The Pennsylvania State University

The Graduate School

College of Medicine

UTILIZATION OF CELLULAR PROTEINS BY ROUS SARCOMA VIRUS

DURING REPLICATION

A Thesis in

Microbiology and Immunology

by

Jared Lynn Spidel

© 2005 Jared Lynn Spidel

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

May 2005

The thesis of Jared Lynn Spidel was reviewed and approved* by the following:

John W. Wills Professor of Microbiology and Immunology Thesis Adviser Chair of Committee

Richard J. Courtney Professor of Microbiology and Immunology Head of the Department of Microbiology and Immunology

Leslie J. Parent Associate Professor of Medicine Associate Professor of Microbiology and Immunology

David J. Spector Associate Professor of Microbiology and Immunology

Vincent Chau Professor of Cellular and Molecular Physiology

*Signatures are on file in the Graduate School ABSTRACT

The goal of this dissertation was to analyze the role of cellular proteins during the final stages of budding leading up to membrane fission. Studies have already identified some of the cellular proteins which interact with various late (L) domains. The L domain of Rous sarcoma virus (RSV) interacts with a ubiquitin (Ub) ligase, a cellular enzyme which conjugates Ub to a target lysine. Indeed, all retroviruses examined so far contain

~100 molecules of Ub, suggesting Ub might have a role during budding. However, unlike all other retroviruses, Ub conjugated to RSV Gag has never been detected. This observation seems inconsistent with the observation that budding of this virus (and many others) is dependent on the presence of free cellular Ub. Therefore, the role of Ub during

RSV budding was examined.

If transient ubiquitination of RSV Gag is required during budding, the five lysines located just upstream of the RSV L domain in matrix (MA) would be the most likely targets of ubiquitination based on known sites of ubiquitination of Gag in other viruses.

These residues were changed to arginines to eliminate the potential for ubiquitination.

As predicted, substitution of these five residues with arginine (mutant 1-5KR) reduced budding by 80-90%. The block to budding was found to be on the plasma membrane, and the few virions released had normal size, morphology, and infectivity. Budding was restored when any one of the residues was changed back to lysine or when lysines were inserted in novel positions within the region of the original five substituted residues or C- terminal in the p10 region. Similar to an L domain mutant, the 1-5KR mutant could be rescued into particles by coexpression of budding-competent Gag molecules. These data suggest that ubiquitination of Gag is likely important for budding.

iii Through examination of lysines involved in RSV budding, a lysine (K244) in capsid (CA) was found to be required for efficient replication. Analysis of the amino acids flanking K244 revealed it resides in a sumoylation consensus sequence ΨKxE, where Ψ is a hydrophobic residue and x is any residue (243IKTE246 in RSV CA). Small

Ub-like MOdifier (SUMO)-1 is a member of the Ub-like protein family that transiently modifies lysines of various target proteins in a manner similar to Ub. Sumoylation of target proteins is important for nuclear events such as nuclear entry, subnuclear structure formation, and modulation of transcriptional activity. The enzyme Ubc9 is capable of conjugating SUMO-1 to a target lysine within the sumoylation consensus sequence.

Indeed, sumoylation has been hypothesized to be involved in the replication of Mason-

Pfizer monkey virus where CA was shown to interact and colocalize with Ubc9 in vivo.

The RSV mutants K244R and E246A were found to be normal for budding and assembly but were reduced in infectivity, revealing a potential role for sumoylation of Gag or CA in replication. The virions contained normal amounts of Pol, Env, and RNA and were normal in core morphology. The defect was during reverse transcription and possibly nuclear import of the preintegration complex (PIC).

Revertant viruses of the mutants K224R and E246A were isolated, and the genomes sequenced to identify second-site suppressors. The K244R phenotype was suppressed by the double mutation R325C/C431R; the E246A phenotype was suppressed by a N343D mutation. C431 is thought to be involved in CA-CA interactions based on its position in the predicted dimerization helix. The involvement of C431 in the suppression of the K244R phenotype suggests the K244R and E246A substitutions may disrupt intra- or intermolecular interactions in CA which is repaired in the suppressors.

iv Further experimentation is required to determine if the K244 and E246 are important in the structural stability of CA or if the suppressors allow for SUMO-independent replication. Whatever the function of K244 and E246, these data support a model whereby CA plays an active role during reverse transcription and/or nuclear import of the

PIC.

The exact function of Ub in retrovirus budding is unknown. Ub has many cellular functions, including involvement in cellular budding events that produce multivesicular bodies (MVBs), and, interestingly, viral budding is similar in topology to this budding.

Therefore, the cellular proteins which interact with Ub to mediate MVB formation could feasibly be involved in viral release. Ubiquitination of a target protein acts to recruit complexes known as ESCRT-I, -II, and -III to facilitate sorting of the target protein into the budding vesicles. To determine the role of the ESCRT complexes during RSV budding, dominant-negative forms of these proteins were coexpressed with RSV Gag.

ESCRT-I protein Tsg101, the ESCRT-III proteins CHMP3, CHMP4A, CHMP4B,

CHMP4C, and CHMP6, and Vps4A inhibit RSV budding implicating a role for these proteins during budding. During the course of this study it was noticed that RSV Gag also contains a YPxL sequence that may allow binding to AIP1/ALIX which itself interacts with ESCRT-III and is important for HIV and EIAV budding. Substituting this sequence with alanines had a modest effect on budding suggesting this sequence may have a slight role during budding. This site may interact with AIP1/ALIX to stabilize

ESCRT complexes with RSV Gag, and failure to do so results in inefficient budding.

Therefore, from these data it appears that RSV and HIV utilize a similar mechanism and cellular proteins for their release.

v TABLE OF CONTENTS

LIST OF FIGURES ...... x LIST OF TABLES...... xii LIST OF ABBREVIATIONS...... xiii ACKNOWLEDGEMENTS...... xvii

CHAPTER I: LITERATURE REVIEW...... 1 Introduction...... 2 Organization of the RSV Genome ...... 6 Particle Morphology and Structure...... 10 The Immature Virion ...... 11 Maturation...... 12 The Mature Virion ...... 15 Entry...... 21 Attachment...... 21 Fusion and Uncoating ...... 24 Reverse Transcription...... 25 Nuclear Targeting and Entry...... 34 Integration...... 36 Transcription...... 37 Translation ...... 41 Virus Assembly...... 42 Gag Trafficking and the M Domain...... 43 Gag-Gag Interactions and the I domain...... 49 RNA Packaging ...... 51 vRNA Used For Packaging...... 52 RNA Dimerization...... 53 Packaging of the tRNA Primer ...... 54 Pol Incorporation ...... 55 Env Incorporation ...... 56 Incorporation of Cellular Proteins ...... 57 Late Budding Events and the L Domain...... 60 Various Types of L Domains and Their Interactions ...... 61 Late Budding Events in Other Enveloped Viruses ...... 64 Arenaviruses ...... 64 Rhabdoviruses...... 64 Filoviruses...... 65 Multivesicular Body Formation...... 65 Ubiquitination and Sumoylation...... 65 MVB Formation and Protein Sorting...... 69 Host Proteins Driving Retrovirus Budding...... 77 Ubiquitin and Retrovirus Budding...... 77 Vps Class E Proteins and Retrovirus Budding ...... 78 Utilization of Cellular Proteins by RSV During Replication ...... 81

vi CHAPTER II: LYSINES CLOSE TO ROUS SARCOMA VIRUS LATE DOMAIN CRITICAL FOR BUDDING...... 82 Abstract...... 83 Introduction...... 84 Materials and Methods...... 86 Expression Vectors ...... 86 Mutagenesis ...... 87 Budding Assays ...... 89 Sucrose Gradient Sedimentation...... 91 Infectivity Assays ...... 91 Confocal Microscopy...... 92 Electron Microscopy...... 92 Results ...... 92 Budding of Mutant 1-5KR...... 95 Subcellular Localization of 1-5KR...... 98 Analysis of 1-5KR Virions ...... 101 Complementation Rescue of 1-5KR ...... 104 An L-Domain-Point Mutant Is Dominant-Negative for Budding ...... 105 Addition of Lysines to 1-5KR ...... 108 Lysine Insertion Mutants of HTLV-1 Gag ...... 113 Discussion...... 116 Acknowledgements...... 120

CHAPTER III: ANALYSIS OF A POTENTIAL SUMOYLATION SITE IN ROUS SARCOMA VIRUS CAPSID IMPORTANT FOR REPLICATION ...... 121 Abstract...... 122 Introduction...... 123 Materials and Methods...... 125 Expression Vectors ...... 125 Mutagenesis ...... 125 Budding Assays ...... 126 Infectivity Assays ...... 127 Confocal Microscopy...... 128 EM...... 128 Detergent Resistance Assay...... 128 CA:RT Ratio Assay ...... 129 Env Incorporation ...... 129 RNA Isolation ...... 130 DNA Isolation...... 130 Endogenous RT Assay...... 131 Quantitative Real Time PCR ...... 131 Isolation of Revertants...... 132 Results ...... 133 Sumoylation Consensus Sequence Required For Replication...... 133 Nuclear Trafficking of K244R...... 138 K244R Virion Characterization...... 139

vii Pol Incorporation ...... 144 Env Incorporation ...... 144 RNA Packaging ...... 147 Analysis of Viral DNA Products in Infected Cells...... 147 Endogenous Reverse Transcription ...... 150 Isolation of Second-Site Suppressors ...... 150 Discussion...... 161 Reverse Transcription Affected by K244R Substitution ...... 161 Suppressors May Allow For SUMO-1 Independent Replication ...... 161 Possible Contribution of Lysine 244 and Glutamate 246 to CA-CA Interactions...... 162 Direct Involvement of Lysine 244 and Glutamate 246 in Reverse Transcription...... 163 Nuclear Import May Be Affected by K244R...... 164 Acknowledgements...... 164

CHAPTER IV: UTILIZATION OF ESCRT PROTEIN COMPLEXES FOR BUDDING OF ROUS SARCOMA VIRUS ...... 166 Abstract...... 167 Introduction...... 168 Materials and Methods...... 171 Expression Vectors ...... 171 Mutagenesis ...... 171 Budding Assays ...... 172 Western Blot Analysis ...... 172 Results ...... 173 Hrs Not Required For Budding...... 173 Dominant-Negative Forms of Tsg101 and RSV Budding...... 173 RSV Budding Mildly Affected by Dominant-Negative ESCRT-II Proteins....178 Overexpression of ESCRT-III Proteins ...... 178 Effect of Vps4A Dominant-Negative on RSV Budding...... 183 Potential AIP1/ALIX Binding Site in RSV Gag ...... 183 Analysis of the Dominant-Negative Effect of PPPY-A When Coexpressed With Wild-Type Gag ...... 186 Discussion...... 192 Utilization of ESCRT Proteins During RSV Budding...... 192 Potential Interaction Between RSV Gag and AIP1/ALIX...... 195 PPPY-A Dominant-Negative Phenotype Due to Overexpression ...... 196 Acknowledgements...... 197

CHAPTER V: DISCUSSION...... 198 Mechanisms of RSV Budding ...... 199 Gag-Driven Budding...... 199 Lysines in MA Required For Release From the Plasma Membrane ...... 200 Possible Budding Independent of Gag Ubiquitination ...... 203 Multiple Roles of Ub During Budding ...... 206

viii ESCRT Protein Complexes and RSV Budding ...... 206 Protein Complexes Downstream of the ESCRT Complexes...... 208 Utilization of CA During Reverse Transcription...... 209 A Sumoylation Consensus Sequence in CA ...... 209 Potential Role of SUMO-1 During Reverse Transcription...... 210 Suppressors of Mutants K244R and E246A ...... 211 Involvement of CA During Reverse Transcription...... 212

REFERENCES ...... 214

ix LIST OF FIGURES

Figure Page

Figure 1.1 Membrane Fission...... 3

Figure 1.2 RSV Genome Organization ...... 7

Figure 1.3 Immature Virion...... 13

Figure 1.4 Mature Virion ...... 16

Figure 1.5 Structure of CA...... 18

Figure 1.6 Retroviral Entry ...... 22

Figure 1.7 Membrane Fusion ...... 26

Figure 1.8 Reverse Transcription...... 30

Figure 1.9 Retrovirus Assembly...... 39

Figure 1.10 RSV Gag and the Assembly Domains...... 46

Figure 1.11 Late Domains...... 62

Figure 1.12 Ubiquitination and Sumoylation...... 67

Figure 1.13 MVB Sorting/Retrovirus Budding Machinery ...... 73

Figure 2.1 RSV Gag Mutants and Chimeras...... 93

Figure 2.2 Requirement of Lysines 95, 115, 124, 138, and 148 For Particle Release...96

Figure 2.3 Intracellular Localization of Mutant 1-5KR ...... 99

Figure 2.4 Characterization of 1-5KR Virions...... 102

Figure 2.5 Complementation of the 1-5KR Defect With Wild-Type Gag...... 106

Figure 2.6 Restoration of Budding With Same-Site Revertants ...... 109

Figure 2.7 Restoration of Budding With Second-Site Revertants ...... 111

Figure 2.8 Introduction of Lysines in HTLV-1...... 114

x Figure 3.1 Positions of Mutants K244R and E246A and Their Suppressor Mutations ...... 134

Figure 3.2 Budding and Infectivity of Sumoylation Consensus Sequence Mutants....136

Figure 3.3 Intracellular Localization of K244R...... 140

Figure 3.4 Characterization of K244R Virions ...... 142

Figure 3.5 Incorporation of Pol, Env, and vRNA Into K244R Virions ...... 145

Figure 3.6 Products of Reverse Transcription...... 148

Figure 3.7 Basic Strategy For Isolating Revertant Viruses...... 151

Figure 3.8 Deletion of egfp in Passaged Viruses ...... 154

Figure 3.9 Isolation of K244R and E246A Revertant Viruses...... 156

Figure 3.10 Infectivity of K244R and E246A Suppressors ...... 159

Figure 4.1 Hrs Not Required For RSV Budding...... 174

Figure 4.2 Tsg101 Inhibition of RSV Budding...... 176

Figure 4.3 ESCRT-II Proteins Not Essential in RSV Budding...... 179

Figure 4.4 Several ESCRT-III Dominant-Negative Proteins Inhibit RSV and HIV Budding...... 181

Figure 4.5 Dominant-Negative Vps4A Inhibition of RSV Budding...... 184

Figure 4.6 Potential AIP1/ALIX Binding Site in RSV Gag Involved in Budding ...... 187

Figure 4.7 Dominant-Negative Effect of PPPY-A on Wild-Type Gag Budding...... 190

Figure 5.1 Model for RSV Budding...... 201

Figure 5.2 Rescue of 1-5KR by the v-Src Membrane Binding Domain ...... 204

xi LIST OF TABLES

Table Page

Table 1.1 Incorporated Cellular Proteins...... 58

Table 1.2 Vps Class E Proteins...... 71

xii LIST OF ABBREVIATIONS

+sssDNA plus-strand strong-stop DNA

–sssDNA minus-strand strong-stop DNA aa amino acid

BLV bovine leukemia virus

CA capsid cDNA copy-DNA

CTD C-terminal domain cpm counts per minute dNTP deoxyribonucleotide triphosphate

DIS dimerization initiation site

DR direct repeat eGFP enhanced green fluorescent protein egfp gene encoding the enhanced green fluorescent protein

EIAV equine infectious anemia virus

Elf-1a elongation factor 1α

Env envelope glycoprotein env gene encoding the envelope glycoprotein

ER endoplasmic reticulum

ESCRT endosomal sorting complex required for transport

FACS fluorescence activated cell sorting

Gag group specific antigen gag gene encoding Gag

xiii GFP green fluorescent protein gfp gene encoding green fluorescent protein

HA hemagglutanin

HFV human foamy virus

HIV human immunodeficiency virus

HTLV human T cell leukemia virus

IAP intracisternal A-type particle

I domain interaction domain

IN integrase

IRES internal ribosomal entry site

L domain late domain

LCMV lymphocytic choriomeningitis virus

LFV Lassa fever virus

LMB leptomycin B

LTR long terminal repeat

M domain membrane binding domain

MA matrix

MHR major homology region

MLV murine leukemia virus

MMTV mouse mammary tumor virus

MPMV Mason-Pfizer monkey virus

MTOC microtubule organizing center

MVB multivesicular body

xiv NC nucleocapsid

NES nuclear export signal

NLS nuclear localization signal

NPC nuclear pore complex nt nucleotide

NTD N-terminal domain

ORF open reading frame

PBP phosphatidylethanolamine-binding protein

PBS primer binding site

PCR polymerase chain reaction

PIC preintegration complex

Pol polymerase pol gene encoding the polymerase protein

PPT polypurine tract pro the gene encoding protease

PR protease

Q-PCR quantitative real-time PCR

Q-RT-PCR quantitative real-time RT-PCR

RER rough endoplasmic reticulum

RNP ribonucleoprotein

RSV Rous sarcoma virus

RT reverse transcriptase

RTC reverse transcription complex

xv RT-PCR reverse transcription-PCR

SA splice acceptor

SD splice donor

SDS sodium dodecyl sulfate

SIV simian immunodeficiency virus

SU surface glycoprotein

SUMO-1 small ubiquitin-like modifier

TEF turkey embryonic fibroblast

TEM transmission electron microscopy

TM transmembrane glycoprotein

Tv tumor virus

Ub ubiquitin

UEV ubiquitin conjugating enzyme E2 variant

UIM ubiquitin interaction motif

VLP virus-like particle

Vps vesicular protein sorting vRNA viral RNA

VSV vesicular stomatitis virus

WDSV walleye dermal sarcoma virus

xvi ACKNOWLEDGMENTS

I believe that success in graduate school requires a productive laboratory/advisor, a supportive spouse/family, and luck. Although luck seemed to be scarce in many of my experiments, I have not lacked a talented lab or a dedicated advisor. I have had the fortunate opportunity to be mentored by an enthusiastic and creative scientist, Dr. John

Wills. John has provided a great environment for a young scientist to develop the skills required to succeed. Thank you for all the wisdom, guidance, and encouragement throughout my years in the lab and for developing me into a competent scientist. A special thank-you goes to past and present members of the Wills lab: Brad, Tina, Akash,

Eric, Josh, Pei-Chun, Nick, David, Amy, and especially Carol, whose technical expertise has been invaluable. Thanks also to Dr. Rebecca Craven for all of her insights. I would like to also thank my committee members Drs. Richard Courtney, Leslie Parent, David

Spector, and Vincent Chau for the time they dedicated to help me grow as a scientist.

Even within a highly productive lab and with a lot of luck, I cannot see how anyone could have a successful and/or fulfilling graduate school career without the support of a spouse, family, or friends. Without them I would dare say that spending five to six years pursuing a degree would be nearly impossible. I fully know that without my wife, Meredith, none of this work would have been possible or even worth it. Her love, support, and encouragement have been essential on every level. Words could never do justice to express my thanks and appreciation for her through these past five and a half years. This dissertation is appropriately dedicated to her. I must also thank my parents,

Jeff and Vickie, for all their support through too many years of schooling and for never hindering, but always channeling the essential element of science – curiosity.

xvii CHAPTER I

LITERATURE REVIEW

INTRODUCTION

Enveloped viruses must acquire a portion of a cellular membrane to complete their synthesis, a process which forces the virus to perform a membrane fission event.

Due to electrostatic repulsion between the bilayers and the steric interaction of membrane proteins, the distance between two biological membranes is usually no closer than ~10-20

nm (382). Therefore, these viruses are faced with the challenge of bringing two

membranes in close enough proximity to allow lipid mixing. Not only must the

membranes be brought together, but an activation energy barrier must be overcome to

allow lipid mixing.

The mechanisms by which viruses perform this function are unknown, but clues

can be taken from the way membrane fission is accomplished at the cellular level. Many

processes within the cell also demand fission of membranes, and a variety of methods are

utilized. Endocytosis and budding of vesicles from the endoplasmic reticulum (ER) and

Golgi apparatus involve invagination of the membrane mediated by a protein coat on the

cytoplasmic side of the membrane, eventually forming a bud with a neck (Figure 1.1).

Proteins are then recruited to the cytoplasmic face of the neck to perform the fission

event. The budding of viruses is morphologically similar to budding of these vesicles,

but are not topologically the same, i.e., viruses coat the inside of their bud with protein

whereas vesicle buds are coated on the outside with proteins. Furthermore, the neck of a

virus bud is not exposed to the cytoplasmic proteins used to constrict the neck of budding vesicles. Therefore, the mechanism of virus budding must be different from these cellular processes.

There is another cellular membrane fission event which is both morphologically

2

Figure 1.1 Membrane fission. Cell surface receptors can be endocytosed upon binding their ligand. The vesicle is released from the plasma membrane into the cytoplasm by a membrane fission event. The endocytic vesicle can then travel to and fuse with an endosome. The receptor is degraded by budding into the endosome thus forming a multivesicular body (MVB). Release of the invaginating vesicle also requires membrane fission. The topology of MVB formation is similar to budding of enveloped virus from the plasma membrane and the mechanisms used by the virus to facilitate its release may be similar to those used during MVB formation.

3 Viral Budding

Membrane Endocytosis Fission

Membrane Fission

MVB Formation

Membrane Fission

4 and topologically similar to virus budding (Figure 1.1). Once endocytosed, vesicles fuse with endosomes and can invaginate and bud into themselves to create a multivesicular body (MVB). Like virus budding, the neck cannot be squeezed from the outside, and this membrane fission must occur by a mechanism different from other cellular vesicles.

Some of the proteins involved in MVB formation have recently been discovered, and it is easy to imagine these proteins being involved in the pinching off of a virion. One protein that is central in MVB formation is ubiquitin (Ub). Ub is covalently linked to a target protein by enzymes termed Ub ligases which form an isopeptide bond between Ub’s C- terminal glycine and the ε-amino group of a lysine on the modified protein. The proposed function of Ub in MVB formation is the recruitment of protein complexes that sort cargo proteins to the site of budding. Ub and/or Ub ligases have been proposed by several labs to be required for budding of retro-, rhabdo-, and filoviruses. Chapter II examines a potential role for Ub in budding of the avian retrovirus Rous sarcoma virus

(RSV). The data suggest that attachment of Ub to Gag (the protein which drives RSV budding) is required for release of RSV from the cell.

During the course of this study a lysine in Gag was identified to be critical for infectivity, but not budding. The lysine resides in a sumoylation consensus sequence. A sumoylation sequence is recognized by an enzyme which covalently modifies a lysine with a Ub-like protein called Small Ub-like MOdifier (SUMO)-1. Analysis of the infectivity defect of the lysine mutant revealed a defect in reverse transcription. Chapter

III discusses the involvement of this lysine in the virus life cycle and what role SUMO-1 may play during retroviral replication.

Chapter IV focuses attention on the connection between MVB formation and

5 retrovirus budding. The data suggest that proteins utilized by the cell to facilitate budding of vesicles into MVBs are also used by RSV for budding from the plasma membrane. Though our understanding of how retroviruses detach themselves from the plasma membrane is incomplete, the studies presented here begin to shed light on the cellular proteins which may be involved in this process.

The data presented in this dissertation encompass many aspects of the retroviral life cycle. Therefore, to understand the work presented in the following chapters it is necessary to review retroviral replication. Different retroviruses utilize similar mechanisms throughout their life cycle of which many aspects will be discussed in general terms. However, specific differences between viruses are noted, and the mechanisms of RSV replication are particularly emphasized.

ORGANIZATION OF THE RSV GENOME

Retroviruses are divided into seven genera based on genome organization and size, the genes expressed, their mode of assembly, virion morphology, and the species they infect. The description of each genus is beyond the scope of this review, but it is useful to list them and highlight key members since differences among genera are discussed below. The genera are alpharetrovirus (RSV), betaretrovirus (mouse mammary tumor virus [MMTV]), gammaretrovirus (murine leukemia virus [MLV]), deltaretrovirus

(bovine leukemia virus [BLV]), epsilonretrovirus (walleye dermal sarcoma virus

[WDSV]), spumavirus (human foamy virus [HFV]), and lentivirus (human immunodeficiency virus [HIV]).

The packaged genome (Figure 1.2) in the virion is two copies (a dimer) of viral

6

Figure 1.2 RSV genome organization. The genome of RSV exists as both DNA and

RNA at various times through replication. The integrated proviral DNA has direct repeats called long terminal repeats (LTRs) at the 5’ and 3’ ends of the genome and encodes all the genes required for replication. The 5’ LTR initiates synthesis of full- length viral transcripts. The RSV genome encodes four genes for the proteins Gag, Pol,

Env, and v-Src. Gag and Gag-Pol are transcribed from unspliced RNA while Env and v-

Src are transcribed from spliced transcripts. The splice donor (SD) and splice acceptor

(SA) sites are indicated. In addition to encoding viral genes, it also contains elements essential for being packaged into the virion (Ψ), for binding the tRNA which initiates reverse transcription (PBS), and for initiating minus-strand synthesis during reverse transcription (PPT).

7 5’ LTR 3’ LTR gag pol env v-src Provirus

S T B P Genome P Y P mRNAgag 5’ mG AAA gag-pol gag pol env v-src mRNA R U5 U3 R Gag (Pr76) Gag

Gag-Pol (Pr180) Gag Pol

SD SA mRNAenv 5’ mG AAA

Env (gPr95) Env

SD SA mRNAv-src 5’ mG AAA

v-Src (pp60) v-Src 8 RNA (vRNA) (482) which, upon infection of the cell, is reverse transcribed and integrated into the host genome to form genomic DNA termed “proviral DNA”. The 5' and 3' ends of proviral DNA are flanked by repeated elements known as long terminal repeats (LTRs) divided into regions termed U5, R, and U3. The promoter is in U3 and drives transcription of the vRNA beginning at the 5' R and ending with the 3' R thus flanking the RNA with direct repeats and a unique region (either U5 or U3 at the 5' or 3' end, respectively). This places the promoter at the 3’ end of the vRNA; through reverse transcription the 3’ U3 is copied to the 5’ end (see below). vRNA (Figure 1.2) is a (+)- strand RNA similar to cellular mRNA with a 5'-methylguanosine cap, internal methylation, and a polyadenylated tail (217, 237) measuring 9312 nt in length for RSV

(19, 28, 428) and is used for translation of the genes as well as the genome packaged by the virion.

All retroviruses encode four genes – gag (group-specific antigen), pro (protease),

pol (polymerase), and env (envelope). Unlike other retroviruses such as HIV where pro is in frame with pol, the RSV pro gene is in frame with gag and is not considered a separate gene. Retroviral genomes often contain genes in addition to gag, pro, pol, and env, and based on the complexity of the genome retroviruses can be subdivided into two categories (482). Complex retroviruses such as lentiviruses contain accessory genes (in addition to gag, pro, pol, and env) involved in activation of transcription, nuclear export of unspliced RNA, etc. and are required for replication. RSV is a simple retrovirus because it lacks accessory genes. However, the genome of RSV also contains the oncogene v-src. Expression of this gene transforms cells and causes tumors in chickens

(218, 404, 467). In a culture of monolayer primary cells, infected cells transformed by v-

9 src lose contact inhibition and continue to divide. The result is a single infected cell

giving rise to a focus. This feature of the virus allows one to determine the amount of

infectious virus in a sample since a single virus gives rise to a single focus (467). v-src is dispensable for the replication cycle and can be replaced, for instance, by a reporter such as a gene encoding a drug resistance marker or fluorescent protein (e.g., 186). The spreading assay used to measure infectivity in Chapters II and III utilize replacement of v-src with the gene for the enhanced green fluorescent protein (egfp) to determine the

number of infected cells by counting green cells.

As indicated in Figure 1.2, the env and v-src genes are translated from spliced

transcripts, and the pol gene product is expressed by a -1 frameshift event during

translation which fuses Gag with Pol (195). Therefore, vRNA must exist in two

populations: spliced RNA for translation of Env and unspliced vRNA for Gag and Gag-

Pol translation and to serve as the packaged genome.

Besides the genes required for replication, vRNA also has other elements essential

for replication. Directly downstream of U5 is the primer binding site (PBS) which binds

a tRNA primer used to initiate reverse transcription. Directly 5' of the U3 is the

polypurine tract (PPT) that serves as the primer for plus-strand DNA synthesis during

reverse transcription.

PARTICLE MORPHOLOGY AND STRUCTURE

Retrovirions are spherical particles which typically range in size from 120-140

nm in diameter (47, 225, 499) and are ~250 MDa in mass (483). A typical virion is

composed of approximately 65% protein, 30% lipid, and 2% RNA (378). About two-

10 thirds of the RNA is genomic vRNA with the remainder being the tRNA primer for reverse transcription and a small portion being cellular RNAs (30, 101, 112).

Approximately 75% of the total protein is the structural protein Gag (483), which upon maturation, is cleaved by protease (PR) into matrix (MA), capsid (CA), nucleocapsid

(NC), and smaller proteins designated by their molecular weights. About 5% of Gag is fused to Pol (or Pro-Pol depending on the virus) as a result of a frameshifting event; Pol is cleaved by PR into reverse transcriptase (RT) and integrase (IN). Various cellular proteins are packaged into the virion during assembly which represent only a few percent of the total protein (e.g., 336, 337, 377). The rest of the protein is accounted for by the glycoprotein Env embedded in the envelope. Env is synthesized as a precursor and cleaved into surface (SU) and transmembrane (TM) glycoproteins held together by either a disulfide bond or non-covalent interactions, depending on the virus. It is generally accepted that the source of the lipid envelope is the plasma membrane of the host cell since the lipid composition of the envelope is similar to that of the plasma membrane.

However, it must be noted that the lipid content of the envelope compared to the plasma membrane is enriched in sphingomyelin and cholesterol with less phosphatidylcholine (5,

378), possibly due to the virus budding from a microdomain of the plasma membrane.

Alternatively, the envelope may be acquired from a membrane other than the plasma membrane (see below).

The Immature Virion

The immature virion is composed of Gag and Gag-Pol; however, Gag-Pol only makes up about 5% of the total Gag proteins. There are about 1500 Gag molecules per

11 virion for RSV (483) and about 5000 Gag molecules for HIV-1 (46) surrounded by a lipid envelope studded with TM and SU trimers (Figure 1.3). Gag and Gag-Pol are arranged radially with the N-termini interacting with the inner leaflet of the bilayer and the C-termini pointing inward (499). Gag is organized into domains corresponding to

MA, the N-terminal domain (NTD) of CA, the C-terminal domain (CTD) of CA, and NC complexed with the vRNA (56, 77, 130, 270) resulting in an electron dense ring and a lucent center when examined by transmission electron microscopy (TEM) (Figure 1.3).

Maturation

Once the immature virion is assembled another round of infection beings.

Through a process called maturation, PR cleaves Gag into MA, CA, NC, and smaller proteins named for their molecular weight (p2a, p2b, and p10 for RSV). Also during maturation, the enzymes which direct reverse transcription and integration (RT and IN, respectively) are released from Pol (71, 449, 484). Maturation is required for infectivity and the timing of it is crucial to the formation of infectious particles. If PR activates before budding, Gag is cleaved and the virion collapses back into the cytoplasm (57, 231,

507).

PR requires homodimerization to function, and it is thought that Gag-Gag and

Gag-Gag-Pol intermolecular interactions initiate this dimerization. Once free, PR cleaves

Gag and Gag-Pol. PR belongs to the family of aspartate proteases that share the active site consensus sequence Asp-Thr/Ser-Gly and use a water molecule to hydrolyze a target peptide bond (239, 301, 471). The target sites within Gag and Pol are typically exposed hydrophobic regions which can fit in the hydrophobic active site of PR.

12

Figure 1.3 Immature virion. The immature virion is composed of a lipid bilayer studded with Env trimers. The interior contains unprocessed Gag and Gag-Pol proteins arranged radially bound to the packaged vRNA dimer. This arrangement results in an electron dense ring and a lucent center when examined in an electron micrograph (I).

13 The Immature Virion

Gag Gag-Pol Env

MA SU p2a/b p10

CA TM I

NC I PR vRNA

RT/IN

14 The Mature Virion

Upon maturation, an electron dense core corresponding to condensation of the core made up of the ribonucleoprotein (RNP) complex can be seen by TEM. This complex consists of the vRNA coated with NC. RT and IN are also located in the core poised for reverse transcription and integration (36, 88, 453). MA remains associated with the envelope (139, 358) (Figure 1.4).

The RNP complex is surrounded by a capsid-like shell made up of CA. There are three capsid shapes which are dependent upon the genus of virus. Capsids from most retroviral genera are somewhat spherical (e.g., RSV), but others are conical (e.g., HIV) or

cylindrical (e.g., Mason-Pfizer monkey virus [MPMV]). Despite poor amino acid

conservation (except for a 20 aa stretch in the CTD known as the major homology region

[MHR]) and differences in the final shape of the capsid between genera, there is great

conservation in the secondary and tertiary structures of CA suggesting similarities in CA-

CA interactions and a common mechanism by which all capsids are formed (308). For

example, all CA proteins are organized into two domains (NTD and CTD) connected by

a flexible linker (24, 64, 197, 219, 224, 306). The NTD of HIV, RSV, MLV, and human

T cell leukemia virus (HTLV)-1 consists of a short two-stranded β-hairpin followed by

six or seven α-helices, and the CTD is comprised of a four-helix bundle (Figure 1.5A).

Within the RSV β-hairpin is a sumoylation consensus sequence. The importance of this sequence during replication is analyzed in Chapter III.

Interactions between CA proteins are required for an infectious virus. Studies using capsids from viral preparations or in vitro capsid-like structures revealed that CA proteins are arranged in hexagonal rings, mediated by six-fold axis interactions between

15

Figure 1.4 Mature virion. The virion matures when protease (PR) activates and cleaves

Gag and Pol into matrix (MA), capsid (CA), nucleocapsid (NC), PR, reverse transcriptase

(RT), integrase (IN), and other smaller proteins designated by their molecular weights

(p2a, p2b, and p10). MA associates with the lipid bilayer while CA forms a capsid shell

around the ribonuleocomplex (RNP) composed of RT, IN, and the RNA coated by NC.

Maturation results in a condensed electron-dense core (M) clearly distinguishable from

the immature (I) virions when examined by electron microscopy.

16 The Mature Virion

MA Env p2a/b M or p10 SU CA

NC TM M PR

RT vRNA

IN

17

Figure 1.5 Structure of CA. (A) The crystal structure of the monomeric RSV N terminal (NTD) and C terminal (CTD) domains are diagramed with a flexible linker connecting them. The NTD consists of a β hairpin followed by seven α helices. The

CTD is comprised of four α helices. The major homology region (MHR) and dimerization domain are indicated. (B) The crystal structure of the MLV NTD hexamer is shown. Individual monomers are colored grey, orange, yellow, green, blue and purple and the β hairpin is highlighted in red in this representation of the molecular surface of the hexamer (Top). Side (middle) and top (bottom) views of the NTD consisting of six α- helices are represented by cylinders and the two β hairpin by ribbons. The molecular dimensions of are indicated. Source: Nature 431:481-485 (2004); used by permission.

18 A B b Hairpin

NTD

N

MHR Flexible Linker

CTD Dimerization Helix

C

19 the NTDs and two-fold interactions between the CTDs (17, 48, 256, 308) (Figure 1.5B).

In the mature CA protein the β-hairpin (formed only upon maturation [316, 462, 486,

487]) is located at the center of a CA hexamer and is involved in intermolecular

interactions with the β-hairpin of adjacent CAs (308). If the formation of the β-hairpin

after maturation is inhibited the capsid cannot form and infectivity is reduced (486, 487)

likely due to a destabilization of the hexamer. The intermolecular interactions between

NTDs are important not only for capsid assembly, but also for disassembly, because

mutations that result in a more stable capsid are not as infectious as the wild type (121).

Interactions between the CTDs also appear to be important for capsid assembly

and stability. The MHR in the CTD is required for proper morphogenesis of the capsid

(83, 276), likely through a network of hydrogen bonds that helps maintain the overall

CTD structure (132). The MHR may also function in Gag-Gag multimerization required

for assembly of the immature particle. A region in the CTD termed the “dimerization

helix” is present in all retroviruses, and, as the name suggests, this helix is at the CA-CA

dimer interface (132) (Figure 1.5). The RSV dimerization helix also contains a cysteine

poised for the formation of an intermolecular disulfide bond between two adjacent CTDs

(63). Two cysteine residues are conserved in lentiviruses and deltaretroviruses, and

mutating the HIV-1 cysteines to serine results in a noninfectious virus (63, 289). For

HIV-1 and equine infectious anemia virus (EIAV) these cysteines are oxidized and form

intramolecular disulfide bonds (132, 197). In RSV and HTLV-1, the cysteine(s) are

reduced in vitro, but upon budding into the oxidizing environment of the bloodstream,

they may become oxidized able to form a disulfide bond between dimerized CA proteins

(63, 220).

20 Interactions between the NTD and CTD may also be critical for an infectious virus. Recent studies using deuterium exchange protection, chemical cross-linking, and an in vitro capsid assembly assay suggest that the NTD makes intermolecular contacts with the CTD of an adjacent molecule in a formed hexamer (133, 238). Further, mutations in the MHR of RSV that reduce infectivity can be suppressed by mutations in the NTD (44). Similarly, data in Chapter III show that mutations in the β-hairpin can be suppressed by substitutions in both the NTD and CTD, again suggesting interactions between the two domains.

ENTRY

Upon maturation, the virion is ready to begin another round of replication (Figure

1.6). In order to make a productive infection, the virus must first gain access to the inside

of the cell. Once there the vRNA genome must be synthesized into a DNA copy

(cDNA), which enters the nucleus and integrates into the host DNA.

Attachment

On the surface of the virion, Env exists as a trimer of SU-TM heterodimers

(Figures 1.3 and 1.4). The receptor-binding SU is exposed at the surface, and the regions

of TM involved in fusion are presumably buried within the trimer bundle (110, 141, 228).

Through an interaction between SU and the cellular receptor, retrovirions attach to the

plasma membrane of a target cell. Every retroviral genus utilizes a different cellular

receptor for attachment. For example, the avian sarcoma and leukemia virus family (of

which RSV is a member) attach to one of three receptors (Tumor virus [Tv]a, Tvb, and

21

Figure 1.6 Retroviral entry. A virion attaches to the plasma membrane of a target cell through an interaction between Env and the receptor/coreceptor. There are three hypothesized routes of entry the virion can take, depending on the virus. 1.) pH independent entry involves fusion of the envelope directly with the plasma membrane.

When Env interacts with its receptor (and coreceptor if necessary), conformational changes in Env occur which drive the membrane fusion process. 2.) In pH-dependent fusion attachment of the virion to the receptor triggers endocytosis followed by acidification of the vesicle which triggers a conformational change in Env resulting in fusion of the envelope and vesicle. 3.) A hybrid of the other two mechanisms has recently been hypothesized involving Env undergoing an activation event upon binding of its receptor followed by endocytosis; at this stage lipid mixing between Env and the plasma membrane can occur. The primed Env is sensitive to acidification of the vesicle and undergoes further conformational changes which mediate fusion. In the cytoplasm the reverse transcription complex (RTC) reverse transcribes the RNA genome into double stranded DNA and is targeted to the nucleus as a preintegration complex (PIC). In the nucleus the proviral DNA then integrates into the host DNA.

22 3. pH independent and dependent fusion 2. pH dependent fusion 1. pH independent fusion Receptor binding Receptor binding - Endocytosis - Endocytosis - Conformational change in Env - Lipid mixing

Receptor binding - Conformational change in Env - Exposure of fusion peptide - Fusion pH pH - Conformational - Exposure of change in Env fusion peptide - Exposure of - Fusion fusion peptide - Fusion

Reverse Transcription

RTC/PIC vRNA

5’LTR gag pol env v-src 3’LTR DNA

Targeting to Nucleus Random Integration

23 Tvc), depending on the subgroup of virus (354, 406, 481), and the receptor for HIV-1 is

CD4 (226, 271, 292). Some viruses also require a co-receptor to trigger conformational changes in Env that expose the fusion peptide (518). HIV-1 entry depends on one of two coreceptors, CXCR4 or CCR5, depending on the strain of virus (73, 96, 100, 118). Both coreceptors are chemokine receptors, and their natural ligands can inhibit HIV-1 replication.

Fusion and Uncoating

Subsequent to binding the receptor (and coreceptor if necessary), the virus can enter the cell by one of three mechanisms (Figure 1.6). The first is to fuse the envelope directly with the plasma membrane (pH-independent); HIV-1 utilizes this mechanism.

When Env interacts with its receptor and coreceptor, conformational changes in Env occur which drive the membrane fusion process (160, 202, 415). A second mechanism

(pH-dependent) involves interaction with a receptor that triggers endocytosis of the virion followed by acidification of the vesicle which initiates fusion of the envelope and vesicle.

This mechanism is utilized by MLV (206). The third mechanism, a hybrid of the other two mechanisms, has recently been hypothesized to be used by avian sarcoma and leukemia viruses. This mechanism is evidenced by the observation that fusion proceeds only to lipid mixing at neutral pH, and low pH is required for uncoating. Env is postulated to undergo an undefined activation or priming event upon binding of its receptor followed by endocytosis. Priming of Env triggers a conformational change and renders it sensitive to acidification of the endocytosed vesicle which induces further conformational changes that mediate fusion (95, 106, 295, 311, 317, 318, 441).

24 Though not all retroviruses attach and initiate fusion the same way, the actual fusion steps are likely to be the same. Indeed, the fusion mechanisms for all enveloped viruses may follow the same mechanism. The most highly studied fusion protein is the hemagglutanin (HA) protein from influenza virus. The current model for Env-mediated fusion is based on the model for HA-mediated fusion (Figure 1.7) (183, 190, 440). By analogy to HA and direct observation, the conformational changes that occur after

receptor/coreceptor binding result in the formation of a prehairpin intermediate with the

buried hydrophobic fusion peptide of TM becoming exposed followed by its insertion

into the cell membrane. A six-helical complex termed the hairpin is then formed

consisting of two antiparallel α-helical domains per TM in a trimer complex (58, 68, 296,

459, 492). This complex pulls the two membranes together allowing the lipids from the

outer leaflets of both membranes to mix resulting in hemifusion. This intermediate is

followed by mixing of the inner leaflets thus forming a fusion pore (230). The fusion

pore then expands allowing release of the virions contents into the cytoplasm leading to

the undefined process of uncoating.

Reverse Transcription

Reverse transcription is not only essential for making a DNA copy of the vRNA,

but is also required to move the promoter from the 3’ end to the 5’ end. Transcription of

the proviral DNA beings at the 5’ R and ends at the 3’ R (see below), and the resulting

transcript is missing the corresponding 5’ U3 and 3’ U5. Given that the promoter is located in U3, if this vRNA transcript is directly copied, the DNA copy would lack a functional promoter. Therefore, the virus must copy the 3’ U3 to the 5’ end to allow

25

Figure 1.7 Membrane fusion. The current model for Env-mediated fusion is illustrated.

(1) Binding of the cellular receptor/coreceptor (black) by Env (blue) results in conformational changes that (2) expose the buried hydrophobic fusion peptide (red) of

TM followed by its insertion into the cell membrane (yellow). (3) The six-helical hairpin complex consisting of antiparallel α-helices (pictured without SU) is then formed and pulls the two membranes together. (4) As the viral (green) and cellular membranes are brought in contact with each other, the fusion pore forms.

