent MoMuLV

particles that encode nuclear-localized β-galactosidase) (Sharkey CM 2001); BHK;

CHO22; CHO18.4 (a mutant cell line derived from CHO22 not expressing HS) (Heil ML

2001, Jan J 1999) [generously donated by Dr. Richard Kuhn’s laboratory] were grown in

Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% heat-inactivated fetal bovine

serum (FBS) and penicillin (10 U/mL ) at 37°C in 5% CO2. NIH 3T3 cells were grown in

DMEM with 10% calf serum (CS) and penicillin (10 U/mL) at 37°C in 5% CO2. Sf9

insect cells were grown in Sf9/III medium with 3% FBS at 28 °C without CO2. C6/36

mosquito cells were grown in DMEM with 10% heat-inactivated-FBS at 28 °C in 5%

CO2, as well as in DMEM with de-lipidated heat-inactivated-FBS at 28 °C in 5% CO2.

De-lipidated serum was obtained by centrifugation of serum in presence of Cab-O-SiL

(Cabot Co.). Cab-O-SiL acts by adsorbing lipids in the serum. ASMase-/- mutant mouse

skin fibroblast cells and ASMase+/+ wild-type mouse skin fibroblast cells (kindly)

23

donated by Dr. Glyn Dawson’s Laboratory) (Qin J 2012), were grown in DMEM with

10% heat-inactivated-FBS at 37 °C in 5% CO2.

3.2 Plasmids

Regions of the RRV cDNA containing amino acid substitutions T216R, T216V, N218R

or N218V (Heil ML 2001) [kind gifts from Dr. Richard Kuhn’s laboratory] were

removed through restriction endonuclease cutting with ScaI and RsrII. These enzymes cut

the genomic RRV clone at nucleotides 9201 and 9568 respectively. Each of these

fragments was individually ligated with ScaI/BglII fragment spanning nucleotides 12-

1766 and the large RsrII/BglII fragment (resulting from a partial RsrII digest) from

nucleotide 2133 to nucleotide 12. These plasmids are designated pRRV-E2E1A-T216R,

pRRV-E1E2A-T216V, pRRV-E2E1A-N218R and pRRV-E2E1A-N218V respectively

according to the single amino acid change in each glycoprotein coding sequence. The

integrity of each cloned plasmid was confirmed by sequence analysis.

Plasmids expressing envelope glycoproteins of alphaviruses namely pRRV-

E1E2A (Sharkey CM 2001), pVEEV-E1E2, pSINV-E1E2 and pSFV-RRV-E1E2 were

constructed by digesting the cDNA of viruses (RRVand SFV- BamHI and XbaI, VEEV-

HindIII and XbaI, SINV-BamHI and XhoI) and ligating the fragment containing E3-E2-

6K-E1 coding region into pcDNA3.1/zeo (+) cut with same enzymes. VSV-G envelope

plasmid, pCMV-G, (Kang Y 2002) and penv1min (Taylor GM 1999) were used for

expression of Vesicular Stomatitis virus envelope glycoproteins and MoMuLV envelope

glycoproteins respectively. pBACgus1-RRV-E1E2 was constructed by digesting pRRV-

E1E2A with BglII and AvrII and inserting the fragment containing the E3-E2-6K-E1

24

coding region into pBACgus1 plasmid cut with the same enzymes. Bacmid bMW033,

which expresses replication-competent baculovirus with intact envelope glycoprotein,

gp64, and bacmid expressing baculovirus cores deficient in gp64, bMW024, were kindly

gifted by Dr. Colin T. Dolphin’s laboratory (Westenberg M 2013).

3.3 Production of virus by transient expression of envelope glycoproteins

4,000,000 producer ØNXnlslacZ cells were grown overnight on 10 cm plates. The

following day they were washed with phosphate-buffered-saline solution (PBS), and 10

mL of fresh growth medium was added to them. Plasmids expressing RRV envelope

glycoproteins: pRRV-E1E2A, pRRV-E1E2A-T216R, pRRV-E1E2A-N218R, pRRV-

E1E2A-T216V and pRRV-E1E2A-N218V were incubated with 12 µL PLUS reagent

(Life Technologies) in DMEM for 15 minutes followed by incubation with 18 µL

Lipofectamine (Life Technologies) for 15 minutes in DMEM at room temperature. Each

of these plasmids was then added to the respective plates containing the producer cells

grown overnight, and the plates were incubated at 37 °C in 5% CO2. 3 hours after

incubation the liquid from each plate was replaced with 10 mL of fresh growth medium.

This was further incubated for 48 hours at 37 °C in 5% CO2 after which the supernatant

medium containing RRV-MoMuLV pseudotyped virus was collected from each of these

plates.

4,000,000 producer ØNXnlslacZ cells were grown overnight in 10 cm plates. The

following day, they were washed with phoshate buffered saline solution (PBS) and 10

mL fresh medium was added to them. Envelope glycoproteins expressing plasmids

pRRV-E1E2A, pVEEV-E1E2, pSINV-E1E2 and pSFV-E1E2 were incubated with 18 µL

25

of Lipofectamine 2000 (Life Technologies) for 15 minutes in DMEM at room

temperature. Each transfection mix containing the envelope plasmids was then added to

the respective plates containing the producer ØNXnlslacZ cells grown overnight, and

then the plates were incubated at 37 °C in 5% CO2. 4 hours after incubation the medium

from each plate was replaced with 10 mL fresh medium and this was further incubated

for 48 hours at 37 °C in 5% CO2. The supernatant media overlying these cells contained

pseudotyped virus. MoMuLV pseudotyped with MoMuLV glycoproteins and MoMuLV

pseudotyped with VSV-G were produced similarly.

3.4 Transduction assay

10 mL of supernatant media overlying producer ØNXnlslacZ cells were collected 48

hours after transfection and passed through a 0.45 µm filter. Hexadimethrine bromide (5

µg/mL) was added to the filtered supernatant media. This media was used to transduce

target cell-lines, NIH 3T3, BHKs, CHO22 and CHO18.4 (a mutant cell line derived from

the parental CHO22 that does not express HS). After 4 hours of incubation, the media

were replaced with fresh media. 48 hours after transduction, cells were stained with X-gal

(Taylor GM 1999).Transduction units were then calculated by the formula: Transduction

Units/ml (TU/mL) = (No. of blue cells/Total number of cells)*(No. of cells

plated/Volume of media added).

3.5 Immunoblot assay

10 mL of supernatant media from producer cells transfected with different plasmids were

passed through a 0.45 µm filter. The virus particles were collected by ultra-centrifugation

of the supernatant media through a 30% sucrose cushion in Beckman 50.2 Titanium rotor

26

at 28000 rpm. The pellets of virus particles accumulated at the bottom were suspended in

SDS-PAGE buffer containing 2% β-mercaptoethanol. The viral pellets were analyzed by

SDS-PAGE gel and then transferred to nitrocellulose paper. Presence of the envelope

glycoproteins RRV-E2 and RRV-E1 along with the MoMuLV-Capsid (p30) proteins

was detected by incubating with rabbit anti-E2 (1:5000), rabbit anti-E1 (1:5000) and goat

anti-Raucher Leukemia virus-Capsid (p30) polyclonal primary antibodies (1:1000)

respectively. Anti-rabbit antibodies (Chemicon) and anti-goat antibodies coupled with

horseradish peroxidase were used as secondary antibodies (1:5000) to detect RRV

envelope glycoproteins and MoMuLV capsid proteins respectively. The amount of

protein present was measured based on chemiluminescence using FluorChem E imaging

system. The lysate and cell-debris samples were obtained by lysing the producer

ØNXnlslacZ cells transfected with different RRV envelope plasmids, using lysis buffer

containing 1% Triton-X 100, 50 mM Tris, 5 mM EDTA and 150 mM NaCl. Immunoblot

analysis of these preparations was also done as above.