26 1. Receptor 2. Exposure of 3. Formation of 4. Pore formation binding fusion peptide hairpin

Fusion peptide

27 transcription of the integrated DNA. As discussed below, the mechanism by which the vRNA is reverse transcribed provides the means by which the promoter is copied to the

5’ end of the proviral DNA.

Synthesis of an integration-competent DNA copy of the vRNA is initiated and performed by the reverse transcription complex (RTC) following the release of the viral core into the cytoplasm (Figure 1.6). This complex contains at least the NC-coated vRNA, RT, and IN. Other viral components of this complex are unclear since RTCs are sensitive to mild detergents and salts, thus making their purification difficult. After purification, the RTCs of simple retroviruses contain MA and CA, and lentiviruses contain MA but are totally void of CA (41, 55, 113, 115, 116, 205, 302). The difference between lentiviruses and simple retroviruses in their ability to infect nondividing cells

(see below) may be an artifact. Lentiviruses readily infect nondividing cells whereas simple viruses poorly infect nondividing cells (188, 189, 254, 255, 398), but the determinant of infection of nondividing cells has been mapped to CA (508). Hence, it appears that at least some CA must remain associated with the RTC. Evidence that CA may participate in reverse transcription comes from studies that show mutations in CA block steps in reverse transcription (59, 83, 389). Data presented in Chapter III also supports an active role for RSV CA in reverse transcription.

The RTC may also contain cellular proteins. Little is known about these potential components, but one candidate may be SUMO-1. SUMO-1 is a member of the Ub-like protein family and functions by covalent conjugation between its C-terminal glycine and the ε-amino group of a target lysine in the consensus sequence ΨKxE/D (where Ψ is a bulky hydrophobic group and x is any amino acid [199, 294, 397, 410]). Due to the

28 similarities between the sumoylation and ubiquitination pathways, details of sumoylation are discussed below with ubiquitin. In brief, SUMO-1 is involved in many nuclear events within the cell: nuclear translocation, subnuclear targeting, transcriptional regulation,

DNA repair, DNA replication, and chromosome condensation, cohesion, and separation during mitosis (reviewed in 199, 429). Since it is involved in regulation of transcription,

SUMO-1 may be required for efficient reverse transcription, if it is indeed a part for the

RTC. It has recently been reported that sumoylation of HIV p6 may have a regulatory role during reverse transcription, although the exact function is unknown (152).

Evidence in Chapter III points to a potential role of sumoylation of CA during RSV reverse transcription.

After the formation of the RTC, reverse transcription begins. For HIV-1 and

MLV this process may begin in the virion prior to entry, given that reverse transcripts have been detected in virions (474). The actual trigger for DNA synthesis initiation is unknown, but could logically be exposure to deoxyribonucleotide triphosphates (dNTPs) since addition of exogenous dNTPs (and a divalent cation) to permeablized purified particles in vitro results in reverse transcription (37, 38, 402).

Whatever the trigger for initiation, reverse transcription begins by RT binding a genome-bound tRNA primer (tRNATrp for RSV) (Figure 1.8). RT is similar to other

DNA polymerases in that it requires a primer with a 3'-OH group to incorporate free

dNTPs via Watson-Crick base pairing with a template strand (437). The catalytic

reaction involves a nucleophilic attack by the proper dNTP to form a 3'-5' phosphodiester

bond with the primer. Unlike other DNA polymerases, RT can use either a DNA or RNA

template. In addition to being a polymerase, RT also contains an RNase H domain

29

Figure 1.8 Reverse transcription. The vRNA genome is shown complexed with the tRNA primer. Minus-strand DNA (light blue arrow) synthesis begins when RT binds the tRNA primer and synthesis continues through R until it is halted by reaching the 5' cap resulting in minus-strand strong-stop DNA (–sssDNA). The RNase H domain of RT cleaves the RNA-DNA duplex resulting in single stranded DNA with the repeated sequence R at the 3' end homologous to R on the 3' end of the vRNA. The DNA is transferred to the 3' end of the vRNA in a process termed first strand transfer. DNA synthesis continues towards the 5' end of the vRNA until the 5' end of the RNA is reached at the PBS. The RNase H domain degrades the RNA from the RNA/DNA duplex, except for the PPT, which is specifically cleaved, leaving a primer for initiation of plus-strand DNA synthesis (light green arrow). The PPT is cleaved by RNase H soon after plus strand DNA synthesis begins. Plus-strand synthesis continues until copying a part of the tRNA primer which is then degraded by RNase H. The resulting product is known as plus-strand strong-stop DNA (+sssDNA). The two compatible ends with homologous PBS sequences hybridize allowing DNA synthesis to restart. DNA synthesis

continues until the viral genome is completely copied with the final product being linear,

blunt-ended cDNA that is subsequently translocated into the nucleus. In the nucleus the

proviral DNA then either integrates into the host genome or forms an aberrant DNA

product resulting from autointegration or formation of 1- or 2-LTR circle DNA.

30 tRNA DNA Synthesis 5' A A A 3' A A A R U5 PBS PPT U3 R R U5 PBS PPT U3 R

RNase H

First Strand Minus-Strand Transfer Strong-Stop DNA R U5 R U5 A A A A A A PBS PPT U3 R PBS PPT U3 R

DNA Synthesis RNase H DNA Synthesis PBS PPT U3 R U5 PPT U3 R U5 RNase H

PBS PPT PPT U3 R U5 RNase H PBS U5 R Second Strand Plus-Strand U3 Transfer U5 PBS Strong-Stop DNA R PBS PPT U3 R U5 U3

U3 R U5 PBS

DNA Synthesis U3 R U5 PBS PPT U3 R U5

U3 R U5 PBS PPT U3 R U5 Proviral DNA

Cytoplasm

Aberrant DNA Nucleus Products OR Integration Into Host Genome U3 R U5 PBS

U3 R R U5 PBS U5 U3 U3 R U5

U5 U3 1-LTR Circle DNA R R or U3 U5 U3 U5 R

Autointegration R U3 U5

P P

2-LTR Circle DNA T

U P

3

P

R

T

U 5

U

3

R

U 5

31 (separate from the polymerase domain, 461), which is capable of hydrolyzing the RNA strand from a RNA/DNA duplex (305), thus degrading the vRNA during reverse transcription.

An active RT functions as a heterodimer with α and β subunits. Upon cleavage

by PR, Pol is processed into either an α polypeptide (63 kDa, containing the polymerase

and RNase H domains) or a β polypeptide (95 kDa, containing the polymerase, RNase H, and integrase domains, but lacking 4.1 kDa at the C terminus; IN is also processed as a separate and functional enzyme). The α subunit provides the catalytic activity whereas the β subunit contributes mainly to the structural stability of the enzyme (496).

RT lacks proofreading activity (18) and is thus highly prone to error with a

fidelity of 10-4/bp for RSV (249). In comparison, the fidelity of T4 DNA polymerase is

10-7/bp. Though this error may not be advantageous to an individual virion, a high mutation rate aids in the survival of the virus population by enabling it to evade the immune system and become insensitive to drug therapies (347). The high error rate of

RT also allows a poorly infectious mutant virus population to regain infectivity by creating a suppressing mutation. Analysis of these second-site suppressors has proven useful in determining functions of regions within the viral proteins or the genome. This approach is utilized in Chapter III.

The current model of reverse transcription is depicted in Figure 1.8 (142). As mentioned, reverse transcription begins by RT binding the tRNA primer bound to the

PBS near the 5' end of the genome. Minus-strand DNA synthesis then proceeds until

synthesis is halted by reaching the 5' cap. The DNA product is known as minus-strand

strong-stop DNA (–sssDNA). During minus strand synthesis, the vRNA template is

32 cleaved by the RNase H domain of RT thus freeing the –sssDNA. The newly synthesized

DNA has the repeated sequence R at the 3' end homologous to R on the 3' end of the vRNA; homology and the structure of the RNA facilitates the –sssDNA transfer to the 3' end of the vRNA (22, 23, 235). This process is known as first strand transfer. DNA synthesis is again initiated and continues towards the 5' end of the vRNA. The RNase H

activity continues to degrade the RNA as the DNA is copied until it reaches the 5' end of

U3. This region contains a stretch of purines known as the polypurine tract (PPT)

recognized by RNase H, which specifically cleaves around it leaving a primer for

initiation of plus strand DNA synthesis. This primer is then cleaved by RNase H soon

after plus strand DNA synthesis begins. Plus strand synthesis continues and stops after

copying a part of the tRNA primer corresponding to the PBS. The tRNA is then

degraded by RNase H. The resulting product is known as plus-strand strong-stop DNA

(+sssDNA). Minus-strand DNA synthesis continues using the RNA as template until the

5' end of the RNA is reached at the PBS. The DNA must now make another transfer to

continue synthesis of the full genome. The two compatible ends with homologous PBS

sequences hybridize allowing DNA synthesis to restart. RT then extends the minus

strand on the plus-strand template; the minus-strand DNA from which the plus-strand

was copied is displaced. At this point the promoter (i.e., the 3’ U3) has been successfully

copied to the 5’ end. DNA synthesis continues until the viral genome is completely

copied with the final product being linear, blunt-ended DNA.

The process of reverse transcription can be detected by polymerase chain reaction

(PCR) amplification. After infection, the DNA products can be isolated, and the amount

of each product can be quantified using quantitative real-time PCR (Q-PCR). This

33 technique is utilized in Chapter III, so a brief description is necessary. –sssDNA is detectable using primers specific to R and U5. Primers specific to U5 and U3 allow amplification of first strand transfer DNA since U3 is synthesized only after strand transfer. Regions 3' of the PBS are synthesized only after the second strand transfer which can be detected using primers specific for R and a region 3' to the PBS.

Nuclear Targeting and Entry

Integration of the DNA into the host genome requires entry into the nucleus. The complex which gains entry into the nucleus and facilitates integration is generally referred to as the preintegration complex (PIC). The PIC is formed in the cytoplasm from the RTC, and both vary little in composition from each other; therefore, their names are used somewhat interchangeably. The exact path a RTC/PIC takes to the nucleus is unknown. Given the viscosity, travel through the cytoplasm cannot be passive (267), and the PIC must therefore associate with cytoskeletal proteins. Indeed, several studies suggest that the PIC associates with actin early after uncoating and is transported to the microtubules where it associates with a minus end-directed motor and targets to the microtubule organizing center (MTOC) near the nuclear membrane where it can gain access to the nucleus (53, 260, 290, 498).

How the PIC gains access into the nucleus differs between retroviruses.

Lentiviruses such as HIV have the ability to infect both dividing and nondividing cells

(254, 491); however, infection by most retroviruses depends on the cell cycle (188, 189,

254, 398). For MLV this restriction depends on the inability of the PIC to enter the nucleus of nondividing cells (398). The PIC is estimated to be ~56 nm in diameter (302),

34 larger than the allowed size for passive diffusion through the nuclear pore complexes

(NPCs) (<25 nm [105]); therefore the PIC must either wait for disassembly of the nuclear envelope to gain access to the nucleoplasm or be actively transported across the nuclear envelope. Lentiviruses overcome this barrier using a nuclear localization signal (NLS) within the PIC (in MA, IN, and/or Vpr) or by interaction with another protein (e.g., importin 7 [114] or LEDGF/p75 [273]) which actively transports it into the nucleus (40,

54, 122, 155, 168, 254, 368, 369, 480, 485). MLV, however, apparently lacks a mechanism to be actively transported and therefore must rely on mitosis.

RSV is also dependent on mitosis to establish a productive infection, similar to

MLV. However, RSV can infect cells arrested in the G1/S phase ~10-fold better than

MLV, though RSV infects those cells 33-fold less than HIV (165), thus suggesting a

difference between RSV and MLV in their dependence on mitosis. Other studies indicate

that RSV gains access to the nucleus and integrates during S phase, and mitosis is

required for replication events after integration (187, 209). What happens after mitosis to

allow replication of the RSV genome is unknown. Nonetheless, the route of nuclear

entry for RSV is believed to be an NLS in IN and/or MA which mediates its translocation

through the NPC (135, 233, 234, 419).

The cellular requirements involved in RSV (and HIV) PIC translocation are

virtually unknown. Recent evidence indicates that nuclear entry of the HIV PIC depends

on importin 7 interacting with the nucleoporins in the NPC (114). RSV PICs may utilize

a similar mechanism.

Alternatively, PICs may gain access to the nucleus through usage of the

sumoylation machinery involved in nuclear and subnuclear targeting of cellular proteins.

35 As mentioned, sumoylation is important for nuclear translocation and subnuclear target of several cellular proteins, thus making it an attractive candidate for the nuclear translocation of the PIC. Indeed, it has recently been reported that MPMV Gag

(specifically CA) interacts with the E2 SUMO-1 conjugating enzyme Ubc9 and overexpression of Ubc9 causes relocalization of a portion of Gag from the cytoplasm to the perinuclear region (494). The potential of SUMO-1 being involved in RSV nuclear localization through sumoylation of CA is discussed in Chapter III.

Integration

After gaining access to the nucleus, the viral cDNA can then be integrated into the host genome (50). Integration involves two distinct steps: DNA processing and DNA joining. DNA processing by IN results in cleavage of the 3' ends of both termini and removal of two nucleotides yielding recessive 3' ends. IN then attacks the phosphate backbone of the host genomic DNA with the free 3' -OH groups of the cDNA. As a result of the joining reaction, gaps are made at the 3' ends of the host DNA. These gaps are then filled by an unknown mechanism. The 5' ends of the cDNA are then ligated with the host DNA resulting in a stable, heritable DNA version of the genome known as a provirus.

In addition to integration, the cDNA can also form at least three dead-end circular products (Figure 1.8). One product is a result of autointegration where the 3' -OH groups attack internal sites of the cDNA (438, 439). A second product is 1-LTR circle DNA resulting from homologous recombination of the 5' and 3' LTRs (431, 432). A third product is 2-LTR circle DNA derived from the ligation of the two ends of the linear

36 cDNA (431). The formation of 1-LTR or 2-LTR circle DNA has typically been a marker for nuclear entry of the PIC (254, 398), though recent data suggests that 2-LTR circle

DNA may for prior to nuclear entry of the MLV PIC (430). 2-LTR circle DNA is readily detectable by PCR amplification using a forward primer in U5 and a reverse primer in U3 and the amount can be quantified by Q-PCR allowing determination of the efficiency of

nuclear entry of the PIC. This assay is utilized in Chapter III to determine entry of a

mutant virus into the nucleus.

TRANSCRIPTION

The proviral DNA must be transcribed into mRNA to continue replication. It is

therefore essential that integration of the cDNA occurs at an accessible region of the host

DNA. Indeed, analysis of integration sites for RSV, MLV, and HIV has shown a

preference for active genes (303, 319, 424, 504) presumably because those regions are

accessible by factors required for transcription of the proviral DNA.

The LTR contains the positive and negative elements which regulate transcription

(379). The enhancer and promoter are located in the 5' U3 region. Cellular transcription

factors bind specific sequences in U3 to positively or negatively regulate transcription.

U3 also contains a TATA box that binds to factors required for the assembly and

positioning of RNA polymerase II on U3 allowing for initiation of transcription at the

first nucleotide of R. The proviral DNA has no termination signal and transcripts

typically terminate outside the template DNA. A polyadenylation signal (AAUAAA) in

R or U3 regulates mRNA processing in the 3' LTR at the R/U5 border, where it is polyadenylated. The mRNA is also capped at the 5' end and, along with the poly(A) tail,

37 this modification ensures mRNA stability and translation.

Spliced and unspliced viral transcripts can be detected in the cytoplasm (Figure

1.9). The unspliced mRNA is used as the packaged genome and for synthesis of Gag and

Gag-Pol; the spliced transcripts are required for the synthesis of Env and other viral proteins (e.g., v-Src for RSV) (379) (Figure 1.2). Using a single splice donor at codon six in RSV gag and splice acceptor sites at the beginning of env and upstream of v-src, cellular spliceosomes generate subgenomic mRNAs which are then exported for translation in the cytoplasm (154, 191).

The viral genome and the synthesis of Gag and Gag-Pol require unspliced transcripts. Therefore, if splicing was unregulated, then little viral genome would be

available for packaging and Gag and Gag-Pol synthesis would be dramatically reduced.

To ensure that unspliced mRNA is exported from the nucleus, the mRNA contains

elements which allow only about one-third of the viral mRNAs to be spliced (210). The

other two-thirds are exported from the nucleus as unspliced transcripts. Complex

retroviruses encode accessory proteins that interact with cis-acting RNA elements and

with nuclear export machinery thus allowing the export of unspliced mRNA (379).

Simple retroviruses contain no accessory protein and rely solely on cis-acting RNA

elements for the export of unspliced mRNAs. RSV mRNA contains a cis-acting negative

regulator of splicing which allows accumulation of unspliced mRNAs in the nucleus.

These mRNAs are believed to be exported by unidentified cellular nuclear export

proteins interacting with one or both of the direct repeat (DR) elements in the 3’ region of

the mRNA (8, 293, 329, 344).

38

Figure 1.9 Retrovirus assembly. vRNA transcripts are synthesized from the integrated proviral DNA and exported to the cytoplasm. Gag and Gag-Pol proteins are synthesized from unspliced mRNA on free ribosomes in the cytoplasm. They may then shuttle into and out of the nucleus for an unknown reason. Gag and Gag-Pol traffic through the plasma membrane, possibly by way of the cytoskeleton, to the site of budding at the plasma membrane. The vRNA genome is brought to the site of budding by Gag. At the plasma membrane, Gag-Gag and Gag-Gag-Pol multimers form and possibly interact with

Env which was synthesized from spliced mRNA in the RER and trafficked through the

Golgi apparatus on the way to the plasma membrane. A bud forms and is then pinched off the membrane by an unknown mechanism. The immature virus subsequently or simultaneously undergoes maturation to form an infectious virion.

39 Mature

Immature

vRNA

spliced, env mRNA

40 TRANSLATION

In the cytoplasm, genomic and subgenomic (i.e., unspliced and spliced, respectively) mRNAs are translated into proteins (Figure 1.9). Since the mRNAs are capped and polyadenylated, they mimic cellular mRNAs and are believed to be translated by a similar mechanism; however, the exact mechanism employed by RSV and several other retroviruses may differ from the traditional ribosome-scanning model (229). For instance, the RSV genome contains three open reading frames (ORFs) upstream of the gag AUG. Therefore, it has been proposed that the RSV mRNA contains an internal ribosomal entry site (IRES) (91).

The Env protein is synthesized in a manner similar to cellular, integral membrane proteins (Figure 1.9) (457). Translation is initiated in the cytoplasm on free ribosomes and soon after the leader peptide is synthesized. The N terminus is translocated into the lumen of the rough endoplasmic reticulum (RER) until the hydrophobic transmembrane domain in TM is anchored into the lipid bilayer. The N-terminal 64 aa leader peptide is cotranslationally cleaved by signal peptidase. Env multimerizes in the ER where it begins to be modified by N-linked sugars on asparagine residues in the canonical sequence N-x-S/T (x is any residue). Env then travels through the Golgi apparatus where it continues the glycosylation process and is also cleaved into the subunits SU and TM by a furin-like protease; SU and TM of RSV remain linked by disulfide bonds whereas SU and TM of other retroviruses are held together by noncovalent interactions (157, 241,

457). Env then follows the secretory pathway to the site of budding where it is incorporated into virions.

Gag and Gag-Pol are synthesized on free ribosomes in the cytoplasm. RSV Gag

41 contains a stop codon at the end of PR, and synthesis of Pol relies on a -1 frameshift relative to the gag reading frame (195). During translation the ribosomal complex moves in the 5' direction one base when it arrives at the last codon of PR resulting in the first codon of pol being in the -1 reading frame. The ribosome then continues translation of

Pol, thus producing the Gag-Pol protein. The frequency of the -1 frameshift is about one in twenty translations, thus resulting in Pol being at the C terminus of ~5% of Gag proteins (166, 333).

Gag proteins are covalently modified several ways during their synthesis. For most Gag proteins, binding to the plasma membrane requires modification of the N- terminal glycine with the 14-carbon fatty acid myristate. RSV is an exception, and instead of being myristylated, Gag is acetylated on the N-terminal methionine; however, this modification not required for replication (111, 291, 346, 502). RSV Gag is also phosphorylated at several positions, but these, too, are not required for replication (236,

320).

VIRUS ASSEMBLY

Retroviruses are efficient when it comes to assembly; only one protein viral protein is required (Gag) and only three domains of that protein are absolutely essential.

The Gag protein carries all the information for assembly of the virion: it is the structural protein which forms the core of the virion, it targets to the site of budding, changes the curvature of the membrane to form a bud, incorporates the enzymes required for reverse transcription and integration, packages the vRNA genome, incorporates Env into the virion, and mediates the pinching off step of budding. Therefore, to study retroviral

42 assembly is to study Gag.

The morphogenesis of Gag-mediated assembly can be divided into three categories (457). The assembly of most retroviruses (including RSV, MLV, and HIV) is

classified as C-type, where aggregates of Gag are not visible by electron microscopy until

after transport to the site of budding, believed to be the plasma membrane (Figure 1.9).

B/D-type assembly is characterized by multimerization of Gag and core formation in the

cytoplasm (an A-type particle) and subsequent targeting of the core to the site of budding,

again believed to be the plasma membrane. A third pattern of assembly is similar to C-

type assembly where the Gag is targeted to the site of budding followed by simultaneous

core and bud formation, but budding in this case is on ER membranes and the virions

pinch off into the lumen of the ER producing intracisternal A-type particles (IAPs).

Gag Trafficking and the M domain

After its synthesis on free ribosomes Gag must go to the site of assembly (Figure

1.9). Based on the composition of the viral envelope (5, 378) and EM data (353), this site

is believed to be the plasma membrane (457). However, recent data for HIV show the

virus may also bud into multivesicular bodies followed by secretion via exocytosis (324,

326, 334, 355, 385). This phenomenon may be cell-type specific as it has only been

observed in macrophages. Given that budding of C-type and B/D-type retroviruses in has

been observed at the plasma membrane of most cell types, this discussion will assume

that assembly of virions occurs at the plasma membrane.

How Gag traffics through the cytoplasm to the plasma membrane is a mystery.

Since passive diffusion through the cytoplasm is unlikely to target Gag directly to the site

43 of assembly, Gag probably utilizes the cytoskeletal machinery for its trafficking.

Evidence for this association is derived from several observations. First, Damsky et al.

(86) reported that RSV and MMTV budding virions associate with filamentous processes containing actin. Second, actin and actin-binding proteins are present in HIV virions

(339, 340). Third, HIV and MLV Gag interacts with actin in vitro and in vivo (108, 109,

193, 260, 307, 361, 392, 498). Fourth, disruption of the actin cytoskeleton by the drugs cytochalasin D or phallacidin results in decreased budding for EIAV, MMTV, and HIV

(70, 274, 414). Fifth, MLV, HIV, SIV (simian immunodeficiency virus), and MPMV

Gag proteins interact in vitro and in vivo with the antrograde microtubule motor KIF-4

(223, 464). Though the exact mechanism for targeting and the exact route of transport is unclear, it appears that the actin cytoskeletal network, rather than the microtubule network, may be the means by which Gag traffics to the plasma membrane since drugs which disrupt the microtubule network have no effect on EIAV budding (70).

It has been observed that RSV Gag trafficking may not be directly to the plasma membrane, but can traverse through the nucleus during assembly (419, 420). Wild-type

Gag is normally localized in the cytoplasm and at the plasma membrane; if CRM-1 mediated nuclear export is inhibited by the drug leptomycin B, Gag is trapped in the nucleus. MA is sufficient to localize Gag to the nucleus, though it does not contain a classical nuclear localization signal (NLS); the nuclear export signal (NES) has been mapped to p10. The significance of this phenomenon is unknown. One possibility is that

Gag traffics through the nucleus to interact with the vRNA packaged into the virion

(419). This possibility seems unlikely since RNA packaging appears to be linked to translation, at least for some retroviruses (see below). In addition, when an MLV

44 packaging cell line is infected by a recombinant vaccinia viral vector encoding a packagable, modified MLV genome, retroviral particles containing the modified genome are produced. This observation indicates that, even though transcription of the modified

MLV genome occurs solely in the cytoplasm, the RNA is able to be packaged into particles (179). Therefore, at least for MLV, Gag does not need to traffic through the nucleus to package its genome. A second hypothesis is that the MA NLS is involved in nuclear import of the PIC upon infection, and as a consequence Gag traffics through the nucleus during assembly; to get out of the nucleus p10 contains an NES so Gag can assemble at the plasma membrane.

Whatever the mechanisms are that Gag uses to traffic through the cytoplasm to the plasma membrane, Gag interacts with the plasma membrane peripherally. This interaction is directed by the membrane binding (M) domain (Figure 1.10) located in

MA. The RSV M domain is composed of the first 85 aa of MA and is highly basic (60,

477); deletions or substitution of basic residues in this region abrogate budding and localization to the plasma membrane (60, 501, 502). Replacement of the M domain with the 10 aa membrane binding domain of v-Src or the HIV M domain is sufficient to target

Gag to the plasma membrane and to facilitate budding (350, 501, 502).

How the M domain specifically targets Gag to the plasma membrane is unknown; however, the mechanism by which Gag binds the plasma membrane is known.

Interactions between the M domain and the plasma membrane are analogous to those utilized by cellular peripheral proteins which bind membranes through a hydrophobic fatty acid modification and/or electrostatic interactions between basic amino acids and acidic phospholipids. Indeed, most Gag proteins are modified with the 14-carbon fatty

45

Figure 1.10 RSV Gag and the assembly domains. RSV Gag is diagramed with sites of cleavage, cleavage products, and domains required for budding are indicated. The crystal structure of the RSV M domain is modeled and its net positive charge is diagramed interacting with the negative charged phospholipids of the plasma membrane. The PPPY sequence of the L domain is shown interacting with a Nedd4-family E3 ligase. The positively charged I domains and the Cys-His boxes involved in RNA binding are also shown.

46 ------H H + + + + + I domain C I domain C + 2+ 2+ Zn Zn

+ + + + C C + + + + + + C C + + +

RNA Binding

Ub Nedd4-Family E3 PPPY 72 175 M L ...1 ... I I

MA a b p10 CA NC PR

p2 SP 701

47 acid myristate, and deletion of the myristate results in the inability of Gag to bind to the plasma membrane (51, 140, 148, 345, 391, 393, 516). However, the presence of a myristate is not sufficient for membrane binding (394, 503), and some Gag proteins (e.g.,

RSV, EIAV, BLV) are not myristylated. Therefore, other regions of the M domain must also interact with the plasma membrane. Like cellular peripheral proteins, M domains contain basic residues which, when folded, are located on the surface of MA (164, 174,

285, 291). The electrostatic interaction between this positively-charged surface and the negatively-charged head groups of acidic phospholipids in membranes govern the ability of Gag to bind (60, 520). However, there must be specific membrane-targeting machinery because acidic phospholipids are not restricted to the plasma membrane. In addition to targeting to and binding the plasma membrane, the M domain may also be involved in targeting to specific areas of budding on the plasma membrane. Indeed, an

RSV Gag mutant defective in the late stages of budding is able to be rescued into particles by a budding competent MLV Gag protein, but only when the M domains of the

RSV and MLV Gags are identical (21). Therefore, in addition to binding the plasma membrane, the M domain is likely to be involved in targeting Gag to discrete areas of budding. These specific regions are believed to be enriched in cellular proteins which drive the budding process (353).

The search for the microdomains where Gag buds has led to the hypothesis that

Gag traffics to and buds from detergent resistant regions of the plasma membrane known as lipid rafts (178, 259, 323, 331, 405, 519). Lipid rafts are typically defined as membranes that are insoluble at 4 °C in a variety of detergents, classically Triton X-100 and Brij98. Rafts are enriched in cholesterol and sphingolipids, and depletion of

48 membrane cholesterol with methyl-β-cyclodextrin leads to a loss of detergent resistance

(49, 364). HIV Gag has indeed been isolated from rafts and disruption of these rafts by

cholesterol depletion decreases budding (178, 259, 323, 331, 519). These data provide

evidence that Gag buds from specific areas of the plasma membrane, but there is much

more to learn about budding from these microdomains. For instance, it is unknown if

Gag targets to existing rafts, or if Gag has the capability to form its own raft. Further, the

cellular proteins within these rafts are unknown, as is the mechanism by which Gag

traffics to and through them.

Gag-Gag Interactions and the I Domain

Gag proteins need to interact and multimerize in order to bud (Figure 1.9). The

primary region responsible for this interaction has been mapped to NC, where deletions

not only result in reduced budding, but also result in virions that are less dense than wild-

type (20, 145, 412, 493). Having a virion of lesser density implies that there are fewer

Gag proteins per virion, likely the result of Gag-Gag interactions being disrupted. A role

for NC in facilitating Gag-Gag interactions is further supported by the observation that

NC deletion mutants cannot be rescued into particles by wild-type Gag (495), whereas M

domain mutants unable to bind the membrane are packaged into virions.

Analysis of the NC sequences of most retroviruses reveals two conserved

characteristics: one or two Cys-His boxes (Cys-X2-Cys-X4-His-X4-Cys) and clusters of basic residues. It is the latter that are essential for facilitating Gag-Gag interactions.

Deletional and mutational analysis of NC revealed that RSV Gag contains two redundant interaction (I) domains (Figure 1.10) (20, 42, 495) which are not the Cys-His boxes

49 (these sequences are involved in specific RNA packaging, see below), but the flanking clusters of basic residues (42). Indeed, replacement of NC with only a short peptide containing six basic residues is sufficient for the production of dense particles (42, 244).

The mechanism by which Gag multimerizes is unknown, but is probably facilitated by the Cys-His boxes binding RNA and is then maintained by electrostatic interactions between the RNA and the basic residues (42, 56, 76, 89, 270, 411, 517). In vitro experiments have demonstrated multimerization and assembly of a CA-SP-NC protein when mixed with RNA (62, 513), and additional experiments using this protein have shown that Gag dimerization is dependent on RNA binding. These in vitro experiments revealed that the NC-NC interaction generated is weak and the CA-CA interactions are also required for stable Gag-Gag interactions (270).

The multimerization of Gag not only allows uniform formation of a viral core, it also is required for efficient binding of Gag to the plasma membrane (60, 330, 411, 412).

Mutants that lack NC or contain non-conservative substitutions are defective both in their

budding activity and the ability to bind membranes. These data suggest that Gag

multimerization is a prerequisite for membrane binding. Indeed, M domain mutants

unable to bind the plasma membrane can be rescued into particles by a coexpressed wild-

type Gag, thereby suggesting Gag-Gag interactions occur prior to membrane binding

(500, 503). This idea is further supported by the observation of small, soluble HIV Gag

oligomers in the cytoplasm which precede budding (248, 473).

The requirement of Gag multimerization for efficient membrane binding suggests

that a single M domain is, by itself, too weak to stably bind the plasma membrane.

Therefore, if Gag can bind the plasma membrane with high enough affinity then there

50 would be no requirement for multimerization of Gag. Indeed, an RSV M domain mutant with two basic residues substituted for two acidic ones (Super M) has an increased rate in budding, presumably due to an increased affinity for the plasma membrane (60). The requirement for a functional I domain was circumvented in this mutant which localized to the plasma membrane even without NC (61). When assayed for infectivity, however,

Super M (with NC) virions were noninfectious. The infectivity defect was attributed to the mutant being unable to package the genomic vRNA. These data suggest that in a wild-type virus there is a balance between membrane binding, Gag multimerization, and genome packaging, and when one is disrupted, assembly of an infectious virion does not occur.

An intriguing model linking RNA binding (i.e., genome packaging) and membrane targeting emerges: Gag-Gag interactions are facilitated by strong interactions between basic residues in NC and RNA. NC-NC interactions are too weak to mediate lateral interactions between themselves, and further assembly steps require CA-CA interactions to stabilize Gag-Gag interactions. The successful multimerization of Gag then allows a strong interaction between the M domain and the plasma membrane. In this model, only multimerized Gag bound to RNA (normally the vRNA) can be efficiently targeted to the site of budding.

RNA Packaging

As mentioned above, Gag binds RNA through NC. This binding is nonspecific given that DNA oligomers with a repeated dG-dT sequence are sufficient for in vitro multimerization of Gag (270). Therefore, Gag requires a means whereby it can

51 specifically bind its genomic vRNA. The specificity of genome packaging is mediated by NC (216, 298, 299, 408) in the context of unprocessed Gag (84, 328, 435, 449).

Replacement of RSV Gag with that of MLV results in packaging of MLV vRNA (103).

Specificity within NC maps to the two Cys-His boxes (Cys-X2-Cys-X4-His-X4-Cys)

flanked by clusters of basic residues, which resemble zinc-fingers found in certain

cellular nucleic acid binding proteins. The secondary and tertiary structure, in addition to

the primary sequence, is highly important for proper function of the Cys-His boxes and

basic residues in RNA recognition and packaging (6, 90, 381).

Recognition of the RNA is not only dependent on the sequence and structure of

NC, but also on the sequence and structure of the vRNA. All full-length, unspliced

vRNAs contain a packaging signal (termed Ψ) in the 5' end of the RNA. In RSV, Ψ is

located in the intron, and is therefore removed from spliced RSV transcripts, thus

providing a means by which the virus can recognize and package only genomic vRNA

(457). The minimal packaging signal for RSV is 160 nucleotides long. Fusion of this

minimal Ψ sequence to the mRNA of gfp enables specific packaging of gfp mRNA into virions. RSV Ψ contains two major stem loops, L3 and O3; deletion of L3 has no effect on packaging. The O3 secondary structure, not the primary sequence, was required for efficient packaging of a heterologous mRNA (16). Similar sequences have also been identified in MLV and HIV (266, 277, 278, 288, 395).

vRNA Used For Packaging

One of the mysteries of RNA packaging is whether genomic vRNA exists in two pools in the cytoplasm (one for translation and one for packaging) or if there is one

52 vRNA pool where mRNAs used for translation are also used for packaging. Early experiments using actinomycin D to inhibit RNA transcription showed that MLV virions synthesized in the absence of newly transcribed RNA contained reduced levels of genomic vRNA (251, 252, 300), suggesting two pools of vRNA exist. However, similar experiments with RSV and HIV suggest that Gag competes with ribosomes for the vRNA

(99, 443, 450). More recent evidence that RNA packaging is linked to translation comes from a study analyzing nonviral proteins packaged into the virion of HIV. Here a cellular proteins linked to translation (elongation factor-1α [Elf-1α]) was identified in virions

(339). A subsequent study utilized cotransfection of two DNAs, one encoding a wild- type HIV-1 genome and one with mutations which inhibited synthesis of HIV-1 Gag without affecting its potential to be packaged into virions. If vRNA is packaged in cis by

HIV-1, the wild-type vRNA should be packaged over the mutant vRNA. If packaging occurs in trans, there should be no preference over which RNA is packaged. The results showed a 6- to 13-fold preference for packaging of wild-type vRNA over the mutant vRNA, thus suggesting HIV-1 Gag preferentially packages the vRNA used for its translation (367). Despite the evidence for cis-packaging of RNA, trans-packaging must be possible given that addition of Ψ to a heterologous RNA is able to be packaged (243,

245).

RNA Dimerization

Two copies vRNA are packaged into virions and dimerize some time during or after budding (27, 128); however, dimerization does not appear to be a prerequisite for

RNA packaging. The stability of the dimers is less in newly released virions as compared

53 to older virions. Maturation of the virion is important for stabilization of dimers, and in

PR-deficient viruses, the packaged vRNA is either a mixture of monomers and dimers

(RSV and HIV [127, 328, 433, 449]) or is extremely unstable (MLV [128]).

Dimerization occurs at the dimerization initiation site (DIS) which partially overlaps Ψ.

Like Ψ, a functional DIS is dependent on its secondary structure (27, 85, 87, 222, 269,

335, 374, 472). This secondary structure is likely recognized by NC to initiate the

dimerization process since NC can facilitate RNA dimerization in vitro (119, 120, 314),

and mutations in NC also affect dimerization of the packaged vRNA (299, 433, 458).

Other regions outside of NC may also influence dimerization. When the v-Src

membrane-binding domain is attached to the N terminus of RSV Gag, the resulting

mutant produces noninfectious virions that are defective forming RNA dimers. How MA

is influencing dimerization is unknown (134, 349).

Packaging of the tRNA Primer

The tRNA primer is also packaged into the virion during assembly. RSV utilizes

tRNATrp for initiation of reverse transcription, and the tRNATrp primer is recognized and specifically packaged into RSV virions by RT (362). Though the primer is the most predominant tRNA packaged, other tRNAs are also present in the virion at various concentrations (466). They, too, may be specifically packaged by RT (362). Therefore, it is not surprising that viruses with the PBS mutated in such a way to allow binding by another tRNA present in the virion are infectious, though not like wild type (497).

Although RT packages the correct tRNA, it does not mediate annealing of the tRNA to the PBS of the genomic vRNA. Given that NC can bind RNA and mediate RNA-RNA

54 interactions, NC is a likely candidate for annealing the tRNA primer to the PBS. Indeed, virions with mutations in NC that do not affect vRNA packaging are noninfectious because the tRNA primer is not bound to the vRNA template (184, 185, 375).

Pol Incorporation

RT and IN are incorporated into virions by their fusion to the C terminus Gag.

These Gag-Pol fusion proteins contain all of the domains required for budding, but are incapable of forming virions when expressed alone (80, 117, 297, 351, 493). Though the reason is unknown, it is possible that the domains are not folded in a context favorable for budding or that the size of the Gag-Pol protein is too large to form a virion due to steric hindrance. Whatever the reason, Gag-Pol must rely on Gag to be incorporated into virions.

The interactions between Gag and Gag-Pol required for incorporation have been mapped to the MHR and the C terminus of CA using a series of deletion and HIV/MLV chimera mutants (182, 446). Recently, however, studies revealed that Pol can be incorporated in the absence of fusion to Gag. Specifically, expression of Pol fused with a small portion of MA at the N terminus is capable of being incorporated into virions (52,

67, 72). The interpretations of these results seem to conflict with the data suggesting CA

incorporates Gag-Pol. Perhaps in the absence of its fusion with Gag, Pol is free to

interact with MA, CA, or NC through interactions used to form the RTC allowing

nonspecific packaging, and the full length Gag-Pol fusion protein is packaged specifically

through interactions involving CA and the MHR.

55 Env Incorporation

The mechanism by which Env is incorporated into virions is largely unknown. It is enticing to assume that Gag plays a critical role in Env incorporation given that Gag does about everything else during assembly. Indeed, interactions between TM and MA have been demonstrated (79, 138, 357, 505), and certain mutations in MA block Env incorporation into virions (98, 125, 126, 280, 332, 514). Further, certain substitutions in

MA actually increase that amount of Env incorporated. These data suggest an active role for Gag in the incorporation of Env. However, there is also evidence that the incorporation of Env is not mediated by Gag. Deletion of the cytoplasmic tail of TM does not prevent its incorporation into the virion (275, 359), and replacement of MA with a general membrane-targeting domain results in Env incorporation (390). Retrovirions can also incorporate envelope proteins from other viruses such as influenza virus HA and vesicular stomatitis virus (VSV) G protein. In addition, RSV can package the human glycoprotein CD4 when expressed in avian cells (512). These observations have led to the hypothesis that budding does not specifically incorporate Env, but specifically excludes normal surface host proteins. These surface proteins are likely tied up by intracellular protein interactions involved in signaling or they are tethered to the cytoskeleton. This model suggests that as Gag begins to bud these host proteins are excluded and since Env is not anchored to the cytoskeleton it (and CD4 expressed in avian cells) can be incorporated into virions.

This latter model, though a good one, does not explain specific budding of retroviruses at the basolateral surface of polarized cells (401). If Gag is expressed alone budding is not restricted to either the apical or basolateral side of the cell. However,

56 coexpression of Gag and Env results in exclusive budding at the basolateral surface (261,

343). The cytoplasmic tail of Env is recognized by the basolateral sorting machinery and is therefore responsible for the location of budding in polarized cells (262). Gag appears to interact with Env prior to targeting to the plasma membrane and both are subsequently targeted to the site of budding. This interaction is further supported by recent data which

show that Gag interacts with Env on endosomal membranes prior to budding (413).

Incorporation of Cellular Proteins

In addition to viral proteins in virions, several cellular proteins have also been

identified (Table 1.1). Since no proteomics approach has been attempted to identify all

proteins (cellular or viral) in retrovirions, the list of proteins presented here is by no

means complete.

The protein cyclophilin A is present in HIV-1 virions, but not in any other

retrovirion. HIV-1 Gag interacts specifically with cyclophilin A through an interaction

with CA. Early experiments suggested that this interaction is required during assembly

(45, 123, 469) to allow for a product infection, but it now appears that the relevant

interaction occurs in the infected target cell (442). Indeed, virions produced from cells

lacking cyclophilin A (and thus the virions themselves lack cyclophilin A) are just as

infectious as virions produced from control cells. However, virions (with and without

packaged cyclophilin A) were unable to infect cells lacking cyclophilin A. Though the

mechanism by which cyclophilin A aids allows for HIV-1 replication, the cyclophilin A-

CA interaction protects the CA from the restriction factors Ref1 and Lv1 (25, 194, 416).

Cytoskeletal proteins have also been detected in HIV-1 virions. These proteins

57

Table 1.1 Incorporated cellular proteins. Known cellular proteins incorporated into retrovirions are listed, and the viruses in which they have been detected are indicated.

58

Incorporated Cellular Proteins

Cellular Protein Detected In

Cyclophilin A HIV-1 Actin HIV-1 Ezrin HIV-1 Moesin HIV-1 Cofilin HIV-1 Elf-1a HIV-1 GAPDH HIV-1 Nm23-H1 HIV-1 HS1 HIV-1 PBP HIV-1 Pin1 HIV-1 Lck HIV-1 RSV, HIV-1, SIV, Ubiquitin MMTV, EIAV, MPMV, MLV Tsg101 HIV-1, MPMV Nedd4 MPMV Vps28 HIV-1 Vps4B HIV-1 AIP1/ALIX EIAV, HIV-1

59 include actin, ezrin, moesin, cofilin, Elf-1α, GAPDH, Nm23-H1, and HS1 (339, 340). As discussed above, the presence of these proteins may be due to their requirement for targeting Gag to the site of budding. The presence of these proteins in other retrovirions has not been confirmed, though their presence seems likely. Other proteins that have been found in HIV-1 virions also include phosphatidylethanolamine-binding protein

(PBP), Pin1, and Lck (339).

One protein found in all retrovirions is Ub at concentration of about 100 molecules per virion (337, 338, 341, 342, 377). Packaged Ub has no known role in the mature virion and may be nonspecifically incorporated due to its function during assembly (see below). Other cellular proteins utilized during budding have also been identified in virions. Tsg101 is present in HIV-1 and MPMV virions (149, 158, 488).

Nedd4 is incorporated into MPMV virions (149). Vps28 and Vps4B are also packaged into HIV-1 virions (455, 488). AIP1/ALIX is packaged into EIAV and HIV-1 virions

(488). The presence of these proteins in the virions of other retroviruses is unknown.

Like Ub, roles for these proteins in the mature virion are unknown, and they may only be present in the virion due to their presence during budding.

Late Budding Events and the L Domain

At the site of budding, everything required for an infectious retrovirus comes together (Figure 1.9). However, everything is useless if the virus cannot be released from the cell. The mechanism by which retroviruses are released is unknown. Pinching off the membrane may either be an active process mediated by a “pinchase” or a passive process due to massive membrane accumulation of Gag forcing separation from the

60 plasma membrane. The latter seems unlikely in light of mutants which accumulate at the plasma membrane and cannot pinch off (147, 181, 500). These “late” mutants form a bud but are tethered to the plasma membrane by a stalk. The region of Gag that is responsible

for pinching off the membrane is just four to eight amino acids, depending on the virus,

and has been termed the late (L) domain (Figure 1.11).

L domains by definition are functionally interchangeable and are, for the most

part, positionally independent (284, 348, 506). For these reasons, it has long been

speculated that L domains function by recruiting host factors that mediate budding (136,

348, 457). In recent years, this hypothesis has been proven to be true (see below) (39,

149, 282, 452, 488).

Various Types of L Domains and Their Interactions

So far, three types of L domains have been identified: PPxY (PPPY, RSV and

MLV [506, 515]), P(T/S)AP (PTAP, HIV [147, 181]), and YPxL (YPDL, EIAV [376])

(Figures 1.10 and 1.11). MMTV Gag does not contain any of these three motifs;

therefore, more L domain motifs are likely to be identified. Some viruses contain two L

domains which may act synergistically or redundantly. Examples of these include

HTLV-1 and MPMV which contain PPxY and P(T/S)AP motifs (39, 149). RSV may also fall into this category since there is a YPxL (181YPSL184) motif downstream of the

PPPY L domain. Chapter IV examines the importance of the YPSL sequence in RSV budding. HIV, too, contains a sequence similar to the YPxL L domain (LYP) which is required for interaction with a host protein involved in budding (see below) (452).

As mentioned, L domains function by recruiting host proteins to facilitate

61

Figure 1.11 Late domains. The L domains of several retroviruses are diagramed. The main L domain(s) is boxed in black, and a known or potential secondary L domain is

boxed in red.

62 RSV TSAPPPPYVGSGLYPSL MPMV FLTRPPPYNKATPSAP HTLV-1 DPQIPPPYVEPTAP

MLV LYPALTP136...161DPPPYR

HIV SRPEPTAPPE460...482ELYPLASLRSLF EIAV TQNLYPDLS 63 budding. Just as all three L domains are quite different, the proteins they bind also are different. PTAP functions by recruiting Tsg101 (93, 137, 283, 478), PPPY binds Nedd4- family Ub ligases (Figure 1.10) (221), and YPxL binds AIP1/ALIX (282, 452, 479, 488).

Though different, the proteins recruited by the L domains are all linked to one function within the cell – MVB formation.

LATE BUDDING EVENTS IN OTHER ENVELOPED VIRUSES

The mechanisms of pinching off the plasma membrane are likely to be similar for all enveloped viruses. Indeed, L domains have been identified in arenaviruses, rhabdoviruses, and filoviruses, and have been shown to interact with components of the

MVB sorting machinery, similar to retroviruses.

Arenaviruses

Arenaviruses are single-stranded, negative-sense RNA viruses. Expression of the

Z protein and a glycoprotein is sufficient to drive budding. The Z protein from lymphocytic choriomeningitis virus (LCMV) contains a PPPY L domain required for budding. Similarly, the Z protein from Lassa fever virus (LFV) contains both PPPY and

PTAP L domains and both are required for efficient budding (360). Interactions have yet to be demonstrated between the Z protein and cellular proteins.

Rhabdoviruses

The M protein of rhabdoviruses is sufficient to drive budding of virus-like particles. Analysis of the primary sequence of the M protein of VSV revealed a PPPY

64 sequence. Mutation of this sequence resulted in a budding defect, suggesting this is an L domain (81, 196). Similar to RSV, the VSV L domain has been shown to interact with a

Nedd4 family Ub ligase, and this interaction results in the ubiquitination of the M protein, thus suggesting a role for Ub in budding (161, 162).

Filoviruses

The VP40 protein of Ebola virus is similar to M proteins and Gag proteins in that its expression results in budding of virus-like particles. Similar to MPMV and HTLV-1,

VP40 contains both PPPY and PTAP sequences. These sequences have been identified as the L domains of VP40 and their mutation results in a budding defect (258, 283). Like other PPxY L domains, the PPPY sequence binds a Nedd4 family Ub ligase (163, 470,

510), and similar to other P(T/S)AP motifs, the PATP sequence interacts with Tsg101

(258, 283, 470).

MULTIVESICULAR BODY FORMATION

The budding of retroviruses is similar in topology to the budding of endosomes into MVBs (Figure 1.1). Since L domains interact with proteins involved in MVB formation, it is conceivable that retroviruses utilize this machinery for their own release from the plasma membrane. To gain a better understanding of the function these proteins have during budding, it is necessary to review their normal cellular functions.

Ubiquitination and Sumoylation

The pathways by which Ub and Ub-like proteins such as SUMO-1 are attached to

65 target proteins are similar. Ub and SUMO-1 are small proteins (76 aa and 97 aa, respectively) which are similar only in their secondary structure. They carry out their various functions by forming an isopeptide bond between the C-terminal glycine of Ub or

SUMO-1 and the ε-amino group of a lysine on the modified protein (Figure 1.12) (75,

171, 257, 279). Ubiquitination and sumoylation occurs through a sequential reaction involving activating (E1), conjugating (E2), and ligase (E3) enzymes. The E1 uses ATP to form a thioester bond between a cysteine and the C-terminal glycine of Ub or SUMO-

1, thus activating it for nucleophilic attack. It is then passed on to a cysteine residue in the active site of an E2 enzyme. At this point the E2 interacts with the E3, which specifically binds the substrate and mediates the transfer of Ub or SUMO-1 to the ε- amino group of a target lysine. The final step in the both pathways is the removal of Ub or SUMO-1 by a specific Ub or SUMO-1 isopeptidase (94, 144, 153, 172, 198, 200, 204,

227, 407, 422).

Though there are similarities between the ubiquitination and sumoylation pathways, there are fundamental differences, the main one being that Ub and SUMO-1 utilize different E1, E2, and E3 enzymes. The ubiquitination enzyme cascade is hierarchical in that there is one E1, many E2s, and many more E3s, thus allowing the cell to efficiently regulate ubiquitination (153, 170, 172). The sumoylation cascade, on the other hand, is not hierarchal. There is one E1 enzyme (heterodimer of AOS1 and UBA2), one E2 (Ubc9), and several E3s (94, 144, 198, 200, 204, 227, 407, 422). Another major difference is that Ub requires an E3 ligase to be conjugated to a target and SUMO-1 can be conjugated to a target directly by the E2, Ubc9 (94, 200). For both systems, the E3 ligases (when used in sumoylation) specifically recognize a target protein. Ubiquitination

66

Figure 1.12 Ubiquitination and sumoylation. The processes of ubiquitination (A) and sumoylation (B) are quite similar. Each process involves Ub or SUMO-1 being relayed from an E1 activating enzyme to an E2 conjugating enzyme which interacts with an E3 ligase to mediate the final transfer onto a target lysine. Both events are transient and specific isopeptidases remove Ub or SUMO-1 from the target proteins. Though the two processes are similar, the enzymes which modulate them are quite different. Every known sumoylation event is mediated by the same E1 (Aos1/Uba2 dimer) and E2 (Ubc9), whereas ubiquitination uses the same one E1, but several different E2s. The specificity of ubiquitination is regulated by the binding of an E3 to the target, and Ub can be attached to any lysine in the binding region. Sumoylation, however, is mostly regulated by a sumoylation consensus sequence (ΨKxE/D) on the target protein. Further, SUMO-1 can be attached to the target without aid of an E3 ligase, whereas ubiquitination requires an E3 ligase.

67 A O H N C Ub O K Ub C OH Protein Ub H Isopeptidase + ATP H N H E1 SH E3 K AMP+PPi Protein O O E1 S C Ub E2 S C Ub E2 SH

B O H N C SUMO O YKxE/D SUMO C OH Protein SUMO-1 isopeptidase H + Aos1 ATP H N H Uba2 SH E3 ? YKxE/D AMP+PPi Protein

Aos1 O O Uba2 S C SUMO Ubc9 S C SUMO

Ubc9 SH

68 occurs on nonspecific lysines close to the E3 binding site, whereas SUMO-1 is usually conjugated to the lysine with the consensus ΨKxE/D (199, 294, 397, 410).

The functions of Ub and SUMO-1 are quite different. As mentioned, SUMO-1 is

involved in nuclear translocation, subnuclear targeting, transcriptional regulation, DNA

repair, DNA replication, and chromosome condensation, cohesion, and separation during

mitosis (199, 429). The most famous role for Ub is in targeting proteins for degradation

by the 26S proteasome (75, 171). Ub is also involved in histone regulation, endocytosis,

protein sorting into MVBs, and regulation of the ribosome to name a few (173). The role

of Ub is different in each process for which it is involved. For instance, addition of poly-

Ub chains to a protein targets that protein for degradation by the proteasome.

Monoubiquitination of a cell surface receptor triggers endocytosis of that receptor and

subsequently targets the protein to be sorted into MVBs. Therefore, tagging a protein

with Ub does not determine the fate of that protein, but how, when, and where that

protein is tagged determines what happens next.

In addition to substrate recognition, Ub (but not SUMO-1) E3 ligases also

determine whether a protein is monoubiquitinated or polyubiquitinated. For example,

Nedd4 (the mammalian homologue of the yeast protein Rsp5) is a Ub ligase responsible

for recognition and monoubiquitination of cell surface receptors, but not the

polyubiquitination of misfolded proteins destined to be degraded by the proteasome

(448).

MVB Formation and Protein Sorting

Many proteins, such as cellular receptors, are endocytosed upon binding of their

69 ligand (Figure 1.1). Endocytosis is triggered in many cases via monoubiquitination by a

Nedd4-family Ub ligase binding to a PPxY motif on the cytoplasmic tail of the receptor

(102, 247, 253, 268, 400, 403, 447, 448, 454, 468). Once endocytosed, the vesicle travels through the endosome on its way to the lysosome, where the receptor is degraded (131).

As the endosome matures, proteins are sorted on the limiting membrane and some of the proteins are targeted for budding into the endosome, thus forming a vesicle within the endosome. At this stage the endosome is referred to as a multivesicular body (146, 156).

Genetic screens in yeast have identified at least 17 proteins, known as class E Vps

(vacuolar protein sorting) proteins, required for protein sorting into, and formation of,

MVBs (213, 386). Yeast expressing dominant-negative forms of class E Vps proteins are blocked in MVB formation and accumulate deformed, multilamellar endosomal compartments known as class E compartments. Human orthologs have been identified for all yeast class E proteins (Table 1.2), and these proteins are necessary for MVB formation in mammalian cells.

There are four major complexes formed by the class E Vps proteins (Figure 1.13).

(1.) The Hrs complex is organized by Hrs and also includes STAM1/STAM2 and Eps15

(9, 15, 304). (2.) Mammalian endosomal sorting complex required for transport

(ESCRT)-I is composed of Tsg101, Vps28, and Vps37 (A-D) with Vps28 and Vps37 each binding to Tsg101 (33, 213, 455). (3.) ESCRT-II is comprised of EAP45, EAP30, and EAP20 (11, 282, 488). (4.) ESCRT-III is not a single, stable complex like ESCRT-I and ESCRT-II. Instead, there are three subclasses of ESCRT-III that form at least two different subcomplexes. Components of the first subclass are CHMP4 and CHMP6 and

form a stable complex; components of the second subclass are CHMP2 and CHMP3 and

70

Table 1.2 Vps class E proteins. The known mammalian Vps class E proteins, their yeast homologues, and the complexes they form are listed. The Hrs complex proteins are boxed in blue, the ESCRT-I proteins are boxed in green, the ESCRT-II complex is boxed in yellow, the ESCRT-III proteins are boxed in orange, the Vps4 complex is boxed in pink, and the two Vps class E proteins associated with no known complex are boxed in white.

71

Vps Class E Proteins

Mammalian Yeast Complex

Hrs Vps27 Hrs STAM Hse1 Hrs Tsg101 Vps23 ESCRT-I Vps28 Vps28 ESCRT-I Vps37 (A-D) Vps37 ESCRT-I EAP30 Vps22 ESCRT-II EAP20 Vps25 ESCRT-II EAP45 Vps26 ESCRT-II CHMP2 (A,B) Vps2 ESCRT-III CHMP6 Vps20 ESCRT-III CHMP3 Vps24 ESCRT-III CHMP4 (A-C) Vps32 ESCRT-III CHMP1 (A,B) Fti1/Did2 ESCRT-III CHMP5 Vps60 ESCRT-III Vps4 (A,B) Vps4 Vps4 AIP1/ALIX Vps31 Unknown SLC9A6 Vps44 Unknown

72

Figure 1.13 MVB sorting/retrovirus budding machinery. There are four major complexes formed by the class E Vps proteins involved in MVB formation. The cargo protein is specifically recognized by and ubiquitinated by a Ub ligase (e.g., Nedd4 family

ligase). The Hrs complex (Hrs, STAM1/STAM2 and Eps15) interacts with the

ubiquitinated cargo protein and recruits ESCRT-I (Tsg101, Vps28, and Vps37), which in

turn recruits ESCRT-II (EAP45, EAP30, and EAP20) which recruits ESCRT-III

(CHMP4/6 and CHMP2/3 subcomplexes and CHMP1 and CHMP5). AIP1/ALIX is also

recruited by CHMP4 and also interacts with Tsg101. Once the cargo is sorted, the

ESCRT complexes are removed by Vps4 and Ub is removed by a Ub isopeptidase.

Retroviral L domains can also interact with the MVB sorting machinery. PPxY L

domains can interact with a Ub ligase, P(T/S)AP L domains interact with Tsg101, and

YPxL L domains and LYP/LxxLF motifs interact with AIP1/ALIX. Black arrows

indicate interactions between MVB sorting proteins. Yellow arrows indicate interactions

between L domains and MVB sorting proteins.

73 Endosomal Lumen Sorting 3 Ub Ub Ub EAP30 Ub 2 Ub 6 4 P P P 2 P

/ Isopeptidase M Hrs M

Tsg101 EAP20 M M 1

H H H H M C C C C A Vps28

T EAP45

S Ub 5

Vps37 1 P P M Ub M H H C Nedd4 Family AIP1/ALIX C E3 VPS4 P(T/S)AP L Domains PPxY L Domains YPxL L Domains LYP/LxxLF Motifs Cytoplasm 74 also form a stable complex; components of the third subclass are CHMP1 and CHMP5 and have not been identified in a complex (10, 282, 488).

The ESCRT complexes are involved in sorting proteins into MVBs in a Ub- dependent manner (212, 388, 475). The monoubiquitination of target proteins required at this stage may be separate from the monoubiquitination event at the plasma membrane since proteins that are endocytosed in a Ub-independent manner are sorted into MVBs in a Ub-dependent manner (281, 396). The emerging role of Ub in sorting appears to be in recruitment of the protein complexes which sort the target protein into the MVB.

Four of the seventeen class E proteins have been shown to interact with Ub: Hrs,

STAM, Tsg101, and yeast Vps26. Hrs and STAM each contain multiple Ub interaction motifs (UIMs) required for sorting of ubiquitinated cargo (29, 304, 312, 366, 380, 436,

456). Tsg101 has a UEV (Ub conjugating enzyme E2 variant) domain which binds Ub in a manner similar to E2 enzymes, but is lacking the catalytic cysteine (31, 212). Vps26 contains a NZF (Npl4 zinc finger) domain that binds Ub (3); however, the human ortholog EAP45 does not contain a NZF, and it is unknown if it can bind Ub.

As mentioned, the pathway which leads to a target protein being sorted into

MVBs begins with Ub, and the recruitment of sorting protein complexes to the ubiquitinated target is sequential (Figure 1.13). Hrs binds Ub and interacts with the endosomal membrane (14, 214, 265, 373). Once bound, the Hrs complex recruits

ESCRT-I by interacting with Tsg101; the UEV domain of Tsg101 binds a PSAP sequence in Hrs (15, 214, 265, 373). Binding to the PSAP activates Tsg101 by an unknown mechanism which allows it to recruit other protein complexes and/or bind the ubiquitin on the target protein. Next, Tsg101 recruits ESCRT-II. ESCRT-II may or may

75 not interact with the ubiquitinated target protein, and the function(s) of ESCRT-II is unknown (11). The ESCRT-III subcomplexes are then recruited to the target protein.

The CHMP4/6 subcomplex first interacts with ESCRT-II via CHMP6 (10, 282, 488).

The CHMP2/3 subcomplex is then recruited by CHMP4/6 (10). CHMP1 and CHMP5 are believed to serve regulatory functions since their deletion causes only modest sorting phenotypes (10). CHMP4 then recruits another class E Vps protein called AIP1/ALIX

(208, 282, 327, 452, 488). AIP1/ALIX can also interact with ESCRT-I via Tsg101, but its recruitment is mediated by CHMP4 (327) and functions late in the MVB sorting

pathway (325), possibly in controlling membrane curvature of the budding vesicle (286).

At some point during sorting, Ub is removed from the target protein by a Ub

isopeptidase, possibly UBPY or AMSH, which have been shown to associate with class E

proteins (207, 460). The ESCRT complexes appear to be solely involved in protein

sorting; further downstream events and the associated proteins which lead to release of

the vesicle from the membrane are unknown. Certainly the “pinchase” has not been

discovered. Given that membrane fission is an energy-driven process, an ATPase would

likely be involved. The only class E proteins with known enzymatic activity are Vps4A

and Vps4B (12). Both enzymes are AAA ATPases and are paralogs with 80% sequence

identity (13, 32, 421). However, their ATPase activity is required for removal of the

ESCRT complexes from the membrane and not pinching off the vesicle from the

membrane (10, 11, 13, 32, 129, 452, 488, 511). Therefore, it appears that not all of the

class E Vps proteins are known and more are likely to be discovered.

76 HOST PROTEINS DRIVING RETROVIRUS BUDDING

Ubiquitin and Retrovirus Budding

The ties between the MVB sorting machinery and retrovirus budding begin with

Ub. Retrovirions contain ~100 molecules of Ub per virion, a level that is higher than in the cytoplasm (337, 338, 341, 342, 377). Therefore, the virion either specifically incorporates Ub during budding or budding occurs at a region enriched in Ub (or both).

There appears to be some specificity in Ub incorporation since 2-5% of the Gag proteins in virions from all retroviruses examined thus far are ubiquitinated (34, 337, 338, 341,

342, 476). The sites of ubiquitination in Gag are close to the L domain (337, 341), suggesting a role for Ub in budding. Indeed, depleting the cell of free Ub through the use of proteasome inhibitors impairs the budding of all retroviruses tested, except EIAV and

MMTV (341, 342, 352, 353, 425, 451). This effect on budding is overcome by expression of Ub in trans or fusion of Ub to the C terminus of Gag, suggesting an active role for ubiquitination of Gag during budding, rather than an indirect effect of the proteasome inhibitors (353). The insensitivity of EIAV to proteasome inhibitors may reflect the ability of EIAV to bypass a requirement for Ub during budding. The region of

Gag responsible for EIAV’s insensitivity to the drugs maps to p9, which contains the L domain. The sequence of p9 contains a region similar to the surface-exposed helix of Ub, and it is possible that this region provides a function in budding similar to the role of Ub, i.e., recruitment of cellular factors (352). However, it has been reported that EIAV mutants which insert PPPY or PTAP L domains in place of the YPDL confer proteasome inhibitor sensitivity to EIAV Gag, suggesting EIAV’s L domain is the cause of resistance

(434). The reason for MMTV’s insensitivity to proteasome inhibitors is unknown.

77 Only recently has RSV Gag been shown to be ubiquitinated, though these results have not been confirmed (476). Prior to these findings it seemed quite likely that RSV

Gag was ubiquitinated during assembly since the L domain of RSV interacts with a Ub ligase (39, 409, 509). Moreover, RSV budding depends on Ub, and this dependence can be overcome by fusion of Gag with Ub. It was not determined whether the ubiquitinated

Gag detected was relevant for assembly. However, the Ub-independent budding of Gag artificially fused with Ub supports a role for ubiquitination of RSV Gag during budding.

Indeed, there are five lysines in close proximity to the L domain making them likely candidates for sites of ubiquitination during budding. Chapter II examines the role these potential sites of ubiquitination have during budding.

Vps Class E Proteins and Retrovirus Budding

The link between MVB formation and retrovirus budding (353) was first shown when yeast two-hybrid screens identified an interaction between the PTAP L domain of

HIV and Tsg101 (137, 283, 315, 478); this interaction is required for budding (92, 137,

143, 283, 315). When Tsg101 expression was knocked down using RNA interference or when dominant-negative forms of Tsg101 were overexpressed, HIV budding was inhibited, suggesting an active role of Tsg101 in HIV budding. The interaction between

Tsg101 and HIV is mediated by the UEV domain of Tsg101 binding to the PTAP L domain sequence in p6 (370), mimicking the interaction between Tsg101 and the PSAP motif in Hrs (373) used to recruit ESCRT-I for protein sorting into MVBs. It therefore appears that HIV taps into the MVB protein sorting pathway by mimicking Hrs.

The utilization of all three ESCRT complexes by HIV has been characterized by

78 using dominant-negative forms of Tsg101, RNA interference to knockdown expression of Tsg101 or VPS37, and creating dominant-negative forms of ESCRT-II and -III proteins by fusing them to fluorescent proteins. HIV budding is inhibited by overexpression of a Tsg101 dominant-negative or by knocking down Tsg101 expression

(92, 137, 283, 478). Likewise, depletion of Vps37B and Vps37C also inhibit budding.

Further, a HIV L domain mutant is rescued by fusion with all or a portion of Vps37B or

Vps37C (107, 455). Therefore, HIV budding requires ESCRT-I. However, HIV budding is not affected by the overexpression of any dominant-negative ESCRT-II protein, but is affected by overexpression of several dominant-negative ESCRT-III proteins. Though there are conflicting reports as to whether CHMP2A or CHMP3 are involved in HIV budding, it is clear that budding requires CHMP1A, CHMP4A, CHMP4B, CHMP4C, and CHMP5, but not CHMP2B or CHMP6 (282, 488). Since ESCRT-III is recruited by

ESCRT-II, why does HIV not require ESCRT-II? HIV Gag can interact directly with

AIP1/ALIX through LYP and LxxLF motifs, and this interaction may bypass ESCRT-II by using AIP1/ALIX to recruit ESCRT-III (282, 488).

Given that HIV utilizes the MVB protein sorting machinery for budding and the L domain of HIV can be functionally replaced by the L domains of other retroviruses, it is likely that other viruses also utilize this machinery. However, through subsequent studies differences between the various L domains became apparent. For instance, the budding of MLV (PPPY L domain) and EIAV (YPDL L domain) were not affected by Tsg101 depletion or dominant-negative expression (92, 137, 282, 434, 465). In addition, HIV

budding driven by PPPY or YPDL L domains in place of PTAP is insensitive to

dominant-negative Tsg101 expression (282). MLV and EIAV probably do utilize the

79 MVB protein sorting machinery because overexpressing a dominant-negative form of

Vps4 inhibits their (and HIV’s) budding (137, 434, 452, 465). EIAV is also inhibited by overexpression of CHMP3 and CHMP4A proteins (452), but other ESCRT-II or -III proteins involved in its budding are unknown. Therefore, all types of L domains likely require the MVB protein sorting machinery with each L domain entering the system at different points. Indeed, EIAV may enter at a different point in the MVB sorting pathway from HIV. The YPDL motif of EIAV interacts directly with AIP1/ALIX allowing it to enter the sorting pathway (452, 488); overexpression of a dominant- negative form of, or depletion of, AIP1/ALIX results in a block to EIAV budding (282,

452). As mentioned, HIV p6 has also been shown to bind to AIP1/ALIX (452, 488), and like EIAV, HIV budding is also inhibited by depletion or overexpression of AIP1/ALIX

(282, 452, 488). The latter observation shows that HIV possibly recruits the MVB sorting machinery through multiple interactions. Other retroviruses may also enter the budding pathway through multiple interactions with MVB sorting machinery. PPPY and

P(T/S)AP L domains have been identified in HTLV-1 and MPMV, and both L domains are required for efficient budding (149, 167, 490). The PPPY and the PTAP L domains in HTLV-1 bind Nedd4 Ub ligase and Tsg101, respectively (34, 39, 167). Further, overexpression of dominant-negative forms of Nedd4 and Tsg101 inhibit HTLV-1 budding (34, 39). In contrast, although MPMV also contains both PPPY and PSAP sequences, overexpression of a dominant-negative form of Tsg101 does not inhibit budding (93). This observation is interesting because Tsg101 has been detected in

MPMV virions, and this packaging depends on the PSAP motif. The reason for this discrepancy is unknown.

80 To summarize, L domains are required for recruiting MVB protein sorting machinery (Figure 1.13). PPPY motifs recruit Ub ligases which presumably ubiquitinate

Gag, thus allowing them to enter the pathway at a point independent of ESCRT-I since

MLV budding is not dependent on Tsg101. P(T/S)AP motifs interact with Tsg101, allowing direct binding to the ESCRT-I complex. YPxL (and LYP/LxxLF) motifs bind

AIP1/ALIX which interacts downstream of ESCRT-I (likely with ESCRT-III) since

EIAV does not require Tsg101, but does use CHMP3, CHMP4A, and Vps4.

Utilization of Cellular Proteins by RSV During Replication

Given the large body of evidence supporting the utilization of cellular proteins by many enveloped viruses to drive budding, it is quite likely that RSV, too, makes use of these same proteins. Chapter IV analyzes the inhibition of RSV budding in the presence of various dominant-negative ESCRT proteins. However, identification of dominant- negative proteins which inhibit budding tells little about the mechanisms by which virions are released from the plasma membrane. Therefore, to begin to understand the mechanisms by which RSV buds, the proteins which function after the L domain during budding have been analyzed in this dissertation.

81 CHAPTER II

LYSINES CLOSE TO THE ROUS SARCOMA VIRUS LATE DOMAIN

CRITICAL FOR BUDDING

Jared L. Spidel, Rebecca C. Craven, Carol B. Wilson, Akash Patnaik,

Huating Wang, Louis M. Mansky, and John W. Wills

Journal of Virology 78:10606-10616

82 ABSTRACT

The release of retroviruses from the plasma membrane requires host factors that are believed to be recruited to the site of budding by the late (L) domain of the virus- encoded Gag protein. The L domain of Rous sarcoma virus (RSV) has been shown to interact with a ubiquitin (Ub) ligase, and budding of this virus is dependent on Ub. RSV is similar to other retroviruses in that it contains ~100 molecules of Ub, but it is unique in that none of these molecules have been found to be conjugated to Gag. If transient ubiquitination of RSV Gag is required for budding, then replacement of the target lysine(s) with arginine should prevent the addition of Ub and reduce budding. Based on known sites of ubiquitination in other viruses, the important lysines would likely reside near the L domain. In RSV, there are five lysines located just upstream of the L domain in a region of the matrix (MA) protein that is dispensable for membrane binding, and substitution of these with arginine (mutant 1-5KR) reduced budding 80 to 90%. The block to budding was found to be on the plasma membrane; however, the few virions that were released had normal size, morphology, and infectivity. Budding was restored when any one of the residues was changed back to lysine or when lysines were inserted in novel positions, either within this region of MA or within the downstream p10 sequence.

Moreover, the 1-5KR mutant could be rescued into particles by coexpression of budding- competent Gag molecules. These data argue that the phenotype of mutant 1-5KR is not due to a conformational defect. Consistent with the idea that efficient budding requires a specific role for lysines, human T cell leukemia virus type 1, which does not bud well compared to RSV and lacks lysines close to its L domain, was found to be released at a higher level upon introduction of lysines near its L domain. This report strongly supports

83 the hypothesis that ubiquitination of the RSV Gag protein (and perhaps those of other retroviruses) is needed for efficient budding.

INTRODUCTION

Ubiquitin (Ub) is a small protein that cells use to modify other proteins to accomplish a remarkably diverse set of tasks that are important at nearly all levels of cellular activity, including DNA replication, transcription, translation, protein folding, sorting, signaling, and degradation. Modification occurs on the ε-amino group of lysines of target proteins and results in either monoubiquitination or polyubiquitination (i.e., chains of Ub). While polyubiquitination usually targets proteins to the proteasome for

degradation, monoubiquitination provides a signal that alters or regulates the function of

the modified protein (173, 387, 423, 427, 444).

Over the past few years, numerous observations have been made that together

seem to indicate an important role for monoubiquitination in the late stages of retrovirus

budding on the plasma membrane. First, all retroviruses that have been examined have

been found to contain ~100 molecules of Ub, an amount that is much higher than can be

explained by random trapping during budding (337, 341, 342, 377). Second, some of

these Ub molecules are individually linked to Gag, the viral protein that drives particle

assembly and budding (337, 341, 342). Third, the identified sites of monoubiquitination

are invariably near the viral late (L) domains (337, 341), which are responsible for

recruiting host factors needed for the late steps in budding (124). Fourth, virus release

has been shown to correlate with the recruitment of ubiquitin ligase activity by L domains

(39, 221, 451, 509). Fifth, Tsg101, a host factor recruited by the L domains of some

84 retroviruses, is a Ub-binding protein (370, 371). Sixth, the release of some retroviruses has been shown to be reduced by proteasome inhibitors, which lower the levels of free

Ub in the cytoplasm (39, 342, 353, 425). Seventh, in the case of Rous sarcoma virus

(RSV), the effects of proteasome inhibitors can be counteracted by overexpressing Ub in trans or by fusing it to the C terminus of Gag (353). Eighth, it has been noted (353) that retrovirus budding bears a topological resemblance to the cellular budding events that lead to the formation of multivesicular bodies (MVBs), and the movement of cellular proteins into these nascent buds on late endosomes requires transient monoubiquitination

(104). Ninth, there is increasing evidence that retrovirus budding makes use of host factors involved in MVB formation (137, 282, 372, 452, 488).

Although the evidence for a role of Ub in retrovirus budding is extensive, there are several observations that would seem to argue against this idea. First, all of the Ub present in RSV is unconjugated (377). Unconjugated Ub could be due to rapid deubiquitination of Gag, either naturally during virus budding or artifactually during lysis of cells or virions. In this regard, it is interesting that Ub-mediated sorting of proteins into MVBs is accompanied by rapid deubiquitination (104), and on the plasma membrane, the photoreceptor of the Drosophila eye is not endocytosed until after Ub is removed, unlike most examples where Ub promotes endocytosis (65). Alternatively, it is possible that RSV Gag is never modified, but instead Ub conjugation might be required on a cellular protein that is essential for budding. Although this is what is believed to be the case for endocytosis of the growth hormone receptor (150), a trans role for Ub is less likely to be the case for RSV given that a Gag-Ub fusion protein can bud even in the presence of proteasome inhibitors, which inhibit wild-type Gag (353). Second, the

85 release of some retroviruses (e.g., equine infectious anemia virus and mouse mammary tumor virus) is not affected by proteasome inhibitors even though their Gag proteins are modified by Ub (341, 342, 352). Such viruses might make use of alternative budding mechanisms or carry redundant “Ub-like” information that enables budding when Ub is limiting (352). Third, elimination of known sites of ubiquitination on the Gag proteins of

HIV-1 and murine leukemia virus has no effect on budding or infectivity (337). The significance of this observation is difficult to ascertain because the Ub literature contains many examples of secondary sites of ubiquitination being used when the primary sites are eliminated (264, 363, 400). Moreover, in light of the many examples of transient monoubiquitination, the sites of Ub-modification that have been mapped on Gag proteins could correspond to secondary sites that happen to be more resistant to deubiquitination.

Alternatively, Ub modification of some Gag proteins might truly be unimportant for budding and may occur on occasion merely because these viral proteins pass by Ub ligases on their way out of the cell.

This study addresses the importance of lysines for the late steps of RSV budding.

Although direct evidence for a Ub-modified Gag protein remains elusive for this virus, the experiments described below unequivocally show that a cluster of five lysines near the L domain are important for budding.

MATERIALS AND METHODS

Expression vectors. The wild-type RSV gag gene was originally derived from the Prague C proviral vector, pATV-8 (211, 428). To analyze budding, gag alleles were transferred either into proviral vector pRC.V8 (83) or pGag.GFP (43), the latter of which

86 contains a gag-egfp fusion. For quantitative infectivity studies, gag alleles were cloned into pRS.V8.eGFP (60), which also carries the coding sequence for eGFP in a nonessential region of the genome. For mutagenesis of the HTLV-1 gag gene, it was subcloned into the pMH vector (Boehringer Mannheim, Indianapolis, IN) to construct pMH-HTLVGag (489).

Mutagenesis. Construction of pT10C.GFP was described previously (353). p∆MA3.GFP was made by cutting pSV.∆MA3 (503) with SstI and BspEI and moving the

5' gag fragment into the equivalent location in pGag.GFP. pGag.3h.GFP was created by

amplifying gag sequence from pRC.V8 using a forward primer (5'-

GATCTCGAGCTCTACTGCAGG-3') spanning the SstI site upstream of the initiation

codon and a reverse primer (5'-CCTAACCAAGGGGGGCCCGAGATGTTCCAT-3') in the

PR coding sequence, thereby generating the 3h deletion mutant described previously

(493) while simultaneously creating an ApaI site at the 3' for subsequent fusion of gag

with egfp in pCMV.N2-eGFP (Clontech). The PCR products and recipient plasmid were

digested with SstI and ApaI and then ligated. To create pGag.3h, the egfp coding sequence was removed from this recombinant by digestion with ApaI and NotI, followed

by incubation with Klenow fragment and ligation. This manipulation resulted in the

introduction of two foreign amino acids (arginine and leucine) in the place of eGFP.

Lysine-to-arginine substitutions in the NC domain were created by three sequential

rounds of mutagenesis by the QuikChange method (Stratagene, La Jolla, CA) using

pGag.3h.GFP as the template. In the first round, the K36, K37 and K39 codons (AAA,

AAA, and AAG) were changed to CGG, AGA, and AGG (respectively), and as a result a

new SstII site overlapping the K36 codon was introduced. The second round of

87 mutagenesis changed the K58 (AAA) and K62 (AAG) codons to CGC and AGG

(respectively), and at the same time, codon A57 was silently changed from GCT to GCG

to introduce a BssHII site. Finally, the G72 and K73 codons were changed from GGA

AAA to GGC CGC, creating another SstII site and the final lysine-to-arginine

substitution. Following mutagenesis, the entire gag gene was sequenced after screening

presumptive clones for the new restriction sites. Lysine-to-arginine, arginine-to-lysine,

and glutamate-to-lysine substitutions in MA, p10, and CA were carried out by using

either M13 mutagenesis with MGAG-S as template (60, 502) or by PCR mutagenesis

with pGag.GFP or p1-5KR.GFP as template. All lysine codons were changed to the

arginine codon "CTG". Arginine and glutamate codons were changed to the "AAA"

lysine codon. Mutations were transferred into pGag.GFP, pRC.V8, or pRS.V8.eGFP

using SstI and FseI, thereby replacing the 5' half of gag. To create the L domain

substitution construct, pPPPY-A.GFP, the proline and tyrosine codons were changed to

four alanine codons (GCT) using PCR mutagenesis. The mutant allele was then

transferred to pGag.GFP using SstI and FseI. To create Gag(-), 1-5KR(-), and T10C(-),

the egfp sequences were removed from pGag.GFP, p1-5KR.GFP, and pT10C.GFP,

respectively, by digesting the vectors with ApaI and NotI, followed by incubation with

Klenow fragment (to create blunt ends) and ligation. This manipulation inserts two

foreign amino acids (arginine and leucine) in the place of eGFP. All of the newly

constructed RSV gag alleles described here were sequenced to confirm that only the

desired mutations were present.

HTLV-1 substitutions at MA amino acids 102 and 109 (P102K and D109K) and

the double mutant R97K/P102K were made by mutagenesis of pMH-HTLVGag using the

88 QuikChange method, as reported previously (489). The following oligonucleotides were used in the mutagenesis reactions: for P102K, 5'-

CCCGTCCCGCGCCACCGAAGCCGTCATCCCCCACC-3' and 5'-

GGTGGGGGATGACGGCTTCGGTGGCGCGGGACGGG-3'; for P109K, 5'-

CCGTCATCCCCCACCCACAAACCCCCGGATTCTGATCC-3' and 5'-

GGATCAGAATCCGGGGGTTTGTGGGTGGGGGATGACGG-3'; for R97K/P102K, 5'-

GCCCAGATCCCGTCCAAACCCGCGCCACCGAAGCCGTCATCCCCCACC-3' and 5'-

GGTGGGGGATGACGGCTTCGGTGGCGCGGGTTTGGACGGGATCTGGGC-3'. For

expression of HTLV-1 Gag, the mutant alleles were transferred from pMH-HTLVGag

into pCMV-HT1 on a DraIII-NheI fragment. All clones were sequenced to confirm the

presence of the desired substitutions and to be certain that no undesired substitutions had

occurred.

Budding assays. To quantitate RSV budding, QT6 cells (310) were transfected

by the CaPO4 method, as previously described (83), and 24 h later, they were

35 35 metabolically radiolabeled with L-[ S]methionine or a L-[ S]methionine/cysteine mix

(50 µCi, >1,000 Ci/mmol). Cells transfected with plasmids expressing Gag proteins

which lack PR (e.g. Gag.GFP) were radiolabeled for 2.5 h, and viral proteins were

immunoprecipitated from detergent-lysed cells and particles using a polyclonal rabbit

serum against whole RSV, as previously described (493). Immunoprecipitates were

separated in sodium dodecyl sulfate (SDS)-12%-polyacrylamide gels, which were

subsequently dried and exposed to Kodak X-Omat AR5 X-ray film. Gag proteins were

also quantitated by PhosphorImager (Molecular Dynamics) analysis. The budding

proficiency was calculated as the amount of Gag in the medium divided by the total

89 amount in the cell lysate and medium.

Cells transfected with proviral constructs (which encode active proteases) were done in duplicate. One plate was pulse-labeled for 5 min, at which time only unprocessed Gag is detected, while the other plate was radiolabeled for 3 h. Gag proteins were immunoprecipitated from detergent-lysates and analyzed by SDS-polyacrylamide electrophoresis. Unprocessed Gag from the 5 min pulse-labeled cells and mature CA protein from the 3 h labeled medium fractions were quantitated by PhosphorImager analysis. Budding proficiency was calculated as the amount of CA in the medium divided by the amount of Gag in the pulse-labeled cell lysate.

For the complementation experiments, QT6 cells were cotransfected with equal amounts of the two Gag constructs to be tested while a parallel plate was cotransfected with pGag.GFP and pGag(-) to provide a normal-budding control for comparison.

Proteins were radiolabeled, immunoprecipitated, analyzed, and quantitated as above.

To analyze HTLV-1 budding, 293T cells were transfected by the CaPO4 precipitation method. Methods for preparing cell lysates and media fractions have been detailed previously (489). Briefly, transfected cells were lysed in RIPA buffer and immunoprecipitated with anti-HTLV1-p19 monoclonal antibody (Zeptometrix, Buffalo,

NY). Media fractions collected from transfected cells were subjected to ultracentrifugation for 1 h and 40,000 x g to obtain particles. Western blotting analysis was performed using anti-HTLV1-p19 monoclonal antibody as primary antibody and horseradish peroxidase-conjugated anti-mouse Ig as secondary antibody with the ECL western analysis kit (Amersham, Arlington Heights, IL). The efficiency of particle production was normalized for cell-associated gp46. Quantitation of band intensities was

90 done using the Quantity One software package with the Chemi Doc 2000 Documentation

System (BioRad, Richmond, CA).

Sucrose gradient sedimentation. The size distribution of 1-5KR mutant particles was evaluated as described previously with only minor variations (232).

6 Briefly, three plates of 1 x 10 QT6 cells in 60 mm dishes were transfected by the CaPO4 method with pRC.V8 (wild-type RSV genome), pRC.1-5KR (mutant), or pGag.3h

(normal-size control). At 18 h posttransfection, the cells were labeled with L-

[35S]methionine/cysteine for 5 h. 300 µl of either wild-type or 1-5KR particles were

mixed with 100 µl of the normal-size control, and the two samples were centrifuged at

26,000 rpm (90,000 x g) for 30 min through 10-30% sucrose gradients in an SW41 rotor

at 4oC. Fractions were collected from each tube by dripping from the bottom, and the

Gag proteins were then immunoprecipitated with anti-RSV rabbit serum, electrophoresed in an SDS-12% polyacrylamide gel, and quantitated by PhosphorImager analysis.

Infectivity assays. QT6 cells were transfected with pRS.V8.eGFP or pRS.1-

5KR.eGFP using the CaPO4 transfection, and virions were allowed to accumulate in the medium for 24 h. The medium fraction was centrifuged at 1000 x g for five min to remove any cells, and half of the cell-free medium was pelleted through a 25% sucrose cushion at 126,000 x g for 40 min at 4oC in a TLA100.4 rotor. Pelleted virions were resuspended in phosphate-buffered saline and analyzed by a reverse transcriptase assay as previously described (83) to determine the amount of virions in the other half of each medium sample. Equal concentrations of virions were place on DF-1 cells (175, 418) for

24 h, after which new medium was added. The numbers of infected (i.e., green) cells

present at subsequent time points were counted by FACS analysis.

91 Confocal microscopy. Live QT6 cells were washed with Tris-buffered saline at

24 h posttransfection cells and overlaid with a glass coverslip. The subcellular locations

of GFP-tagged Gag proteins were observed by confocal microscopy using a Zeiss laser

scanning microscope following excitation with a helium-argon laser (488-nm peak

excitation).

Electron Microscopy. QT6 cells were seeded in 60mm Permanox dishes

(Electron Microscopy Sciences, Ft. Washington, PA) at 3x106 per plate, transfected with proviral plasmids by the CaPO4 method, and processed for electron microscopy (EM) as described previously (83).

RESULTS

The RSV Gag protein (Figure 2.1) contains 31 lysines, and the functions of many of these are known. The MA sequence is rich in lysines, containing nearly 50% of the total. Of the nine lysines in the first half of MA, all are critical for the interaction of the membrane-binding (M) domain with phospholipids in the plasma membrane; however, binding appears to be dependent on charge rather than lysines per se (60, 61). The second half of MA contains five lysines positioned between the M and L domains. Small deletions in this region have no effect on budding or infectivity suggesting this region contains little structure. However, some large deletions result in budding defects, but this could have been due to conformational defects of surrounding regions (321). There are no lysines within p2 (which contains the L domain) or p10. Capsid (CA) contains eight lysines, but this part of Gag can be deleted in its entirety with minimal effects on budding

(495). The six lysines in nucleocapsid (NC) are thought to be involved in RNA binding

92

Figure 2.1 RSV Gag mutants and chimeras. Sites of cleavage, cleavage products, and domains required for budding are indicated. The C-terminal half of MA contains five lysine residues (amino acids 95, 115, 124, 138, and 148) that are potential sites of ubiquitination. For simplicity, the lysines are referred to as 1, 2, 3, 4, and 5 respectively.