3.6 Immunofluorescence assay

48 hours after transfection of CHO22 and CHO18.4 cell lines with RRV-envelope

encoding plasmids, cells were washed with PBS, fixed with 2% formaldehyde and

permeabilized with 0.1% Triton-X 100. This was followed by blocking them with 1%

BSA solution after which they were incubated overnight at 4°C with (1:100) primary

Anti-E2 (rabbit) antibodies. After washing off the excess primary antibodies, cells were

incubated with (1:500) Alexa Fluor 488-coupled anti-rabbit antibodies for 1-2 hours at

27

room temperature followed by removal of excess antibodies by washing. The cells were

visualized by fluorescence microscopy (Olympus IX) using MetaMorph software.

3.7 Addition of Heparinase I to Producer cells

44 hours after transfecting producer ØNXnlslacZ cells with RRV envelope plasmids, the

supernatant media overlying the cells were replaced with fresh media, and the cells were

incubated with 6 µg/mL Heparinase I at 37°C. After 4 hours the supernatant media were

collected and passed through a 0.45 µm filter. Hexadimethrine bromide (5 µg/mL) was

added to the filtered supernatant media. These media were used to transduce target cell

lines (NIH 3T3, BHKs, CHO22 and CHO18.4)

3.8 Addition of Heparinase I to target cells

Target cells (BHK, CHO22 and CHO18.4) grown overnight were incubated with

increasing concentrations (0, 2, 4, 6 µg/mL) of Heparinase I, an enzyme that cleaves HS

from the cell surface, for one hour at 37°C. After being washed with PBS, these cells

were transduced using supernatant media from producer ØNXnlslacZ cells as explained

earlier.

3.9 Normalization following quantitative analysis of the immunoblot assay

10 mL supernatant media from producer cells transfected with different plasmids were

collected and replaced with 10 mL fresh media. Collected media were passed through a

0.45 µm filter and subjected to ultracentrifugation in a Beckman 50.2 Titanium rotor at

28000 rpm with a 30% sucrose cushion to obtain pellets of viral particles. An

immunoblot assay of virus pellets using anti-E2 antibodies was performed. Using Image J

software, the amount of RRV-E2 glycoprotein in each virus pellet was quantified. Based

28

on these values the media overlying the producer cells was normalized across all the

samples and used for transduction of target cells (NIH 3T3, BHK, CHO22 and

CHO18.4). Normalization was performed by appropriate dilutions of the media

containing WT-RRV, T216R-RRV, T216V-RRV, N218R-RRV and N218V-RRV

pseudotyped viruses.

3.10 Transduction assays in cells treated with lipid-lowering drugs

The target cells (BHK and NIH-3T3) were incubated with 4 µg/mL of lovastatin (Sigma-

Aldrich) for 12 hours followed by another 12 hours of incubation with 2.5 mM of methyl-

beta-cyclodextrin (ß-mCD) (Sigma-Aldrich) 4 µg/mL of lovastatin, after which they were

washed with phosphate-buffered-saline solution (PBS). Supernatant media overlying the

producer ØNXnlslacZ cells were replaced with serum-free media, and the cells were

incubated for 5 hours. These serum-free media containing the pseudotyped viruses were

collected after passing them through a 0.45 µm filter. Hexadimethrine bromide (5 µg/mL)

was added to filtered supernatant media. These media were added to the target cells,

NIH-3T3 and BHK for transduction. After 24 hours of incubation, the media were

replaced with fresh media. 48 hours after transduction, cells were stained with X-gal.

Transduction units were then calculated by the formula (TU/mL) = (No. of blue cells/

Total number of cells)*No. of cells plated/ Volume of media added. These experiments

were also performed by replenishing the cholesterol content of cholesterol-depleted cells.

Repletion of cholesterol was done by incubating cholesterol-depleted cells in media

containing 50 µg/mL of cholesterol for 1 hour.

29

3.11 Production of baculovirus virus pseudotyped with alphavirus envelope

glycoproteins

500,000 Sf9 cells were grown for an hour on 6 cm plates. Following that the media were

replaced with 6 mL of fresh Sf9/III + 3% FBS media. Plasmid pBACgus1-RRV-E1E2

was coincubated with bMW024 and 18 µL of Cellfectin (Life technologies) for 30

minutes in 500 µL Sf9/III medium at room temperature. The transfection mix was added

to the plate containing Sf9 cells. 24 hours after incubation the medium from the plate was

replaced with 6mL fresh medium and this was further incubated for 4 days at 28 °C. The

supernatant medium containing baculovirus pseudotypes was passed through a 0.45 µm

filter and used for the transduction assay. Baculovirus incorporating VSV-G was

produced by co-transfection of pBACgus1-VSV-G and bMW024. Replication-competent

baculovirus was produced by transfection of cells with bMW033.

3.12 Transduction assay in C6/36 cells grown in sterol-free conditions

5 µg/mL of hexadimethrine bromide was added to filtered supernatant media containing

the baculovirus pseudotypes. These media were added to the C6/36 cells grown in 6 well

plates. After 12 hours of incubation, the media were replaced with fresh media. 72 hours

after transduction, cells were visualized under a fluorescent microscope (Olympus IX)

using MetaMorph software. Transduction units were calculated using the formula stated

above after counting the fluorescent cells.

3.13 Transduction of ASMase-/- cells

MoMuLV pseudotypes with alphavirus glycoproteins were produced as mentioned

above. The supernatent media overlying producer ØNXnlslacZ cells were used to

30

transduce target ASMase-/- and ASMase+/+ cells. The media were replaced with fresh

media after 24 hours. 48 hours after transduction, cells were stained with X-gal and

counted. Transduction units were then calculated.

3.14 Verification of cholesterol concentrations in ASMase-/- cells and ASMase+/+

cells

The difference in the concentration of cholesterol in ASMase-/- and ASMase+/+ cells

was verified by a fluorometric cholesterol assay. Cholesterol was extracted from one

million cells by lysing them using 200 µL of chloroform: methanol solution (v/v 2:1).

These samples were dried by allowing the solvent to evaporate at room temperature and

resolubilized using cholesterol-assay buffer. The concentrations of cholesterol in these

cells were determined using fluorometric cholesterol assay kit (Caymen Chemicals) and

96-well FLX microplate reader.

31

CHAPTER 4. ROLE OF HEPARAN SULFATE IN ENTRY AND EXIT OF ROSS

RIVER VIRUS GLYCOPROTEIN-PSEUDOTYPED RETROVIRAL VECTORS

4.1 Summary

The role of heparan sulfate (HS) in alphavirus entry is a topic of considerable ongoing

investigation. Variants of Ross River virus (RRV) that bind to HS have previously been

selected by serial passaging in cell culture. To explore the effects of mutations that

convey HS utilization specifically upon the process of entry, we pseudotyped Moloney

Murine Leukemia virus (MoMuLV), with the envelope of RRV. We substituted amino-

acid residues 216 and 218 on RRV-E2-envelope glycoprotein with basic amino-acid

residues, because these mutations were previously shown to confer upon the virus an

ability to bind HS. However, pseudotyped virus incorporating the basic amino-acid

substitutions possessed lower transduction titers. Using immunoblot and

immunofluorescence assays, we demonstrate that the affinity towards HS impeded

release of pseudotyped virus from producer cells. Addition of heparinase to the HS-

expressing target cells reduces the transduction efficiency of the virus carrying the basic

amino-acid substitutions, whereas no such effect is seen in cells lacking HS. These virus

particles had enhanced transduction capacity that was dependent upon utilization of HS

as an attachment factor. However, increased affinity towards HS also affected viral egress

32

negatively. This is reminiscent of the interaction between influenza virus and sialic acid,

in which the former utilizes the latter as an attachment factor for its entry, but the same

sialic acid, in the absence of neuraminidase, hinders viral release. General principles

concerning viral adaptation to the use of attachment factors and improving pseudotyped

virus titers through modifying cell membrane components can be derived from these

results.

4.2 Introduction

Viruses are commonly used as vectors to deliver a desired gene to the target cells

for the purpose of gene therapy or gene transfer. Retroviruses, such as human

immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), and Moloney

murine leukemia virus (MoMuLV) may be useful as gene-delivery vectors, because they

allow stable transfer of genes under the influence of strong promoters to cells (Naldini L

1996, Kafri T 1997, Poeschla EM 1998, Noh MJ 2010). Whereas the recognition and

infection of host cells by viruses depends on their envelope proteins, incorporating the

envelope proteins from other viruses can convey to the parent virus the ability to enter

different cells than those it normally infects. These chimeric viruses are called

pseudotyped viruses. Recombinant retroviruses and lentiviruses bearing alphavirus

glycoproteins have been created and have been shown to be efficient gene

transfer/therapy vectors (Sharkey CM 2001, Kang Y 2002). Apart from their utility as

gene transfer/therapy vectors, alphavirus pseudotypes have been used as tools to study

the entry of alphaviruses.

33

Alphaviruses are arthropod-borne viruses that cause severe infections in a wide

range of vertebrates. The clinical manifestations in infected humans include fever, rash,

arthralgia, myalgia, headaches and polyarthritis (Strauss J H 1994). Alphaviruses are

classified into two categories: (i) Old World viruses, including Ross River virus (RRV),

Semliki Forest virus (SFV), Chikungunya virus (CHKV) and Sindbis virus (SINV) and

(ii) New World viruses, Venezuelan equine encephalitis virus (VEEV) and Eastern

equine encephalitis virus (EEEV) (Strauss J H 1994).

An alphavirus is an enveloped, single-stranded, positive-sense RNA virus

(11.5kb) (Strauss J H 1994). The RNA genome is surrounded by the capsid shell, which

is made up of 240 units of the capsid protein. This in turn is surrounded by an envelope

consisting of the glycoproteins E1, E2, and E3 embedded in a cell-membrane-derived

lipid bilayer. Each spike is a trimer of an E1/E2 heterodimer (Cheng RH 1995), and E3 is

non-stoichiometrically abundant. The envelope and the inner nucleocapsid core share

icosahedral symmetries with triangulation number T=4 (Strauss J H 1994, Cheng RH

1995).

RRV, like other alphaviruses, has a wide host range and tissue tropism, as their

infectious cycle requires infecting both mammals and insects (Jose J 2009, C.-V. C.

Kielian MC 2010, Kahl CA 2004). Alphaviruses enter the cells via receptor-mediated

endocytosis (DeTulleo L 1998, Sharkey CM 2001, C.-V. C. Kielian MC 2010). Binding

to receptors and attachment factors is mediated by E2 glycoproteins, whereas E1

glycoproteins mediate fusion of the viral and endosomal membranes (Strauss J H 1994,

Smith TJ 1995). Many proteinaceous receptors are thought to be responsible for

internalization of alphaviruses particles. However, these receptors have not been

34

characterized completely (C.-V. C. Kielian MC 2010, Jose J 2009). Cell-surface

attachment factors, which facilitate the initial binding of alphaviruses and thus increase

the concentration of virus particles on the cell surface, such as DC-SIGN and L-SIGN

have been suggested to play a role in alphaviral entry (N. E. Klimstra WB 2003).

Heparan sulfate (HS), an attachment factor used by a number of viruses, such as human

papilloma virus, hepatitis C virus, most herpes viruses, adeno-associated virus, dengue

virus, vaccinia virus, yellow fever virus and human respiratory syncytial virus (Giroglou

T 2001, Barth H 2003, S. P. Shukla D 2001, Summerford C 1998, Chen Y 1997, Chung

CS 1998, Germi R 2002, Feldman SA 2000), has also been proposed to be involved in

alphaviral entry (G. D. Byrnes AP 1998, Zhu W 2010, C.-N. J. Gardner CL 2013). In

particular, it has been proposed that capacity to use HS as an attachment factor plays a

role in the tissue tropism of Eastern Equine Encephalitis virus (E. G. Gardner CL 2011).

HS is ubiquitously found in the human body on the cell surfaces and in the

extracellular matrix. It is a glycosaminoglycan made up of repeating units of uronic acid

and glucosamine with varying degrees of sulfation (Figure 4.1), which confers a negative

charge (Lopes CC 2006). This negative charge allows ionic interaction with cationic

molecules.

35

Figure 4.1 Structure of Heparan Sulfate

HS is glycosaminoglycan made up of repeating units of uronic acid and glucosamine with varying degrees of sulfation.

Sindbis virus has been shown to use HS as an attachment factor (G. D. Byrnes AP

1998). It was later shown that serial passaging in cell culture led to adaptive mutations in

Sindbis virus, which led to its selection of HS as an attachment factor (R. K. Klimstra

WB 1998). Similar studies done in RRV revealed that serial passaging in chick embryo

fibroblasts led to mutations at position 218 on the E2 envelope glycoprotein that resulted

in replacement of the original Asparagine residue with Lysine (Kerr PJ 1993).

Incorporation of these N218R and N218K mutations in RRV was shown to permit RRV

to utilize HS as an attachment factor (Heil ML 2001). Similarly, basic amino-acid

substitutions of residue 216 of RRV leads to utilization of HS as attachment factor (ML

2001). It was also shown that even though substitutions did not result in formation of the

HS-binding motifs XBBXBX or XBXBBBX (Figure 4.2), the electrostatic interaction

between negatively charged HS and basic amino-acids residues clustered around the

receptor-binding region of the E2 glycoprotein (Figure 4.3 A and B) made utilization of

HS as an attachment factor possible (Heil ML 2001, Zhang W 2005).

36

Figure 4.2 RRV-E2 Envelope glycoprotein amino-acid sequence

Amino acid residue T216 and N218 (arrows) of RRV-E2 envelope glycoprotein were substituted with basic amino-acid residues, namely arginine. The amino-acid sequence of the RRV-E2 envelope glycoprotein (Figure 4.2A) contains other basic amino-acid residues in the vicinity of these two amino-acid residues (asterisk).

37

Figure 4.3 RRV-E2 Envelope glycoprotein and HS

We have previously described pseudotyping of recombinant retroviruses and

lentiviruses with alphaviral glycoproteins, including those of RRV (Sharkey CM 2001,

Kang Y 2002, Kahl CA 2004). In vivo gene transfer to hepatocytes and neuroglial cells

was successfully achieved by pseudotyping lentivirus with RRV envelope glycoproteins

Substitution of amino-acid residues T216 and N218 with basic amino-acid residues does not result in formation of either of the HS binding motifs, XBBXBX or XBXBBBX. However, these residues along with other basic amino-acid residues cluster around the peak of the RRV-E2 envelope glycoprotein (Figure 4.3A) (Zhang W 2005). Thus, the receptor-binding region of RRV-E2 glycoprotein develops electropositive charge around itself. This results in increased affinity of RRV-E2 glycoprotein towards negatively charged heparan sulfate. Cryo-electron microscopy imaging shows HS bound to N218R RRV-E2 glycoprotein (Figure 4.3B). Figure from Zhang et al., Virology, 2005 (Zhang W 2005).

A

B

38

(Kang Y 2002). In order to explore additional potential advantages of alterations to the

viral glycoproteins for transduction by RRV-MoMuLV pseudotyped virus, we created

RRV-envelope glycoproteins with the substitutions of basic amino-acid residues that had

been previously demonstrated to promote utilization of HS as an attachment factor for

RRV. We demonstrate that these substitutions promote the utilization of HS by the RRV

glycoprotein-pseudotyped retrovirus. Interestingly, these substitutions also appear to

reduce the release of pseudotyped virus from producer cells. Incorporation of

substitutions therefore has both advantages and disadvantages for gene transduction by

alphavirus-pseudotyped retroviruses and lentiviruses.