The lysines were all changed to arginine (1-5KR), changed in combinations (e.g.

1,2,3,5KR) or changed individually (e.g., 2KR). ∆MA3, an M domain mutant, has a deletion in the N-terminal half of MA which does not allow membrane binding. T10C deletes the L domain plus a portion of MA and CA resulting in a "late" phenotype.

Gag.GFP is a C-terminal fusion between Gag and eGFP. The resulting fusion occurs in

NC and eliminates the last seven amino acids of NC. Gag(-) deletes eGFP and therefore the resulting Gag protein is truncated 7 amino acids from the end of NC, but the deletion resulted in the addition of an arginine and leucine at the C terminus.

93 M L I I

RSV Gag MA a b p10 CA NC PR

p2 SP 577 701

PPPY

MA a b p10 CA

K1 K2K3 K4K5 K244

R RR R R 1-5KR 15 37 DMA3 121 337 T10C AAAA PPPY-A

Gag.GFP MA p10 CA NC GFP 818

Gag(-) MA p10 CA NC -Arg-Leu 571

94 events that promote tight interactions among Gag proteins, but functionality of the interaction (I) domain appears to be dependent more on charge than lysines per se (42), and substitution of all six of these residues with arginine had no effect of budding (data not shown). Protease (PR) contains three lysines, but deletion of PR has little effect on budding (495).

Given what is known about the lysines in RSV Gag and the location of known sites of ubiquitination in other retroviruses, the lysines most likely to play a role in the late steps of budding seemed to be those located in the second half of MA. To ascertain their importance, these five residues were changed to arginine to eliminate the potential for ubiquitination while preserving structure.

Budding of mutant 1-5KR. The first mutant made was 1-5KR in which all five of the lysines in the second half of MA were replaced with arginine in a budding- competent derivative of Gag that has eGFP linked to its C-terminus (Gag.GFP; Figure

2.1). QT6 cells were transfected with plasmids encoding the parental Gag.GFP (wild type), ∆MA3.GFP (M domain deletion mutant), T10C.GFP (L domain deletion mutant),

or 1-5KR.GFP. As expected from previous studies (500, 503), ∆MA3.GFP and

T10C.GFP were released at levels <5% of wild type (Figure 2.2A). Release of mutant 1-

5KR.GFP was also reduced but slightly higher than the M- and L-domain mutants at 8%

of wild type. Replacement of the closest downstream lysine in CA (K244R), which

resides in a consensus sumoylation site (397, 410), had no effect on budding either alone

or in the context of the 1-5KR mutant (data not shown). These results suggest that one or

more of the lysines immediately upstream of the L domain are critical for budding.

The effect of the 1-5KR substitution on budding was also examined in the context

95

Figure 2.2 Requirement of lysines 95, 115, 124, 138, and 148 for particle release. (A)

Transfected QT6 cells were metabolically labeled for 2.5 h 24 h posttransfection. Gag

proteins were immunoprecipitated from the cell lysates and the labeling media using an

anti-RSV serum. Gag proteins were visualized by autoradiography and quantitated by

PhosphorImager analysis (see Materials and Methods). Mutants were compared to the

wild type which was normalized to 100%. The graph represents three independent

experiments. (Inset) An autoradiograph with cell lysates and medium fractions shows

that 1-5KR.GFP migrates at the expected size of 88 kDa. (B) The 1-5KR substitutions

were introduced into two proviral constructs (RC.V8 and RS.V8.eGFP) to create RC.1-

5KR and RS.1-5KR.eGFP, respectively. Gag proteins were expressed, radiolabeled,

immunoprecipitated, visualized, and quantitated (see Materials and Methods). The

experiments using RC.V8 and RC.1-5KR were repeated three times, whereas those using

RS.V8.eGFP and RS.1-5KR.eGFP were repeated nine times.

96 A

e 100 p P P

y F F T .G 3 C R .G 3 C R

d g A K g A K

l 0 0 i 80 a M 1 -5 a M 1 -5 G D T 1 G D T 1 W

o t

e 60 v i t a l e 40 R

e Lysates Media s a e

l 20 e R

% 0 P 3 C R F A 0 K .G M 1 -5 g D T 1 a G B

e 100 p RCV8 y T

d l

i 80 W

o t

e 60 v i t a l e 40 R

e s a

e 20 l e R

% 0 8 R P P .V K F F C -5 G G R .1 .e .e C 8 R R .V K S -5 R .1 S R

97 of two different proviral genomes. pRC.V8 is a vector in which the nonessential v-src gene of RSV has been replaced with a gene for hygromycin resistance, whereas in pRS.V8.eGFP, v-src has been replaced with the gene for eGFP. The gag genes in both vectors encode active PR. Upon transient transfection of QT6 cells, Gag protein processing for the 1-5KR derivatives was unaffected (data not shown), but budding was once again seen to be reduced compared to the wild type: 80% reduction for RC.1-5KR and 53% reduction for RS.1-5KR.eGFP (Figure 2.2B). The apparent difference between proviral mutants was observed only in transient transfection experiments; cells infected with RS.1-5KR.eGFP exhibited an 80% reduction in budding compared to the wild type

(see the description of the infectivity experiments below). Therefore, the budding defect originally seen for 1-5KR.GFP appears not to be an artifact of expression of Gag in the absence of the other viral genes.

Subcellular localization of 1-5KR. In cells treated with proteasome inhibitors,

Gag proteins accumulate at the plasma membrane (353). If one or more of the five lysines in MA are involved in a ubiquitination event that is important for budding, then the 1-5KR mutant should also accumulate at the plasma membrane. On the other hand, due to their close proximity to the M domain, substitutions at these five residues could conceivably interfere with transport to the membrane. To ascertain where the mutant proteins accumulate, QT6 cells were transfected with small amounts (1 µg) of the various pGag.GFP constructs and analyzed by confocal microscopy (Figure 2.3A-E). Wild-type

Gag.GFP was seen in punctate fluorescence at the plasma membrane (arrowheads) while

∆MA3.GFP, unable to bind to the plasma membrane, was found in large aggregates in the cytoplasm, and an L domain mutant, T10C.GFP, was found at the plasma membrane.

98

Figure 2.3 Intracellular localization of mutant 1-5KR. peGFP, pGag.GFP, p1-

5KR.GFP, p∆MA3.GFP, and pT10C.GFP were transfected into QT6 cells using 1 µg (A

to E) or 10 µg (F to J) of DNA and examined using confocal microscopy 24 h after

transfection.

99 A F

eGFP eGFP B G

Gag.GFP Gag.GFP C H

DMA3.GFP DMA3.GFP D I

T10C.GFP T10C.GFP E J

1-5KR.GFP 1-5KR.GFP

100 1-5KR.GFP, like the wild type and the L domain mutant, was also localized to the plasma membrane. This phenotype was seen more clearly in cells transfected with larger amounts (10 µg) of DNA (Figure 2.3F to J); however, under these conditions the cells typically round up. It is clear from all of these results that the 1-5KR mutant has no obvious membrane-targeting defect and the substitutions likely affect a later step in budding.

Analysis of 1-5KR virions. Retroviruses lacking their L domains form particles that accumulate at the plasma membrane, unable to be released efficiently (124).

Similarly, when RSV-infected cells are treated with proteasome inhibitors to deplete free

Ub levels, large clusters of virions are often seen stuck to the cell surface (353).

Surprisingly, analysis of RC.1-5KR-infected cells by transmission electron microscopy

revealed no hints of clusters tethered to the surface, but instead only particles of normal

size and morphology were seen (Figure 2.4A), even though parallel budding assays

revealed the expected defect in particle release (data not shown). Scanning EM of cells

expressing 1-5RK Gag also showed that clusters of particles do not form on the cell

surface, but there was an increased number of normal-size particles blocked to budding

compared to the wild type, similar to an L domain mutant (M. Johnson and V. Vogt,

personal communication). To test the possibility that particle clusters were rapidly

released, medium from RC.1-5KR-transfected cells was subjected to sucrose gradient

centrifugation. A small population of 1-5KR particles indeed was found to sediment

faster than normal; however, most were of normal size (Figure 2.4B). If the 1-5KR

mutations affect ubiquitination, then it may be the case that low- level usage of

alternative lysines provides sufficient activity to destabilize any clusters that form on the

101 Figure 2.4 Characterization of 1-5KR virions. (A) QT6 cells infected with RC.V8 or

RC.1-5KR were thin sectioned and examined by EM. 1-5KR virions are visible with a morphology similar to that of the wild type. (Magnification, x50,000; bar, 100nm) (B)

RC.V8 and RC.1-5KR particle size was determined by sedimentation of virions through a

10 to 30% sucrose gradient for 30 min. Fractions were collected and proteins were separated by SDS-PAGE. For RC.V8 and RC.1-5KR the amount of CA present in each fraction was determined by PhosphorImager analysis and is shown on the left y axis as arbitrary units. The amount of internal control (Gag.3h) present in each fraction was also determined by PhosphorImager analysis and is shown as arbitrary units on the right y axis. The graph is representative of three independent experiments. (C) QT6 cells were transfected with pRS.V8.eGFP or pRS.1-5KR.eGFP, and virions were collected in the medium for 24 h. Equal amounts of virions were added to DF-1 cells for 24 h. The cells were analyzed by FACS at various times postinfection to determine the percentage of green cells, indicating an infection. (Inset) An autoradiograph showing Gag proteins from cells infected for 14 days, obtained as in Figure 2.2. Lanes 1 and 2 are cell lysate

fractions from the wild type and 1-5KR, respectively, and lanes 3 and 4 are medium

fractions from the wild type and 1-5KR, respectively. The graph shows the average of

three independent experiments. (D) Lysine 244 is part of the consensus sumoylation

sequence, ΨxKE, where Ψ is a hydrophobic residue and x is any residue. To destroy the

potential sumoylation site lysine 244 was changed to arginine (K244R) and cloned into

RS.V8.eGFP. Two K244R clones were analyzed for infectivity as described above and

were compared to wild type and 1-5KR. A representative graph of the results of three experiments is shown.

102 80 A B 30 WT WT Wild Type Gag.3h 60 20 R

40 e l a t y i t

10 v i e s 20

n b e a t n n

i 0

0 d

d i

1600 n n t a 600 e b n

s 1-5KR 1-5KR e 1200 i v t i 1-5KR y t

a Gag.3h l 400 e 800 R

200 400

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 fraction number C D 100 Wild Type Wild Type 1-5KR s 1-5KR 1 2 3 4 l l 80 K244R c1 e

C gag K244R c5 Pr76 n 60 e e r G

t 40 n e c r 20 e

P CA 0 2 3 4 5 6 7 8 9 10 11 12 13 14 2 3 4 5 6 7 8 9 10 11 12 13 14 Days Postinfection Days Postinfection

103 cell surface (see Discussion).

To determine whether 1-5KR particles have any infectivity, QT6 cells were transfected with pRS.V8.eGFP, pRS.1-5KR.eGFP, or pRS.K244R.eGFP, and particles were allowed to accumulate in the media for 24 h. Equal amounts of released reverse transcriptase activity were used to infect fresh cultures of DF-1 cells, and at various days postinfection, these were trypsinized and sorted by FACS to count the number of green

(i.e., infected) cells. The half-time of spreading with wild-type virus was 5.3 days, while the 1-5KR half-time was 8.3 days (Figure 2.4C). In contrast, two clones of mutant

K244R were found to be largely noninfectious (Figure 2.4D), even though they bud with the same efficiency as the wild type (data not shown). At the end of the experiment, when the cells were fully infected with mutant 1-5KR (day 14), budding assays revealed that particle release was still only ~20% the wild-type efficiency (Figure 2.4C, inset), and thus, the ability of this mutant to spread through the culture was not due to reversion or suppressor mutations. All together, these experiments show that the 1-5KR mutant is infectious and suggest that its slower rate of spread is simply due to a reduced rate of budding.

Complementation rescue of 1-5KR. The L domain mutant T10C can heteromultimerize with and be rescued into particles by coexpressing budding-competent

Gag molecules from RSV or MLV (21, 500). If the 1-5KR mutant is blocked at a late step in budding, then it, too, should be rescued by complementation; however, if it instead has a severe conformational defect or has been directed into a different pathway, then rescue might not be likely. To be able to distinguish the Gag derivatives used in the complementation assays, three constructs were made that lack the eGFP tag (Figure 2.1)

104 and hence are detectably smaller than their counterparts: Gag(-), 1-5KR(-), and T10C(-).

Control experiments showed that each of these alone behaved as expected: i.e., the first buds like the wild type, while the latter two are defective (data not shown).

Cells were cotransfected with the pairs of DNAs to be tested, while a control plate was cotransfected with pGag.GFP and pGag(-). In this control, release of Gag.GFP was monitored to provide a measure of normal-budding, and pGag(-) was included in the transfection to keep the total amount of DNA equal to that used in the test plates. As expected, release of the L domain mutant, T10C(-), was greatly increased to 50% of the control when coexpressed with Gag.GFP, and an inhibitory effect was not exerted upon the rescuing molecule (Figure 2.5A). Mutant 1-5KR(-) was even more efficiently rescued (80%). Switching the eGFP tag from the rescuing Gag construct to the 1-5KR mutant reduced the efficiency, but complementation still occurred. Coexpression of

T10C(-) and 1-5KR.GFP did not result in rescue of either defective mutant. Together with the ability of 1-5KR to reach the plasma membrane, these results suggest that conformational defects do not account for the block to budding.

An L-domain-point mutant is dominant-negative for budding. To ascertain whether the inability of T10C and 1-5KR to rescue each other was due to the size of the deletion in T10C, the complementation experiment was repeated with the L domain point mutant, PPPY-A.GFP. As expected, budding of PPPY-A.GFP was low when expressed alone (15%; data not shown) or when coexpressed with the L domain deletion mutant,

T10C(-) (~7%) (Figure 2.5B). Likewise, mutant 1-5KR(-) could not be rescued by

PPPY-A.GFP. Surprisingly, budding of Gag(-) was strongly inhibited (75%) and PPPY-

A.GFP budding was not increased. Elucidation of the mechanism behind this dominant-

105

Figure 2.5 Complementation of the 1-5KR defect with wild-type Gag. Wild-type

(WT) and mutant Gag proteins were coexpressed. (A) To differentiate between the two proteins, eGFP was deleted from Gag.GFP and 1-5KR.GFP [Gag(-) and 1-5KR(-), respectively] and the proteins were coexpressed with 1-5KR.GFP and Gag.GFP, respectively. Gag.GFP was also coexpressed with T10C(-), and 1-5KR.GFP was also coexpressed with T10C(-). (B) PPPY-A.GFP was coexpressed with Gag(-), 1-5KR(-), or

T10C(-). Cotransfected cells were metabolically radiolabeled, and Gag proteins were immunoprecipitated, separated by SDS-PAGE, and quantitated as in the legend to Figure

2.2. The percent release of Gag proteins was determined by normalizing the release of

Gag.GFP when coexpressed with Gag(-) to 100% (single solid bar) and comparing the release of Gag protein in wild type-mutant (paired solid and hashed bars) and mutant- mutant (paired hashed bars) coexpressions. Solid bars represent release of wild-type Gag proteins, and hashed bars represent release of mutant Gag proteins. The results of three independent experiments are shown.

106 P A F 120 G . g a 100 G

o t 80 e v i t a

l 60 e R

e 40 s a e l 20 e R

% 0 P P (-) P R(-) FP C(-) FP C(-) .GF .GF .GF Gag R.G T10 R.GT10 Gag Gag 1-5K Gag 1-5K 1-5K P

F 100 G

B . g

a 80 G

o t 60 e v i t a

l 40 e R 20 e s a e l 0 e

R P (-) FP FP R(-) FP C(-)

% .GF Gag -A.G -A.G -A.GT10 Gag Y Y 1-5K Y PPP PPP PPP

107 negative phenotype will require further experimentation (see Discussion).

Addition of lysines to 1-5KR. Further evidence that loss of budding is not the result of conformational defects caused by one or more of the five substitutions was obtained by adding lysines back into mutant 1-5KR. This was done in two ways. First, lysines were reintroduced at each of the five original positions, either singly or in groups of two, three, or four (Figure 2.6). The reintroduction of just one lysine at any one of the five positions brought budding back to levels that are similar to wild type, although the effect seemed to diminish somewhat with changes furthest from the L domain. Lysines reintroduced in various combinations of two or more also restored budding and showed that no single lysine is indispensable. Moreover, several lysine-restoration mutants (1-

4KR, 2-4KR, 2-3KR, and 2KR) were cloned into the provirus, and again budding was found to be restored (data not shown). Because the original five substitutions are scattered through a large region of the protein (54 amino acids), it is difficult to imagine how a gross conformational defect could be corrected simply by restoring any of the positions back to lysine.

The second way that lysines were added back was by targeting novel positions within the 1-5KR mutant, both upstream and downstream of the L domain (Figure 2.7A).

Some of these were conservative changes in which arginines at positions 121 and 135 were replaced with lysine, both individually (1RK and 2RK) and together (1,2RK). Two sets of nonconservative changes also were made by inserting lysines in place of glutamates 111 and 143 (1,2EK) upstream of the L domain or in place of arginine 205 and glutamine 213 downstream of the L domain (3RQ/KK). By themselves, these new lysines had only minor effects on budding (Figure 2.7B), but all suppressed the defect of

108

Figure 2.6 Restoration of budding with same-site revertants. Mutants were created that restore one, two, three, or four of the five lysines. The percent release of mutants compared to the wild type was determined as in the legend to Figure 2.2. A graphical representation of the results of three independent experiments is shown.

109 e p 150 y T

One lysine d l i 125 restored W o t 100 e v i t 75 a l e R

50 e s a

e 25 l e R 0 % R R R GFP ,5KR ,5KR 1-5K 1-4K ,5KR 2-5K Gag. 1,2,3 1,2,4 1,3,4 e p 150 y T

Two lysines d l i 125 restored W o t 100 e v i t 75 a l e R

50 e s a

e 25 l e R 0 % P R R R R GF 5K 3K 4K 5K g. 1- 1- 2- 3- Ga e p 150 y T

Three lysines d l i 125 restored W o t 100 e v i t 75 a l e R

50 e s a

e 25 l e R 0

% R FP KR KR KR KR 5K g.G 1- 1,2 2,3 3,4 4,5 Ga e p 150 y T Four lysines d l i 125 restored W o t 100 e v i t 75 a l e R 50 e s a e 25 l e R 0 % R R R R R R FP 5K 1K 2K 3K 4K 5K g.G 1- Ga

110

Figure 2.7 Restoration of budding with second-site revertants. (A) Lysines were

substituted for arginines at positions 121 (1RK), 135 (2RK), or both (1,2RK), for

glutamates at amino acid positions 111 and 143 (1,2EK), and downstream of the L

domain in p10 at R205 and Q213 (3RQ/KK). Substitutions were also created in the

context of 1-5KR. (B) The percent release of mutants with additional lysines in

otherwise wild-type Gag compared to Gag.GFP was determined as in the legend to

Figure 2.2. (C) The budding of 1-5KR with additional lysines was determined as percent

release of each construct relative to its parent containing the five lysines: e.g., the percent

release of 5KR;1,2EK was relative to 1,2EK normalized as 100%. Transfection,

metabolic radiolabeling, immunoprecipitation, and quantitation of Gag proteins were

done as in the legend to Figure 2.2.

111 A M L K1 K2K3 K4 K5 Wild type MA a b p10 CA R Q E1 R1 R2 E2 3

R R R R R 1-5KR

K K 1,2RK

K K 1,2EK

K K 3RQ/KK

e 125 p

B y T

d l i 100 W

o t

e 75 v i t a l e 50 R

e s a

e 25 l e R

% 0 P K K K K P K F R R R E F /K .G 1 2 ,2 ,2 .G g 1 1 g Q a a R G G 3

C t 125 n e r a

P 100

o t

e v

i 75 t a l e R

50 e s a e l

e 25 R

% 0 R K K K K R K K K 5 R R R E /K - ;1 ;2 ,2 ,2 -5 Q 1 R R ;1 ;1 1 R K K R R ;3 -5 -5 K K R 1 1 -5 -5 K 1 1 -5 1

112 1-5KR mutant (Figure 2.7C). Although novel lysines were not introduced at positions more distant to the L domain, it is clear that the presence of eGFP and its 20 lysines to the

C terminus of Gag does not suppress the 1-5KR phenotype (Figure 2.2). Collectively, these results demonstrate that one or more lysines must be present in the vicinity of the L domain for efficient particle release. Whatever the role that lysines play (e.g.,

ubiquitination), it appears to require the L domain to be present on the same molecule

because 1-5KR.GFP, which contains an L domain, was unable to rescue T10C(-), which

retains two lysines that would normally be sufficient for budding (Figure 2.5).

Lysine insertion mutants of HTLV-1 Gag. If lysines near late domains are

important for efficient release of retroviruses, then the scarcity of such lysines might

explain why HTLV-1 does not produce particles as well as RSV. The nearest lysine

upstream from the L domain (PPPY) of this virus is 44 residues away (K74). Based on

the three-dimensional structure of the highly similar MA sequence of HTLV-2 (74), this

residue is predicted to be part of the M domain (240). The nearest downstream lysine is

only 27 residues away (K148), but it resides in the CA domain rather than an

unstructured region of Gag (78, 219).

The poor release properties of HTLV-1 provided an opportunity for exploring the

need for lysines in a gain-of-function approach. For this, lysines were introduced at

positions R97, P102, and D109K upstream of the L domain (Figure 2.8A). Consistent

with the hypothesis, insertion of a single lysine at a position close to the L domain

(D109K) resulted in a substantial (64%) increase of particle release over that of the wild-

type control. For unknown reasons, budding of mutant P102K was reduced to nearly half that of the wild type; however, this phenotype was also suppressed by the insertion of

113

Figure 2.8 Introduction of lysines in HTLV-1. (A) HTLV-1 Gag is shown with the

MA, CA, and NC domains indicated. The amino acids at the C terminus of MA are shown with the residues changed to lysine (i.e., residues R97, P102, and D109) underlined and the PPPY and PTAP motifs in boldface letters. (B) Immunoprecipitation and Western blot analysis of cell- and virus-like particle (VLP)-associated proteins.

293T cells were transfected with the wild type (WT) or derivatives containing the

indicated HTLV MA mutations. VLPs were pelleted by ultracentrifugation and then

subjected to immunoprecipitation-Western blot analysis. Cell-associated material was

immunoprecipitated with an antibody to HTLV-1 p19 (MA) prior to Western blot

analysis (see Materials and Methods). The relative levels of VLP-associated p19

(normalized for cell-associated Pr53gag) are indicated under each lane of the VLP panel.

The positions of Pr53gag, Gag-Pol, p19, and incompletely processed intermediate MA-CA

are shown. The experiment was repeated three times, and representative data are shown.

114 A

HTLV-1 Gag MA CA NC

94 130 IPSRPAPPPPSSPTHDPPDSDPQIPLPPPYVEPTAPQVL

B K 2 0 1 P / K K 9 K 2 0 7 0 9 T 1 1 W P D R

Gag-Pol Cell Pr 53gag

MA-CA VLP MA

% Particle production 100% 64% 165% 96% (relative to WT)

115 another lysine to create mutant R97K/P102K (Figure 2.8B). Although attempts to create single-substitution mutant R97K were unsuccessful, these results nevertheless offer further support for the conclusion that lysines near the L domain are required for efficient retrovirus budding.

DISCUSSION

Lysines serve many roles in the functions of proteins. Structurally, they may contribute to proper folding by providing hydrophilic, positive charges that interface with the aqueous environment or interact with negatively charged moieties. In general, these structural roles can also be satisfied by arginine. In RSV, for example, interaction of the

M domain with membranes requires a critical number of basic residues but not lysines per se (60), and all six of the lysines in NC could be replaced with arginine without affecting budding (data not shown). In contrast, the studies described here revealed a strict requirement for lysines in the vicinity of the L domain for efficient budding of

RSV. The precise location and number of lysines were not important factors, suggesting that the block to budding seen in the absence of lysines (mutant 1-5KR) is not due to conformational or structural defects in the structure of Gag. The increase in budding seen when additional lysines were inserted near the L domain of HTLV-1 provides further support for a nonstructural role for these residues.

Lysines can also provide sites for at least five different types of modification to modulate the functions of proteins. For example, acetylation of lysines is known to regulate the activity of histones, HMG (high-mobility group) proteins, nuclear import factors, and transcription factors, including the Tat protein of HIV-1 (203, 365).

116 Methylation of specific lysines in histones further regulates chromatin structure and gene expression (159). Hydroxylation of lysines in collagen is needed for cross-linking of triple helices, and some of the resulting hydroxylysines are subsequently glycosylated

(263). The identification of phospholysine phosphatases suggests that phosphorylation of lysines can also occur (287). Finally, modification of lysines by Ub or Ub-like molecules serves a multitude of functions for a variety of different proteins (173, 387, 423, 427,

444). The data presented here do not exclude any of these various posttranslation modifications with regard to the lysines near the L domain, but the hypothesis that monoubiquitination is involved seems most likely given numerous other lines of supporting evidence (see the introduction). Although a previous report showed that known sites of ubiquitination in the p6 protein of HIV-1 can be eliminated without affecting budding or infectivity (337), it did not take into account the presence of two additional lysines located just 13 and 19 residues upstream from the L domain. In light of the redundancy and positional independence of lysines described here for RSV, the

HIV-1 data should be interpreted with caution.

While it is clear that lysines near the L domain are important for RSV budding,

there are reasons for doubting that they are involved in ubiquitination. Ub modification

has never been detected for Gag or MA from this virus (356, 377). Moreover, in the

course of this study, attempts were made to prevent postlysis removal of Ub from Gag

molecules present in cell extracts using strong denaturatants such as guanidinium

chloride or guanidinium thiocyanate in the presence of N-ethymaleimide to inactivate

deubiquitination enzymes, but modified Gag products still could not be detected by

immunoblotting with Ub-specific antisera (data not shown). Nevertheless, this absence

117 of evidence does not eliminate the possibility that transient Ub modifications take place on Gag, as is the case for proteins that bud into multivesicular bodies (104). Attempts were also made to rescue mutant 1-5KR by placing Ub at its C-terminus, but the chimera was equally defective for budding (data not shown). This result is especially perplexing since a similar Ub fusion was capable of repressing the negative effects of proteasome inhibitors on RSV budding (353). Attempts to rescue mutant 1-5KR by fusing the HIV-1 p6 sequence to its C terminus were unsuccessful too. While an explanation for this remains to be found, one possibility is that host factors bound to the L domain, waiting to modify lysines that are no longer present, result in a dominant-negative over p6. Further investigation of the role of the lysines near the L domain is needed.

Whatever the role of the lysines near the RSV L domain, the block to budding observed when they are missing appears to be at a late step. The evidence for this is several fold. First, cell-associated proteolytic processing of the 1-5KR mutant was similar to that of the wild type with no build-up of Pr76gag or intermediate cleavage

products. The decreased levels of mature cleavage products seen with the mutant are

possibly the result of degradation following aborted budding attempts (Figure 2.4C) (data

not shown). This is unlike M domain mutants of RSV and MLV in which proteolysis is

reduced, presumably because dimerization of the viral protease is limited in the absence

of membrane binding (391, 426, 502, 503) but similar to RSV L domain mutants (500).

Second, confocal microscopy showed that the mutant is present at the plasma membrane.

Third, EM showed that mutant virus particles are formed on the cell surface. Unlike

what has been seen in cells treated with proteasome inhibitors (353), an increased number of particles or clusters of particles compared to wild type was not observed by

118 transmission EM. This could be due either to the limited selection of electron micrographs that were observed or to collapse of nascent particles as a result of viral protease activity. Studies done by scanning EM found 1-5KR blocked at the cell surface, similar to an L domain mutant, but not in clusters (M. Johnson and V. Vogt, personal communication). The fact that no particle clusters like those seen in Ub-depleted cells were observed may indicate an additional requirement for ubiquitination of a cellular protein during budding. In any case, the ability to rescue mutant 1-5KR into particles

using budding-competent Gag proteins suggests that not every molecule in the population requires lysines near the L domain. The inability of 1-5KR and mutant T10C to rescue each other suggests that lysines and L domains may be required on the same Gag protein.

The lack of complementation is not likely due to a failure to heteromultimerize with

T10C, since even MLV Gag can rescue this L-domain-deletion mutant when the M domains are the same (21). Thus, it appears that the RSV L domain requires lysines to function.

During the course of this study, it was found that a budding-competent Gag molecule can rescue mutant 1-5KR and the L domain deletion mutant T10C, but it cannot rescue the point mutant PPPY-A. Indeed, the rescuing Gag molecule is strongly inhibited

when coexpressed with the point mutant. The dominant-negative activity of mutant

PPPY-A is puzzling, but may be due to the binding of previously unrecognized host

factors on regions of Gag that are missing in the large deletion mutant. For example,

there is a YPSL motif just downstream of the RSV L domain, in the p10 sequence, and

this is remarkably similar to the YPDL motif in equine infectious anemia virus, which

provides L domain activity and binds the host protein AIP1/ALIX (452, 488). Perhaps

119 binding of AIP1/ALIX to the RSV Gag protein results in a dominant-negative phenotype when the L domain is absent. Further experimentation is required to determine the nature of the block.

The studies described here also revealed a potential involvement of SUMO-1 in

RSV replication. This Ub-like protein has been shown to play roles in protein translocation, subnuclear structure formation, and modulation of transcriptional activity, and has an antagonistic role against Ub (429). The only lysine in the RSV Gag protein that resides in a clear sumoylation consensus sequence (ΨKxE, where Ψ is a hydrophobic residue and x is any residue [397, 410]) is the one at position 244 in CA (IK244TE).

Although this residue was found to be unimportant for budding, it proved to be critical

for infectivity. Further experimentation will be required to ascertain whether the loss of

infectivity seen with mutant K244R is due to loss of sumoylation or the result of

structural defects.

ACKNOWLEDGEMENTS

C. B. Wilson provided technical assistance in mutagenesis, R. C. Craven

provided Figure 2.4A and 2.4B, H. Wang provided Figure 2.8, and A. Patnaik provided

experimental planning and discussions. Thanks are extended to Marc Johnson and

Volker Vogt for providing their scanning EM data prior to publication. Thanks also to

Eric Callahan for construct p∆MA3.GFP, Roland Meyers for thin-sectioning and EM,

and Nate Sheaffer of the PSU Cell Science/Flow Cytometry Core Facility for assistance

with the FACS analyses. This work was supported by National Institutes of Health

grants CA47482 to J.W.W. and AI053155 to L.M.M.

120 CHAPTER III

ANALYSIS OF A POTENTIAL SUMOYLATION SITE IN ROUS

SARCOMA VIRUS CAPSID IMPORTANT FOR REPLICATION

121 ABSTRACT

SUMO-1 is typically conjugated to a target lysine within the consensus sequence

ΨKxE/D (Ψ is a hydrophobic residue and x is any residue). It was previously noted that

the Rous sarcoma virus (RSV) capsid (CA) protein contains a sumoylation consensus

sequence (243IKTE246) in the β hairpin region, and destruction of the consensus with the substitution K244R had no effect on virus budding, but the released virions were greatly reduced in their infectivity, revealing a potential role for sumoylation of Gag or CA during replication. Indeed, interaction between sumoylation machinery and Mason-

Pfizer monkey virus Gag has been observed. To further explore the importance of the

243IKTE246 sequence, mutants T245I, E246D, and E246A were created. As predicted,

E246D had no effect on infectivity and T245I had only a mild effect; however, E246A

had a defect similar to that of K244R. The cause for the infectivity defect of K244R was not due to Pol or Env incorporation, RNA packaging, core stability, or nuclear import or export of Gag during assembly. There was, however, a ~2-fold decrease in the amount of

DNA synthesized in an endogenous reverse transcription assay and in newly infected cells. This decrease was attributed to only half the amount of the virions being able to initiate reverse transcription as determined by a focus assay. To gain further insight into the role of this consensus sequence during replication, revertant viruses were isolated by passaging K244R or E246A virions in uninfected cells multiple times. K244R was suppressed by the double substitution R325C/C431R and E246A was suppressed by a

N343D substitution. The suppressor mutations did not create a new sumoylation consensus sequence as expected, but instead may provide a way for the virion to replicate independent of sumoylation. Alternatively, lysine 244 and glutamate 246 may be

122 involved in formation and/or stabilization of the CA hexamer in the mature capsid, as suggested by their location in the β hairpin. The suppressors, then, would compensate for any structural defects caused by the mutations. Though the exact function of these residues during replication is unknown, the inability of mutant virions to initiate reverse transcription provides further evidence that CA is involved in this essential process.

INTRODUCTION

Although the capsid (CA) proteins of retroviruses have been extensively studied, their role in replication remains poorly understood. CA is first synthesized as part of the

Gag polyprotein and is required for particle morphology and infectivity, but not release

(1, 417, 495). Upon maturation of the virion, CA encloses the ribonucleoprotein complex of the viral RNA (vRNA), the tRNA primer, nucleocapsid (NC), reverse transcriptase

(RT), and integrase (IN) that is responsible for viral DNA synthesis in the infected cell.

Mutants of CA have been identified that reduce or block infectivity without affecting assembly and budding (4, 59, 83, 463, 487) suggesting that the function of this protein is not merely structural but is actively involved in one or more still undefined roles during the early stages of infection. CA may also play a role in regulating the nuclear transport of the preintegration complex (PIC). HIV and other lentiviruses are able to infect dividing as well as nondividing cells, whereas simple retroviruses such as RSV and MLV primarily infect dividing cells. This difference has been mapped to CA where replacement of HIV MA with MLV MA and p12 had no effect on HIV’s ability to infect nondividing cells, but replacement of HIV MA and CA with MLV MA, p12, and CA abrogated the ability of HIV to infect nondividing cells (508). CA has also been reported

123 to be a part of the reverse transcription complex (RTC) and PIC of MLV (41, 115), but not of HIV (116, 302). Other evidence suggests that HIV reverse transcription may initiate in the intact capsid shell which dissociates before reverse transcription is complete (290).

The function of CA in reverse transcription and nuclear transport is unknown, but given that an infectious retrovirion relies on cellular proteins for much of its lifecycle, one of its functions may be to recruit cellular proteins required at various postentry stages. For instance, it was recently observed that CA of MPMV interacts with the cellular protein Ubc9, the E2 enzyme required for conjugation of SUMO (Small

Ubiquitin-like MOdifier)-1 to a lysine of a target protein (494). SUMO-1 is a 97 aa polypeptide typically conjugated to a target lysine in the consensus sequence ΨKxE/D,

where Ψ is a hydrophobic residue and x is any residue (199, 294, 397, 410). The exact

role of sumoylation is not well understood, but it is utilized in many nuclear events such as nuclear entry, subnuclear structure formation, and modulation of transcriptional activity (199, 429). Its role in various nuclear events makes SUMO-1 an attractive candidate for regulation of reverse transcription and nuclear entry. This hypothesis is supported by a recent report that suggested that sumoylation of HIV p6 has a regulatory

role during reverse transcription (152). Further, the interaction between sumoylation machinery and viruses has also been observed for herpes viruses (2, 69, 151, 177, 246,

313, 322, 445), papilloma viruses (383, 384), vaccinia virus (399), adenovirus (250), hantavirus (215, 242, 272), and geminiviruses (66) where it is important for nuclear localization and regulation of transcription.

It was noted in Chapter II that a consensus sumoylation sequence (243IKTE246)

124 resides in CA of RSV. When the lysine was changed to arginine, infectivity was reduced compared to wild-type, suggesting a possible role for sumoylation machinery during RSV replication. In the studies described below, the involvement of the consensus was further characterized through substitutions at the threonine and glutamate positions. The reason for the infectivity defect for K244R was determined by analyzing the relative amount of

RNA, Pol, and Env packaged, the stability and morphology of the core, and the ability of the virion to reverse transcribe its genome. Revertant virions were also isolated which suppress the K244R phenotype.

MATERIALS AND METHODS

Expression vectors. The wild-type RSV gag gene was originally derived from the Prague C proviral vector, pATV-8 (211, 428). To analyze subcellular localization, gag alleles were transferred into pGag.GFP (43) which contains a gag-egfp fusion. For all other assays, gag alleles were cloned into pJD100 or pRS.V8.eGFP (60).

Mutagenesis. Construction of pK244R.GFP and pRS.K244R.eGFP was described previously (Chapter II). The K244R substitution was transferred to pJD100 using SacII which cuts twice, once in MA and once in CA. The vector was treated with shrimp alkaline phosphatase and then ligated with the mutant fragment; proper orientation of the insert was confirmed by digestion with BglII. T245I, E246A, and

E246D substitutions were generated by PCR mutagenesis with pGag.GFP as template.

The threonine codon was changed from “ACA” to the “ATA” isoleucine codon, the glutamate codon was changed from the “GAG” codon to either the “GCG” alanine codon or the “GAC” aspartate codon. Mutations were transferred into pGag.GFP or

125 pRS.V8.eGFP using SstI and FseI, thereby replacing the 5' half of gag. R325C, C431R,

R325C/C431R, and N343D substitutions were also generated using PCR mutagenesis.

The arginine codon was changed from “CGC” to the cysteine codon “TGC”. The

cysteine codon was changed from “TGC” to the arginine codon “CGC”. The asparagine

codon was changed from “AAT” to the aspartate codon “GAT”. The C431R mutation

was transferred into pRS.V8.eGFP or pRS.K244R.eGFP using SdaI and BstXI replacing

the C-terminal half of gag and most of pol. R325C and N343D mutations were

transferred into pRS.V8.eGFP, pRS.K244R.eGFP, pRS.C431R.eGFP,

pRS.RS.K244R,C431R.eGFP, or pRS.E246D using SstI and SdaI, thereby replacing the

N-terminal half of gag. All clones were sequenced to confirm the presence of the desired

substitutions and to be certain that no undesired substitutions had occurred.

Budding assays. Budding of wild-type and mutant constructs was analyzed by

transfecting QT6 cells (310) by the CaPO4 method, as previously described (83),

35 followed by metabolic radiolabeling with a L-[ S]methionine/cysteine mix (50 µCi,

>1,000 Ci/mmol) 24 h after transfection. QT6 cells were transfected with proviral constructs in duplicate; one plate was pulse-labeled for 5 min, at which time only unprocessed Gag is detected, while the other plate was radiolabeled for 3 h. The viral proteins were immunoprecipitated from detergent-lysed cells and particles using a polyclonal rabbit serum against whole RSV, as previously described (493).

Immunoprecipitates were separated in sodium dodecyl sulfate (SDS)-12%- polyacrylamide gels, which were subsequently dried and exposed to Kodak X-Omat AR5

X-ray film. Unprocessed Gag from the 5 min pulse-labeled cells and mature CA protein from the 3 h labeled medium fractions were quantified by PhosphorImager analysis.

126 Budding proficiency was calculated as the amount of CA in the medium divided by the amount of Gag in the pulse-labeled cell lysate.

Infectivity assays. Spreading assays were performed as previously described in

Chapter II. QT6 cells were transfected with pRS.V8.eGFP, pRS.K244R.eGFP, pRS.T245I.eGFP, pRS.E246A.eGFP, or pRS.E246D.eGFP, and virions were allowed to accumulate in the medium for 24 h. Cells were removed from the medium fraction by centrifugation at 1000 x g for five min, and virions from half of the cell-free medium were pelleted through a 25% sucrose cushion at 126,000 x g for 40 min at 4 °C in a

TLA100.4 rotor. Pelleted virions were resuspended in phosphate-buffered saline (PBS) and analyzed by a reverse transcriptase assay as previously described (83) to determine the amount of virions in the other half of each medium sample. Equal concentrations of virions were place on DF-1 cells for 24 h, after which new medium was added. The numbers of infected (i.e., green) cells present at subsequent time points were counted by

FACS analysis.

Focus forming assays were performed essentially as previously described (192).

QT6 cells were transfected with pJD100 or pJD.K244R using the CaPO4 transfection, and virions were allowed to accumulate in the medium for 24 h. The medium fraction was centrifuged at 1000 x g for five min followed by passage through a 0.45µm filter to remove any cells, and virions from a fraction of the cell-free medium were pelleted through a 25% sucrose cushion at 126,000 x g for 40 min at 4 °C in a TLA100.4 rotor.

Pelleted virions were resuspended in PBS and analyzed by a reverse transcriptase assay as above for normalization of the amount of virions per ml medium. Ten-fold dilutions were made of each virus stock and were placed on primary turkey embryonic fibroblasts

127 (TEFs) for 16 h, the cells were washed with Tris-buffered saline (TBS) followed by addition of an agar overlay (F-10, 10% tryptose phosphate broth, 0.7% bactoagar, 0.05% sodium bicarbonate, 5% fetal bovine serum, 1% DMSO, penicillin, and streptomycin).

At various days postinfection foci were counted to determine the focus forming units per ml.

Confocal microscopy. At 24 h posttransfection QT6 or DF-1 cells seeded on coverslips were treated with DMSO or 10 µg/ml leptomycin B for 2.5 h. Cells were fixed with 3% paraformaldehyde for 45 min. The subcellular locations of Gag.GFP or

K244R.GFP were observed by confocal microscopy using a Leica TCS SP2 AOBS confocal microscope following excitation with a helium-argon laser (488-nm peak excitation).

EM. Stably infected DF-1 cells were seeded in 60mm Permanox dishes (Electron

Microscopy Sciences, Ft. Washington, PA) and upon confluency were processed for EM as described previously (83).

Detergent resistance assay. The detergent resistance assay was performed as previously described (44). The medium fraction from radiolabeled stably infected DF-1 cells was centrifuged at 1000 x g for five min to remove any cells. Step gradients were prepared with 0.5 ml of either 5% sucrose or 5% sucrose plus 1% Triton X-100 layered on top of 1.5 ml of 10% sucrose. The cell-free medium samples were split in half and layered onto a sucrose gradient with or without Triton X-100. Both sets of gradients were spun at 126,000 × g for 40 min at 4 °C. The supernatant was transferred to another tube, and the pellet was resuspended in lysis buffer. Gag proteins were immunoprecipitated from the supernatant and pellet fractions and resolved on SDS-12%-

128 polyacrylamide gels, which were subsequently dried and exposed to film as described above. The amount of CA protein present in each fraction was determined by

PhosphorImager analysis as above. For each half of the experiment (i.e., with detergent or without) the amount of CA in either the pellet or supernatant was expressed as a percentage of total CA.

CA:RT ratio assay. The medium fraction from radiolabeled stably infected DF-

1 cells was centrifuged at 1000 x g for five min to remove any cells, and virions from the cell-free medium were pelleted through a 25% sucrose cushion at 126,000 x g for 40 min at 4 °C in a TLA100.4 rotor. Pelleted virions were resuspended in PBS and split into two samples. One sample was analyzed by a reverse transcriptase assay as above to determine the relative amount of Pol present. CA proteins were immunoprecipitated from the other sample and resolved on a SDS-12%-polyacrylamide gel, which was subsequently dried and exposed to film as described above. The amount of CA protein present in each fraction was determined by PhosphorImager analysis. For each virus the amount of CA was divided by the counts per minute (cpm) in the RT assay to give a

CA:RT ratio.

Env incorporation. The relative amount of Env was determined by collecting virions for 24 h in the medium of stably infected cells, removing cells by centrifugation at

1000 x g followed by passage through a 0.45µm filter, and pelleting the virions from the medium through a 25% sucrose cushion at 126,000 x g for 40 min at 4 °C in a TLA100.4 rotor. Viral proteins were separated on a SDS-12%-polyacrylamide gel followed by

Western blot analysis using a rabbit anti-TM antibody (a gift from J. M. White,

University of Virginia, Charlottesville, VA) as the primary antibody and visualized by

129 enhanced chemiluminescence after incubation of the membranes with a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase. The membrane was stripped and reprobed using a rabbit anti-Gag antibody as the primary antibody and again visualized by enhanced chemiluminescence after incubation of the membranes with a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase. Visualized bands were quantified by densitometry.

RNA isolation. Virions from stably infected DF-1 cells were collected in the medium for 24 h. Cells were removed from the medium fraction by centrifugation at

1000 x g followed by passage through a 0.45µm filter. Virions from the cell-free medium were pelleted through 20% sucrose at 20,000 x g for 3 h and resuspended in PBS. A fraction of the resuspended virions was used to perform an RT assay to determine the relative amount of virions in the sample. Equal amounts of RNA from wild-type and mutant virions were isolated using QIAmp® Viral RNA mini kit (Qiagen) according to

the manufacturer’s instructions. Purified RNA was DNase-treated with DNA-free DNase

kit® (Ambion).

DNA isolation. Virions from stably infected DF-1 cells were collected in the medium for 24 h. Cells were removed from the medium fraction by centrifugation at

1000 x g followed by passage through a 0.45µm filter. Virions from a fraction of the cell-free medium were pelleted through a 20% sucrose cushion at 20,000 x g for 3 h and

resuspended in PBS. The relative amount of virions per ml was determined by

performing an RT assay on the pelleted virions. Equal amounts of virions were added to

uninfected DF-1 cells and low molecular weight DNA was isolated at 14 h postinfection

as previously described (59, 176).

130 Endogenous RT (ERT) assay. Virions from stably infected DF-1 cells were collected in the medium for 24 h. Cells were removed from the medium fraction by centrifugation at 1000 x g followed by passage through a 0.45µm filter. Virions from a fraction of the cell-free medium were pelleted through a 20% sucrose cushion at 20,000 x g for 3 h and resuspended in PBS. A fraction of the resuspended virions was used to perform an exogenous RT assay to determine the relative amount of virions in the sample. Equal amounts of virions from wild-type and mutant virions were used in an endogenous RT assay performed as previously described (59). Briefly, virions were incubated in an ERT reaction buffer with 125 µg/ml melittin for 3h at 42 °C. DNA was extracted using a QIAquick® purification kit (Qiagen) according to the manufacturer’s instructions.

Quantitative real time PCR. To determine the amount of reverse transcription products present per sample primers corresponding to the RSV LTR and the 5' and 3' untranslated regions were designed to detect various DNA products of reverse transcription: primer A (5'-GCCATTTGACCATTCACCA-3') and primer B (5'-

AATGAAGCCTTCTGCTTCATG-3') for minus-strand strong stop DNA; primer C (5'-

ATTCCGCATTGCAGAGATATTG-3') and primer B for first strand transfer; primer A and primer D (5'-GATGGAGACAGGATCGCCAC-3') for second strand transfer; and primer

A and E (5'-CATGTTGCTAACTCATCGTTACCA-3') for 2LTR circles. Additional primers (5'-CCTCCCCCTCTTAACCAAAAC-3' and 5'-

TGCTATTTCATCTTTCCCTTGC-3') were used to amplify chicken mitochondrial DNA to normalize the amount of input cells used to isolate reverse transcription products from infected cells. FAM/TAMRA dual-labeled probes (Sigma-Genosys) specific for the RSV

131 LTR (5'-CCATCAACCCAGGTGCACACCAATG-3') or chicken mitochondrial DNA

(5'-CAGTATAGGCGATAGAAAAGACTACCCCGGC-3') were used for the quantitative-

PCR (Q-PCR) reaction using a QuantiTect® Probe PCR kit (Qiagen) according to the

manufacturer’s directions.

For determining the amount of RNA isolated from virions, Q-reverse transcription

(RT)-PCR was performed on purified viral RNA using primer pairs A+D, the RSV LTR

dual-labeled probe, and a QuantiTect® Probe RT-PCR kit (Qiagen) according to the manufacturer’s directions.

Isolation of revertants. Medium from cells stably infected with RS.V8,

RS.K244R.eGFP, or RS.E246A.eGFP was collected and the cells were removed by a low speed centrifugation as above. This cell-free medium was placed onto uninfected DF-1 cells. Cells were maintained for one week to two months and again the medium was collected for ~24 h, the cells were removed, and the medium was placed onto uninfected

DF-1 cells. This process was repeated twelve times. After various passages, medium was collected for 24 h and cells were removed. Virions from half of the cell-free medium were pelleted as described above. Pelleted virions were resuspended in PBS and analyzed by a reverse transcriptase assay as above to determine the amount of virions in the other half of each medium sample. Equal concentrations of virions were placed on

DF-1 cells for 24 h, after which new medium was added. At various days postinfection virions were pelleted from the medium of infected cells and the amount of RT activity in the medium was determined by a RT assay to analyze the rate of spread throughout the cell culture over time.

Virions isolated from passage twelve were pelleted from cell-free medium and the

132 RNA was isolated as above. Purified RNA was DNase-treated with DNA-free DNase kit® (Ambion). The RNA was reverse transcribed in ~1.4 kb segments from U3 through env using Invitrogen’s SuperScript III® RT-PCR kit according the manufacturer’s instructions. DNA fragments were then sequenced with the same primers used for amplification.

RESULTS

Sumoylation consensus sequence required for replication. If the 243IKTE246 sequence in RSV CA (Figure 3.1) is indeed a sumoylation site then other substitutions within it can be made with the following predictions: First, the threonine (which resides in the “x” position) should be able to be substituted with any amino acid such as isoleucine and have little effect on replication. Second, substitution of the glutamate with an aspartate ought to have no real effect on replication since either a glutamate or aspartate give a consensus sequence. Third, substitution of the glutamate with an alanine would be expected to have an effect on RSV replication. The resulting mutants (T245,

E246D, and E246A) were engineered into the proviral plasmid pRS.V8.eGFP where v- src has been replaced with the gene for eGFP.

Wild type (RS.V8.eGFP) and mutants RS.K244R.eGFP, RS.T245I.eGFP,

RS.E246A.eGFP, and RS.E246D.eGFP were expressed in QT6 cells to determine their budding efficiency. As shown in Figure 3.2A budding was unaffected by any of the substitutions.

To determine the infectivity of each mutant, QT6 cells were transfected with pRS.V8.eGFP, pRS.K244R.eGFP, pRS.T245I.eGFP, pRS.E246D.eGFP, or

133

Figure 3.1 Position of mutants K244R and E246A and their suppressor mutations.

RSV Gag is illustrated with the locations of the major cleavage products indicated. CA is enlarged to show the NTD and CTD along with the location of mutants K244R and

E246A connected by arrows to the location of their respective suppressor mutations.

134 p2 243IKTE246 SP Gag MA p10 CA NC PR 701

NTD CTD CA

K244R R325C C431R E246A N343D

135

Figure 3.2 Budding and infectivity of sumoylation consensus sequence mutants. (A)

Gag proteins were expressed in QT6 cells, radiolabeled, and immunoprecipitated (see

Materials and Methods). Gag proteins and cleavage products were visualized by autoradiography and quantified by PhosphorImager analysis. The ratio of CA protein in the media versus the amount of Gag expressed in the pulse labeled cells was used to determine the budding efficiency. Mutants were compared to the wild type, which was normalized to 100%. The graph represents the results of four independent experiments.

QT6 cells were transfected with pRS.V8.eGFP, pRS.K244R.eGFP, pRS.T245I.eGFP, pRS.E246D.eGFP, or pRS.E246A.eGFP, and virions were collected in the medium for 24 h. Equal amounts of virions from undiluted (B) or 1:20 diluted (C) medium were added to DF-1 cells for 24 h. The cells were analyzed by FACS at various times postinfection to determine the percentage of green cells, indicating an infection. A representative graph of the results of three experiments is shown. (D) QT6 cells were transfected with pJD100 (wild type) or pJD.K244R, and virions were collected in the medium for 24 h.

Equal amounts of virions from a series of 10-fold dilutions were added to primary TEF cells for 16 h. At 16 d postinfection foci were counted to determine the amount of focus forming units per ml. K244R was compared to wild-type to determine the fold defect in infectivity. The graph is an average of the results of three independent experiments.

136 A 140 e

p 120 y T

d l

i 100 W

o

t 80

e v i t

a 60 l e R 40 e s a e l 20 e R

% 0 e I p R A 5 D y 4 6 4 6 4 4 2 4 T 2 2 T 2 ild K E E B W 100 s l l 80 e C

n

e 60 e r G

Wild Type t 40 n E246D e c

r T245I

e 20 K244R P E246A 0 3 5 7 9 11 13 15 17 19 21 23 25 27 C Days Postinfection 100 s l

l 80 e C

n 60 e e r

G Wild Type 40 t

n E246D e

c T245I r 20

e K244R

P E246A 0 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 Days Postinfection D

U 1.0 F F

T

n 0.8 i

W

n o t o i

t 0.6 e c v i u t d

a 0.4 l e e R

R d

l 0.2 o F 0 Wild Type K244R

137 pRS.E246A.eGFP, and particles were allowed to accumulate in the media for 24 h.

Equal amounts of released RT activity were used to infect fresh cultures of DF-1 cells, and at various days postinfection, these were trypsinized and sorted by FACS to count the number of green (i.e., infected) cells. The half-time of spreading for mutants K244R and

E246A as typically almost three times longer than wild type and E246D (Figure 3.2B).

T245I had an intermediate effect with a half-time of spreading about twice that of the wild type. The same effect was seen when cells were infected with a lower MOI (Figure

3.2C): the half-time of spreading of T245I virions was about twice that of the wild type and that of K244R and E246A was about three times as long. These results suggest that this region of CA is important for efficient replication of the virus.

A focus forming assay was used to quantify the defect in infectivity seen by the

K244R substitution. QT6 cells were transfected with pJD100 (wild type) or pJD.K244R

(which encode the v-src oncogene) and particles were allowed to accumulate in the media for 24 h. Equal amounts of released RT activity were used to infect primary TEFs. At 16 h postinfection, the medium was removed and cells were washed with TBS to remove any virus. An agar overlay was added to inhibit the spread of virus through the culture.

The cells were observed for foci formation, each presumably formed from a single infected cell. Cultures infected with wild-type virions started forming foci about seven days postinfection whereas foci did not appear in cultures infected with K244R for another three to five days (data not shown). At day sixteen postinfection foci on all cultures were fully formed and cultures infected with K244R virions had 2-fold fewer foci than those infected by wild type (Figure 3.2D).

Nuclear trafficking of K244R. RSV Gag traffics into and out of the nucleus

138 during assembly (419) and, in one report, a mutant which does not traffic through the nucleus had reduced amounts of vRNA packaged (61). Since sumoylation is involved in nuclear transport of cellular proteins and MPMV Gag has been shown to partially colocalize with Ubc9 around the nuclear rim (494) it is possible mutant K244R Gag is unable to traffic into or out of the nucleus. To determine if nuclear trafficking of Gag was affected by the K244R substitution, QT6 cells (Figure 3.3) or DF-1 cells (data not shown) were transfected with pGag.GFP or pK244R.GFP. One set of cells was treated with 10 µg/ml leptomycin B (LMB) to inhibit CRM1-mediated nuclear export and another set was treated with the solvent DMSO. Cells were fixed with 3% paraformaldehyde and analyzed by confocal microscopy. In cells treated with DMSO, wild-type and mutant Gag were localized to the cytoplasm and the plasma membrane. In

LMB-treated cells wild-type and mutant Gag proteins were both localized to the nucleus.

Therefore, lysine 244 is not involved in Gag trafficking into or out of the nucleus during

assembly.

K244R virion characterization. Substitutions in CA have been shown to affect

Gag processing, particle size, core morphology, and core stability (44, 82, 83, 97, 121,

232, 487) and a defect in any of these could explain the reduction of K244R infectivity.

QT6 cells transfected with pRS.V8.eGFP, pRS.K244R.eGFP, pRS.T245I.eGFP,

pRS.E246D.eGFP, or pRS.E246A.eGFP were radiolabeled with 35S-Met/Cys. Gag proteins were immunoprecipitated from the cell lysate and medium fractions with an anti-

RSV antibody. Processing of all mutant Gag proteins was similar to wild type (Figure

3.4A). Cells infected with the wild type or K244R mutant were thin sectioned and analyzed by EM to determine if the K244R substitution affects virion size or core

139

Figure 3.3 Intracellular localization of K244R. pGag.GFP or pK244R.GFP was transfected into duplicate plates of QT6 cells using 1µg of DNA. 16 h posttransfection, cells were treated with DMSO or 10 µg/ml leptomycin B for 2.5 h followed by paraformaldehyde fixation. The subcellular localization of Gag proteins was examined using confocal microscopy.

140 DMSO LMB

WT

K244R

141

Figure 3.4 Characterization of K244R virions. (A) QT6 cells expressing pRS.V8.eGFP, pRS.K244R.eGFP, pRS.T245I.eGFP, pRS.E246D.eGFP, or pRS.E246A.eGFP were metabolically radiolabeled, and Gag proteins were immunoprecipitated, separated by SDS-PAGE, and visualized as in Figure 3.2. (B) DF-1 cells stably infected with RS.V8.eGFP or RS.K244R.eGFP were thin sectioned and examined by EM (Magnification, x37,000; bar, 100nm). (C) Cell-free medium from radiolabeled DF-1 cells infected with RS.V8.eGFP or RS.K244R.eGFP was layered over a sucrose step gradient comprised of 0.5% sucrose or 0.5% sucrose + 1% Triton X-100 layered over 10% sucrose and was centrifuged at 126,000 x g for 40 min. Gag cleavage products were immunoprecipitated, separated by SDS-PAGE, visualized, and quantified as described in the Material and Methods. The amount of CA in either the pellet or supernatant was expressed as a percentage of total CA. The graph is an average of the results of three independent experiments.

142 R A I D R A I D 4 6 5 6 k 4 6 5 6 k 4 4 4 4 c 4 4 4 4 c T 2 2 2 2 o T 2 2 2 2 o A W K E T E M W K E T E M

Gag

CA

B Lysates Media

WT WT

K244R K244R C Supe e

p 100

y Pellet T

d l i 80 W

o t 60 e v i t a l

e 40 R

A

N 20 D

% 0 Triton-X 100 - + - + Wild Type K244R

143 morphology. Particle size and morphology were normal for K244R (Figure 3.4B).

Despite normal Gag processing and core morphology, the stability of the core may still be compromised by the substitution. The stability of wild-type and mutant cores was determined by pelleting virions through sucrose step gradients in the presence and absence of 1% Triton X-100. In the absence of detergent approximately 90% of wild- type or mutant CA could be immunoprecipitated from the pellet (Figure 3.4C). When compared to the wild type there was a slight, but insignificant (P=0.3534) decrease in the amount of pelletable mutant CA in the presence of detergent. Therefore, the K244R substitution had no real effect on Gag processing, core morphology, particle size, or core

sensitivity to detergent.

Pol incorporation. CA has been reported to be important for incorporation of

Gag-Pol into virions (182, 446). To determine if there are similar levels of Pol

incorporated into wild-type and mutant virions, infected DF-1 cells were radiolabeled and

virions were pelleted from cell-free medium. The pellet was resuspended in PBS and

half was used in an exogenous RT assay to determine the relative amount of RT; CA

from the other half was immunoprecipitated. The amount of CA was determined by

PhosphorImager analysis and was divided by the cpm in the RT assay; the mutant ratio

was compared to the normalized wild type. This experiment demonstrated that there was

no defect in the amount of Pol incorporated into K244R virions (Figure 3.5A).

Env incorporation. Since it was also possible that the reduction to infectivity of

K244R virions was due to there being less Env present on the virion, the relative amount

of Env was determined. Mutant and wild-type virions were analyzed by Western blot

using an antibody against TM and quantified by densitometry. Membranes were stripped

144

Figure 3.5 Incorporation of Pol, Env, and vRNA into K244R virions. (A) The relative amount of Pol incorporated into virions was determined by analyzing the ratio of

CA to RT. Virions from the cell-free medium of radiolabeled DF-1 cells infected with

RS.V8.eGFP or RS.K244R.eGFP were pelleted through a 25% sucrose cushion at

126,000 x g for 40 min and were resuspended in PBS. The sample was split in two and an exogenous RT assay was performed on one, whereas CA proteins were immunoprecipitated, separated by SDS-PAGE, and quantified. The amount of CA was divided by the cpm in the RT assay to give a relative CA:RT ratio. The graph is an average of the results of three independent experiments. (B) Virions from the cell-free medium from DF-1 cells infected with RS.V8.eGFP or RS.K244R.eGFP were pelleted as described in (A). The pellet was dissolved in sample buffer, separated by SDS-PAGE, transferred to nitrocellulose, and visualized by Western blot analysis using a primary antibody against TM. The nitrocellulose was stripped and reprobed using an anti-Gag primary antibody. The amount of CA and TM was determined by densitometry. The graph is an average of the results of three independent experiments. (C) Virions from cell-free medium from DF-1 cells infected with RS.V8.eGFP or RS.K244R.eGFP were pelleted (see Materials and Methods) and resuspended in PBS. RNA was isolated from normalized virions using QIAmp® viral RNA mini kit. The relative amount of RNA

present was determined by Q-RT-PCR using primers and a dual-labeled probe specific

for regions in the LTR. The graph is an average of the results of three independent

experiments done in duplicate.

145 A 1.2 T R

1.0 o t

A 0.8 C

f 0.6 o

o i t 0.4 a R 0.2 0 e R p 4 y 4 T 2 ild K W B 1.2 v T

n 1.0 W E

f o

t 0.8 o

t e n v i t u 0.6 a o l e m 0.4 R A 0.2 0 e R p 4 y 4 T 2 ild K W C 1.2 A T 1.0 N W

R

o f

t 0.8

o

e t v n i

t 0.6 u a o l e

m 0.4 R A 0.2 0 e R p 4 y 4 T 2 ild K W

146 and reprobed to determine the relative amount of CA using an antibody against Gag followed by densitometry. The ratio of Gag to Env per sample was determined and samples were normalized to the wild type. Using this semiquantitative method, there appeared to be little difference in the relative amount of Env in mutant and wild-type virions (Figure 3.5B).

RNA packaging. To determine if the infectivity defect of K244R was due to less vRNA being packaged, RNA was isolated from normalized amounts of wild-type and mutant virions using Qiagen’s QIAmp® Viral RNA mini kit. The relative amount of vRNA was determined by Q-RT-PCR. K244R and wild-type virions packaged similar amounts of vRNA (Figure 3.5C). Therefore, a defect in vRNA packaging was not the reason for the defect.

Analysis of viral DNA products in infected cells. The above results indicate that mutant K244R virions were not defective in assembly, release, or virion morphology.

To determine if mutant virions were competent for entry into cells, infected DF-1 cells were analyzed at 16 h postinfection for the presence of viral DNA. Low molecular weight DNA was isolated and the amount of minus-strand strong-stop, first strand

transfer, and second strand transfer DNA products was determined by Q-PCR using

primers specific for each stage of reverse transcription (see Materials and Methods). The

amount of DNA isolated per sample was normalized using primers specific for chicken

mitochondrial DNA. Compared to the wild type, the amount of mutant minus-strand

strong-stop DNA was decreased by ~45% (Figure 3.6A); first strand transfer and second

strand transfer were decreased ~25-35% for the mutant virus. These results suggest an

early block to reverse transcription, prior to minus-strand strong-stop synthesis. The

147

Figure 3.6 Products of reverse transcription. (A) Medium from DF-1 cells infected with RS.V8.eGFP or RS.K244R.eGFP was collected for 24 h. Equal amounts of virions normalized by an RT assay were added to uninfected DF-1 cells for 16 h at which time low molecular weight DNA was isolated (see Materials and Methods). The relative amount of minus-strand strong-stop, first strand transfer, second strand transfer, and 2

LTR circle DNA products was determined by Q-PCR using primers and a dual-labeled probe as described in the Materials and Methods. The graph is an average of the results of five independent experiments. (B) Virions from the cell-free medium from DF-1 cells infected with RS.V8.eGFP or RS.K244R.eGFP were pelleted as in Figure 3.5 and resuspended in PBS. Normalized amounts of virions were permeablized with melittin and incubated with dNTPs at 42 °C for 3 h. The DNA was then extracted and subjected to Q-PCR as described in (A) to determine the relative amount of minus-strand strong- stop and first strand transfer DNAs. The graph is an average of the results of five independent experiments.

148 A Strong Stop 1st Strand Transfer e

p 100 100 y T

d l i 80 80 W

o t 60 60 e v i t a l

e 40 40 R

A

N 20 20 D

% 0 0 e R e R p 4 p 4 y 4 y 4 T 2 T 2 ild K ild K W W

2nd Strand 2 LTR Circle Transfer

e 100

p 100 y T

d l i 80 80 W

o t 60 60 e v i t a l 40 40 e R

A

N 20 20 D

% 0 0 e R e R p 4 p 4 y 4 y 4 T 2 T 2 ild K ild K W W

B Strong Stop 1st Strand Transfer e

p 100 100 y T

d l i 80 80 W

o t 60 60 e v i t a l

e 40 40 R

A

N 20 20 D

% 0 0 e R e R p 4 p 4 y 4 y 4 T 2 T 2 ild K ild K W W

149 ability of the PIC to enter the nucleus was determined by analyzing the presence of 2

LTR circles, a hallmark of nuclear entry. The amount of mutant 2 LTR circle DNA compared to wild type was decreased by ~65%.

Endogenous reverse transcription. To determine if the defect in reverse transcription was inherent to the virion an ERT assay was performed. Normalized virions were permeablized with melittin and allowed to undergo reverse transcription in the presence of dNTPs for three hours at 42 °C. The DNA was isolated and the presence of minus-strand strong-stop and first strand transfer DNAs was detected by Q-PCR. The

synthesis of both minus-strand strong-stop and first strand transfer DNAs was decreased

by ~50% for the mutant virus (Figure 3.6B). The defect in making minus-strand strong-

stop DNA was similar to the defect seen in infected cells suggesting the reverse

transcription defect was inherent to the virion.

Isolation of second-site suppressors. To isolate second-site suppressors the

medium was collected from cells stably infected with wild-type, K244R, or E246A virus

and subjected to a low-speed centrifugation to remove any cells (Figure 3.7). The cell-

free medium was then added to uninfected DF-1 cells. One week to one month

postinfection the medium was again collected and cell-free medium was added to

uninfected cells. This process was repeated and the virus was passaged onto uninfected

cells twelve times. The infectivity of the virus was analyzed by placing the cell-free

medium on duplicate plates. One plate was used to continually passage the virus, and the

rate of spreading was analyzed on the other plate. Infectivity at passages one through

five was monitored by FACS for the accumulation of green cells as described above.

However, by passage three, the percent of green cells plateaued at ~65% for wild-type

150

Figure 3.7 Basic strategy for isolating revertant viruses. To isolate revertant viruses, the medium from infected DF-1 cells was added to duplicate uninfected DF-1 cells. The spread of infection on one plate was monitored over time, while the virus was allowed to spread on the other plate. The medium from the later plate was again added to duplicate plates, and the process was repeated. See text for details.

151 Monitored for infectivity Monitored for infectivity

Medium added to Medium added to uninfected cells uninfected cells

Etc...

Stably infected Passage 1 Passage 2 ...Passage 12 cells Cell-Free Cell-Free Medium Medium

152 and less then 40% for K244R (Figure 3.8A), indicating only a percentage of cells infected were expressing eGFP. By the fifth passage virtually no cells expressed eGFP, even fourteen days postinfection (Figure 3.8B). When infectivity was analyzed by monitoring the accumulation of green cells over time versus the accumulation of RT activity in the medium over time it was clear that although there was virtually no accumulation of green cells between days two and fourteen, the RT activity in the medium during this time increased (Figure 3.8C). Therefore, it appeared that cells were being infected by virions which had lost the nonessential egfp gene. For that reason, the spread of virus through cell culture was monitored by the increase in RT activity in the medium over time. At passages six (Figure 3.9A), eleven (data not shown), and twelve (Figure 3.9B) the rate of the virus spreading through the culture was monitored by this method. At passage six the rate of spread for the E246A virus was virtually identical to the wild type. K244R spread through culture at a rate better than unpassaged virus (data not shown), but not like the

wild type. At passage twelve the spread of the passaged K244R was similar, though not

identical, to the spread of the wild type.

The gain of infectivity seen by the E246A and K244R viruses could be due to contamination by the wild type virus, a reversion back to the original residue (glutamate or lysine, respectively), or a second site suppressor somewhere in the genome. To distinguish between these two possibilities, RNA from viruses at passage twelve was isolated, reverse transcribed, PCR amplified, and sequenced from the 5' U3 through env.

The original mutations were present in both of the E246A and K244R samples and therefore had neither been contaminated by wild-type virus nor undergone a true reversion. No nucleotide changes were detected in any region except in the CA region of

153

Figure 3.8 Deletion of egfp in passaged viruses. (A) At passage 3, the spread of virus through culture was analyzed by FACS at days 9 and 30. (B) Similarly, at passage 5, the spread of virus through culture was analyzed by FACS at days 2, 8, and 14. (C) The

spread of virus through culture at passage 5 was also monitored by the amount of RT

activity present in the medium at days 3, 5, 10, and 17.

154 A 100 Wild Type K244R s l

l 80 e C

n 60 e e r G

40 t n e c

r 20 e P 0 7 9 11 13 15 17 19 21 23 25 27 29 31 Days Postinfection B 100 Wild Type K244R s l

l 80 e C

n 60 e e r G

40 t n e c

r 20 e P 0 2 3 4 5 6 7 8 9 10 11 12 13 14 Days Postinfection C 300

250 0

0 200 1

x 150 m p c 100

50 Wild Type K244R 0 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Days Postinfection

155

Figure 3.9 Isolation of K244R and E246A revertant viruses. The medium from cells stably infect with wild type, K244R, or E246A virus was passaged onto uninfected cells twelve times. On the sixth (A) and twelfth (B) passage the amount of RT released in the medium over time was determined by an exogenous RT assay to measure the rate of spread of the virus through cell culture. (C) The crystal structures of the RSV NTD and

CTD are combined using PBD files 1EM9 and 1EOQ, respectively. The flexible linker is illustrated by the dashed line. The location of lysine 244 and glutamate 246 are marked and connected by an arrow to their respective suppressors.

156 A 180

150

0 120 0 0 1

x 90

m

p 60 c Wild Type 30 K244R E246A 0 3 4 5 6 7 8 9 B Days Postinfection 1600

1400

1200 0

0 1000 0 1

x 800

m

p 600 c 400 Wild Type 200 K244R E246A 0 3 4 5 6 7 8 9 10 11 12 13 Days Postinfection

C E246 K244 N343

R325

C431

157 gag. The E246A gag gene had an A to G change at nucleotide 1407 resulting in a

N343D substitution (data not shown, Figures 3.1 and 3.9C). The K244R gag gene had

two nucleotide changes per genome, a C to T change at nucleotide 1353 and a T to C

change at nucleotide 1671, yielding R325C and C431R substitutions, respectively (data

not shown, Figures 3.1 and 3.9C).

The N343D substitution was cloned into pRS.V8.eGFP and pRS.E246A.eGFP to

determine if it was responsible for the rescue of E246A infectivity. The spread of these

mutants through cell culture was monitored by FACS analysis as above. The N343D

substitution did not affect budding (data not shown), nor did it affect the infectivity of the

virus (Figure 3.10A). Further, the N343D substitution was able to completely restore the

infectivity of the E246A mutant (Figure 3.10A).

Likewise, the single substitutions R325C or C431R or the double substitution

R325C/C431R were cloned into pRS.V8.eGFP and pRS.K244R.eGFP to determine if

these substitutions suppressed the K244R phenotype. The R325C and C431R

substitutions by themselves or in combination with each other did not affect budding

(data not shown). The R325C substitution had a slight effect on the infectivity of the

virus and was able to weakly suppress the K244R phenotype (Figure 3.10C). Likewise,

the C431R substitution had no affect on budding (data not shown), had a mild effect on

infectivity (Figure 3.10B), and was able to slightly suppress the K244R phenotype

(Figure 3.10C). The R325C/C431R double mutant had no affect on budding (data not

shown), but the mutant had a slight infectivity defect (Figure 3.10B). The double

mutation was, however, able to strongly suppress the K244R phenotype similar to the

passaged virus (Figure 3.10C).

158

Figure 3.10 Infectivity of K244R and E246A suppressors. (A) QT6 cells were transfected with pRS.V8.eGFP, pRS.N343D.eGFP, pRS.E246A.eGFP, or

pRS.E246A,N343D.eGFP, and virions were collected in the medium for 24 h. Equal

amounts of virions were added to DF-1 cells for 24 h. The cells were analyzed by FACS

at various times postinfection to determine the percentage of green cells, indicating an

infection. A representative graph of the results of three experiments is shown. (B) QT6

cells were transfected with pRS.V8.eGFP, pRS.R325C.eGFP, pRS.C431R.eGFP, or

pRS.R325C,C431R.eGFP, and virions were collected in the medium for 24 h. Equal

amounts of virions were added to DF-1 cells for 24 h. The cells were analyzed by FACS

at various times postinfection to determine the percentage of green cells, indicating an

infection. A representative graph of the results of three experiments is shown. (C) QT6

cells were transfected with pRS.V8.eGFP, pRS.K244R.eGFP, pRS.K244R,R325C.eGFP,

pRS.K244R,C431R.eGFP, or pRS.K244R,R325C,C431R.eGFP, and virions were

collected in the medium for 24 h. Equal amounts of virions were added to DF-1 cells for

24 h. The cells were analyzed by FACS at various times postinfection to determine the

percentage of green cells, indicating an infection. A representative graph of the results of

three experiments is shown.

159 A 100

Wild Type s

l 80 l N343D e

C E246A

n E246A/ e 60

e N343D r G

t

n 40 e c r e

P 20

0 3 4 5 6 7 8 9 10 11 12 Days Postinfection B 100 s l l 80 e C

n

e 60 Wild Type e

r R325C G

t C431R

n 40

e R325C/ c

r C431R e

P 20

0 3 4 5 6 7 8 9 10 11 12 13 14 C Days Postinfection

100 s l l 80 e C

n

e 60 e

r Wild Type

G K244R

t

n 40 K244R/

e R325C c r K244R/ e

P 20 C431R K244R/ R325C/C431R 0 3 4 5 6 7 8 9 10 11 12 13 14 Days Postinfection

160 DISCUSSION

Reverse transcription affected by K244R substitution. The reduced infectivity of the K244R virions can be attributed to a defect in reverse transcription, as a ~2-fold reduction was seen in the amount of DNA products produced by mutant virions in infected cells compared to the wild type. This defect could be due to every virion being equally impaired in reverse transcription resulting in viral DNA being synthesized at a slower rate. If true, then all virions should give rise to productive infections, given enough time. However, when analyzed by a focus assay, only half the amount of foci were formed from mutant virions compared to the wild type. Together, these two results suggest that half of the mutant virions are defective in initiating reverse transcription and half are able to complete reverse transcription resulting in a productive infection. It is interesting that even though they are produced in the same cells from the same viral transcripts, half of the virions are noninfectious. This observation is possibly due to a threshold effect where the K244R mutants are on the verge of being noninfectious, and only half are able to initiate reverse transcription. The contribution of this residue (and glutamate 246) to reverse transcription is unknown, but given that it is in a sumoylation consensus sequence lysine 244 may be a substrate for sumoylation which may be involved in initiation of reverse transcription.

Suppressors may allow for SUMO-1-independent replication. If this sequence truly is a sumoylation sequence, then creation of an alternative sumoylation site should rescue the infectivity of mutants K244R or E246A. Surprisingly, the revertants had mutations in CA which did not create a new sumoylation consensus sequence. There are three interpretations of how the revertants suppress the K244R and E246A phenotypes.

161 First, though the second-site suppressors do not create new sumoylation consensus sites,

it is possible that the suppressors provide a way for the virus to replicate independent of

sumoylation. It is of interest to determine if the suppressors are allele specific, i.e. if

N343D can suppress K244R or if R325C/C431R can suppress E246A. If the suppressors

are not allele specific, that supports the hypothesis that the suppressors allow for reverse

transcription independent of sumoylation, since it is unlikely that a conformational defect caused by K244R would be compensated for by an E246A suppressor. Given that it is not known whether lysine 244 is sumoylated (or even if CA is sumoylated at another position) this explanation is difficult to test. Further experimentation is required to determine if the wild-type CA is sumoylated, and if so where.

Possible contribution of lysine 244 and glutamate 246 to CA-CA interactions.

A second possibility is that lysine 244 and glutamate 246 contribute to the overall structure of the capsid which is disrupted when mutated; this disruption was than compensated by the suppressors. Indeed, the location of lysine 244 and glutamate 246 are suggestive that they may be involved in CA-CA interactions since both residues are located in the β hairpin region of CA central in the formation of the CA hexamer found in the mature capsid shell (308) and mutations in this region of HIV CA result in a loss in infectivity (486, 487). The suppressors themselves suggest that these residues are involved in CA-CA interactions. Both arginine 325 and asparagine 343 are on the same surface of the NTD (Figure 3.9C) available for interactions with adjacent CA proteins, and cysteine 431 in the CTD is in the dimerization domain and has also been postulated to be involved in forming intermolecular disulfide bonds which stabilize the capsid (63).

The K244R suppressors also give further insight to the intramolecular interactions

162 between the NTD and the CTD. If the structural integrity of the capsid is compromised by K244R, then suppression by a substitution in both the NTD and the CTD (Figure 3.1 and 3.9C) supports the hypothesis that the NTD and CTD interact within the capsid.

These suppressors are reminiscent of the NTD substitutions which suppress mutations in the MHR (in the CTD) that cause a dramatic decrease in the infectivity (44). This study proposed NTD-CTD interactions in the mature capsid are important for replication.

Little detail is known about all of the biochemical intermolecular interactions involved in the formation of the mature capsid, but it appears that interactions between the NTD and

CTD of adjacent CA proteins is required for hexamer formation. Data based on amide hydrogen exchange rates and chemical crosslinking also indicate that in the HIV capsid the NTD of one CA interacts with the CTD of an adjacent CA (238) suggesting that these interactions are important for proper capsid assembly which itself is required replication.

If the CA-CA intermolecular interactions were disrupted in the K244R mutant core, then one might expect it to be less stabile. However, the mutant K244R core was just as stable as the wild type in a detergent sensitivity assay, and the mutant and wild type cores were indistinguishable when examined by EM. These data suggest that either these assays are not sensitive enough to detect any change in core stability or that a simple structural defect may not be the cause of the infectivity defect.

Direct involvement of lysine 244 and glutamate 246 in reverse transcription.

A third possibility is that lysine 244 and glutamate 246 are involved in neither a structural role nor in sumoylation, but are involved in regulation of reverse transcription through an unknown means. Mutations in the RSV MHR that do not affect assembly have defects in reverse transcription leading to the hypothesis that CA may be involved in RSV reverse

163 transcription (59, 83). Indeed, CA has been identified in the reverse transcription complex of another simple retrovirus, MLV (115). If this possibility is the case, then

lysine 244 and glutamate 246 may be interacting with other viral proteins or even with

the RNA and when they are mutated reverse, transcription is inefficient; the second-site

suppressors are able to restore the required interactions, thus allowing efficient reverse

transcription.

Whatever the role of lysine 244 or glutamate 246 in reverse transcription, it is

clear that these residues are important for replication of the virus. Though further

experimentation is required to elucidate how exactly these two residues contribute to

reverse transcription, mutants K244R and E246A (among others previously published)

support a model in which CA has an active role in reverse transcribing the vRNA

genome.

Nuclear import may be affected by K244R. The K244R substitution may affect

more than reverse transcription given that the decrease in the amount of 2-LTR circle

DNA present in newly infected cells was greater than the decrease in second-strand

transfer DNA. Since 2-LTR circle DNA is a marker for nuclear entry, lysine 244 may

also contribute to the transport of the PIC into the nucleus. This observation could also

explain the delay seen in focus formation by mutant K244R; if there is a defect in

targeting the PIC to the nucleus, or to the correct place in the nucleus, then it would take

longer to establish a productive infection and therefore longer to form a focus.

ACKNOWLEDGMENTS

Special thanks to Judith M. White for the anti-TM antibody, Roland Meyers for

164 thin-sectioning and electron microscopy, Terry Ruger and Dan Krissinger of the PSU

Functional Genomics Core Facility for assistance with the Q-PCR, and Nate Sheaffer of the PSU Cell Science/Flow Cytometry Core Facility for assistance with the FACS analyses. This work was supported by National Institutes of Health grant CA47482 to

J.W.W.

165 CHAPTER IV

UTILIZATION OF ESCRT PROTEIN COMPLEXES FOR BUDDING OF ROUS

SARCOMA VIRUS

166 ABSTRACT

Retroviral assembly is mediated by the viral protein Gag. The late (L) domain required for budding has been hypothesized to function in the recruitment of cellular proteins which are needed budding. The L domain of Rous sarcoma virus (RSV) has been shown to interact with a Nedd4-family E3 ubiquitin (Ub) ligase, and five lysines

close to the L domain are required for budding; both observations suggest ubiquitination

of Gag is required for budding. Therefore, it is possible that the ubiquitin moiety on Gag

recruits other cellular proteins that then direct budding similar to ubiquitinated endosomal

proteins, which are directly recognized by the Hrs and ESCRT-I complexes for budding

into endosomes by subsequent recruitment of ESCRT-II, ESCRT-III, AIP1/ALIX, and

Vps4. To determine whether these same endosomal protein complexes are important for

RSV budding, dominant-negative forms of Hrs, ESCRT-I (Tsg101), ESCRT-II (EAP20,

EAP30, EAP45), ESCRT-III (CHMP1A, -2A, -2B, -3, -4A, -4B, -4C, -5, and -6), and

Vps4A were coexpressed with Gag. RSV budding was inhibited by dominant-negative

expression of Tsg101, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, and Vps4A,

similar to what has been published for HIV. RSV Gag also has a potential AIP1/ALIX binding site (181YPSL184) which when mutated resulted in a minor budding defect indicating a potential role for AIP1/ALIX in RSV budding. Therefore, from these data, a model for RSV budding emerges where Gag recruits a Ub ligase to ubiquitinate Gag, enabling subsequent recognition by Tsg101 which, along with the YPSL sequence, recruits AIP1/ALIX to recruit CHMP4 in a complex with CHMP6 which recruits

CHMP3. These ESCRT proteins are released from the site of budding by Vps4A to allow membrane fission, thus releasing the virion from the cell surface.

167 INTRODUCTION

The budding of retroviruses is mediated by the viral protein Gag, which contains all the necessary information required for assembly and release of particles. There are three domains within Gag required for budding: the membrane binding (M) domain targets Gag to and binds the plasma membrane, the interaction (I) domain mediates Gag multimerization necessary for budding, and the late (L) domain is essential for release of the virion from the plasma membrane. There are three types of L domains found in retroviruses: P(T/S)AP, PPxY, and YPxL (124). Since it is unlikely these short stretches of amino acids solely facilitate release of the virus from the plasma membrane, it has been speculated that their primary function is to recruit cellular proteins, which facilitate retroviral budding. The PTAP motif of HIV has been shown to interact with the cellular protein Tsg101 (93, 137, 283, 478), a component of the endosomal sorting complex required for transport (ESCRT)-I (93) involved in sorting cargo proteins into vesicles which bud into the lumen of endosomes forming multivesicular bodies (MVBs). The topology of vesicles budding into the endosome and the budding of retroviruses from the plasma membrane are quite similar. Therefore, Tsg101 may provide a link between the machinery involved in MVB formation and HIV budding.

The current model for the sorting of cargo proteins into budding vesicles begins with ubiquitination of the cargo protein. A ubiquitin (Ub) ligase specifically recognizes a protein destined for the budding vesicle and ubiquitinates it. The monoubiquitin moiety is recognized by the Hrs protein complex (consisting of Hrs, STAM1/STAM2, and

Eps15) which serves to recruit the ESCRT-I complex (Tsg101, Vps28, and Vps37) through specific interactions between Tsg101 and a PSAP motif in Hrs (15, 214, 265,

168 373). Next, ESCRT-II (EAP20, EAP30, and EAP45) is recruited to the endosomal membrane through interactions between Tsg101 and EAP45 and EAP30 (11, 282, 488).

ESCRT-III is then brought to the endosomal membrane through an interaction with

ESCRT-II. Unlike ESCRT-I and -II complexes which form single stable complexes,

ESCRT-III is a dynamic complex made of several smaller complexes. There are six nonhomologous groups of ESCRT-III proteins (CHMP1 [A and B], CHMP2 [A and B],

CHMP3, CHMP4 [A, B, and C], CHMP5, and CHMP6), that form CHMP4/6 and

CHMP2/3 subcomplexes. CHMP4/6 is first recruited to the endosomal membrane and subsequently recruits CHMP2/3 (10, 282, 488). CHMP1 and CHMP5 are not in a known complex and are likely involved in regulation of the other two complexes. The ESCRT-

III complex then interacts with AIP1/ALIX, which appears to be involved in facilitating membrane curvature of the budding vesicle (286). Finally, the AAA-ATPase Vps4 proteins facilitate release of the ESCRT complexes from the endosomal membrane (10,

11, 13, 32, 452, 488). The mechanism of how the ESCRT complexes contribute to vesicle formation and budding is unknown, and it appears they are primarily responsible for sorting and targeting the proteins to the site of budding. Therefore, it is likely there are undiscovered protein complexes involved in the later events of budding, including those which actually facilitate release of the vesicle from the membrane.

Recent reports have shown the dependence of HIV budding on several components of the ESCRT complexes. Specifically, HIV budding is inhibited by overexpression of a Tsg101 dominant-negative or by knocking down Tsg101 expression

(93, 137, 143, 283, 315). Likewise, depletion of Vps37B and Vps37C also inhibits budding. Further, a HIV L domain mutant is rescued by fusion with all or a portion of

169 Vps37B or Vps37C (107, 455). Therefore, HIV release requires ESCRT-I. The role of

ESCRT-II and ESCRT-III proteins during HIV budding was also examined by overexpressing fluorescent-tagged proteins which presumably cause a dominant-negative phenotype (180). (It must be noted that, except for DsRed-CHMP4B, the dominant- negative nature of these proteins was never tested [488].) HIV budding is not affected by the overexpression of any dominant-negative ESCRT-II protein, but several dominant- negative ESCRT-III proteins inhibit budding, specifically CHMP1A, CHMP4A,

CHMP4B, CHMP4C, and CHMP5 (282, 488). HIV Gag also interacts directly with

AIP1/ALIX through LYP and LxxLF motifs in p6. Similarly, EIAV’s YPDL L domain

is linked to the ESCRT complexes through an interaction with AIP1/ALIX (282, 452,

488).