4.3 Results

4.4.1 Transduction by T216R-RRV and N218R-RRV pseudotyped viruses is lower

in both HS expressing as well as non-expressing cell lines

To determine whether substituting amino-acid residues of RRV-envelope glycoproteins

with basic amino-acid residues made the virus utilize HS and thereby increase its

transduction efficiency, T216R-RRV and N218R-RRV pseudotyped viruses were

studied. Pseudotypes with no substitutions in envelope glycoproteins, WT-RRV, and

pseudotypes with envelope glycoproteins containing substitutions of residues 216 or 218

with Valine, T216V-RRV and N218V-RRV, were used as controls. Transduction assays

were performed using these pseudotyped viruses. The data show that the transduction

titers of WT-RRV, T216V-RRV, N218V-RRV pseudotyped viruses were higher than

those of pseudotyped viruses with basic amino-acid substitutions (T216R-RRV and

N218R-RRV) in both HS expressing as well as non-expressing cell lines (Figure 4.4).

39

Figure 4.4 T216R-RRV and N218R-RRV pseudotypes have lower transduction titers

Supernatant media overlying producer ØNXnlslacZ cells producing WT-RRV, T216R-RRV, T216V-RRV, N218R-RRV and N218V-RRV pseudotyped viruses respectively were collected and passed through a 0.45 µm filter. Hexadimethrine bromide (5 µg/mL) was added to the filtered supernatant media. This media was used to transduce target cell-lines (A) NIH 3T3, (B) BHKs, (C) CHO22 (parent cell-line) and (D) CHO18.4 (cell-line not expressing HS). The transduced target cells were stained with X-gal and transduction titers were calculated. The experiments were repeated thrice. The values shown by the graph represent the mean TU/mL values (± SD) calculated from these experiments.

0

5000

10000

15000

20000

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L)

BHK

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CHO18.4

01000200030004000500060007000

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CHO22

A B

C D

40

4.3.2 T216R-RRV and N218R-RRV envelope glycoproteins are retained in the

producer cells

To investigate the reason for lower transduction titers in T216R-RRV and N218R-RRV,

immunoblot assays were carried out. Immunoblot analysis (Figure 4.5) of the pellets

containing the viral particles that were collected after ultra-centrifugation showed that

there was very little incorporation of the T216R-RRV and N216R-RRV-envelope

glycoproteins as compared to that of the WT-RRV, T216V-RRV and N218V-RRV-

envelope glycoproteins. The amount of capsid protein in the viral pellets observed

however was equal across all the samples. Thus the virus particles from T216R-RRV and

N218R-RRV samples were lacking the envelope glycoprotein around the retroviral cores.

This explains the reason for lower TU/mL in these samples. Further, the amounts of

envelope glycoproteins and capsid proteins in the lysates obtained from producer

ØNXnlslacZ cells were the same across in all samples. This shows that the mutations did

not affect the production of the T216R-RRV and N218R-RRV envelope glycoprotein.

The cell debris, which is the insoluble part of producer cell (parts of the cells resistant to

1% Triton-X 100 lysis solution), however, contained higher levels of the glycoproteins in

the T216R-RRV and N218R-RRV expressing cells as compared to wild-type, T216V-

RRV and N218V-RRV expressing cells. The quantities of the capsid proteins were same

across all the samples. This could suggest that the envelope glycoproteins of T216R-

RRV, N218R-RRV are being retained within the producer cells. Thus even though

envelope and capsid proteins were produced in equal quantities in all samples, greatly

reduced quantities of complete pseudotyped virus particles were not formed in T216R-

RRVand N218R-RRV samples. Envelope glycoproteins transfected with plasmids

41

encoding envelopes with these mutations, were retained within producer ØNXnlslacZ

cells and only naked MoMuLV cores lacking the envelope were released.

Figure 4.5 T216R-RRV and N218R-RRV envelope glycoproteins are inefficiently

incorporated in pseudotyped virus

Virus pellets (A) were spun down from supernatant media overlying producer ØNXnlslacZ cells transfected with respective RRV-envelope encoding plasmids, while the lysate (B) and the cell debris (C) were obtained by lysing the same producer ØNXnlslacZ cells. These preparations were run on SDS-PAGE gels and transferred on to nitrocellulose paper. RRV-envelope glycoproteins (E1 and E2) and MoMuLV Capsid proteins in these samples were detected by immunoblotting with rabbit anti-E1, rabbit anti-E2 and goat anti-Capsid (p30) polyclonal primary antibodies (1:5000) respectively, followed by secondary antibodies coupled with horseradish peroxidase. These experiments were performed thrice.

42

4.4.3 Attachment of T216R-RRV and N218R-RRV envelope glycoproteins to HS

leads to retention of these glycoproteins within the cells producing them.

Immunoblot assays showed that the envelope glycoproteins of T216R-RRV and N218R-

RRV were retained within the producer ØNXnlslacZ cells. To show that the retention of

glycoproteins could be due to their affinity towards HS expressed by the producer cells,

an immunofluorescence assay was performed. Since virus particles released from

producer ØNXnlslacZ can reenter those same cells, the study of this exit process cannot

be isolated from the entry process using immunofluorescence assay in these cells. Hence

to study the role of HS in the retention of the envelope glycoproteins, the glycoprotein

expressing plasmids were transfected in to CHO22 and CHO18.4 cell-lines. Results

showed that there was excess retention of T216R-RRV and N218R-RRV envelope

proteins in CHO22 cells along the secretory pathway during transport to the plasma

membrane as compared to WT-RRV, T216V-RRV and N218V-RRV envelope

glycoproteins (Fig. 3 A and Supplemental Fig. 1). In CHO 18.4, a cell line that does not

express HS, no excess retention of T216R-RRV and N218R-RRV envelope glycoproteins

was seen in comparison to rest of the samples (Fig. 3 B). It can be concluded that the

affinity of T216R-RRV and N218R-RRV glycoproteins towards HS leads to their

intracellular retention in virus-producing cells, and predominantly naked MoMuLV

cores, incapable of transducing target cells, are released from cells producing these

mutant envelope glycoproteins.

43

Figure 4.6 Intracellular retention of T216R-RRV and N218R-RRV in HS-expressing

cells

(A) CHO22 and (B) CHO18.4 were transfected with respective RRV-envelope plasmids. After fixing and permeabilizing the cells, they were incubated with primary Anti-E2 (rabbit) antibodies. After washing off the excess primary antibodies, cells were incubated with Alexa Fluor 488-coupled anti-rabbit antibodies and visualized under fluorescent microscope (rendered red color). (C) CHO22 cells expressing T216R and N218R RRV E2-glycoproteins (green fluorescence) were probed with Golgi-specific marker BODIPY® TR-C5-ceramide complexed to BSA (red fluorescence)

44

CHO22

CHO18.4

CHO22 with golgi marker

Figure 4.6

T216R

A

B

T216V

N218R

N218V WT

Control

Control T216R N218R

WT T216V N218V

C

T216R N218R

45

4.3.4 Attachment of T216R-RRV and N218R-RRV Envelope Glycoproteins to HS

hinders the release of virus particles

To determine whether the affinity of RRV envelope glycoproteins towards HS expressed

on the surface of the producer cells affects the release of virus particles Heparinase I was

added to the producer ØNXnlslacZ cells. The aim of this experiment was to see whether

cleaving of HS from the surface of these producer cells facilitates the release of T216R-

RRV and N218R-RRV pseudotyped viruses and thus increases their transduction

efficiency. T216R-RRV and N218R-RRV pseudotyped viruses collected from producer

ØNXnlslacZ cells treated with Heparinase I have higher transduction titers as compared

to the ones not treated with Heparinase I (Figure 4.7). Addition of Heparinase I to

producer ØNXnlslacZ cells producing WT-RRV, T216V-RRV and N218V-RRV

pseudotyped viruses shows no effect on their respective transduction efficiency. Thus HS

expressed on surface of producer ØNXnlslacZ cells hinders the process of release of

T216R-RRV and N218R-RRV pseudotyped viruses.