The cellular proteins utilized by Rous sarcoma virus (RSV) to facilitate budding

have not been identified, but the RSV PPPY L domain has been shown to interact with a

Ub ligase (221). Since the RSV L domain is interchangeable with the L domains of

EIAV and HIV (284, 348, 506), it is likely to utilize similar, if not the same, proteins.

Further, RSV Gag also contains a YPSL sequence (similar to the EIAV L domain)

downstream of the L domain. This sequence may allow RSV to interact directly with

AIP1/ALIX. To determine whether the ESCRT complexes are utilized by RSV during

budding, dominant-negative forms of these proteins were overexpressed with RSV Gag.

The YPSL sequence was also mutated to examine the involvement of the potential

interaction with AIP1/ALIX during budding.

170 MATERIALS AND METHODS

Expression vectors. The wild-type RSV gag gene was originally derived from the Prague C proviral vector, pATV-8 (211, 428). To analyze budding, gag alleles were transferred into pGag.GFP (44) or pGag.3h.GFP (Chapter II) which contain a gag-egfp fusion. pGag.3h, which removes egfp, was created by digesting pGag.3h.GFP with ApaI and NotI, followed by incubation with Klenow fragment (to create blunt ends) and

ligation. This manipulation inserts five foreign amino acids (KDRPL) in the place of

eGFP. pHIV.Gag.GFP was a gift from M. D. Resh (169). pTsg101 WT was a gift from

S. C. Sun. pTsg101 5' and pTsg101 3' were gifts from E. O. Freed (93, 143). pYFP-

EAP20, pYFP-EAP30, pYFP-EAP45, pYFP-CHMP1A, pYFP-CHMP2B, pYFP-

CHMP4A, pYFP-CHMP5, and pYFP-CHMP6 were gifts from P. D. Bienaisz (282).

pDsRed-CHMP2A, pCHMP3-YFP, pDsRed-CHMP4B, and pCFP-CHMP4C were gifts

from W. I. Sundquist (488). pVps4(WT).GFP and pVps4(EQ).GFP were gifts from P.

Woodman (32). The control plasmid (pcDNA3.1), pHrs WT, pHrs 1.4, and pHrs 3.8

were gifts from R. C. Piper.

Mutagenesis. Construction of pPPPY-A.GFP (Chapter II), pGag(-) (Chapter II),

and p∆QM1.GFP (419) were previously described. Individual (Y181A, P182A, S183A,

and L184A) or all residues (YPSL-A) within the YPSL sequence in p10 were changed to

alanines by changing the codons to the “GCT” alanine codon by PCR mutagenesis using

pGag.GFP as a template. Construction of pPPPY-A.YPSL-A.GFP was accomplished just

like the construction of pPPPY-A.GFP, except pYPSL-A.GFP was used as the template

during PCR amplification. Mutations were transferred into pGag.GFP using SstI and

FseI, thereby replacing the 5' half of gag. All of the newly constructed RSV gag alleles

171 described here were sequenced to confirm that only the desired mutations were present.

Budding assays. Budding of RSV or HIV Gag was analyzed by transfecting DF-

1 cells (175, 418) using FuGene6 according to the manufacturer’s instructions or by the

CaPO4 method followed by metabolically radiolabeled for 2.5 or 3 h with a L-

[35S]methionine/cysteine mix (50 µCi, >1,000 Ci/mmol) 24 h after transfection. Viral

proteins were immunoprecipitated from detergent-lysed cells and particles using a

polyclonal rabbit serum against whole RSV or using a purified Ig derived from AIDS

patients (anti-HIV Ig, obtained from the NIH AIDS Research and Reference Reagent

Program), as previously described (493). Immunoprecipitates were separated in sodium

dodecyl sulfate (SDS)-12%-polyacrylamide gels, which were subsequently dried and

exposed to Kodak X-Omat AR5 X-ray film. Gag proteins were also quantitated by

PhosphorImager (Molecular Dynamics) analysis. The budding proficiency was

calculated as the amount of Gag in the medium divided by the total amount in the cell

lysate and medium.

Western blot analysis. Transfected cells were washed with PBS and removed

from the plate. Cells were pelleted by centrifugation at 1000 x g for 5 min followed by

resuspension in sample buffer. Whole-cell lysates were sonicated and proteins were

separated in SDS-12%-polyacrylamide gels, and western blot analysis was performed

using anti-HA (Sigma), anti-GFP (Clonetech), or anti-DsRed (Clonetech) antibodies as

the primary antibody and horseradish peroxidase-conjugated anti-rabbit Ig as secondary

antibody with an ECL western analysis kit (Amersham).

172 RESULTS

Hrs not required for budding. It has been hypothesized that ubiquitination of

RSV Gag is required for budding and that the Ub moiety recruits cellular proteins which drive budding (Chapter II, 353). Hrs contains a Ub interaction motif (UIM) in the N terminus, and it is possible that Gag recruits ESCRT complexes through an interaction with Hrs. RSV Gag.3h (a protease deletion mutant) was coexpressed with wild-type

(WT) or forms of mammalian Hrs with a deletion in the N terminus (Hrs 1.4) or the C terminus (Hrs 3.8) that are presumably dominant-negative based on deletions in the yeast homologue, Vps27. Expression of Hrs proteins was confirmed by western blot analysis using an anti-HA antibody (data not shown). Figure 4.1 shows that coexpression of either WT or dominant-negative forms of Hrs with RSV Gag had no effect on budding.

Therefore, it is unlikely RSV Gag utilizes an interaction with Hrs to facilitate budding.

Dominant-negative forms of Tsg101 and RSV budding. The ESCRT-I complex protein Tsg101 can also interact with Ub. To determine whether budding of

RSV Gag involves Tsg101, WT or dominant-negative forms of Tsg101 with deletions in the C terminus (Tsg101 5') or the N terminus (Tsg101 3') were coexpressed with Gag.3h in DF-1 cells following cotransfection at a 1:1 DNA ratio. Expression of Tsg101 proteins was confirmed by western blot analysis using an anti-HA antibody (data not shown).

Budding of RSV Gag was mildly affected when coexpressed with any of the Tsg101 proteins at a 1:1 DNA ratio (Figure 4.2). However, budding was decreased as the ratio of transfected Tsg101 to Gag was increased. WT and dominant-negative Tsg101 expression affected budding to the same degree, similar to what has been reported for HIV, but different from MLV which was inhibited only by Tsg101 3' (143).

173

Figure 4.1 Hrs not required for RSV budding. DF-1 cells cotransfected with pGag.3h

and a control plasmid (pcNDA3.1), pHrs WT, pHrs 1.4, or pHrs 3.8 were metabolically

radiolabeled for 2.5 h 24 h posttransfection. Gag proteins were immunoprecipitated from

the cell lysates and the labeling media using an anti-RSV serum. Gag proteins were

visualized by autoradiography and quantified by PhosphorImager analysis (see Materials

and Methods). Budding of Gag coexpressed with the Hrs proteins was compared to

budding of Gag coexpressed with the control plasmid normalized to 100%. The graph

represents three independent experiments.

174 l 120 o r t n o

C 100 o t e v 80 i t a l e

R 60 e s a e l 40 e R t n 20 e c r e

P 0 l o T .4 .8 tr 3 n W s 1 s o rs r r c H H H

175

Figure 4.2 Tsg101 inhibition of RSV budding. DF-1 cells cotransfected with pGag.3h

(1 µg) and a control plasmid (4 µg) or various amounts of pTsg101 WT (1, 2, or 4 µg),

pTsg101 5' (1, 2, or 4 µg), or pTsg101 3' (1, 2, or 4 µg) were metabolically radiolabeled

for 2.5 h 24 h posttransfection, and Gag proteins were immunoprecipitated, visualized,

and quantified as in the legend to Figure 4.1. Budding of Gag coexpressed with the

Tsg101 proteins was compared to budding of Gag coexpressed with the control plasmid

normalized to 100%. Solid bars represent cells transfected with 1 µg pGag.3h and 4 µg

control or 1 µg pTsg101 WT, pTsg101 5' or pTsg101 3'. Single hatched bars represent

cells transfected with 1 µg pGag.3h and 2 µg pTsg101 WT, pTsg101 5' or pTsg101 3'.

Crosshatched bars represent cells transfected with 1 µg pGag.3h and 4 µg pTsg101 WT,

pTsg101 5' or pTsg101 3'. The graph represents three independent experiments.

176 l o r t n 100

o 1:1 1:2 1:4 C o t 80 e v i t a l

e 60 R e s 40 a e l e R

20 t n e c r

e 0 P l ’ ’ ’ ’ ’ ’ o T 5 3 T 5 3 T 5 3 tr W 1 1 W 1 1 W 1 1 n 1 0 0 1 0 0 1 0 0 co 0 1 1 0 1 1 0 1 1 1 g g 1 g g 1 g g g Ts Ts g Ts Ts g Ts Ts Ts Ts Ts

177 RSV budding mildly affected by dominant-negative ESCRT-II proteins. The proteins which comprise the ESCRT-II protein complex are not believed to be involved in HIV budding given that overexpression of dominant-negative versions had little effect on budding. To determine whether these proteins are involved in RSV budding, DF-1 cells were cotransfected with equal amounts of pGag.3h and pYFP-EAP20, pYFP-

EAP30, or pYFP-EAP45. The budding of HIV was also analyzed as a control. The expression of EAP20, EAP30, and EAP45 proteins was confirmed by western blot analysis using an anti-GFP antibody (data not shown). In DF-1 cells, RSV budding was decreased by only 15-35% (Figure 4.3), whereas HIV budding was inhibited by 35-50% of control. The minor inhibition of HIV budding by the ESCRT-II proteins in DF-1 differs from what had been reported in human cells where no inhibition of budding was detected (282).

Overexpression of ESCRT-III proteins. Overexpression of dominant-negative forms of several ESCRT-III complex proteins inhibit HIV budding. The budding of RSV in the presence of dominant-negative ESCRT-III proteins was determined and compared to HIV budding in the presence of the same proteins. DF-1 cells were cotransfected with equal amounts of pGag.3h or pHIV.Gag.GFP and pYFP-CHMP1A, pDsRed-CHMP2A, pYFP-CHMP2B, pCHMP3-YFP, pYFP-CHMP4A, pDsRed-CHMP4B, pCFP-CHMP4C, pYFP-CHMP5, or pYFP-CHMP6. The expression of the ESCRT proteins was confirmed by western blot analysis using an anti-GFP or anti-DsRed antibodies (data not shown).

Expression of dominant-negative forms of CHMP1A and CHMP5 had little effect on the budding of RSV and HIV (Figure 4.4). Similarly, expressing the dominant-negative forms of the CHMP2 (CHMP2A or CHMP2B) component of the CHMP2/3 complex had

178

Figure 4.3 ESCRT-II proteins not essential in RSV budding. DF-1 cells cotransfected with pGag.3h (black bars) or pHIV Gag.GFP (grey bars) and a control plasmid, pYFP-

EAP20, pYFP-EAP30, or pYFP-EAP45 were metabolically radiolabeled for 3 h 24 h posttransfection, and Gag proteins were immunoprecipitated (with either anti-RSV or anti-HIV serum), visualized, and quantified as in the legend to Figure 4.1. Budding of

Gag coexpressed with the ESCRT-II proteins was compared to budding of Gag coexpressed with the control plasmid normalized to 100%. The graph represents three independent experiments.

179 l

o 120 r t n o

C 100 o t e v i 80 t a l e R

60 e s a e l 40 e R t n

e 20 c r e P 0 control EAP20 EAP30 EAP45

180

Figure 4.4 Several ESCRT-III dominant-negative proteins inhibit RSV and HIV budding. DF-1 cells cotransfected with pGag.3h (black bars) or pHIV Gag.GFP (grey bars) and a control plasmid, pYFP-CHMP1A, pDsRed-CHMP2A, pYFP-CHMP2B, pCHMP3-YFP, pYFP-CHMP4A, pDsRed-CHMP4B, pCFP-CHMP4C, pYFP-CHMP5, or pYFP-CHMP6 were metabolically radiolabeled for 3 h 24 h posttransfection, and Gag proteins were immunoprecipitated (with either anti-RSV or anti-HIV serum), visualized, and quantified as in the legend to Figure 4.1. Budding of Gag coexpressed with the

ESCRT-III proteins was compared to budding of Gag coexpressed with the control plasmid normalized to 100%. The graph represents three independent experiments.

181 l o r t n o 100 C o t e

v 80 i t a l e

R 60 e s a e 40 l e R t n 20 e c r e

P 0 l o A A B 3 A B C 5 6 tr 1 2 2 P 4 4 4 P P n P P P M P P P M M o M M M H M M M H H c H H H C H H H C C C C C C C C

182 very little affect on budding. Expression of the CHMP3 dominant-negative, however, reduced RSV and HIV budding 70%. The CHMP4/6 complex was also examined for importance during budding of RSV and HIV. Whereas expression of a dominant- negative CHMP6 protein reduced budding 50% and 70% for RSV and HIV, respectively, expressing dominant-negative CHMP4 proteins (CHMP4A, CHMP4B, and CHMP4C) reduced budding ~80% for RSV and ~95% for HIV. From these data, it appears that

RSV and HIV budding are most sensitive to dominant-negative forms of CHMP3,

CHMP4, and CHMP6.

Effect of Vps4A dominant-negative on RSV budding. Vps4 is required for the removal of ESCRT complexes from the endosomal membrane and is essential for endosomal budding. To determine whether budding of RSV Gag requires the AAA

ATPase Vps4, WT [Vps4(WT).GFP] or a dominant-negative form of Vps4A with an active site mutation [Vps4(EQ).GFP] was coexpressed with Gag.3h. DF-1 cells were cotransfected with pGag.3h and pVps4(WT).GFP or pVps4(EQ).GFP at a 1:1 DNA ratio.

Expression of the Vps4 proteins was confirmed by western blot analysis using an anti-

GFP antibody (data not shown). Budding of RSV Gag was severely inhibited (95% reduction) by Vps4(EQ) (Figure 4.5), and Vps4(WT) also had a mild effect (40% reduction) on budding.

Potential AIP1/ALIX binding site in RSV Gag. HIV and EIAV Gag proteins have been shown to interact with AIP1/ALIX, and this interaction is required for their budding (282, 452, 488). EIAV facilitates this interaction through its YPDL L domain.

Just downstream of the RSV L domain is a YPxL motif (181YPSL184) similar to EIAV’s L domain (Figure 1.11). To determine whether this sequence is important for RSV budding

183

Figure 4.5 Dominant-negative Vps4A inhibition of RSV budding. DF-1 cells cotransfected with pGag.3h and a control plasmid, pVps4(WT).GFP, or pVps4(EQ).GFP were metabolically radiolabeled for 2.5 h 24 h posttransfection, and Gag proteins were immunoprecipitated, visualized, and quantified as in the legend to Figure 4.1. Budding of Gag coexpressed with the Vps4A proteins was compared to budding of Gag coexpressed with the control plasmid normalized to 100%. The graph represents three independent experiments.

184 l o r t n 100 o C o t 80 e v i t a l

e 60 R e s 40 a e l e R

20 t n e c r

e 0

P l o T Q tr E n W 4 o s4 s c p p V V

185 (possibly by interacting with AIP1/ALIX), this sequence was changed to all alanines

(YPSL-A.GFP, Figure 4.6A). Budding of YPSL-A.GFP was not reduced as much as the

L domain mutant PPPY-A.GFP, but it was reduced 60% (Figure 4.6B). If this sequence is a true YPxL motif then single alanine substitutions at the tyrosine, proline, and leucine positions should affect budding, but a substitution at the serine position should not affect budding. Indeed, budding of S183A.GFP was not greatly reduced, but budding of

Y181A.GFP, P182A.GFP, and L184A.GFP were reduced as much as YPSL-A.GFP

(Figure 4.6B). These data suggest that these residues are important for budding.

However, when these residues are deleted in the mutant ∆QM1 (which deletes the N- terminal half of p10, Figure 4.6A) budding was only slightly reduced (see Discussion).

The defect in budding for the YPSL-A mutant was at the plasma membrane when analyzed by confocal microscopy (data not shown), thus indicating a late block to budding. The budding defects of other mutants defective in the late stages in budding

(T10C and 1-5KR) are rescueable by coexpression with a wild-type Gag protein. To determine if the YPSL-A mutation can be complemented, YPSL-A.GFP was coexpressed with Gag(-) at a 1:1 ratio as determined by PhosphorImager quantitation. The budding of the YPSL-A mutant was rescued to >70% of the wild type (Figure 4.6C) indicating that the wild type can rescue the YPSL-A mutant into particles.

Analysis of the dominant-negative effect of PPPY-A when coexpressed with wild-type Gag. It was previously reported that coexpression of mutant PPPY-A with a wild-type Gag caused an inhibition of wild-type budding. This observation is contrary to the mutant T10C (Figure 4.6A) which was readily rescued by wild-type Gag (Chapter II).

It was hypothesized that the dominant-negative affect could be due to host factors, such

186 Figure 4.6 Potential AIP1/ALIX binding site in RSV Gag involved in budding. (A)

Sites of cleavage, cleavage products, and domains required for budding are indicated.

The PPPY L domain and the putative AIP1/ALIX binding site YPSL are indicated.

PPPY-A substitutes PPPY with four alanines; likewise, YPSL-A substitutes YPSL with four alanines. The double mutant PPPY-A.YPSL-A substitutes both motifs with alanines. T10C deletes the L domain plus a portion of MA and CA resulting in a "late" phenotype. ∆QM1 deletes the last two residues of p2b (but no residues within the L domain) and the first 21 residues in p10, including the YPSL motif. (B) QT6 cells transfected with wild-type or mutant Gag proteins with substitutions in or a deletion of the potential AIP1/ALIX binding site were metabolically radiolabeled for 2.5 h 24 h posttransfection, and Gag proteins were immunoprecipitated, visualized, and quantified as in the legend to Figure 4.1. Budding of mutant Gag proteins was compared to budding of the wild type Gag normalized to 100%. The graph represents three independent experiments. (C) Wild-type and mutant Gag proteins were coexpressed. To differentiate between the two proteins, eGFP was deleted from Gag.GFP [Gag(-)] and was coexpressed with YPSL-A.GFP at a 1:1 ratio. Cotransfected cells were metabolically

radiolabeled, and Gag proteins were immunoprecipitated, visualized, and quantified as in

the legend to Figure 4.1. The percent release of Gag proteins was determined by

normalizing the release of Gag(-) when coexpressed with Gag.GFP to 100% (single solid

bar) and comparing the release of Gag protein in wild type-mutant (paired solid and

hashed bars) coexpressions. Solid bars represent release of wild-type Gag proteins, and

hashed bars represent release of mutant Gag proteins. The results of two independent

experiments are shown.

187 A PPPY YPSL

RSV Gag MA a b p10 CA NC PR

p2 SP 577 701 PPPY YPSL

MA a b p10 CA

AAAA PPPY-A AAAA YPSL-A AAAA AAAA PPPY-A. YPSL-A 121 337 T10C

175 199 DQM1

B C 140

120

e 100 100 p e y s T

a 80 80 d e l l i e

W 60 60

R

o t t

n e e 40 40 v c i r t e a

l 20 20 P e

R 0 0 e 1 A A A A p -A 1 2 3 4 ) ) y M L 8 8 8 8 (- P (- P T Q S 1 1 1 1 g F g F d D P Y P S L a a il Y .G .G G -A G -A W L L S S P P Y Y

188 as AIP1/ALIX, binding to the YPSL sequence, unable to be released when the L domain is absent, thus inhibiting budding of the interacting mutant and wild-type Gag proteins.

The YPSL motif is deleted in T10C, and T10C would be bound by no host factors, thus rescue of T10C by wild-type Gag can occur. To address if the dominant-negative affect is due to the presence of the YPSL sequence in mutant PPPY-A, the double mutant

PPPY-A.YPSL-A.GFP (Figure 4.6A) was constructed. Budding of this mutant was further decreased (2% of the wild type) compared to PPPY-A.GFP (9% of the wild-type)

(Figure 4.7A). The effect of this double mutant on wild-type budding when coexpressed was determined by expressing varying ratios of mutant to wild-type Gag proteins. At a wild-type to mutant ratio of 1:0.5, PPPY-A.YPSL-A.GFP was rescued to 35% and the wild type was only reduced to 75% of the control (Figure 4.7A). However, when the ratio of wild-type to mutant Gag proteins was increased to a 1:1 ratio or 1:3 ratio, mutant budding was rescued to only 25% and wild-type budding was reduced to 45-55%.

Therefore, it appears that the YPSL sequence does not account for the difference in rescue between T10C and PPPY-A.

Given that the PPPY-A.YPSL-A mutant inhibits the wild type Gag in a dose dependent manner, it is possible that the difference in the rescue of T10C and PPPY-A was due solely to differences in expression. Indeed, in the previous study which identified the difference in the rescue of these mutants by wild type Gag, T10C was expressed 80-90% fold less than the coexpressed wild-type while PPPY-A was expressed

3-6 fold more than the coexpressed wild-type (unpublished data). Coexpression of various amounts of T10C with wild-type Gag was attempted, but for an unknown reason

T10C could not be expressed at a level equal to or greater than wild-type Gag. It was

189

Figure 4.7 Dominant-negative effect of PPPY-A on wild-type Gag budding. (A)

Gag(-) and PPPY-A.YPSL-A.GFP proteins were coexpressed at 1:0.5, 1:1, or 1:3 ratios or PPPY-A.YPSL-A.GFP was expressed by itself. Transfected cells were metabolically radiolabeled, and Gag proteins were immunoprecipitated, visualized, and quantified as in the legend to Figure 4.1. The percent release of Gag proteins was determined by

normalizing the release of Gag(-) when coexpressed with Gag.GFP to 100% (single solid

bar) and comparing the release of Gag protein in wild type-mutant (paired solid and

hashed bars) coexpressions. Solid bars represent release of wild-type Gag proteins, and

hashed bars represent release of mutant Gag proteins. The results of two independent

experiments are shown. (B) Gag(-) and PPPY-A.GFP proteins were coexpressed at 1:0.5,

1:1, or 1:3 ratios or PPPY-A.GFP was coexpressed with PPPY-A(-) (not shown).

Transfected cells were metabolically radiolabeled, and Gag proteins were

immunoprecipitated, visualized, and quantified as in the legend to Figure 4.1. The

percent release of Gag proteins was determined by normalizing the release of Gag(-)

when coexpressed with Gag.GFP to 100% (single solid bar) and comparing the release of

Gag protein in wild type-mutant (paired solid and hashed bars) coexpressions. Solid bars represent release of wild-type Gag proteins, and hashed bars represent release of mutant

Gag proteins. The results of two independent experiments are shown.

190 A

e 100 p e y 1:0.5 s T

a 80 d e l l i

e 1:3 W

R 60 1:1

o t t

n e e 40 v c i r t e a l

P 20 e R 0 ) ) ) ) (- P (- P (- P (- P g F g F g F g F a .G a .G a .G a .G G -A G -A G -A G -A L L L L S S S S P P P P .Y .Y .Y .Y -A -A -A -A Y Y Y Y P P P P P P P P P P P P B 120

e 1:0.5 p 100 e y s T

a d e l 80 l i

e 1:1 W

R

60 o t t

n e e 1:3 v c 40 i r t e a l P

e 20 R 0 ) ) ) ) (- P (- P (- P (- P g F g F g F g F a a .G a .G a .G .G G G G G -A -A -A -A Y Y Y Y P P P P P P P P P P P P

191 then determined whether the dominant-negative effect of the PPPY-A mutant is alleviated when less mutant Gag is coexpressed with wild-type Gag. Wild-type to mutant

Gag proteins were expressed at 1:0.5, 1:1, and 1:3 ratios, and similar to the PPPY-

A.YPSL-A double mutant, the PPPY-A mutant was dominant-negative only when expressed at a 1:1 or 1:3 ratio (Figure 4.7B).

DISCUSSION

Utilization of ESCRT proteins during RSV budding. Before discussing the preceding data, it is important to mention the caveats of the experiments performed. The

ESCRT proteins used in these studies were human proteins expressed in avian cells. One obvious criticism is that they may not behave the same as in human cells. For this reason budding of HIV was also examined in DF-1 cells expressing dominant-negative ESCRT-

II and -III proteins. The HIV results obtained in DF-1 cells were similar to those previously published using human cells (282, 488). Therefore, these ESCRT proteins appear to be functioning similarly in chicken and human cells (at least during retroviral budding). Another criticism of the results is that it is unknown if the ESCRT fusion proteins used (except for CHMP4B) yield a dominant-negative phenotype in DF-1 cells

(282, 488). Experiments confirming a dominant-negative phenotype would be especially useful in determining if the proteins which do not block budding are dominant-negative for their normal cellular function. Yet another criticism is that, assuming these proteins are all dominant-negative, it is difficult to determine if the budding defects caused by these proteins are direct or indirect. For example, Tsg101 and dominant-negative forms inhibited RSV budding in a dose-dependent manner. However, it has been observed that

192 overexpression of Tsg101 3' causes formation of aggresome-like structures to which RSV is localized (201) suggesting that the effect of Tsg101 3' may be indirect for RSV. This hypothesis is further supported by the observation that overexpression of Tsg101 3' inhibits MLV budding, whereas Tsg101 5' overexpression and Tsg101 depletion have only minor effects on budding (93) suggesting the effect of Tsg101 3' may be indirect.

However, it may be that all of the proteins tested actually are dominant-negative and specific for budding, but until that can be absolutely proven, the data must be interpreted with caution.

HIV Gag is believed to mimic the Hrs protein to interact with Tsg101, thereby providing a means by which Gag recruits the ESCRT protein complexes that participate in budding (373). Though the PPPY L domain of RSV has not been shown to interact with ESCRT protein complexes, it does interact with a Ub ligase (221). Ubiquitination of cargo proteins is required for their targeting into MVBs and several ESCRT proteins interact with Ub. It is reasonable, then, to hypothesize that RSV gains access to the

ESCRT proteins through ubiquitination of Gag. The involvement of the Ub-binding protein Hrs was analyzed for a role during RSV budding, but overexpression dominant- negative forms of the protein had no effect on budding. Therefore, if RSV utilizes the

MVB sorting pathway, it does so downstream of Hrs.

Tsg101 also contains a Ub binding domain, and overexpression of either WT

Tsg101 or dominant-negative forms inhibited RSV budding in a dose-dependent manner, similar to that published from HIV (93, 143), but contrary to what has been observed for

MLV (93, 137). This observation is puzzling because the RSV and MLV L domains are both PPPY, and the insensitivity of MLV to Tsg101 depletion was attributed to the MLV

193 L domain (137). Therefore, if the requirement for specific ESCRT proteins for budding is determined by the L domain, there seems to be some inconsistency between the RSV and MLV data. It could be that although RSV and MLV share a common L domain motif, they utilize different proteins during budding. This idea is supported by the observation that MLV and RSV cannot be copackaged into the same virion unless they share the same M domain (21), thus suggesting fundamental differences to how these viruses bud.

If indeed all of the ESCRT-II and -III fusion proteins expressed in DF-1 cells are dominant-negative, if their effects are direct, and if there is a direct correlation between a dominant-negative protein inhibition of and its utilization during budding, then it appears that ESCRT-II complexes may have a minor role during RSV and HIV budding, whereas only a subset of ESCRT-III proteins (CHMP4/6 and possibly CHMP2/3) are used by the viruses. Further, it appears that despite having quite different L domains, HIV and RSV vary little in the cellular proteins they utilize for budding.

It seems reasonable that if HIV or RSV utilizes one of the ESCRT-III proteins, than all would be utilized. That is to say, since overexpression of the CHMP4 proteins inhibit budding, the CHMP2 proteins also ought to be involved in budding, given their interaction during MVB formation. Assuming again that the expressed proteins are dominant-negative, it is possible that HIV and RSV exploit only certain ESCRT proteins during budding. If this is indeed true, then another question arises: how can certain

ESCRT-III proteins be absolutely required for RSV and HIV budding, but ESCRT-II have only a minor role? It seems logical that since HIV requires ESCRT-I, it enters the

ESCRT pathway early, but these data suggest that HIV budding does not absolutely

194 depend on the ESCRT-II complex and can mostly occur in the presence of only ESCRT-I and ESCRT-III. It could be that the LYP and LxxLF motifs in HIV p6 that interact with

AIP1/ALIX, can interact with directly Tsg101 and CHMP4, bridging Tsg101 and

CHMP4 with AIP1/ALIX and thereby bypassing the requirement for ESCRT-II.

Potential interaction between RSV Gag and AIP1/ALIX. RSV, too, may interact with AIP1/ALIX through its YPSL sequence, which may subsequently interact with the ESCRT-III proteins, thus bypassing an absolute requirement for ESCRT-II.

When the YPSL sequence was changed to alanines, budding was reduced about 60% indicating a potential role for these residues in budding. Given that mutation of the

YPSL sequence mildly affects budding, it is possible that the YPSL is involved in stabilizing ESCRT complexes through an AIP1/ALIX interaction. Although a structural defect seems rather unlikely since changing the tyrosine, proline, or leucine individually to alanine also reduced budding to an extent similar to YPSL-A, the alanine substitutions could may be affecting the conformation of the nearby L domain, resulting in the L domain being unable to efficiently function. This idea is supported by the observation that deletion of the N-terminal third of p10 (∆QM1) had little effect on budding, suggesting that the YPSL is not important for budding. However, it is possible that the effect of the point mutants is a result of AIP1/ALIX being unable to interact with Gag.

The ∆QM1 deletion then shuttles Gag into an AIP1/ALIX-independent budding pathway or moves up a downstream region that can now interact with AIP1/ALIX. Further experimentation is required to determine whether RSV Gag can directly interact with

AIP1/ALIX, and whether budding of RSV is inhibited by depletion of AIP1/ALIX from the cell or by overexpression of a dominant-negative form of the protein.

195 PPPY-A dominant-negative phenotype due to overexpression. Chapter II described a difference in the ability of two L domain mutants to be rescued by wild-type

Gag; the deletion T10C was readily rescued, but the point mutant PPPY-A was not rescued and actually inhibited budding of the wild type. It was noted that the difference may be due to the binding of previously unrecognized host factors on regions of Gag that are missing in the large deletion mutant; specifically the YPSL sequence is absent in the

T10C deletion and perhaps binding of AIP1/ALIX to the RSV Gag protein results in a dominant-negative phenotype in the PPPY-A mutant. This possibility was tested using the double-mutant PPPY-A.YPSL-A. When the wild type and the mutant were coexpressed at equal levels or when the mutant expression was greater than the wild type, the mutant exhibited a dominant-negative effect similar to the PPPY-A mutant.

However, when less mutant protein was expressed compared to the wild type, the mutant was rescued into particles and the wild type budding was not inhibited. This observation suggested that the dominant-negative phenotype of the PPPY-A mutant was also dose dependent. Indeed, the PPPY-A mutant was not dominant-negative when it was expressed at a level less than the wild type. Though technical problems (see Results)

hindered examination of T10C in this system, the data generated from the previous study

were from cells expressing more PPPY-A Gag than wild-type and more wild-type than

T10C. Therefore, the PPPY-A dominant-negative phenotype is likely due to

overexpression of mutant Gag proteins.

There are a couple explanations as to why L domain mutants inhibit budding of

coexpressed wild-type Gag. First, mutant Gag proteins may be mislocalized to an area of

the plasma membrane which does not allow for budding. When more mutant than wild-

196 type Gag is expressed, the mutant interacts and sequesters the wild type, not allowing budding to occur. Second, mutant and wild-type Gag proteins may be at the proper site of assembly. However, the mutant Gag may not be able to interact with the budding machinery in order to recruit it to the site of budding. Though the wild type can recruit the cellular proteins required for budding, there simply are not enough cellular proteins to mediate budding.

ACKNOWLEDGEMENTS

Special thanks to Carol B. Wilson for technical assistance in mutagenesis and in preparation of Figure 4.7. Thanks also to Marilyn D. Resh, Shao-Cong Sun, Eric O.

Freed, Paul D. Bienaisz, Wes I. Sundquist, Philip Woodman, and Richard C. Piper for providing reagents. This work was supported by National Institutes of Health grant

CA47482 to J.W.W.

197 CHAPTER V

DISCUSSION

198 MECHANISMS OF RSV BUDDING

The mechanisms of how retroviruses bud are relatively unknown despite the recent explosion of data identifying protein networks consisting of cellular proteins and retroviral L domains. The studies in this dissertation attempted to extend our understanding of the mechanism by which RSV buds from the cell.

Gag-driven budding

The process of Gag-driven budding can be separated into early and late events.

The early events consist of Gag trafficking and binding to the plasma membrane and the formation of Gag multimers through Gag-Gag interactions. These events involve the M and I domains of Gag, and when either are altered, budding is inhibited before Gag traffics to the site of budding. The late events consist of submembrane trafficking to the correct site of budding, deformation of the plasma membrane forming a bud, and finally pinching off from the membrane. Little is known about submembrane trafficking, i.e., where exactly on the plasma membrane budding occurs and what are all the regions of

Gag responsible for this trafficking. The site of budding seems to differ between viruses, and this specificity is determined by the M domain. For instance, MLV and RSV Gag cannot be copackaged into the same virion when coexpressed, but can when they share the same M domain (21). This observation is intriguing since despite both viruses having the same L domain (and presumably utilizing the same cellular proteins to facilitate budding), they appear to bud at different areas of the plasma membrane. Curvature of the membrane is thought to be facilitated by Gag, but the exact mechanism is unknown. The pinching off of the virus from the membrane is initiated by the L domain, and when the L

199 domain is mutated virions line the plasma membrane, unable to be released.

The pinching off event of budding can also be subdivided into its own pathway with early and late stages. The earliest stage is likely the recruitment of a cellular factor(s) by the L domain. In the case of RSV, the L domain interacts with a Nedd4- family E3 ligase (221). The function of the E3 ligase during budding is unknown, but

given the cellular function of E3 ligases it is most logically for ubiquitination of Gag.

This idea is supported by the observation that Ub is essential for budding of most

retroviruses (including RSV), and that all genera of Gag proteins have been shown to be ubiquitinated (34, 337, 338, 341, 342, 377, 476).

Lysines in MA required for release from the plasma membrane

Data presented in Chapter II further support a role for ubiquitination of RSV Gag during budding. When lysines close to the L domain were mutated (mutant 1-5KR), budding was decreased. The phenotype of mutant 1-5KR was similar to an L domain mutant in that budding was blocked at the plasma membrane. Lysines around the L domain can provide their function in a positionally-independent manner, thus indicating that the role of the lysine is most likely for a posttranslational modification. Lysines can

provide sites for at least five different types of modification to modulate the functions of

proteins: acetylation, methylation, hydroxylation, phosphorylation, and modification by

Ub or Ub-like molecules. Though these five lysines may be substrates for any of these modifications, the observations that Ub is required for budding and that an E3 ligase binds Gag very close to these lysines suggests that monoubiquitination seems most likely.

If this is indeed the role of these lysines during budding, a pathway (Figure 5.1) towards

200

Figure 5.1 Model for RSV budding. A model of RSV budding based off experiments analyzing budding in the presence of dominant negative proteins normally functioning in endosomal budding is proposed. In this model Gag is ubiquitinated either while trafficking to or at the plasma membrane. The ubiquitinated Gag recruits ESCRT-I through Tsg101 interacting with Ub. Tsg101 and the YPSL sequence in Gag then recruit

AIP1/ALIX which also brings CHMP4/6; CHMP2/3 may have a minor role. Gag is then sorted to the proper site of budding and the ESCRT complexes are released by Vps4. A

Ub isopeptidase then deubiquitinates Gag. The budding particle is released from the plasma membrane by unidentified proteins through an unknown mechanism.

201 Sorting??

Ub Ub Ub Ub 6 Pinching off by 4 P Tsg101 Tsg101 P Ub an unknown M M H H Ub mechanism C Vps28 Vps28 C 3

2 Isopeptidase P Tsg101 Vps37 Vps37 P M M

H VPS4 Vps28 H C C Ub Vps37 AIP1/ALIX Nedd4 Family OR E3 AIP1/ALIX Ub VPS4 3 2 P Tsg101 P 6 4 M M P P H H M Vps28 M C C H H C Vps37 C EAP30 5 1 P EAP20 P M M

2 MVB / H Hrs H 1 C

EAP45 C M A 202 T S release of the virion begins to unfold. In this pathway Gag is ubiquitinated by an E3 ligase recruited by the L domain. This modification may occur as Gag traffics to the plasma membrane or it may occur at the plasma membrane. The ubiquitin moiety (and possibly the E3 ligase) recruits other cellular proteins which then facilitate the final stages of release.

Possible budding independent of Gag ubiquitination

Mutant 1-5KR is thought to have a budding defect due to its inability to be ubiquitinated. However, when the first 10 aa of the v-Src membrane binding domain replaced the first 10 aa of 1-5KR (Myr1.1-5KR; Figure 5.2A), budding was restored to wild-type (Figure 5.2B). The rescue was still sensitive to proteasome inhibitors, thereby indicating that budding still requires free Ub (Figure 5.2C). The rescue may have been

due to the introduction of three new lysines in the v-Src membrane binding domain, but

changing them to arginine (SrcKR) had no affect on budding (Figure 5.2B). It is thought

that the addition of the v-Src membrane binding domain disrupts the rest of the RSV M

domain which has not been replaced (349). Lysines within the M domain, normally

structured and unavailable for ubiquitination, would now be free for ubiquitination.

Therefore, the entire RSV M domain was replaced by the v-Src membrane-binding

domain, and the v-Src lysines were changed to arginine (SrcKR.∆MA1e). In mutant

SrcKR.∆MA1e.1-5KR, the closest lysine available for ubiquitination is lysine 244, which

has been shown in Chapter II to not be involved. Compared to SrcKR.∆MA1e,

SrcKR.∆MA1e.1-5KR had no budding defect (Figure 5.2D). Though it is possible that

the v-Src membrane binding domain causes ubiquitination of Gag at a lysine elsewhere in

203 Figure 5.2 Rescue of 1-5KR by the v-Src membrane binding domain. (A) The N terminal third of Gag is shown with the sites of cleavage, cleavage products, M domain, and the five lysines required for budding indicated. The 10 aa of the v-Src membrane binding domain replaced the first 10 aa of the M domain to create Myr1. The v-Src membrane binding domain contains three lysines which were changed to arginine in the mutant SrcKR. The M domain was replaced by the v-Src membrane binding domain in the construct Myr1.∆MA1e and the three lysines in v-Src were replaced by arginine in

SrcKR.∆MA1e. (B) Transfected QT6 cells were metabolically labeled for 2.5 h 24 h posttransfection. Gag proteins were immunoprecipitated from the cell lysates and the labeling media using an anti-RSV serum. Gag proteins were visualized by autoradiography and quantified by PhosphorImager analysis. Mutants were compared to the wild type which was normalized to 100%. The graph represents six independent experiments. (C) Duplicated plates of transfected QT6 cells were pretreated with 10 µM

MG132 or DMSO (untreated) for 90 min followed by metabolic labeling and MG132 or

DMSO treatment for 2.5 h 24 h posttransfection. Gag proteins were immunoprecipitated,

visualized, and quantified as in (B). Gag budding from treated cells was compared to

budding from the untreated cells which was normalized to 100%. The graph represents

three independent experiments. (D) Transfected QT6 cells were metabolically labeled for

2.5 h 24 h posttransfection. Gag proteins were immunoprecipitated, visualized, and quantified as in (B). 1-5KR mutants were compared to their parental construct (e.g.,

Myr1.∆MA1e.1-5KR was compared to Myr1.∆MA1e) which was normalized to 100%.

The graph represents two independent experiments.

204 A M MA a b p10

K1 K 2K 3 K4 K 5 KK K Myr1 10

RRR SrcKR

KK K 85 Myr1.DMA1e

RRR 85 SrcKR.DMA1e B C d e e p

140 t 140 y a T

e r d t l 120 120 i n U W

100 100 o o t t

e e v v 80 80 i i t t a a l l e e 60 60 R R

e e 40 40 s s a a e e l l 20 20 e e R R

0 0 % % e e R r1 R R R R r1 R yp K y K K K yp K y K T -5 M -5 rc -5 T -5 M -5 ild 1 .1 S .1 ild 1 .1 R r1 W yr1 K W y M rc M S D

t 160 n e r 140 a P

o

t 120

e v i

t 100 a l

e 80 R

e

s 60 a e l

e 40 R

% 20 0 e e 1 R 1 R A K A K M -5 M -5 D .1 D .1 . e . e r1 1 R 1 y A K A M M rc M .D S .D r1 R y K M rc S

205 Gag, this seems unlikely since it is postulated that the L domain is responsible for Gag ubiquitination, not the M domain (34, 451, 476). Taken together these data suggest that the membrane binding domain of v-Src rescues mutant 1-5KR by shuttling it into a different pathway which allows for budding independent of ubiquitination of Gag, yet still dependent upon the presence of free Ub (as indicated by the sensitivity to proteasome inhibitors), perhaps for ubiquitination of a host factor which mediates budding.

Multiple roles of Ub during budding

Though it seems that the main role of Ub during RSV budding is for ubiquitination of Gag and subsequent recruitment of host proteins, Ub may have other functions during budding as implied by mutant Myr1.1-5KR. This possibility may be the case for HIV budding which requires free cellular Ub for budding, but does not depend on ubiquitination of the lysines in p6 known to be ubiquitinated (337). Though other lysines in p1 or NC may become substrates for ubiquitination, ubiquitination of HIV Gag could be a byproduct of there being ubiquitination machinery present at the site of budding to ubiquitinate a host factor such as Tsg101 or another ESCRT protein (7).

ESCRT protein complexes and RSV budding

It is believed that HIV plugs into the MVB protein sorting pathway by mimicking proteins that interact with ESCRT complexes. Specifically, the HIV L domain mimics

Hrs to interact with Tsg101 thus recruiting the ESCRT-I complex (373). It is likely that

RSV also utilizes this same pathway. This possibility is further supported by evidence linking Nedd4-family E3 ligases to MVB formation (309). RSV likely enters the

206 pathway at a different point than HIV since the L domains of these viruses interact with different components of the MVB pathway. Although HIV mimics the Hrs complex, which interacts with the ubiquitinated MVB cargo proteins, RSV may mimic the cargo proteins themselves. Proteins that are sorted into MVBs are typically ubiquitinated, followed by recruitment of the Hrs complex by an interaction between Hrs, STAM1/2, and Ub (29, 212, 304, 312, 366, 380, 388, 436, 456, 475). The Tsg101 subunit of

ESCRT-I also interacts with Ub, as does the yeast homologue of EAP45, Vps26p. If

RSV Gag does indeed mimic the cargo proteins to gain access to the MVB sorting machinery to facilitate budding, then at least one of these Ub-binding proteins ought to be required for RSV budding.

With the disclaimer discussed in Chapter IV in mind, let us assume that the data observed are correct, at least for discussion purposes. These results ultimately suggest that RSV and HIV budding depend on the same ESCRT proteins downstream of Hrs, specifically Tsg101, CHMP3, CHMP4A, CHMP4B, CHMP4C, and CHMP6. These data also imply that RSV and HIV Gag both can interact with Tsg101 by different mechanisms. HIV interacts with Tsg101 through the PTAP L domain, but RSV Gag may interact through Ub (Figure 5.1). Retroviral budding depends little on a functional

ESCRT-II complex, but does require certain ESCRT-III components. Therefore, Gag could have a mechanism by which it bypasses ESCRT-II. The ESCRT-III binding protein AIP1/ALIX may be able to accomplish such a task. This protein has been shown to interact directly with HIV Gag and is required for budding (282, 452, 488). RSV contains a motif (YPSL) which is similar to the YPDL L domain of EAIV which has also been shown to interact with AIP1/ALIX (452, 488). Mutation of the YPSL sequence in

207 RSV results in a 60% reduction in budding indicating a possible role. Indeed,

AIP1/ALIX may be the link between Gag and ESCRT-III in HIV and RSV budding, thus bypassing a requirement of ESCRT-II.

Once the ESCRT proteins perform their function, they are removed from the membrane by Vps4. This ATPase is also required for RSV budding. Again, one must consider the relevance of this result. Since a dominant-negative Vps4 does not allow release of ESCRT proteins from the endosomal membrane, the ESCRT-I or -III proteins involved in budding may not be localized to the proper site of viral assembly, thereby inhibiting budding.

Protein complexes downstream of the ESCRT complexes

The identification of the ESCRT proteins is only the beginning of determining the cellular machinery involved in retroviral budding. It is believed that the role of the

ESCRT complexes in MVB budding is for sorting the cargo proteins to the proper site of budding, not for pinching the bud off the endosomal membrane. Therefore, the role of these complexes during retroviral budding may also be to sort the Gag proteins to the proper site of budding. If sorting is the function of the ESCRT proteins, then they do not directly release the virions from the plasma membrane. Since it is believed that budding occurs at distinct regions of the plasma membrane (e.g., rafts), the ESCRT complexes may be essential for shuttling Gag to these regions, and once there Gag may meet up with the proteins which are actually responsible for the pinching off step. These as-of-yet unidentified proteins are likely to be the same ones involved in MVB formation acting downstream of the ESCRT complexes to complete budding. Clearly the mechanisms of

208 retroviral release are far from being understood.

UTILIZATION OF CA DURING REVERSE TRANSCRIPTION

The true function of CA is unknown. It is easy to assume that its sole purpose is formation of the capsid shell around the RNP, but it appears that CA has a more active role during replication. The role of CA during replication is most evident in the restriction of replication of HIV and MLV by certain cell types (25, 26, 194, 416). The target of this restriction is capsid indicating that CA is quite important for the replication cycle. Mutational analysis of CA also revealed a role during replication, specifically during reverse transcription (4, 59, 83, 389). Though the regions of CA involved in reverse transcription are only partially known, the mechanisms by which CA functions during reverse transcription are completely unknown.

A sumoylation consensus sequence in CA

Chapter III examined the sumoylation consensus sequence in RSV CA.

Mutations resulted in a defect in infectivity. Specifically, about half of the K244R and

E246A mutant-virions were unable to initiate reverse transcription. Taken at face value, these results suggest a role for sumoylation at some point during replication. However, the reduction in infectivity may be due to a structural defect causing the over stabilization or destabilization of the viral core or compromising intermolecular interactions between adjacent CA proteins. Though the stability of the core was tested by pelleting cores though Triton-X 100, and the stability of the mutant core was essentially the same as the wild type, this crude assay may not be able to detect more subtle changes in stability that

209 may have an impact on initiation of reverse transcription.

Determining whether sumoylation of CA is required for replication may prove to be a difficult task. In vitro and in vivo sumoylation assays would be useful to determine whether CA can be sumoylated, but these assays do not address the relevant question if

CA is sumoylated during replication. However, determining whether CA is sumoylated during infection may be very difficult since the amount of CA present in a cell infected by one virion is quite small (~1500 molecules of CA), and the amount of sumoylated CA may be less. Therefore, to determine the importance of SUMO-1 during replication, it would be ideal to produce virions from cells lacking SUMO-1 and/or infect cells lacking

SUMO-1. Unlike Ub, there are no known drugs that reduce the amount of free SUMO-1 in the cell. It was recently reported that expression of the adenoviral protein Gam1 inhibits the expression of SUMO-1 by inactivating the E1 protein complex (35). It would be interesting to produce RSV virions in cells expressing Gam1 to determine whether they are infectious. Since the endogenous RT activity is inhibited in half of the K244R mutant-virions, it is most likely that if sumoylation has a role during infectivity, it is during assembly of the virions. It would also be advantageous to infect cells expressing

Gam1 to determine whether SUMO-1 is important for replication in the infected cell.

Potential role of SUMO-1 during reverse transcription

SUMO-1 is involved in many processes within the cell and is utilized during the

replication of several viruses. Most of the viral and cellular processes involving

sumoylation are in some way related to the nucleus, whether it is nuclear transport,

transcriptional regulation, or subnuclear targeting. Sumoylation is essential for nuclear

210 localization of viral proteins and regulation of transcription herpes viruses (2, 69, 151,

177, 246, 313, 322, 445), papilloma viruses (383, 384), vaccinia virus (399), adenovirus

(250), hantavirus (215, 242, 272), and geminiviruses (66). Given that the exact function of SUMO-1 is unknown, it is difficult to know the exact function of sumoylation during reverse transcription, but there are several possibilities. Sumoylation of CA may be involved in upregulating the activity of RT, and when CA is not sumoylated, reverse transcription simply is not as efficient. Sumoylation of PML is important for facilitating protein-protein interactions that form the subnuclear structures known as PML bodies

(199, 429). Therefore, it is possible that sumoylation of CA is important for facilitating the formation of the RTC. A third possibility is that sumoylation of CA in the infected cell recruits unknown cellular factors which help modulate reverse transcription. A final possibility is that sumoylation of CA targets the RTC to a specific region within the cell where reverse transcription is most efficient. The latter possibilities are the least likely since the defect in reverse transcription is seen in the endogenous RT assay, thereby indicating if sumoylation of CA is important, it is important apart from the infected cell.

Suppressors of mutants K244R and E246A

In light of the difficulties in determining whether CA is sumoylated and if this modification is required for replication, it was advantageous to isolate revertant viruses with the hypothesis that if sumoylation is important, a new sumoylation site would be created. Unexpectedly, a sumoylation consensus sequence was not seen in the revertant viruses. Rather, the K244R mutant was suppressed by the double mutation R325C and

C431R; the E246A mutant was suppressed by a N343D mutation.

211 Out of the three amino acids involved in suppressing the mutants, only the cysteine has been hypothesized to have a function, which is in CA-CA interactions. It is therefore hard to envision how these reversions are suppressing the mutants. One interpretation of how the K244R and E246A are suppressed by their respective reversions is that the overall structure of the capsid may be compromised by the initial mutation and the second-site suppressors compensate for this change in structure. This idea is supported by the location of lysine 244 and glutamate 246 in the β hairpin of CA known to be essential for capsid assembly and replication (121, 486, 487). To test this hypothesis further an in vitro capsid assembly assay would be useful in determining whether hexamer formation is impaired or whether the hexamer is as stable as the wild type.

A second interpretation of the suppressors is that they allow for SUMO-1 independent replication. This interpretation assumes that the function of lysine 244 in replication is for sumoylation of CA involved during reverse transcription. Further experimentation is required to determine if the suppressors shuttle reverse transcription into a sumoylation independent pathway.

A third interpretation is that the CA protein contributes to reverse transcription in some way that involves lysine 244 and glutamate 246 directly. Since it is not known that

CA is directly involved in reverse transcription it is difficult to speculate on how these two residues contribute to the process.

Involvement of CA during reverse transcription

Despite the lack of evidence allowing a solid interpretation of the role of lysine

212 244 and glutamate 246 during replication, it is clear that CA is important for reverse transcription. This importance is not made evident by these mutants alone, but other mutations in CA are required for efficient reverse transcription (4, 59, 83, 389).

Specifically, mutations in the MHR have been characterized as having defects in the early stages of reverse transcription. The mechanisms by which this region is involved in reverse transcription are unknown. It could be that individual CA proteins can interact with the polymerase, thus enhancing its activity or stabilizing the RT complex allowing for efficient reverse transcription. Alternatively, CA may interact with the RNA and, in conjunction with NC, stabilize the interaction between RT and the RNA. Another possibility is that the capsid shell somehow participates in initiating reverse transcription, and when the three-dimensional structure of the capsid is altered even slightly, initiation of reverse transcription is inhibited. Given our ignorance of the intermolecular interactions between the components of the RNP it is easy to speculate on the various roles of CA during reverse transcription, but difficult to make a solid hypothesis as to its function.

213 REFERENCES

1. Accola, M. A., B. Strack, and H. G. Gottlinger. 2000. Efficient Particle Production by Minimal Gag Constructs Which Retain the Carboxy-Terminal Domain of Human Immunodeficiency Virus Type 1 Capsid-p2 and a Late Assembly Domain. J. Virol. 74:5395-5402.

2. Adamson, A. L. and S. Kenney. 2001. Epstein-Barr Virus Immediate-Early Protein BZLF1 Is SUMO-1 Modified and Disrupts Promyelocytic Leukemia Bodies. J. Virol. 75:2388-2399.

3. Alam, S. L., J. Sun, M. Payne, B. D. Welch, B. K. Blake, D. R. Davis, H. H. Meyer, S. D. Emr, and W. I. Sundquist. 2004. Ubiquitin interactions of NZF zinc fingers. EMBO J. 23:1411-1421.

4. Alin, K. and S. P. Goff. 1996. Mutational analysis of interactions between the Gag precursor proteins of murine leukemia viruses. Virology 216:418-424.

5. Aloia, R. C., H. Tian, and F. C. Jensen. 1993. Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes. Proc. Natl. Acad. Sci. 90:5181-5185.

6. Amarasinghe, G. K., J. Zhou, M. Miskimon, K. J. Chancellor, J. A. McDonald, A. G. Matthews, R. R. Miller, M. D. Rouse, and M. F. Summers. 2001. Stem-loop SL4 of the HIV-1 psi RNA packaging signal exhibits weak affinity for the nucleocapsid protein. structural studies and implications for genome recognition. J. Mol. Biol. 314:961-970.

7. Amit, I., L. Yakir, M. Katz, Y. Zwang, M. D. Marmor, A. Citri, K. Shtiegman, I. Alroy, S. Tuvia, Y. Reiss, E. Roubini, M. Cohen, R. Wides, E. Bacharach, U. Schubert, and Y. Yarden. 2004. Tal, a Tsg101-specific E3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding. Genes Dev. 18:1737-1752.

8. Arrigo, S. and K. Beemon. 1988. Regulation of Rous sarcoma virus RNA splicing and stability. Mol. Cell Biol. 8:4858-4867.

9. Asao, H., Y. Sasaki, T. Arita, N. Tanaka, K. Endo, H. Kasai, T. Takeshita, Y. Endo, T. Fujita, and K. Sugamura. 1997. Hrs Is Associated with STAM, a Signal-transducing Adaptor Molecule. Its suppressive effect on cytokine- induced cell growth. J. Biol. Chem. 272:32785-32791.

10. Babst, M., D. J. Katzmann, E. J. Estepa-Sabal, T. Meerloo, and S. D. Emr. 2002. ESCRT-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell 3:271-282.

11. Babst, M., D. J. Katzmann, W. B. Snyder, B. Wendland, and S. D. Emr. 2002. Endosome-associated complex, ESCRT-II, recruits transport machinery

214 for protein sorting at the multivesicular body. Dev. Cell 3:283-289.

12. Babst, M., T. K. Sato, L. M. Banta, and S. D. Emr. 1997. Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p. EMBO J 16:1820-1831.

13. Babst, M., B. Wendland, E. J. Estepa, and S. D. Emr. 1998. The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J 17:2982-2993.

14. Bache, K. G., A. Brech, A. Mehlum, and H. Stenmark. 2003. Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. The J. Cell Biol. 162:435-442.

15. Bache, K. G., C. Raiborg, A. Mehlum, and H. Stenmark. 2003. STAM and Hrs Are Subunits of a Multivalent Ubiquitin-binding Complex on Early Endosomes. J. Biol. Chem. 278:12513-12521.

16. Banks, J. D. and M. L. Linial. 2000. Secondary structure analysis of a minimal avian leukosis-sarcoma virus packaging signal. J. Virol. 74:456-464.

17. Barklis, E., J. McDermott, S. Wilkens, S. Fuller, and D. Thompson. 1998. Organization of HIV-1 Capsid Proteins on a Lipid Monolayer. J. Biol. Chem. 273:7177-7180.

18. Battula, N. and L. A. Loeb. 1976. On the fidelity of DNA replication. Lack of exodeoxyribonuclease activity and error-correcting function in avian myeloblastosis virus DNA polymerase. J. Biol. Chem. 251:982-986.

19. Beemon, K., P. Duesberg, and P. Vogt. 1974. Evidence for crossing-over between avian tumor viruses based on analysis of viral RNAs. Proc. Natl. Acad. Sci. 71:4254-4258.

20. Bennett, R. P., T. D. Nelle, and J. W. Wills. 1993. Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus Gag proteins. J. Virol. 67:6487-6498.

21. Bennett, R. P. and J. W. Wills. 1999. Conditions for copackaging Rous sarcoma virus and murine leukemia virus Gag proteins during retroviral budding. J. Virol. 73:2045-2051.

22. Berkhout, B., A. T. Das, and J. L. van Wamel. 1998. The native structure of the human immunodeficiency virus type 1 RNA genome is required for the first strand transfer of reverse transcription. Virology 249:211-218.

23. Berkhout, B., N. L. Vastenhouw, B. I. Klasens, and H. Huthoff. 2001. Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription. RNA 7:1097-1114.

215 24. Berthet-Colominas, C., S. Monaco, A. Novelli, G. Sibai, F. Mallet, and S. Cusack. 1999. Head-to-tail dimers and interdomain flexibility revealed by the crystal structure of HIV-1 capsid protein (p24) complexed with a monoclonal antibody Fab. EMBO J. 18:1124-1136.

25. Berthoux, L., S. Sebastian, E. Sokolskaja, and J. Luban. 2004. Lv1 Inhibition of Human Immunodeficiency Virus Type 1 Is Counteracted by Factors That Stimulate Synthesis or Nuclear Translocation of Viral cDNA. J. Virol. 78:11739-11750.

26. Bieniasz, P. D. 2003. Restriction factors: a defense against retroviral infection. Trends in Microbiology 11:286-291.

27. Bieth, E., C. Gabus, and J. L. Darlix. 1990. A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro. Nucleic Acids Res. 18:119-127.

28. Billeter, M. A., J. T. Parsons, and J. M. Coffin. 1974. The nucleotide sequence complexity of avian tumor virus RNA. Proc. Natl. Acad. Sci. 71:3560-3564.

29. Bilodeau, P. S., J. L. Urbanowski, S. C. Winistorfer, and R. C. Piper. 2002. The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting. Nat. Cell Biol. 4:534-539.

30. Bishop, J. M., W. E. Levinson, N. Quintrell, D. Sullivan, L. Fanshier, and J. Jackson. 1970. The low molecular weight RNAs of Rous sarcoma virus. I. The 4 S RNA. Virology 42:182-195.

31. Bishop, N., A. Horman, and P. Woodman. 2002. Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein- ubiquitin conjugates. J. Cell Biol. 157:91-102.

32. Bishop, N. and P. Woodman. 2000. ATPase-defective Mammalian VPS4 Localizes to Aberrant Endosomes and Impairs Cholesterol Trafficking. Mol. Biol. Cell 11:227-239.

33. Bishop, N. and P. Woodman. 2001. TSG101/Mammalian VPS23 and Mammalian VPS28 Interact Directly and Are Recruited to VPS4-induced Endosomes. J. Biol. Chem. 276:11735-11742.

34. Blot, V., F. Perugi, B. Gay, M. C. Prevost, L. Briant, F. Tangy, H. Abriel, O. Staub, M. C. Dokhelar, and C. Pique. 2004. Nedd4.1-mediated ubiquitination and subsequent recruitment of Tsg101 ensure HTLV-1 Gag trafficking towards the multivesicular body pathway prior to virus budding. J. Cell Sci. 117:2357-2367.

35. Boggio, R., R. Colombo, R. T. Hay, G. F. Draetta, and S. Chiocca. 2004. A

216 mechanism for inhibiting the SUMO pathway. Mol. Cell 19;16:549-561.

36. Bolognesi, D. P., R. Luftig, and J. H. Shaper. 1973. Localization of RNA tumor virus polypeptides. I. Isolation of further virus substructures. Virology 56:549-564.

37. Boone, L. R. and A. Skalka. 1980. Two species of full-length cDNA are synthesized in high yield by melittin-treated avian retrovirus particles. Proc. Natl. Acad. Sci. 77:847-851.

38. Boone, L. R. and A. M. Skalka. 1981. Viral DNA synthesis in vitro by avian retrovirus particles permeabilized with melittin. J. Virol. 37:109-116.

39. Bouamr, F., J. A. Melillo, M. Q. Wang, K. Nagashima, S. M. de Los, A. Rein, and S. P. Goff. 2003. PPPYEPTAP motif is the late domain of human T- cell leukemia virus type 1 Gag and mediates its functional interaction with cellular proteins Nedd4 and Tsg101. J. Virol. 77:11882-11895.

40. Bouyac-Bertoia, M., J. D. Dvorin, R. A. Fouchier, Y. Jenkins, B. E. Meyer, L. I. Wu, M. Emerman, and M. H. Malim. 2001. HIV-1 infection requires a functional integrase NLS. Mol. Cell 7:1025-1035.

41. Bowerman, B., P. O. Brown, J. M. Bishop, and H. E. Varmus. 1989. A nucleoprotein complex mediates the integration of retroviral DNA. Genes and Dev. 3:469-478.

42. Bowzard, J. B., R. P. Bennett, N. K. Krishna, S. M. Ernst, A. Rein, and J. W. Wills. 1998. Importance of basic residues in the nucleocapsid sequence for retrovirus Gag assembly and complementation rescue. J. Virol. 72:9034-9044.

43. Bowzard, J. B., R. J. Visalli, C. B. Wilson, J. S. Loomis, E. M. Callahan, R. J. Courtney, and J. W. Wills. 2000. Membrane targeting properties of a herpesvirus tegument protein-retrovirus Gag chimera. J. Virol. 74:8692-8699.

44. Bowzard, J. B., J. W. Wills, and R. C. Craven. 2001. Second-site suppressors of Rous sarcoma virus CA mutations: evidence for interdomain interactions. J. Virol. 75:6850-6856.

45. Braaten, D., E. K. Franke, and J. Luban. 1996. Cyclophilin A is required for an early step in the life cycle of human immunodeficiency virus type 1 before the initiation of reverse transcription. J. Virol. 70:3551-3560.

46. Briggs, J. A., M. N. Simon, I. Gross, H. G. Krausslich, S. D. Fuller, V. M. Vogt, and M. C. Johnson. 2004. The stoichiometry of Gag protein in HIV-1. Nat. Struct. Mol. Biol. 11:672-675.

47. Briggs, J. A., B. E. Watson, B. E. Gowen, and S. D. Fuller. 2004. Cryoelectron microscopy of mouse mammary tumor virus. J. Virol. 78:2606-

217 2608.

48. Briggs, J. A., T. Wilk, R. Welker, H. G. Krausslich, and S. D. Fuller. 2003. Structural organization of authentic, mature HIV-1 virions and cores. EMBO J. 22:1707-1715.

49. Brown, D. A. and E. London. 1998. Structure and origin of ordered lipid domains in biological membranes. J. Membr. Biol. 164:103-114.

50. Brown,P.O. 1997. Integration, p. 161-204. In J.M.Coffin, S.H.Hughes, and H.E.Varmus (eds.), Retroviruses. Cold Spring Harbor Laboratory Press, Plainview, NY.

51. Bryant, M. and L. Ratner. 1990. Myristoylation-dependent replication and assembly of human immunodeficiency virus 1. Proc. Natl. Acad. Sci. 87:523- 527.

52. Buchschacher, G. L., Jr., L. Yu, F. Murai, T. Friedmann, and A. Miyanohara. 1999. Association of Murine Leukemia Virus Pol with Virions, Independent of Gag-Pol Expression. J. Virol. 73:9632-9637.

53. Bukrinskaya, A., B. Brichacek, A. Mann, and M. Stevenson. 1998. Establishment of a Functional Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcription Complex Involves the Cytoskeleton. J. Exp. Med. 188:2113-2125.

54. Bukrinsky, M. I., S. Haggerty, M. P. Dempsey, N. Sharova, A. Adzhubel, L. Spitz, P. Lewis, D. Goldfarb, M. Emerman, and M. Stevenson. 1993. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365:666-669.

55. Bukrinsky, M. I., N. Sharova, T. L. McDonald, T. Pushkarskaya, W. G. Tarpley, and M. Stevenson. 1993. Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infeciton. Proc. Natl. Acad. Sci. 90:6125- 6129.

56. Burniston, M. T., A. Cimarelli, J. Colgan, S. P. Curtis, and J. Luban. 1999. Human immunodeficiency virus type 1 Gag polyprotein multimerization requires the nucleocapsid domain and RNA and is promoted by the capsid- dimer interface and the basic region of matrix protein. J. Virol. 73:8527-8540.

57. Burstein, H., D. Bizub, and A. M. Skalka. 1991. Assembly and processing of avian retroviral Gag polyproteins containing linked protease dimers. J. Virol. 65:6165-6172.

58. Caffrey, M., M. Cai, J. Kaufman, S. J. Stahl, P. T. Wingfield, D. G. Covell, A. M. Gronenborn, and G. M. Clore. 1998. Three-dimensional solution

218 structure of the 44 kDa ectodomain of SIV gp41. EMBO J 17:4572-4584.

59. Cairns, T. M. and R. C. Craven. 2001. Viral DNA synthesis defects in assembly-competent Rous sarcoma virus CA mutants. J. Virol. 75:242-250.

60. Callahan, E. M. and J. W. Wills. 2000. Repositioning basic residues in the M domain of the Rous sarcoma virus Gag protein. J. Virol. 74:11222-11229.

61. Callahan, E. M. and J. W. Wills. 2003. Link between genome packaging and rate of budding for Rous sarcoma virus. J. Virol. 77:9388-9398.

62. Campbell, S. and V. M. Vogt. 1995. Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1. J. Virol. 69:6487-6497.

63. Campos-Olivas, R., J. L. Newman, Y. Ndassa, and M. F. Summers. 1999. 1H, 13C and 15N chemical shift assignments of the capsid protein from Rous sarcoma virus. J. Biomol. NMR 15:267-268.

64. Campos-Olivas, R., J. L. Newman, and M. F. Summers. 2000. Solution structure and dynamics of the Rous sarcoma virus capsid protein and comparison with capsid proteins of other retroviruses. J. Mol. Biol. 296:633- 649.

65. Carthew, R. W. and C. Xu. 2000. Endocytosis: why not wait to deubiquitinate? Curr. Biol. 10:R532-R534

66. Castillo, A. G., L. J. Kong, L. Hanley-Bowdoin, and E. R. Bejarano. 2004. Interaction between a Geminivirus Replication Protein and the Plant Sumoylation System. J. Virol. 78:2758-2769.

67. Cen, S., M. Niu, J. Saadatmand, F. Guo, Y. Huang, G. J. Nabel, and L. Kleiman. 2004. Incorporation of Pol into Human Immunodeficiency Virus Type 1 Gag Virus-Like Particles Occurs Independently of the Upstream Gag Domain in Gag-Pol. J. Virol. 78:1042-1049.

68. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89:263-273.

69. Chang, L. K., Y. H. Lee, T. S. Cheng, Y. R. Hong, P. J. Lu, J. J. Wang, W. H. Wang, C. W. Kuo, S. S. L. Li, and S. T. Liu. 2004. Post-translational Modification of Rta of Epstein-Barr Virus by SUMO-1. J. Biol. Chem. 279:38803-38812.

70. Chen, C., O. A. Weisz, D. B. Stolz, S. C. Watkins, and R. C. Montelaro. 2004. Differential Effects of Actin Cytoskeleton Dynamics on Equine Infectious Anemia Virus Particle Production. J. Virol. 78:882-891.

219 71. Cheung, O.-S., R. E. Smith, M. P. Stone, and W. K. Joklik. 1972. Comparison of immature (rapid harvest) and mature Rous sarcoma virus particles. Virology 50:851-864.

72. Chiu, H. C., S. Y. Yao, and C. T. Wang. 2002. Coding Sequences Upstream of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Domain in Gag-Pol Are Not Essential for Incorporation of the Pr160gag-pol into Virus Particles. J. Virol. 76:3221-3231.

73. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J. Sodroski. 1996. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135-1148.

74. Christensen, A. M., M. A. Massiah, B. G. Turner, W. I. Sundquist, and M. F. Summers. 1996. Three-dimensional structure of the HTLV-II matrix protein and comparative analysis of matrix proteins from the different classes of pathogenic human retroviruses. J. Mol. Biol. 264:1117-1131.

75. Ciechanover, A., H. Heller, S. Elias, A. L. Haas, and A. Hershko. 1980. ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation. Proc. Natl. Acad. Sci. 77:1365-1368.

76. Cimarelli, A. and J. Luban. 2000. Human immunodeficiency virus type 1 virion density is not determined by nucleocapsid basic residues. J. Virol. 74:6734-6740.

77. Cimarelli, A., S. Sandin, S. Hoglund, and J. Luban. 2000. Basic residues in human immunodeficiency virus type 1 nucleocapsid promote virion assembly via interaction with RNA. J. Virol. 74:3046-3057.

78. Cornilescu, C. C., F. Bouamr, X. Yao, C. Carter, and N. Tjandra. 2001. Structural analysis of the N-terminal domain of the human T-cell leukemia virus capsid protein. J. Mol. Biol. 306:783-797.

79. Cosson, P. 1996. Direct interaction between the envelope and matrix proteins of HIV-1. EMBO J. 15:5783-5788.

80. Craven, R. C., R. P. Bennett, and J. W. Wills. 1991. Role of the avian retroviral protease in the activation of reverse transcriptase during virion assembly. J. Virol. 65:6205-6217.

81. Craven, R. C., R. N. Harty, J. Paragas, P. Palese, and J. W. Wills. 1999. Late domain function identified in the vesicular stomatitis virus M protein by use of rhabdovirus-retrovirus chimeras. J. Virol. 73:3359-3365.

82. Craven, R. C., A. E. Leure-duPree, C. R. Erdie, C. B. Wilson, and J. W. Wills. 1993. Necessity of the spacer peptide between CA and NC in the Rous

220 sarcoma virus Gag protein. J. Virol. 67:6246-6252.

83. Craven, R. C., A. E. Leure-duPree, R. A. Weldon, and J. W. Wills. 1995. Genetic analysis of the major homology region of the Rous sarcoma virus Gag protein. J. Virol. 69:4213-4227.

84. Crawford, S. and S. P. Goff. 1985. A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the Gag and Pol polyproteins. J. Virol. 53:899-907.

85. D'Souza, V. and M. F. Summers. 2004. Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus. Nature 431:586-590.

86. Damsky, C. H., J. B. Sheffield, G. P. Tuszynski, and L. Warren. 1977. Is there a role for actin in virus budding? J. Cell Biol. 75:593-605.

87. Darlix, J. L., C. Gabus, M. T. Nugeyre, F. Clavel, and F. Barre-Sinoussi. 1990. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1. J. Mol. Biol. 216:689-699.

88. Davis, N. L. and R. R. Rueckert. 1972. Properties of a ribonucleoprotein particle isolated from Nonidet P-40-treated Rous sarcoma virus. J. Virol. 10:1010-1020.

89. Dawson, L. and X. F. Yu. 1998. The Role of Nucleocapsid of HIV-1 in Virus Assembly. Virology 251:141-157.

90. De Guzman, R. N., Z. R. Wu, C. C. Stalling, L. Pappalardo, P. N. Borer, and M. F. Summers. 1998. Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. Science 279:384-388.

91. Deffaud, C. and J. L. Darlix. 2000. Rous sarcoma virus translation revisited: characterization of an internal ribosome entry segment in the 5' leader of the genomic RNA. J. Virol. 74:11581-11588.

92. Demirov, D. G., A. Ono, J. M. Orenstein, and E. O. Freed. 2002. Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function. Proc. Natl. Acad. Sci. 99:955-960.

93. Demirov, D. G., J. M. Orenstein, and E. O. Freed. 2002. The late domain of human immunodeficiency virus type 1 p6 promotes virus release in a cell type- dependent manner. J. Virol. 76:105-117.

94. Desterro, J. M. P., J. Thomson, and R. T. Hay. 1997. Ubch9 conjugates SUMO but not ubiquitin. FEBS Letters 417:297-300.

95. Diaz-Griffero, F., S. A. Hoschander, and J. Brojatsch. 2002. Endocytosis Is a Critical Step in Entry of Subgroup B Avian Leukosis Viruses. J. Virol.

221 76:12866-12876.

96. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M. Parmentier, R. G. Collman, and R. W. Doms. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149-1158.

97. Dorfman, T., A. Bukovsky, A. Ohagen, S. Hoglund, and H. G. Gottlinger. 1994. Functional domains of the capsid protein of human immunodeficiency virus type 1. J. Virol. 68:8180-8187.

98. Dorfman, T., F. Mammano, W. A. Haseltine, and H. G. Gottlinger. 1994. Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein. J. Virol. 68:1689-1696.

99. Dorman, N. and A. Lever. 2000. Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus. J. Virol. 74:11413-11417.

100. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A. Paxton. 1996. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 20;381:667-673.

101. Duesberg, P. H. 1968. Physical properties of Rous Sarcoma Virus RNA. Proc. Natl. Acad. Sci. 60:1511-1518.

102. Dunn, R. and L. Hicke. 2001. Multiple Roles for Rsp5p-dependent Ubiquitination at the Internalization Step of Endocytosis. J. Biol. Chem. 276:25974-25981.

103. Dupraz, P. and P. F. Spahr. 1992. Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging. J. Virol. 66:4662-4670.

104. Dupre, S., C. Volland, and R. Haguenauer-Tsapis. 2001. Membrane transport: ubiquitylation in endosomal sorting. Current Biology 11:R932-R934

105. Dworetzky, S. I., R. E. Lanford, and C. M. Feldherr. 1988. The effects of variations in the number and sequence of targeting signals on nuclear uptake. J. Cell Biol. 107:1279-1287.

106. Earp, L. J., S. E. Delos, R. C. Netter, P. Bates, and J. M. White. 2003. The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH. J. Virol. 77:3058-3066.

107. Eastman, S. W., J. Martin-Serrano, W. Chung, T. Zang, and P. D. Bieniasz. 2004. Identification of human VPS37C, a component of ESCRT-I important for viral budding. J. Biol. Chem. M410384200

222 108. Edbauer, C. A. and R. B. Naso. 1983. Cytoskeleton-associated Pr65gag and retrovirus assembly. Virology 130:415-426.

109. Edbauer, C. A. and R. B. Naso. 1984. Cytoskeleton-associated Pr65gag and assembly of retrovirus temperature-sensitive mutants in chronically infected cells. Virology 134:389-397.

110. Einfeld, D. and E. Hunter. 1988. Oligomeric structure of a prototype retrovirus glycoprotein. Proc. Natl. Acad. Sci. 85:8688-8692.

111. Erdie, C. R. and J. W. Wills. 1990. Myristylation of Rous sarcoma virus Gag protein does not prevent replication in avian cells. J. Virol. 64:5204-5208.

112. Erikson, E. and R. L. Erikson. 1970. Isolation of amino acid acceptor RNA from purified avian myeloblastosis virus. J Mol. Biol. 52:387-390.

113. Farnet, C. M. and W. A. Haseltine. 1991. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J. Virol. 65:1910-1915.

114. Fassati, A., D. Gorlich, I. Harrison, L. Zaytseva, and J. M. Mingot. 2003. Nuclear import of HIV-1 intracellular reverse transcription complexes is mediated by importin 7. EMBO J 22:3675-3685.

115. Fassati, A. and S. P. Goff. 1999. Characterization of Intracellular Reverse Transcription Complexes of Moloney Murine Leukemia Virus. J. Virol. 73:8919-8925.

116. Fassati, A. and S. P. Goff. 2001. Characterization of Intracellular Reverse Transcription Complexes of Human Immunodeficiency Virus Type 1. J. Virol. 75:3626-3635.

117. Felsenstein, K. M. and S. P. Goff. 1988. Expression of the Gag-Pol fusion protein of Moloney murine leukemia virus without Gag protein does not induce virion formation or proteolytic processing. J. Virol. 62:2179-2182.

118. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein- coupled receptor. Science 272:872-877.

119. Feng, Y. X., T. D. Copeland, L. E. Henderson, R. J. Gorelick, W. J. Bosche, J. G. Levin, and A. Rein. 1996. HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro. Proc. Natl. Acad. Sci. 93:7577-7581.

120. Feng, Y. X., S. Campbell, D. Harvin, B. Ehresmann, C. Ehresmann, and A. Rein. 1999. The Human Immunodeficiency Virus Type 1 Gag Polyprotein Has Nucleic Acid Chaperone Activity: Possible Role in Dimerization of Genomic

223 RNA and Placement of tRNA on the Primer Binding Site. J. Virol. 73:4251- 4256.

121. Forshey, B. M., U. von Schwedler, W. I. Sundquist, and C. Aiken. 2002. Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J. Virol. 76:5667-5677.

122. Fouchier, R. A., B. E. Meyer, J. H. Simon, U. Fischer, A. V. Albright, F. Gonzalez-Scarano, and M. H. Malim. 1998. Interaction of the Human Immunodeficiency Virus Type 1 Vpr Protein with the Nuclear Pore Complex. J. Virol. 72:6004-6013.

123. Franke, E. K., H. E. H. Yuan, K. L. Bossolt, S. P. Goff, and J. Luban. 1994. Specificity and sequence requirements for interactions between various retroviral Gag proteins. J. Virol. 68:5300-5305.

124. Freed, E. O. 2002. Viral late domains. J. Virol. 76:4679-4687.

125. Freed, E. O. and M. A. Martin. 1995. Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix. J. Virol. 69:1984-1989.

126. Freed, E. O. and M. A. Martin. 1996. Domains of the human immunodeficiency virus type 1 matrix and gp41 cytoplasmic tail required for envelope incorporation into virions. J. Virol. 70:341-351.

127. Fu, W., R. J. Gorelick, and A. Rein. 1994. Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease- defective virions. J. Virol. 68:5013-5018.

128. Fu, W. and A. Rein. 1993. Maturation of dimeric viral RNA of Moloney murine leukemia virus. J. Virol. 67:5443-5449.

129. Fujita, H., M. Yamanaka, K. Imamura, Y. Tanaka, A. Nara, T. Yoshimori, S. Yokota, and M. Himeno. 2003. A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class E vps phenotype in mammalian cells. J. Cell Sci. 116:401- 414.

130. Fuller, S. D., T. Wilk, B. E. Gowen, H. G. Krausslich, and V. M. Vogt. 1997. Cryo-electron microscopy reveals ordered domains in the immature HIV- 1 particle. Current Biology 7:729-738.

131. Futter, C. E., A. Pearse, L. J. Hewlett, and C. R. Hopkins. 1996. Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J. Cell Biol. 132:1011-1023.

224 132. Gamble, T. R., S. Yoo, F. F. Vajdos, U. K. von Schwedler, D. K. Worthylake, H. Wang, J. P. McCutcheon, W. I. Sunquist, and C. P. Hill. 1997. Structure of the carboxy-terminal dimerization domain of the HIV- 1 capsid protein. Science 278:849-853.

133. Ganser-Pornillos, B. K., U. K. von Schwedler, K. M. Stray, C. Aiken, and W. I. Sundquist. 2004. Assembly properties of the human immunodeficiency virus type 1 CA protein. J. Virol. 78:2545-2552.

134. Garbitt, R. A., J. A. Albert, M. D. Kessler, and L. J. Parent. 2001. trans- acting inhibition of genomic RNA dimerization by Rous sarcoma virus matrix mutants. J. Virol. 75:260-268.

135. Garbitt, R. A., K. R. Bone, and L. J. Parent. 2004. Insertion of a classical nuclear import signal into the matrix domain of the Rous sarcoma virus Gag protein interferes with virus replication. J. Virol. 78:13534-13542.

136. Garnier, L., J. W. Wills, M. F. Verderame, and M. Sudol. 1996. WW domains and retrovirus budding. Nature 381:744-745.

137. Garrus, J. E., U. K. von Schwedler, O. W. Pornillos, S. G. Morham, K. H. Zavitz, H. E. Wang, D. A. Wettstein, K. M. Stray, M. Cote, R. L. Rich, D. G. Myszka, and W. I. Sundquist. 2001. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107:55-65.

138. Gebhardt, A., J. V. Bosch, A. Ziemiecki, and R. R. Friis. 1984. Rous sarcoma virus p19 and gp35 can be chemically crosslinked to high molecular weight complexes. An insight into virus assembly. J. Mol. Biol. 174:297-317.

139. Gelderblom,H. 1990. Morphogenesis, maturation and fine structure of lentiviruses, p. 159-180. In L.H.Pearl (ed.), Retroviral proteases: Maturation and morphogenesis. Stockton Press, New York, NY.

140. Gheysen, D., E. Jacobs, F. de Foresta, C. Thiriart, M. Francotte, D. Thines, and M. De Wilde. 1989. Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells. Cell 59:103-112.

141. Gilbert, J. M., L. D. Hernandez, J. W. Balliet, P. Bates, and J. M. White. 1995. Receptor-induced conformational changes in the subgroup A avian leukosis and sarcoma virus envelope glycoprotein. J. Virol. 69:7410-7415.

142. Gilboa, E., S. W. Mitra, S. Goff, and D. Baltimore. 1979. A detailed model of reverse transcription and tests of crucial aspects. Cell 18:93-100.

143. Goila-Gaur, R., D. G. Demirov, J. M. Orenstein, A. Ono, and E. O. Freed. 2003. Defects in Human Immunodeficiency Virus Budding and Endosomal Sorting Induced by TSG101 Overexpression. J. Virol. 77:6507-6519.

225 144. Gong, L., T. Kamitani, K. Fujise, L. S. Caskey, and E. T. Yeh. 1997. Preferential Interaction of Sentrin with a Ubiquitin-conjugating Enzyme, Ubc9. J. Biol. Chem. 272:28198-28201.

145. Gonzalez, S. A., J. L. Affranchino, H. R. Gelderblom, and A. Burny. 1993. Assembly of the matrix protein of simian immunodeficiency virus into virus- like particles. Virology 194:548-556.

146. Gorden, P., J. L. Carpentier, S. Cohen, and L. Orci. 1978. Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. Proc. Natl. Acad. Sci. 75:5025-5029.

147. Gottlinger, H. G., T. Dorfman, J. G. Sodroski, and W. A. Haseltine. 1991. Effect of mutations affecting the p6 Gag protein on human immunodeficiency virus particle release. Proc. Natl. Acad. Sci. 88:3195-3199.

148. Gottlinger, H. G., J. G. Sodroski, and W. A. Haseltine. 1989. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. 86:5781-5785.

149. Gottwein, E., J. Bodem, B. Muller, A. Schmechel, H. Zentgraf, and H. G. Krausslich. 2003. The Mason-Pfizer Monkey Virus PPPY and PSAP Motifs Both Contribute to Virus Release. J. Virol. 77:9474-9485.

150. Govers, R., T. ten Broeke, P. van Kerkhof, A. L. Schwartz, and G. J. Strous. 1999. Identification of a novel ubiquitin conjugation motif, required for ligand-induced internalization of the growth hormone receptor. EMBO J. 18:28-36.

151. Gravel, A., V. Dion, N. Cloutier, J. Gosselin, and L. Flamand. 2004. Characterization of human herpesvirus 6 variant B immediate-early 1 protein modifications by small ubiquitin-related modifiers. J. Gen. Virol. 85:1319- 1328.

152. Gurer, C., L. Berthoux, and J. Luban. 2005. Covalent Modification of Human Immunodeficiency Virus Type 1 p6 by SUMO-1. J. Virol. 79:910-917.

153. Haas, A. L., J. V. Warms, A. Hershko, and I. A. Rose. 1982. Ubiquitin- activating enzyme. Mechanism and role in protein-ubiquitin conjugation. J. Biol. Chem. 257:2543-2548.

154. Hackett, P. B., R. Swanstrom, H. E. Varmus, and J. M. Bishop. 1982. The leader sequence of the subgenomic mRNA's of Rous sarcoma virus is approximately 390 nucleotides. J. Virol. 41:527-534.

155. Haffar, O. K., S. Popov, L. Dubrovsky, I. Agostini, H. Tang, T. Pushkarsky, S. G. Nadler, and M. Bukrinsky. 2000. Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration

226 complex. J. Mol. Biol. 299:359-368.

156. Haigler, H. T., J. A. McKanna, and S. Cohen. 1979. Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J. Cell Biol. 81:382-395.

157. Hallenberger, S., V. Bosch, H. Angliker, E. Shaw, H. D. Klenk, and W. Garten. 1992. Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160. Nature 360:358-361.

158. Hammarstedt, M. and H. Garoff. 2004. Passive and Active Inclusion of Host Proteins in Human Immunodeficiency Virus Type 1 Gag Particles during Budding at the Plasma Membrane. J. Virol. 78:5686-5697.

159. Hampsey, M. and D. Reinberg. 2003. Tails of intrigue: phosphorylation of RNA polymerase II mediates histone methylation. Cell 113:429-432.

160. Hart, T. K., R. Kirsh, H. Ellens, R. W. Sweet, D. M. Lambert, S. R. Petteway, Jr., J. Leary, and P. J. Bugelski. 1991. Binding of Soluble CD4 Proteins to Human Immunodeficiency Virus Type 1 and Infected Cells Induces Release of Envelope Glycoprotein gp120. Proc. Natl. Acad. Sci. 88:2189-2193.

161. Harty, R. N., M. E. Brown, J. P. McGettigan, G. Wang, H. R. Jayakar, J. M. Huibregtse, M. A. Whitt, and M. J. Schnell. 2001. Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction. J. Virol. 75:10623- 10629.

162. Harty, R. N., J. Paragas, M. Sudol, and P. Palese. 1999. A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding. J. Virol. 73:2921-2929.

163. Harty, R. N., M. E. Brown, G. Wang, J. Huibregtse, and F. P. Hayes. 2000. A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: Implications for filovirus budding. PROC. NATL. ACAD. SCI. 97:13871-13876.

164. Hatanaka, H., O. Iourin, Z. Rao, E. Fry, A. Kingsman, and D. I. Stuart. 2002. Structure of equine infectious anemia virus matrix protein. J. Virol. 76:1876-1883.

165. Hatziioannou, T. and S. P. Goff. 2001. Infection of nondividing cells by Rous sarcoma virus. J. Virol. 75:9526-9531.

166. Hayman, M. J. 1978. Viral polyproteins in chick embryo fibroblasts infected with avian sarcoma leukosis viruses. Virology 85:241-252.

167. Heidecker, G., P. A. Lloyd, K. Fox, K. Nagashima, and D. Derse. 2004. Late

227 Assembly Motifs of Human T-Cell Leukemia Virus Type 1 and Their Relative Roles in Particle Release. J. Virol. 78:6636-6648.