46

Table 4.1 Paired T-test

Figure 4.7 Addition of Heparinase I to Producer ØNXnlslacZ cells releases T216R-RRV

and N218R-RRV pseudotyped viruses

0

20

40

60

80

100

120

140

160

Control WildType

T216R T216V N218R N218V

% T

ran

sdu

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n (

% T

U/m

L)

No Heparinase

Heparinase

Sample T-value P-value

WT 1.24 0.342

T216R -10.55 0.009 *

T216V 0.38 0.741

N218R -4.82 0.040*

N218V -0.69 0.561

Producer ØNXnlslacZ cells transfected with respective RRV-envelope plasmids are treated with Heparinase I. Supernatant media were collected from these cells and used for transduction of target cells (BHK) as described earlier. Transduction titers were calculated for each experiment which was repeated thrice. The graph represents transduction titers, where 100% TU/mL for WT, T216R, T216V, N218R and N218V are 6090, 1650, 3860, 1760 and 4510 TU/mL respectively. The effect of addition of Heparinase I to producer cells is shown statistically using the paired T-Test in the table alongside (Table 4.1).

47

4.3.5 T216R-RRV and N218R-RRV pseudotyped viruses utilize HS as an

attachment factor

To investigate whether T216R-RRV and N218R-RRV pseudotyped viruses utilize HA as

an attachment factor, Heparinase I was added to the target cells. Heparinase I cleaves HS

from the cell surface by breaking the α (1-4) glycosidic bond of HS. The data

demonstrate (Figure 4.8) that in BHK and CHO22 cells, as the concentration of

Heparinase I increases, the transduction titers of T216R-RRV and N218R-RRV

pseudotyped viruses decrease, whereas transduction titers of WT-RRV, T216V-RRV and

T218V-RRV pseudotyped viruses do not change drastically. However, in CHO18.4 the

transduction titers remains equal across all samples irrespective of Heparinase I

concentration. In addition, whereas transduction by WT-RRV, T216V-RRV and T218V-

RRV pseudotyped viruses in CHO22 and CHO18.4 cells is approximately equivalent,

transduction by T216R-RRV and N218R-RRV pseudotyped viruses is higher in CHO22

cells than in CHO18.4 cells. Furthermore, treatment of the CHO22 cells with heparinase

reduces transduction by the T216R-RRV and N218R-RRV pseudotyped viruses

approximately to the level seen in CHO18.4 cells. Therefore in cell lines expressing HS,

the transduction efficiency of T216R-RRV and N218R-RRV pseudotyped viruses

decreases, as the amount of cell surface HS decreases, whereas no such effect is seen in

cell lines not expressing HS. Thus, these data demonstrate that T216R-RRV and N218R-

RRV pseudotyped viruses utilize HS as an attachment factor.

48

Figure 4.8 Heparinase treatment of target cells reduces transduction by T216R-RRV and

N218R-RRV pseudotyped viruses

Target cells (A) BHK, (B) CHO22 and (C) CHO18.4 were treated with increasing concentrations of Heparinase I for 1 hour before transducing them with WT-RRV and mutant pseudotyped virus, respectively. The experiments were performed in triplicate. The correlation between concentrations of Heparinase I and transduction titers for each sample was determined by calculating Pearson coefficients using Minitab software in (Table 4.2) BHK, (Table 4.3) CHO22 and (Table 4.4) CHO18.4 cell lines.

49

Figure 4.8

01000200030004000500060007000

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Heparinase Concentration

BHK

Control

Wild Type

T216R

T216V

N218R

N218V

0

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CHO22

Control

Wild Type

T216R

T216V

N218R

N218V

Type Pearson

Coefficient P value

WT 0.008 0.976

T216R -0.842 *0.001

T216V 0.085 0.792

N218R -0.896 *0.0001

N218V -0.052 0.865

Type Pearson

Coefficient P value

WT -0.159 0.707

T216R -0.737 *0.037

T216V -0.105 0.805

N218R -0.848 *0.008

N218V -0.055 0.898

Type Pearson

Coefficient P value

WT 0.365 0.374

T216R 0.132 0.755

T216V -0.129 0.761

N218R 0.143 0.763

N218V 0.257 0.539

Table 4.2 Pearson Coefficient BHK

0

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CHO 18.4

Control

Wild Type

T216R

T216V

N218R

N218V

Table 4.2 Pearson Coefficient CHO22

Table 4.2 Pearson Coefficient CHO18.4

50

4.3.6 Transduction efficiency of T216R-RRV and N218R-RRV is higher in cell lines

expressing HS

Intracellular retention of the T216R-RRV and N218R-RRV glycoproteins results in

markedly reduced transduction efficiencies by the pseudotyped virus recovered from the

supernatant medium of producer cells. It may be that there are fewer virus particles that

bear alphavirus glycoproteins and/or that virus particles bear fewer glycoproteins. To

investigate whether attachment to HS expressed on target cells increases the transduction

efficiency by the virus particles bearing the low quantities of T216R-RRV and N218R-

RRV glycoproteins that are released, normalization of virus concentration by

glycoprotein content was performed. The RRV-E2 envelope glycoprotein content of the

virus particles collected by ultracentrifugation of the supernatant media of each of the

pseudotyped-virus producing cells was measured by immunoblotting. The data show

(Figure 4.9) that after normalization based on the amount of RRV-E2 glycoprotein, the

transduction efficiency for T216R-RRV and N218R-RRV pseudotyped viruses were

significantly higher than that of wild type, T216V-RRV and T218V-RRV pseudotyped

viruses in NIH 3T3, BHK and CHO22 cells. In CHO18.4, however, there was no

significant difference in the transduction efficiency across these samples. Thus, in cell

lines expressing HS, when the amount of RRV-E2 glycoprotein protein is equal across all

the samples, T216R-RRV and N218R-RRV pseudotyped viruses have higher

transduction efficiency than wild-type, T216V-RRV and N218V-RRV pseudotyped

viruses.

51

Figure 4.9 T216R-RRV and N218R-RRV pseudotyped viruses possess higher

transduction efficiencies when transductions by viruses are normalized by glycoprotein

content

Virus was collected by ultracentrifugation from supernatant media overlying producer ØNXnlslacZ cells transfected with respective RRV-envelope encoding plasmids. The virus-containing samples were analyzed by SDS-PAGE and transferred onto nitrocellulose paper. RRV-E2 glycoprotein was detected by immunoblotting with rabbit anti-E2 primary and anti-rabbit secondary antibodies coupled with horseradish peroxidase. Using Image J software, (A) the amount of RRV-E2 glycoprotein in each virus sample was quantified. A representative image is shown. The amount of media added to target cells (B) NIH-3T3, (C) BHK, (D) CHO22 and (E) CHO18.4, for transduction was normalized against the amount of RRV-E2 glycoprotein present in the given sample. The transduction of the CHO22 and CHO18.4 cells was performed with different starting samples of the five viruses, so the transduction efficiencies measured in this experiment between the two cell lines cannot be directly compared. The experiments were repeated thrice. The values shown by the graph represent the mean TU/mL values (± SD) calculated from these experiments.

52

Figure 4.9

A

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53

4.4 Discussion

Pseudotyped viruses have many experimental and clinical applications. Gene

therapy/transfer is one of the important applications of these pseudotyped viruses. Based

on the therapeutic need, the range of cells targeted by the viral vector for gene

therapy/transfer can be either expanded or narrowed by pseudotyping the virus with the

envelope of another virus (Sanders 2002). For the treatment of systemic genetic disorders

a viral vector needs to be pseudotyped with an envelope of a virus that has a broad tissue

tropism. Since RRV, an alphavirus, has wide host range and tissue tropism, it was

incorporated in ØNX pseudotyped system to produce RRV-MoMuLV pseudotyped virus

(Sharkey CM 2001). Previous studies showed that pseudotyping with RRV was far less

cytotoxic but equally efficient as VSV-G (Kang Y 2002). Retrovirus pseudotyped with

RRV envelope glycoproteins was successfully used for in vivo gene transfer in

hepatocytes, glial cells, muscle tissue and airway epithelial tissue (Kang Y 2002). To

further enhance the transduction efficiency and further broaden the range of cells that can

be targeted by the pseudotyped viral vector additional modifications of these envelope

glycoproteins are needed.

Earlier studies in RRV show that amino acid T216 on RRV-E2 glycoprotein is the

site involved in interaction with the receptors (Smith TJ 1995). The RRV48 strain of

RRV normally does not utilize HS as an attachment factor. Serial passaging of this strain

led to mutations on the E2 envelope glycoprotein in which the original amino-acid

residues were replaced with basic amino-acid residues (Kerr PJ 1993). Further studies

showed that site-directed mutagenesis involving replacement of threonine residue at 216

or the neighboring asparagine residue at 218 with a basic amino acid residue confers

54

upon the virus an affinity towards a negatively charged molecule like HS (Heil ML

2001). Cryo-electron microscopy and image reconstruction studies of RRV-E2

glycoprotein bound to heparin further confirmed this (Zhang W 2005).

Substituting the amino-acid residues at 216 and 218 sites on RRV-E2 glycoprotein with

basic amino acid residues in the RRV-MoMuLV pseudotyped virus, allows the

pseudotyped virus to use HS, an ubiquitous molecule present on most of the cell surfaces,

as an attachment factor. Using HS as an attachment factor increases the concentration of

the virus on the cell surface, which can later be internalized by cell surface receptors via

endocytosis. Thus the three-dimensional search for the receptor is reduced to a two-

dimensional search, which increases the efficiency of cell entry, thereby enhancing the

transduction efficiency of the pseudotyped vector. HS binding ability could also

potentially alter the tissue tropism of this viral vector (Kang Y 2002, E. G. Gardner CL

2011).

Ability to use HS as an attachment factor however affects the assembly and

release process of the virus particles. Due to the increased affinity of RRV-E2

glycoproteins towards HS, envelope glycoproteins get retained along the secretory

pathway, especially in the Golgi, since HS based glycosylation of other proteins takes

place in the Golgi (DK 2011). Thus the envelope glycoproteins with basic amino-acid

mutations are prevented from reaching the plasma membrane. Hence they are not

efficiently incorporated into the budding pseudotyped virus. Secondly, even if the

glycoproteins reach the plasma membrane and get incorporated into budding pseudotyped

virus, the data indicate that their affinity towards HS prevents the release of virus particle

from the cell surface. Studies done in Sindbis virus have shown that virus particles

55

utilizing HS had lesser viremia resulting in lower pathogenicity. This led to the reversion

of virus particles adapted to utilize HS to that wild-type form that had reduced affinity

towards HS. It was proposed that one reason for lesser viremia seen in Sindbis virus

utilizing HS was decreased dissemination of the virus (G. D. Byrnes AP 2000). Similar

studies in Venezuela equine encephalitis virus and Chikungunya virus show attenuation

of HS-binding mutant viruses due to rapid clearance and decrease in the spread of virus

particles (Bernard KA 2000, H. J. Gardner CL 2014).

This interaction between the HS-utilizing viruses and HS is reminiscent of the

interaction between influenza virus and its attachment factor, sialic acid. Sialic acid

facilitates the entry of influenza virus by attaching to the viral and increases their

concentration on the cell surface (Skehel JJ 2000, Wagner R 2002). However, the same

attachment factors, when expressed on the cells producing the virus particles, hinder the

release of the viral particles from the cell surface (Wagner R 2002). The release of

influenza virus from cell surfaces is facilitated by the Neuraminidase protein intrinsic to

the virus, which cleaves the sialic acid (Air GM 1989, Bucher D 1975). Because such an

intrinsic process for facilitating the release of virus particles is not present in our

pseudotyped viral vector, one of our future goals is to develop a producer cell line that

does not express HS for the production of this pseudotyped viral vector that utilizes HS

as an attachment factor. A general principle arising from these studies is that altering

producer cell membrane components may improve pseudotyped virus production through

enhancing viral particle release.

The attachment of viruses to cell-surface heparan sulfate has been proposed both

to be an inherent component of viral entry in vivo and an adaptation to propagation in cell

56

culture. An important consequence of our studies is the conclusion that utilization of

heparan sulfate as an attachment factor can have deleterious effects on viral release either

through intracellular retention of the heparan-sulfate-binding viral proteins or adsorption

of the viral particles to producer cells. HSV-1 utilizes heparan sulfate as an attachment

factor for infection of host cells (S. P. Shukla D 2001, L. J. Shukla D 1999). Recently it

has been demonstrated that increased host heparinase expression is a consequence of

herpes simplex virus-1 infection and that augmentation of heparinase expression

correlates with enhanced HSV-1 release (Hadigal SR 2015). These data reinforce our

conclusion that if a virus utilizes an ubiquitous and abundant cell component for entry

then there must be a mechanism for release of the virus from an infected cell if efficient

infection is to occur. This issue is likely to be particularly critical when the utilization of

the attachment factor appears to arise from an adaptation to growth in cell culture

57

CHAPTER 5. ROLE OF CHOLESTEROL IN ENTRY OF PSEUDOTYPES

INCORPORATING ALPHAVIRAL GLYCOPROTEINS AND NIEMANN PICK’S

DISEASE

5.1 Summary

The tissue tropism of a virus can be modified by replacing the envelope-glycoproteins of

that virus with those of other viruses, through the process of pseudotyping. Such

pseudotyped viruses are good candidates as vectors for gene therapy/ transfer. In this

study, we propose that retroviruses incorporating alphaviral glycoproteins will be suitable

gene delivery vectors in Niemann Pick’s disease, type A (NPD-A). NPD-A is a genetic

lipid-storage disorder characterized by severe deficiency of acid sphingomyelinase

(ASMase), which results in excess accumulation of cholesterol and sphingolipids in the

lysosomes and late endosomes. Previously it has been shown that cholesterol is required

by alphaviruses for fusion process during the entry. We show that alphavirus-

glycoprotein retrovirus pseudotypes also require cholesterol for entry into the cells.

Furthermore, excess cholesterol in cells facilitates the entry of alphavirus-glycoprotein

retroviral pseudotypes. Alphavirus-glycoprotein pseudotypes transduce the acid

sphingomyelinase deficient (ASMase-/-) cells derived from Niemann Pick’s disease-

model mice more efficiently than the cells from healthy mice (ASMase+/+). Thus

58

alphavirus-glycoprotein pseudotypes hold a potential as suitable vector for gene therapy/

transfer in NPD-A.

5.2 Introduction

Neimann Pick’ disease (NPD) is a lysosomal storage disease. Types A and B of

this disease are caused by mutations in the SMPD1 gene, leading to severe deficiency of

ASMase activity. Deficiency in this enzyme leads to excess accumulation of

sphingolipids in lysosomes and late endosomes (Schuchman EH 2001). Accumulation of

sphingolipids is coupled with the accumulation of cholesterol in lysosomes and

endosomes (Puri V 1999, Choudhury A 2004). NPD-A is a more severe form this

disease, than the other three types NPD-B, NPD-C and NPD-D. Clinical manifestations

in NPD-A include persistent early jaundice, hepatosplenomegaly, enlarged abdomen,

delay in developmental milestones, intellectual developmental disorders, stunted growth,

hypotonia and rigidity (Crocker AC 1958). NPD-A has poor prognosis leading to an early

death, usually by 2 to 4 years of age. Currently there is no cure available for this disease.

However, gene transfer studies in cultured NPD cells as well as diseased-mouse models

have shown to correct the metabolic defect (Suchi M 1992, Dodge JC 2005).

Viruses are frequently used as gene therapy/ gene transfer vectors to introduce the

gene of choice into cells. Since entry of an enveloped virus depends on its envelope

proteins, the range of target cells that a virus can enter into can be modified by replacing

the envelope glycoproteins of that virus with those of other viruses having the desired

range of tissue tropism, through the process of pseudotyping (Sanders 2002). Such

pseudotyped viruses are good candidates as vectors for gene therapy/transfer. Therapeutic

59

need would determine the choice of virus whose envelope glycoproteins need to be

incorporated in the pseudotyped virus (Sanders 2002).

Retroviruses are commonly used as gene delivery vectors because they allow

stable transfer and efficient expression of the genetic material under the influence of their

strong promoter. However, they have limited host range and tissue tropism. With the

purpose of broadening the tissue tropism and further enhancing the transduction

efficiency of this virus, we pseudotyped Moloney Murine Leukemia Virus (MoMuLV), a

model retrovirus, with envelope glycoproteins of Ross River Virus (RRV), an alphavirus

(Sharkey CM 2001). Successful in vivo gene transfer studies have been conducted

previously using lentiviral pseudotypes incorporating alphaviral envelope glycoproteins

(Kang Y 2002).

Alphaviruses are mosquito-borne viruses and are classified into Old World

viruses, which include Ross River Virus (RRV), Sindbis Virus (SINV), Semliki Forest

virus (SFV) and New World viruses, which include Venezuelan Equine Encephalitis

virus (VEEV), Eastern Equine Encephalitis virus (EEEV) and Western Equine

Encephalitis virus (WEEV). Alphaviruses have wide host-range and tissue tropism.

Alphaviruses enter the cells via receptor-mediated endocytosis. Two of the alphaviral

glycoproteins, E1 and E2 are required for this entry process (C.-V. C. Kielian MC 2010).

The E2-glycoprotein is involved in the initial receptor-binding phase, whereas the E1

glycoprotein is responsible for fusion of viral and cellular membranes (C.-V. C. Kielian

MC 2010, Jose J 2009). Once the virus gets internalized, the acidic pH of endosomes

leads to dissociation of the E1-E2 heterodimer exposing the hidden fusion loop of E1 (C.-

V. C. Kielian MC 2010). The insertion of fusion loop into the endosomal membrane

60

initiates the fusion process (C.-V. C. Kielian MC 2010). Studies in alphaviruses like

Sindbis Virus (SINV) and Semliki Forest Virus (SFV) using liposomes show that

cholesterol and sphingolipids are required for this step of fusion of the viral membrane

with the cellular membrane (Helenius A 1984) (Nieva JL 1994). Micro-domains of

cholesterol and sphingolipids are called lipid-rafts (Figure 5.1). These cholesterol-rich

lipid rafts have also been shown to be required for glycoprotein transport and the exit

process (G. D. Ng CG 2006, C. I. Ng CG 2008). Furthermore, studies have shown that

SINV causes fatal encephalitis in knock-out mice deficient in acid sphingomyelinase

(ASMase) as compared to healthy mice (G. D. Ng CG 2006).

61

Figure 5.1 Cholesterol and Sphingolipid rich lipid rafts

Compartmentalization of lipid bilayer forms micro-domains called as ‘lipid rafts’ which are rich in cholesterol and sphingolipids content. The factors contributing to formation and stability of lipid rafts are hydrogen bonding between polar heads, interaction between long hydrophobic chains and complementarity between the shapes of lipid molecules. Various proteins are embedded within these lipid-rafts.

62

Alphaviruses mainly infect brain, liver, bones, muscles, lungs and reticulo-

endothelial tissue and these sites can be targeted using pseudotyped viruses incorporating

alphaviral glycoproteins (Kang Y 2002). An overlap exists between the organs infected

by alphaviruses and the organs that get affected in Niemann Pick’s Disease. At the

cellular level, the fusion process of alphaviruses, which requires cholesterol, takes place

in endosomes. In NPD-A, accumulation of sphingolipids and cholesterol also takes place

in endosomes and lysosomes (Puri V 1999, Choudhury A 2004). By incorporating the

alphaviral glycoproteins in pseudotype system, we are trying to investigate if cholesterol

can have an effect on transduction efficiency of MoMuLV pseudotyped with alphavirus

envelope glycoproteins. We demonstrate that these pseudotyped viruses have higher

transduction efficiency in Niemann Pick-Type A model cells. These pseudotypes thus

hold a potential as vectors for gene transfer/therapy studies in Niemann Pick’s disease.

5.3 Results

5.3.1 Alphavirus-MoMuLV pseudotypes require cholesterol-rich lipid rafts for entry

in mammalian cells

Since the entry process of envelope forming virus in a pseudotyped virus can be studied

in isolation, alphavirus-MoMuLV pseudotypes were used to determine if cholesterol-rich

lipid rafts are required for cell entry. To show that lipid rafts are required for entry, the

lipid-rafts in target cells (BHK and NIH3T3) were disrupted using cholesterol lowering

drugs, namely lovastatin and ß-mCD. The experiment was also performed on cholesterol-

depleted cells, which were replenished with cholesterol. MoMuLV pseudotyped with

MoMuLV glycoproteins was used as positive control (S. J. Lu X 2000), whereas

63

MoMuLV pseudotyped with Vesicular stomatitis virus glycoprotein was used as negative

control (Yonezawa A 2005).

The data shown in Figure 5.2A and Figure 5.2B suggest that, the transduction

titers for pseudotypes with alphaviral glycoproteins are lower in cells treated with lipid-

lowering drugs as compared to non-treated cells. On replenishing the cells with

cholesterol, higher values of transduction units/mL are obtained. No such difference is

noted in transduction efficiency of pseudotypes incorporating VSV glycoproteins. Thus,

cholesterol-laden lipid rafts are essential for entry of alphavirus-MoMuLV pseudotyped

virus.

64

Figure 5.2 Alphavirus-MoMuLV pseudotypes require cholesterol for entry in mammalian

cells

0

20

40

60

80

100

120

VSV MoMuLV RRV SFV SINV VEEV

% T

ran

sdu

ctio

n

NIH-3T3

Untreated cells

Cells treated with Lovastatin andmßCD

Repletion with Cholesterol

0

20

40

60

80

100

120

VSV MoMuLV RRV SFV SINV VEEV

% T

ran

sdu

ctio

n

BHK

Untreated cells

Cells treated with Lovastatin andmßCD

Repletion with Cholesterol

Supernatant media overlying producer ØNXnlslacZ cells containing the pseudotyped virus particles were passed through a 0.45 µm filter and 5 µg/mL of hexadimethrine bromide was added to these filtered media. These media were used to transduce target cells, NIH-3T3 (Figure 5.1A) and BHK (Figure 5.1B). Cholesterol depletion was achieved in target cells by incubating the target cells with cholesterol-lowering drugs, namely lovastatin and methyl-beta-cyclodextrin (ß-mCD). The transduced target cells were stained with X-gal and transduction titers were calculated. Results shown are averages ± SD of three independent assays. The values shown in the graph represent transduction titers in percentages, where 100% TU/mL for VSV, MoMuLV, RRV, SFV, SINV and VEEV pseudotypes are 15000, 15000, 15000, 13000, 16000, 11000 and 15000 in NIH-3T3 cells, and 4000, 0, 5500, 5000,3500, 7000 in BHK cells respectively. The experiment was also performed following repletion of the cholesterol-depleted cells with 50 µg/mL cholesterol (β-sterol).

A

B

65

5.3.2 Alphavirus-baculovirus pseudotypes require cholesterol for entry into insect

cells

To rule out any direct effect of lipid-lowering drugs on the transduction values,

transduction assays were carried out in mosquito-derived C6/36 cells. The cholesterol

content of these cells can be reduced to as low as 1- 2% of their actual value, when grown

in sterol-free conditions (X. Y. Lu X 2002). Since pseudotyped viruses grown from

ØNXnlslacZ cells cannot transduce insect cells, baculovirus pseudotypes were produced.

Baculovirus pseudotypes incorporating baculovirus glycoprotein, gp64 was used as

positive control (Kataoka C 2001).The data show that transduction titers of these

baculovirus pseudotyped viruses incorporating alphaviral glycoproteins are lower in cells

grown in sterol-free condition as compared to cells not grown in sterol restricted

conditions (Figure 5.3). Repletion of cells grown in sterol-free conditions with

cholesterol leads to increase in the transduction efficiency of alphavirus-baculovirus

pseudotypes.

66

Figure 5.3 Alphavirus-baculovirus pseudotypes require cholesterol for entry in insect

cells

5 µg/mL of hexadimethrine bromide was added to the filtered supernatant media overlying SF9 cells. These media containing the baculovirus pseudotypes were used to transduce target C6/36 cells grown in DMEM+10% FBS (a), cholesterol depleted media (b) and media replenished with cholesterol (c). Cholesterol depletion was achieved in C6/36 cells by growing these cells in sterol-free condition. Experiments were also performed in these cells after replenishing them with β-sterol by adding 50 µg/mL cholesterol. The transduced target cells were identified using the fluorescent microscopy and transduction titers were calculated. The experiments were repeated thrice and atleast 10 fields (one field represented) were inspected per plate for each sample (Figure 5.2A). The graph shows averages±SD of transduction titers in percentages from three independent assays (Figure 5.2B), where100% TU/mL for Baculovirus, VSV and RRV pseudotypes are 1800, 450 and 450 respectively.

67

Figure 5.3

0

20

40

60

80

100

120

gp64 VSV RRV

%Tr

ansd

uct

ion

DMEM +10% FBS

Cholesterol-depletedMedia

Media replinished withCholesterol

gp64

VSV-G

RRV-Env

(a) (b) (c) A

Control

B

68

5.3.3 Alphavirus-MoMuLV pseudotypes transduce ASMase-/- cells more efficiently

than ASMase+/+ cells

To see whether excess cholesterol facilitates the entry of these pseudotyped viruses into

the cells, transduction assay was carried out in ASMase-/- cells and ASMase+/+ cells.

Absence of acid sphingomyelinase leads to excess accumulating of sphingomyelin and

cholesterol in the cells. Fluorometric assay confirmed that the cholesterol levels in

ASMase-/- cells were higher than ASM+/+ cells (Figure 5.4B). The data show (Figure

5.4A) that transduction efficiency of pseudotypes with alphaviral glycoproteins is higher

in ASMase-/- cells than in ASMase+/+ cells while not much difference was noted in

transduction efficiency of pseudotypes with VSV glycoproteins across these two cell-

lines.

69

Figure 5.4 Transduction efficiency of alphaviral pseudotypes is higher in ASMase-/- cells

0

100

200

300

400

500

600

VSV MoMuLV RRV VEEV SFV SINV

% T

ran

sdu

ctio

n

ASMase+/+ vs. ASMase-/- cells

ASMase+/+

ASMase-/-

0

20

40

60

80

100

120

Asmase+/+ Asmase-/-

% C

ho

lest

ero

l C

on

cen

trat

ion

Supernatant media overlying producer ØNXnlslacZ cells containing the pseudotyped virus particles were passed through a 0.45 µm filter. Hexadimethrine bromide (5 µg/mL) was added to these filtered media. These media were used to transduce target ASMase+/+ and ASMase-/- cells. The transduced target cells were stained with X-gal and transduction titers were calculated. Results shown are averages±SD of three independent assays represented in percentages (Figure 5.3A). The difference in the concentration of cholesterol in ASMase-/- and ASMase+/+ cells was verified using a fluorometric cholesterol assay kit (Figure 5.3B).

A

B

70

5.4 Discussion

NPD-Type A is an autosomal recessive genetic disorder caused by mutation in

SMPD1 gene and is characterized by excess accumulation of sphingolipids and

cholesterol in the cells. The treatment is mainly symptomatic and there is no cure

available for this disease. However, gene transfer studies performed using viral vectors

have shown to correct the metabolic defect in cultured cells as well as mouse models

(Suchi M 1992, Dodge JC 2005).

In our earlier study, we produced a pseudotyped virus by replacing the envelope

of Moloney Murine Leukemia Virus (MoMuLV) with the envelope of Ross River Virus

(RRV). Pseudotyping helps us in combining features of retrovirus, that are ideal for gene

transfer such as ability to allow fast and stable transfer of transgene to cells and efficient

expression of the transgene for prolonged period of time under the influence of its strong

promoter system, along with the features of alphaviruses which is to allow entry into

wide range of cells. This makes these pseudotyped viruses a potential gene

therapy/transfer vector in systemic genetic disorders. Successful in vivo gene transfer

studies have been conducted using lentiviral pseudotypes incorporating alphaviral

glycoproteins (Kang Y 2002). The cells specifically targeted by pseudotypes

incorporating alphaviral glycoproteins are neuronal cells, neuroglia, hepatocytes, Kupffer

cells and reticulo-endothelial cells (Kang Y 2002). Modest transductions are also seen in

myocytes and airway epithelial cell (Kang Y 2002). Interestingly, the tissues targeted by

the pseudotypes with alphaviral glycoproteins are similar to the ones affected in NPD-A

(Hua G 2013).

71

Since the entry of the virus depends on the envelope of the virus, the pseudotyped

virus has the entry mechanism similar to that of envelope forming virus and this entry

mechanism can be studied in isolation using the pseudotyped system. Using lipid-

lowering drugs and sterol-free conditions to grow cells, we show that pseudotypes

incorporating alphaviral glycoproteins require cholesterol for entry into the cells just like

wild-type alphaviruses. Studies in alphaviruses have shown that ASMase-/- mice were

more susceptible to fatal encephalitis (G. D. Ng CG 2006). Studies done in human NPD

cells which lacked ASMase enzyme attributed the higher virulence to rapid replication of

the virus in ASMase deficient cells and to production of more infectious viral particles

from these cells (C. I. Ng CG 2008). Here, using pseudotyped viruses, in which entry of

the envelope forming virus can be exclusively studied in isolation, we show that the

excess cholesterol in these cells facilitates the entry of pseudotyped viruses incorporating

alphaviral glycoproteins. Thus, efficient entry in ASMases deficient cells could be

another factor responsible for higher virulence and pathogenicity of alphaviruses in these

cells.

These characteristics make pseudotyped viruses incorporating alphaviral

glycoproteins suitable vectors for gene transfer/therapy in NPD-A. Further studies using

animal disease-models need to be done in order to validate their application in clinical

field.

72

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VITA

Dr. Aditi Kesari was born in Ahmednagar, India on October 4, 1984. Within a

couple of years, Aditi, her parents, Mr. Shashikant Kesari and Mrs. Shubhada Kesari, and

her elder brother, Mr. Amit Kesari, moved to Pune. She did her schooling at Modern

High School. Aditi graduated from High School by standing 20th in the Merit List

amongst 200,000 students in Secondary School Board Certificate Examination, India.

She then went to Fergusson College for Higher Secondary education. Aditi appeared for

the State-level Common Entrance Test for Medicine in 2002, through which she secured

admission into B.J. Medical College, one of India’s top Medical Colleges. She received

Bachelor of Medicine and Bachelor of Surgery (MBBS) degree in 2008. She worked at

Jehangir hospital in the Cardiology department before coming to Purdue to pursue PhD.

At Purdue, Aditi joined Dr. David Sanders’ laboratory in August 2010. Her work

involved application of virology in the field of gene therapy. She studied entry

mechanism of Alphaviral glycoprotein pseudotyped viruses, which can be used as

potential gene therapy vectors. Aditi presented her work with a lecture at European

Molecular Biology Laboratory Symposium held at Germany in 2013, and at American

Society of Virology conference held at Canada in 2015. She also presented a poster at

American Society of Gene and Cell Therapy Conference in 2014.

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Aditi met her husband, Aditya Josyula while at Purdue. They have recently

become parents to their son, Aariv and are enjoying their parenthood.

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