168. Heinzinger, N. K., M. I. Bukrinsky, S. A. Haggerty, A. M. Ragland, V. Kewalramani, M. Lee, H. E. Gendelman, L. Ratner, M. Stevenson, and M. Emerman. 1994. The Vpr Protein of Human Immunodeficiency Virus Type 1 Influences Nuclear Localization of Viral Nucleic Acids in Nondividing Host Cells. PROC. NATL. ACAD. SCI. 91:7311-7315.

169. Hermida-Matsumoto, L. and M. D. Resh. 2000. Localization of Human Immunodeficiency Virus Type 1 Gag and Env at the Plasma Membrane by Confocal Imaging. J. Virol. 74:8670-8679.

170. Hershko, A. and A. Ciechanover. 1998. The ubiquitin system. Annu. Rev. Biochem. 67:425-479.

171. Hershko, A., A. Ciechanover, H. Heller, A. L. Haas, and I. A. Rose. 1980. Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc. Natl. Acad. Sci. 77:1783-1786.

172. Hershko, A., H. Heller, S. Elias, and A. Ciechanover. 1983. Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258:8206-8214.

173. Hicke, L. 2001. Protein regulation by monoubiquitin. Nat. Rev. Mol. Cell Biol. 2:195-201.

174. Hill, C. P., D. Worthylake, D. P. Bancroft, A. M. Christensen, and W. I. Sundquist. 1996. Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly. Proc. Natl. Acad. Sci. 93:3099-3104.

175. Himly, M., D. N. Foster, I. Bottoli, J. S. Iacovoni, and P. K. Vogt. 1998. The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses. Virology 248:295-304.

176. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mous cell cultures. J. Mol. Biol. 26:365-3369.

177. Hofmann, H., S. Floss, and T. Stamminger. 2000. Covalent Modification of the Transactivator Protein IE2-p86 of Human Cytomegalovirus by Conjugation to the Ubiquitin-Homologous Proteins SUMO-1 and hSMT3b. J. Virol. 74:2510-2524.

178. Holm, K., K. Weclewicz, R. Hewson, and M. Suomalainen. 2003. Human Immunodeficiency Virus Type 1 Assembly and Lipid Rafts: Pr55gag Associates

228 with Membrane Domains That Are Largely Resistant to Brij98 but Sensitive to Triton X-100. J. Virol. 77:4805-4817.

179. Holzer, G. W., J. A. Mayrhofer, W. Gritschenberger, F. Dorner, and F. G. Falkner. 1999. Poxviral/retroviral chimeric vectors allow cytoplasmic production of transducing defective retroviral particles. Virology 253:107-114.

180. Howard, T. L., D. R. Stauffer, C. R. Degnin, and S. M. Hollenberg. 2001. CHMP1 functions as a member of a newly defined family of vesicle trafficking proteins. J. Cell Sci. 114:2395-2404.

181. Huang, M., Z. Hanna, and P. Jolicoeur. 1995. Mutational analysis of the murine AIDS-defective viral genome reveals a high reversion rate in vivo and a requirement for an intact Pr60gag protein for efficient induction of disease. J. Virol. 69:60-68.

182. Huang, M. and M. A. Martin. 1997. Incorporation of Pr160gag-pol into virus particles requires the presence of both the major homology region and adjacent C-terminal capsid sequences within the Gag-Pol polyprotein. J. Virol. 71:4472- 4478.

183. Huang, Q., R. P. Sivaramakrishna, K. Ludwig, T. Korte, C. Bottcher, and A. Herrmann. 2003. Early steps of the conformational change of influenza virus hemagglutinin to a fusion active state: Stability and energetics of the hemagglutinin. Biochimica et Biophysica Acta (BBA) - Biomembranes 1614:3- 13.

184. Huang, Y., A. Khorchid, J. Wang, M. A. Parniak, J. L. Darlix, M. A. Wainberg, and L. Kleiman. 1997. Effect of mutations in the nucleocapsid protein (NCp7) upon Pr160(gag-pol) and tRNA(Lys) incorporation into human immunodeficiency virus type 1. J. Virol. 71:4378-4384.

185. Huang, Y., A. Khorchid, J. Gabor, J. Wang, X. Li, J. L. Darlix, M. A. Wainberg, and L. Kleiman. 1998. The Role of Nucleocapsid and U5 Stem/A- Rich Loop Sequences in tRNA3Lys Genomic Placement and Initiation of Reverse Transcription in Human Immunodeficiency Virus Type 1. J. Virol. 72:3907-3915.

186. Hughes, S. H., J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave. 1987. Adaptor plasmids simplify the insertion of foreign DNA into helper- independent retroviral vectors. J. Virol. 61:3004-3012.

187. Humphries, E. H., C. Glover, and M. E. Reichmann. 1981. Rous sarcoma virus infection of synchronized cells establishes provirus integration during S- phase DNA synthesis prior to cellular division. Proc. Natl. Acad. Sci. 78:2601- 2605.

188. Humphries, E. H. and H. M. Temin. 1972. Cell cycle-dependent activation of

229 Rous sarcoma virus-infected stationary chicken cells: avian leukosis virus group-specific antigens and ribonucleic acid. J. Virol. 10:82-87.

189. Humphries, E. H. and H. M. Temin. 1974. Requirement for cell division for initiation of transcription of Rous sarcoma virus RNA. J. Virol. 14:531-546.

190. Hunter,E. 1997. Virus entry and receptors, J.M.Coffin, S.H.Hughes, and H.Varmus (eds.), Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

191. Hunter, E., E. Hill, M. Hardwick, A. Bhown, D. E. Schwartz, and R. Tizard. 1983. Complete sequence of the Rous sarcoma virus env gene: identification of structural and functional regions of its product. J. Virol. 46:920-936.

192. Hunter,E. 1979. Biological Techniques for Avian Sarcoma Viruses, p. 379- 393. In W.B.Jakoby and I.H.Pastan (eds.), Methods in Enzymology v. 58. Academic Press, Inc., New York, NY.

193. Ibarrondo, F. J., R. Choi, Y. Z. Geng, J. Canon, O. Rey, G. C. Baldwin, and P. Krogstad. 2001. HIV type 1 Gag and nucleocapsid proteins: cytoskeletal localization and effects on cell motility. AIDS Res. Hum. Retroviruses 17:1489-1500.

194. Ikeda, Y., L. M. J. Ylinen, M. Kahar-Bador, and G. J. Towers. 2004. Influence of Gag on Human Immunodeficiency Virus Type 1 Species-Specific Tropism. J. Virol. 78:11816-11822.

195. Jacks, T., H. D. Madhani, F. R. Masiarz, and H. E. Varmus. 1988. Signal for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell 55:447-458.

196. Jayakar, H. R., K. G. Murti, and M. A. Whitt. 2000. Mutations in the PPPY Motif of Vesicular Stomatitis Virus Matrix Protein Reduce Virus Budding by Inhibiting a Late Step in Virion Release. J. Virol. 74:9818-9827.

197. Jin, Z., L. Jin, D. L. Peterson, and C. L. Lawson. 1999. Model for lentivirus capsid core assembly based on crystal dimers of EIAV p26. J. Mol. Biol. 286:83-93.

198. Johnson, E. S., I. Schwienhorst, R. J. Dohmen, and G. Blobel. 1997. The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J 16:5509-5519.

199. Johnson, E. S. 2004. Protein modification by SUMO. Annu. Rev. Biochem. 73:355-382.

200. Johnson, E. S. and G. Blobel. 1997. Ubc9p Is the Conjugating Enzyme for the

230 Ubiquitin-like Protein Smt3p. J. Biol. Chem. 272:26799-26802.

201. Johnson, M. C., J. L. Spidel, D. Ako-Adjei, J. W. Wills, and V. M. Vogt. 2005. The C-terminal half of TSG101 blocks Rous sarcoma virus budding and sequesters Gag into unique non-endosomal structures. J. Virol. In Press:

202. Jones, P. L., T. Korte, and R. Blumenthal. 1998. Conformational Changes in Cell Surface HIV-1 Envelope Glycoproteins Are Triggered by Cooperation between Cell Surface CD4 and Co-receptors. J. Biol. Chem. 273:404-409.

203. Kaehlcke, K., A. Dorr, C. Hetzer-Egger, V. Kiermer, P. Henklein, M. Schnoelzer, E. Loret, P. A. Cole, E. Verdin, and M. Ott. 2003. Acetylation of Tat defines a cyclinT1-independent step in HIV transactivation. Mol. Cell 12:167-176.

204. Kahyo, T., T. Nishida, and H. Yasuda. 2001. Involvement of PIAS1 in the Sumoylation of Tumor Suppressor p53. Mol. Cell 8:713-718.

205. Karageorgos, L., P. Li, and C. Burrell. 1993. Characterization of HIV replication complexes early after cell-to-cell infection. AIDS Res. Hum. Retroviruses 9:817-823.

206. Katen, L. J., M. M. Januszeski, W. F. Anderson, K. J. Hasenkrug, and L. H. Evans. 2001. Infectious Entry by Amphotropic as well as Ecotropic Murine Leukemia Viruses Occurs through an Endocytic Pathway. J. Virol. 75:5018- 5026.

207. Kato, M., K. Miyazawa, and N. Kitamura. 2000. A Deubiquitinating Enzyme UBPY Interacts with the Src Homology 3 Domain of Hrs-binding Protein via a Novel Binding Motif PX(V/I)(D/N)RXXKP. J. Biol. Chem. 275:37481-37487.

208. Katoh, K., H. Shibata, H. Suzuki, A. Nara, K. Ishidoh, E. Kominami, T. Yoshimori, and M. Maki. 2003. The ALG-2-interacting Protein Alix Associates with CHMP4b, a Human Homologue of Yeast Snf7 That Is Involved in Multivesicular Body Sorting. J. Biol. Chem. 278:39104-39113.

209. Katz, R. A., J. G. Greger, K. Darby, P. Boimel, G. F. Rall, and A. M. Skalka. 2002. Transduction of interphase cells by avian sarcoma virus. J. Virol. 76:5422-5434.

210. Katz, R. A., M. Kotler, and A. M. Skalka. 1988. cis-acting intron mutations that affect the efficiency of avian retroviral RNA splicing: implication for mechanisms of control. J. Virol. 62:2686-2695.

211. Katz, R. A., J. H. Omer, J. H. Weis, S. A. Mitsialis, A. J. Fara, and R. V. Guntaka. 1982. Restriction endonuclease and nucleotide sequence analysis of molecularly cloned unintegrated avian tumor virus DNA: structure of large terminal repeats in circle junctions. J. Virol. 42:346-351.

231 212. Katzmann, D. J., M. Babst, and S. D. Emr. 2001. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106:145-155.

213. Katzmann, D. J., G. Odorizzi, and S. D. Emr. 2002. Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell Biol. 3:893-905.

214. Katzmann, D. J., C. J. Stefan, M. Babst, and S. D. Emr. 2003. Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J. Cell Biol. 162:413- 423.

215. Kaukinen, P., A. Vaheri, and A. Plyusnin. 2003. Non-covalent interaction between nucleocapsid protein of Tula hantavirus and small ubiquitin-related modifier-1, SUMO-1. Virus Research 92:37-45.

216. Kaye, J. F. and A. M. Lever. 1996. trans-acting proteins involved in RNA encapsidation and viral assembly in human immunodeficiency virus type 1. J. Virol. 70:880-886.

217. Keith, J. and H. Fraenkel-Conrat. 1975. Identification of the 5' end of Rous sarcoma virus RNA. Proc. Natl. Acad. Sci. 72:3347-3350.

218. Keogh, E. V. 1938. Ectodermal lesions produced by the virus of Rous sarcoma. Br. J. Exp. Pathol. 19:1-9.

219. Khorasanizadeh, S., R. Campos-Olivas, C. A. Clark, and M. F. Summers. 1999. Sequence-specific 1H, 13C and 15N chemical shift assignment and secondary structure of the HTLV-I capsid protein. J. Biomol. NMR 14:199- 200.

220. Khorasanizadeh, S., R. Campos-Olivas, and M. F. Summers. 1999. Solution structure of the capsid protein from the human T-cell leukemia virus type-I. J. Mol. Biol. 291:491-505.

221. Kikonyogo, A., F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis. 2001. Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for Gag budding from cells. Proc. Natl. Acad. Sci. 98:11199-11204.

222. Kim, C. H. and I. Tinoco, Jr. 2000. A retroviral RNA kissing complex containing only two G.C base pairs. Proc. Natl. Acad. Sci. 97:9396-9401.

223. Kim, W., Y. Tang, Y. Okada, T. A. Torrey, S. K. Chattopadhyay, M. Pfleiderer, F. G. Falkner, F. Dorner, W. Choi, N. Hirokawa, and H. C. Morse. 1998. Binding of murine leukemia virus Gag polyproteins to KIF4, a microtubule-based motor protein. J. Virol. 72:6898-6901.

224. Kingston, R. L., T. Fitzon-Ostendorp, E. Z. Eisenmesser, G. W. Schatz, V.

232 M. Vogt, C. B. Post, and M. G. Rossmann. 2000. Structure and self- association of the Rous sarcoma virus capsid protein. Structure. Fold. Des 8:617-628.

225. Kingston, R. L., N. H. Olson, and V. M. Vogt. 2001. The organization of mature Rous sarcoma virus as studied by cryoelectron microscopy. J. Struct. Biol. 136:67-80.

226. Klatzman, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J.-C. Gluckman, and L. Montagnier. 1984. T-lymphocyte T4 molecule behaves as a receptor for human retrovirus LAV. Nature 312:767- 768.

227. Kotaja, N., U. Karvonen, O. A. Janne, and J. J. Palvimo. 2002. PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases. Mol. Cell. Biol. 22:5222-5234.

228. Kowalski, M., J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski. 1987. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237:1351-1355.

229. Kozak, M. 1989. The scanning model for translation: an update. J. Cell Biol. 108:229-241.

230. Kozlovsky, Y., L. V. Chernomordik, and M. M. Kozlov. 2002. Lipid Intermediates in Membrane Fusion: Formation, Structure, and Decay of Hemifusion Diaphragm. Biophys. J. 83:2634-2651.

231. Krausslich, H. G., A. M. Traenckner, and F. Rippmann. 1991. Expression and characterization of genetically linked homo- and hetero -dimers of HIV proteinase. Adv. Exp. Med. Biol. 306:417-428.

232. Krishna, N. K., S. Campbell, V. M. Vogt, and J. W. Wills. 1998. Genetic determinants of Rous sarcoma virus particle size. J. Virol. 72:564-577.

233. Kukolj, G., K. S. Jones, and A. M. Skalka. 1997. Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases. J. Virol. 71:843-847.

234. Kukolj, G., R. A. Katz, and A. M. Skalka. 1998. Characterization of the nuclear localization signal in the avian sarcoma virus integrase. Gene 223:157- 163.

235. Kulpa, D., R. Topping, and A. Telesnitsky. 1997. Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors. EMBO J 16:856-865.

233 236. Lai, M. M. 1976. Phosphoproteins of Rous sarcoma viruses. Virology 74:287- 301.

237. Lai, M. M. and P. H. Duesberg. 1972. Adenylic acid-rich sequence in RNAs of Rous sarcoma virus and Rauscher mouse leukaemia virus. Nature 235:383- 386.

238. Lanman, J., T. T. Lam, S. Barnes, M. Sakalian, M. R. Emmett, A. G. Marshall, and P. E. Prevelige, Jr. 2003. Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry. J. Mol. Biol. 325:759-772.

239. Lapatto, R., T. Blundell, A. Hemmings, J. Overington, A. Wilderspin, S. Wood, J. R. Merson, P. J. Whittle, D. E. Danley, and K. F. Geoghegan. 1989. X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes. Nature 342:299-302.

240. Le, B., I, A. R. Rosenberg, and M. C. Dokhelar. 1999. Multiple functions for the basic amino acids of the human T-cell leukemia virus type 1 matrix protein in viral transmission. J. Virol. 73:1860-1867.

241. Leamnson, R. N. and M. S. Halpern. 1976. Subunit structure of the glycoprotein complex of avian tumor virus. J. Virol. 18:956-968.

242. Lee, B. H., K. Yoshimatsu, A. Maeda, K. Ochiai, M. Morimatsu, K. Araki, M. Ogino, S. Morikawa, and J. Arikawa. 2003. Association of the nucleocapsid protein of the Seoul and Hantaan hantaviruses with small ubiquitin-like modifier-1-related molecules. Virus Research 98:83-91.

243. Lee, E., A. Yeo, B. Kraemer, M. Wickens, and M. L. Linial. 1999. The Gag domains required for avian retroviral RNA encapsidation determined by using two independent assays. J. Virol. 73:6282-6292.

244. Lee, E. G. and M. L. Linial. 2004. Basic residues of the retroviral nucleocapsid play different roles in Gag-Gag and Gag-Psi RNA interactions. J. Virol. 78:8486-8495.

245. Lee, E. g., A. Alidina, C. May, and M. L. Linial. 2003. Importance of Basic Residues in Binding of Rous Sarcoma Virus Nucleocapsid to the RNA Packaging Signal. J. Virol. 77:2010-2020.

246. Lee, J. M., H. J. Kang, H. R. Lee, C. Y. Choi, W. J. Jang, and J. H. Ahn. 2003. PIAS1 enhances SUMO-1 modification and the transactivation activity of the major immediate-early IE2 protein of human cytomegalovirus. FEBS Lett. 555:322-328.

247. Lee, P. S., Y. Wang, M. G. Dominguez, Y. G. Yeung, M. A. Murphy, D. D. Bowtell, and E. R. Stanley. 1999. The Cbl protooncoprotein stimulates CSF-1

234 receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation. EMBO J. 18:3616-3628.

248. Lee, Y. M., B. Liu, and X. F. Yu. 1999. Formation of virus assembly intermediate complexes in the cytoplasm by wild-type and assembly-defective mutant human immunodeficiency virus type 1 and their association with membranes. J. Virol. 73:5654-5662.

249. Leider, J. M., P. Palese, and F. I. Smith. 1988. Determination of the mutation rate of a retrovirus. J. Virol. 62:3084-3091.

250. Lethbridge, K. J., G. E. Scott, and K. N. Leppard. 2003. Nuclear matrix localization and SUMO-1 modification of adenovirus type 5 E1b 55K protein are controlled by E4 Orf6 protein. J. Gen. Virol. 84:259-268.

251. Levin, J. G., P. M. Grimley, J. M. Ramseur, and I. K. Berezesky. 1974. Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D. J. Virol. 14:152-161.

252. Levin, J. G. and M. J. Rosenak. 1976. Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA. Proc. Natl. Acad. Sci. 73:1154-1158.

253. Levkowitz, G., H. Waterman, E. Zamir, Z. Kam, S. Oved, W. Y. Langdon, L. Beguinot, B. Geiger, and Y. Yarden. 1998. c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12:3663-3674.

254. Lewis, P. F. and M. Emerman. 1994. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J. Virol. 68:510-516.

255. Lewis, P. F., M. Hensel, and M. Emerman. 1992. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J. 11:3053-3058.

256. Li, S., C. P. Hill, W. I. Sundquist, and J. T. Finch. 2000. Image reconstructions of helical assemblies of the HIV-1 CA protein. Nature 407:409- 413.

257. Li, S. J. and M. Hochstrasser. 1999. A new protease required for cell-cycle progression in yeast. Nature 398:246-251.

258. Licata, J. M., M. Simpson-Holley, N. T. Wright, Z. Han, J. Paragas, and R. N. Harty. 2003. Overlapping Motifs (PTAP and PPEY) within the Ebola Virus VP40 Protein Function Independently as Late Budding Domains: Involvement of Host Proteins TSG101 and VPS-4. J. Virol. 77:1812-1819.

235 259. Lindwasser, O. W. and M. D. Resh. 2001. Multimerization of human immunodeficiency virus type 1 Gag promotes its localization to barges, raft-like membrane microdomains. J. Virol. 75:7913-7924.

260. Liu, B., R. Dai, C. J. Tian, L. Dawson, R. Gorelick, and X. F. Yu. 1999. Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin. J. Virol. 73:2901-2908.

261. Lodge, R., H. Gottlinger, D. Gabuzda, E. A. Cohen, and G. Lemay. 1994. The intracytoplasmic domain of gp41 mediates polarized budding of human immunodeficiency virus type 1 in MDCK cells. J. Virol. 68:4857-4861.

262. Lodge, R., J. P. Lalonde, G. Lemay, and E. A. Cohen. 1997. The membrane- proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells. EMBO J. 16:695-705.

263. Lodish,H., A.Berk, L.Zipursky, P.Matsudaira, D.Baltimore, and J.Darnell. 2000. Mol. Cell Biol. W.H. Freeman and Company, New York, NY.

264. Lohrum, M. A., D. B. Woods, R. L. Ludwig, E. Balint, and K. H. Vousden. 2001. C-terminal ubiquitination of p53 contributes to nuclear export. Mol. Cell Biol. 21:8521-8532.

265. Lu, Q., L. W. Hope, M. Brasch, C. Reinhard, and S. N. Cohen. 2003. TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation. PROC. NATL. ACAD. SCI. 100:7626-7631.

266. Luban, J. and S. P. Goff. 1994. Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. J. Virol. 68:3784-3793.

267. Luby-Phelps, K. 2000. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int. Rev. Cytol. 192:189-221.:189-221.

268. Lucero, P., E. Penalver, L. Vela, and R. Lagunas. 2000. Monoubiquitination is sufficient to signal internalization of the maltose transporter in Saccharomyces cerevisiae. J. Bacteriol. 182:241-243.

269. Ly, H. and T. G. Parslow. 2002. Bipartite Signal for Genomic RNA Dimerization in Moloney Murine Leukemia Virus. J. Virol. 76:3135-3144.

270. Ma, Y. M. and V. M. Vogt. 2004. Nucleic acid binding-induced Gag dimerization in the assembly of Rous sarcoma virus particles in vitro. J. Virol. 78:52-60.

271. Maddon, P. J., A. G. Dalgleish, J. S. McDougal, P. R. Clapham, R. A. Weiss, and R. Axel. 1986. The T4 gene encodes the AIDS virus receptor and is

236 expressed in the immune system and the brain. Cell 47:333-348.

272. Maeda, A., B. H. Lee, K. Yoshimatsu, M. Saijo, I. Kurane, J. Arikawa, and S. Morikawa. 2003. The Intracellular Association of the Nucleocapsid Protein (NP) of Hantaan Virus (HTNV) with Small Ubiquitin-like Modifier-1 (SUMO- 1) Conjugating Enzyme 9 (Ubc9). Virology 305:288-297.

273. Maertens, G., P. Cherepanov, Z. Debyser, Y. Engelborghs, and A. Engelman. 2004. Identification and Characterization of a Functional Nuclear Localization Signal in the HIV-1 Integrase Interactor LEDGF/p75. J. Biol. Chem. 279:33421-33429.

274. Maldarelli, F., J. King, and M. Jane Yagi. 1987. Effects of cytoskeletal disrupting agents on mouse mammary tumor virus replication. Virus Research 7:281-295.

275. Mammano, F., E. Kondo, J. Sodroski, A. Bukovsky, and H. G. Gottlinger. 1995. Rescue of human immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins with short cytoplasmic domains. J. Virol. 69:3824- 3830.

276. Mammano, F., A. Ohagen, S. Hoglund, and H. G. Gottlinger. 1994. Role of the major homology region of human immunodeficiency virus type 1 in virion morphogenesis. J. Virol. 68:4927-4936.

277. Mann, R. and D. Baltimore. 1985. Varying the position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAs. J. Virol. 54:401-407.

278. Mann, R., R. C. Mulligan, and D. Baltimore. 1983. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33:153-159.

279. Mannen, H., H. M. Tseng, C. l. Cho, and S. S. Li. 1996. Cloning and Expression of Human Homolog HSMT3 to Yeast SMT3 Suppressor of MIF2 Mutations in a Centromere Protein Gene. Biochemical and Biophysical Research Communications 222:178-180.

280. Manrique, J. M., C. C. P. Celma, E. Hunter, J. L. Affranchino, and S. A. Gonzalez. 2003. Positive and Negative Modulation of Virus Infectivity and Envelope Glycoprotein Incorporation into Virions by Amino Acid Substitutions at the N Terminus of the Simian Immunodeficiency Virus Matrix Protein. J. Virol. 77:10881-10888.

281. Marchese, A. and J. L. Benovic. 2001. Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting. J. Biol. Chem. 276:45509-45512.

237 282. Martin-Serrano, J., A. Yaravoy, D. Perez-Caballero, and P. D. Bieniasz. 2003. Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins. Proc. Natl. Acad. Sci. USA 100:12414-12419.

283. Martin-Serrano, J., T. Zang, and P. D. Bieniasz. 2001. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat. Med. 7:1313-1319.

284. Martin-Serrano, J., D. Perez-Caballero, and P. D. Bieniasz. 2004. Context- Dependent Effects of L Domains and Ubiquitination on Viral Budding. J. Virol. 78:5554-5563.

285. Massiah, M. A., D. Worthylake, A. M. Christensen, W. I. Sundquist, C. P. Hill, and M. F. Summers. 1996. Comparison of the NMR and X-ray structures of the HIV-1 matrix protein: Evidence for conformational changes during viral assembly. Protein Sci 5:2391-2398.

286. Matsuo, H., J. Chevallier, N. Mayran, I. Le Blanc, C. Ferguson, J. Faure, N. S. Blanc, S. Matile, J. Dubochet, R. Sadoul, R. G. Parton, F. Vilbois, and J. Gruenberg. 2004. Role of LBPA and Alix in Multivesicular Liposome Formation and Endosome Organization. Science 303:531-534.

287. Matthews, H. R. 1995. Protein kinases and phosphatases that act on histidine, lysine, or arginine residues in eukaryotic proteins: a possible regulator of the mitogen-activated protein kinase cascade. Pharmacol. Ther. 67:323-350.

288. McBride, M. S., M. D. Schwartz, and A. T. Panganiban. 1997. Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation. J. Virol. 71:4544- 4554.

289. McDermott, J., L. Farrell, R. Ross, and E. Barklis. 1996. Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents. J. Virol. 70:5106-5114.

290. McDonald, D., M. A. Vodicka, G. Lucero, T. M. Svitkina, G. G. Borisy, M. Emerman, and T. J. Hope. 2002. Visualization of the intracellular behavior of HIV in living cells. J. Cell Biol. 159:441-452.

291. McDonnell, J. M., D. Fushman, S. M. Cahill, W. Zhou, A. Wolven, C. B. Wilson, T. D. Nelle, M. D. Resh, J. Wills, and D. Cowburn. 1998. Solution structure and dynamics of the bioactive retroviral M domain from Rous sarcoma virus. J. Mol. Biol. 279:921-928.

292. McDougal, J. S., P. J. Maddon, A. G. Dalgleish, P. R. Clapham, D. R. Littman, M. Godfrey, D. E. Maddon, L. Chess, R. A. Weiss, and R. Axel. 1986. The T4 glycoprotein is a cell-surface receptor for the AIDS virus. Cold

238 Spring Harb. Symp. Quant. Biol 51 Pt 2:703-11.:703-711.

293. McNally, M. T. and K. Beemon. 1992. Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing. J. Virol. 66:6-11.

294. Melchior, F. 2000. SUMO-Nonclassical Ubiquitin. Annu. Rev. of Cell Dev. Biol. 16:591-626.

295. Melikyan, G. B., R. J. O. Barnard, R. M. Markosyan, J. A. T. Young, and F. S. Cohen. 2004. Low pH Is Required for Avian Sarcoma and Leukosis Virus Env-Induced Hemifusion and Fusion Pore Formation but Not for Pore Growth. J. Virol. 78:3753-3762.

296. Melikyan, G. B., R. M. Markosyan, H. Hemmati, M. K. Delmedico, D. M. Lambert, and F. S. Cohen. 2000. Evidence That the Transition of HIV-1 gp41 into a Six-Helix Bundle, Not the Bundle Configuration, Induces Membrane Fusion. J. Cell Biol. 151:413-424.

297. Mergener, K., M. Facke, R. Welker, V. Brinkmann, H. R. Gelderblom, and H. G. Krausslich. 1992. Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells. Virology 186:25-39.

298. Meric, C., E. Gouilloud, and P. F. Spahr. 1988. Mutations in Rous sarcoma virus nucleocapsid protein p12 (NC): deletions of Cys-His boxes. J. Virol. 62:3328-3333.

299. Meric, C. and P.-F. Spahr. 1986. Rous sarcoma virus nucleic acid-binding protein p12 is necessary for viral 70S RNA dimer formation and packaging. J. Virol. 60:450-459.

300. Messer, L. I., J. G. Levin, and S. K. Chattopadhyay. 1981. Metabolism of viral RNA in murine leukemia virus-infected cells; evidence for differential stability of viral message and virion precursor RNA. J. Virol. 40:683-690.

301. Miller, M., M. Jaskolski, J. K. Rao, J. Leis, and A. Wlodawer. 1989. Crystal structure of a retroviral protease proves relationship to aspartic protease family. Nature 337:576-579.

302. Miller, M. D., C. M. Farnet, and F. D. Bushman. 1997. Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition. J. Virol. 71:5382-5390.

303. Mitchell, R. S., B. F. Beitzel, A. R. Schroder, P. Shinn, H. Chen, C. C. Berry, J. R. Ecker, and F. D. Bushman. 2004. Retroviral DNA Integration: ASLV, HIV, and MLV Show Distinct Target Site Preferences. PLoS. Biol. 2:E234

304. Mizuno, E., K. Kawahata, M. Kato, N. Kitamura, and M. Komada. 2003.

239 STAM Proteins Bind Ubiquitinated Proteins on the Early Endosome via the VHS Domain and Ubiquitin-interacting Motif. Mol. Biol.Cell 14:3675-3689.

305. Molling, K., D. P. Bolognesi, H. Bauer, W. Busen, H. W. Plassmann, and P. Hausen. 1971. Association of viral reverse transcriptase with an enzyme degrading the RNA moiety of RNA-DNA hybrids. Nat New Biol 234:240-243.

306. Momany, C., L. C. Kovari, A. J. Prongay, W. Keller, R. K. Gitti, B. M. Lee, A. E. Gorbalenya, L. Tong, J. McClure, L. S. Ehrlich, M. F. Summers, C. Carter, and M. G. Rossmann. 1996. Crystal structure of dimeric HIV-1 capsid protein. Nat. Struct. Biol. 3:763-770.

307. Mortara, R. A. and G. L. Koch. 1989. An association between actin and nucleocapsid polypeptides in isolated murine retroviral particles. J. Submicrosc. Cytol. Pathol. 21:295-306.

308. Mortuza, G. B., L. F. Haire, A. Stevens, S. J. Smerdon, J. P. Stoye, and I. A. Taylor. 2004. High-resolution structure of a retroviral capsid hexameric amino- terminal domain. Nature 431:481-485.

309. Morvan, J., M. Froissard, R. Haguenauer-Tsapis, and D. Urban-Grimal. 2004. The Ubiquitin Ligase Rsp5p is Required for Modification and Sorting of Membrane Proteins into Multivesicular Bodies. Traffic 5:383-392.

310. Moscovici, C., M. G. Moscovici, H. Jimenez, M. M. C. Lai, M. J. Hayman, and P. K. Vogt. 1977. Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail. Cell 11:95-103.

311. Mothes, W., A. L. Boerger, S. Narayan, J. M. Cunningham, and J. A. T. Young. 2000. Retroviral Entry Mediated by Receptor Priming and Low pH Triggering of an Envelope Glycoprotein. Cell 103:679-689.

312. Mueller, T. D. and J. Feigon. 2003. Structural determinants for the binding of ubiquitin-like domains to the proteasome. EMBO J. 22:4634-4645.

313. Muller, S. and A. Dejean. 1999. Viral immediate-early proteins abrogate the modification by SUMO-1 of PML and Sp100 proteins, correlating with nuclear body disruption. J. Virol. 73:5137-5143.

314. Muriaux, D., H. De Rocquigny, B. P. Roques, and J. Paoletti. 1996. NCp7 Activates HIV-1Lai RNA Dimerization by Converting a Transient Loop-Loop Complex into a Stable Dimer. J. Biol. Chem. 271:33686-33692.

315. Myers, E. L. and J. F. Allen. 2002. Tsg101, an inactive homologue of ubiquitin ligase e2, interacts specifically with human immunodeficiency virus type 2 Gag polyprotein and results in increased levels of ubiquitinated Gag. J. Virol. 76:11226-11235.

240 316. Nandhagopal, N., A. A. Simpson, M. C. Johnson, A. B. Francisco, G. W. Schatz, M. G. Rossmann, and V. M. Vogt. 2004. Dimeric Rous sarcoma virus capsid protein structure relevant to immature Gag assembly. J. Mol. Biol. 335:275-282.

317. Narayan, S., R. J. O. Barnard, and J. A. T. Young. 2003. Two Retroviral Entry Pathways Distinguished by Lipid Raft Association of the Viral Receptor and Differences in Viral Infectivity. J. Virol. 77:1977-1983.

318. Narayan, S. and J. A. T. Young. 2004. Reconstitution of retroviral fusion and uncoating in a cell-free system. PROC. NATL. ACAD. SCI. 101:7721-7726.

319. Narezkina, A., K. D. Taganov, S. Litwin, R. Stoyanova, J. Hayashi, C. Seeger, A. M. Skalka, and R. A. Katz. 2004. Genome-wide analyses of avian sarcoma virus integration sites. J. Virol. 78:11656-11663.

320. Nelle, T. D., M. F. Verderame, J. Leis, and J. W. Wills. 1998. The major site of phosphorylation within the Rous sarcoma virus matrix protein is not required for replication. J. Virol. 72:

321. Nelle, T. D. and J. W. Wills. 1996. A large region within the Rous sarcoma virus matrix protein is dispensable for budding and infectivity. J. Virol. 70:2269-2276.

322. Nevels, M., W. Brune, and T. Shenk. 2004. SUMOylation of the Human Cytomegalovirus 72-Kilodalton IE1 Protein Facilitates Expression of the 86- Kilodalton IE2 Protein and Promotes Viral Replication. J. Virol. 78:7803-7812.

323. Nguyen, D. H. and J. E. Hildreth. 2000. Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. J. Virol. 74:3264-3272.

324. Nguyen, D. G., A. Booth, S. J. Gould, and J. E. K. Hildreth. 2003. Evidence That HIV Budding in Primary Macrophages Occurs through the Exosome Release Pathway. J. Biol. Chem. 278:52347-52354.

325. Nikko, E., A. M. Marini, and B. Andre. 2003. Permease Recycling and Ubiquitination Status Reveal a Particular Role for Bro1 in the Multivesicular Body Pathway. J. Biol. Chem. 278:50732-50743.

326. Nydegger, S., M. Foti, A. Derdowski, P. Spearman, and M. Thali. 2003. HIV-1 Egress is Gated Through Late Endosomal Membranes. Traffic 4:902- 910.

327. Odorizzi, G., D. J. Katzmann, M. Babst, A. Audhya, and S. D. Emr. 2003. Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae. J. Cell Sci. 116:1893-1903.

241 328. Oertle, S. and P. F. Spahr. 1990. Role of the Gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA. J. Virol. 64:5757-5763.

329. Ogert, R. A., L. H. Lee, and K. L. Beemon. 1996. Avian retroviral RNA element promotes unspliced RNA accumulation in the cytoplasm. J. Virol. 70:3834-3843.

330. Ono, A., D. Demirov, and E. O. Freed. 2000. Relationship between human immunodeficiency virus type 1 Gag multimerization and membrane binding. J. Virol. 74:5142-5150.

331. Ono, A. and E. O. Freed. 2001. Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc. Natl. Acad. Sci. 98:13925-13930.

332. Ono, A., M. Huang, and E. O. Freed. 1997. Characterization of human immunodeficiency virus type 1 matrix revertants: effects on virus assembly, Gag processing, and Env incorporation into virions. J. Virol. 71:4409-4418.

333. Oppermann, H., J. M. Bishop, H. E. Varmus, and L. Levintow. 1977. A joint produce of the genes Gag and Pol of avian sarcoma virus: a possible precursor of reverse transcriptase. Cell 12:993-1005.

334. Orenstein, J. M., M. S. Meltzer, T. Phipps, and H. E. Gendelman. 1988. Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study. J. Virol. 62:2578-2586.

335. Oroudjev, E. M., P. C. E. Kang, and L. A. Kohlstaedt. 1999. An additional dimer linkage structure in moloney murine leukemia virus RNA. J. Mol. Biol. 291:603-613.

336. Ott, D. E. 1997. Cellular proteins in HIV virions. Rev. Med. Virol. 7:167-180.

337. Ott, D. E., L. V. Coren, E. N. Chertova, T. D. Gagliardi, and U. Schubert. 2000. Ubiquitination of HIV-1 and MuLV Gag. Virology 278:111-121.

338. Ott, D. E., L. V. Coren, T. D. Copeland, B. P. Kane, D. G. Johnson, R. C. Sowder, Y. Yoshinaka, S. Oroszlan, L. O. Arthur, and L. E. Henderson. 1998. Ubiquitin is covalently attached to the p6 Gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12Gag protein of Moloney murine leukemia virus. J. Virol. 72:2962-2968.

339. Ott, D. E., L. V. Coren, D. G. Johnson, B. P. Kane, R. C. Sowder, Y. D. Kim, R. J. Fisher, X. Z. Zhou, K. P. Lu, and L. E. Henderson. 2000. Actin- binding cellular proteins inside human immunodeficiency virus type 1. Virology 266:42-51.

242 340. Ott, D. E., L. V. Coren, B. P. Kane, L. K. Busch, D. G. Johnson, I. I. Sowder, E. N. Chertova, L. O. Arthur, and L. E. Henderson. 1996. Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J. Virol. 70:7734-7743.

341. Ott, D. E., L. V. Coren, R. C. Sowder, J. Adams, K. Nagashima, and U. Schubert. 2002. Equine infectious anemia virus and the ubiquitin-proteasome system. J. Virol. 76:3038-3044.

342. Ott, D. E., L. V. Coren, R. C. Sowder, J. Adams, and U. Schubert. 2003. Retroviruses have differing requirements for proteasome function in the budding process. J. Virol. 77:3384-3393.

343. Owens, R. J., J. W. Dubay, E. Hunter, and R. W. Compans. 1991. Human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells. Proc. Natl. Acad. Sci. 88:3987-3991.

344. Paca, R. E., R. A. Ogert, C. S. Hibbert, E. Izaurralde, and K. L. Beemon. 2000. Rous sarcoma virus DR posttranscriptional elements use a novel RNA export pathway. J. Virol. 74:9507-9514.

345. Pal, R., M. S. Reitz, Jr., E. Tschachler, R. C. Gallo, M. G. Sarngadharan, and F. D. Veronese. 1990. Myristoylation of Gag proteins of HIV-1 plays an important role in virus assembly. AIDS Res. & Human Retrovir. 6:721-730.

346. Palmiter, R. D., J. Gagnon, V. M. Vogt, S. Ripley, and R. N. Eisenman. 1978. The NH2-terminal sequence of the avian oncovirus Gag precursor polyprotein (Pr76gag). Virology 91:423-433.

347. Paolucci, S., F. Baldanti, R. Maserati, F. Castelli, F. Suter, F. Maggiolo, A. Pan, and G. Gerna. 2000. Quantification of the impact of HIV-1 reverse transcriptase and protease mutations on the efficacy of rescue HAART. Antiviral. Res. 45:101-114.

348. Parent, L. J., R. P. Bennett, R. C. Craven, T. D. Nelle, N. K. Krishna, J. B. Bowzard, C. B. Wilson, B. A. Puffer, R. C. Montelaro, and J. W. Wills. 1995. Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins. J. Virol. 69:5455-5460.

349. Parent, L. J., T. M. Cairns, J. A. Albert, C. B. Wilson, J. W. Wills, and R. C. Craven. 2000. RNA dimerization defect in a Rous sarcoma virus matrix mutant. J. Virol. 74:164-172.

350. Parent, L. J., C. B. Wilson, M. D. Resh, and J. W. Wills. 1996. Evidence for a second function of the MA sequence in the Rous sarcoma virus Gag protein. J. Virol. 70:1016-1026.

243 351. Park, J. and C. D. Morrow. 1991. Overexpression of the Gag-Pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production. J. Virol. 65:5111-5117.

352. Patnaik, A., V. Chau, F. Li, R. C. Montelaro, and J. W. Wills. 2002. Budding of equine infectious anemia virus is insensitive to proteasome inhibitors. J. Virol. 76:2641-2647.

353. Patnaik, A., V. Chau, and J. W. Wills. 2000. Ubiquitin is part of the retrovirus budding machinery. Proc. Natl. Acad. Sci. 97:13069-13074.

354. Payne, L. N. and P. M. Biggs. 1966. Genetic basis of cellular susceptibility to the Schmidt-Ruppin and Harris strains of Rous sarcoma virus. Virology 29:190-198.

355. Pelchen-Matthews, A., B. Kramer, and M. Marsh. 2003. Infectious HIV-1 assembles in late endosomes in primary macrophages. J. Cell Biol. 162:443- 455.

356. Pepinsky, R. B., I. A. Papayannopoulos, S. Campbell, and V. M. Vogt. 1996. Analysis of Rous sarcoma virus Gag protein by mass spectrometry indicates trimming by host exopeptidase. J. Virol. 70:3313-3318.

357. Pepinsky, R. B. and V. M. Vogt. 1979. Identification of retrovirus matrix proteins by lipid-protein cross-linking. J. Mol. Biol. 131:819-837.

358. Pepinsky, R. B. and V. M. Vogt. 1984. Fine-structure analyses of lipid-protein and protein-protein interactions of Gag protein p19 of the avian sarcoma and leukemia viruses by cyanogen bromide mapping. J. Virol. 52:145-153.

359. Perez, L. G., G. L. Davis, and E. Hunter. 1987. Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions. J. Virol. 61:2981-2988.

360. Perez, M., R. C. Craven, and J. C. de la Torre. 2003. The small RING finger protein Z drives arenavirus budding: Implications for antiviral strategies. PROC. NATL. ACAD. SCI. 100:12978-12983.

361. Perrin-Tricaud, C., J. Davoust, and I. M. Jones. 1999. Tagging the human immunodeficiency virus Gag protein with green fluorescent protein. Minimal evidence for colocalisation with actin. Virology 255:20-25.

362. Peters, G. G. and J. Hu. 1980. Reverse transcriptase as the major determinant for selective packaging of tRNA's into Avian sarcoma virus particles. J. Virol. 36:692-700.

244 363. Pickart, C. M. 2001. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70:503-33.:503-533.

364. Pike, L. J. 2004. Lipid rafts: heterogeneity on the high seas. Biochem. J. 378:281-292.

365. Polevoda, B. and F. Sherman. 2002. The diversity of acetylated proteins. Genome Biol. 3:reviews0006.1-reviews0006.6

366. Polo, S., S. Sigismund, M. Faretta, M. Guidi, M. R. Capua, G. Bossi, H. Chen, P. De Camilli, and P. P. Di Fiore. 2002. A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins. Nature 416:451-455.

367. Poon, D. T. K., E. N. Chertova, and D. E. Ott. 2002. Human Immunodeficiency Virus Type 1 Preferentially Encapsidates Genomic RNAs That Encode Pr55Gag: Functional Linkage between Translation and RNA Packaging. Virology 293:368-378.

368. Popov, S., M. Rexach, G. Zybarth, N. Reiling, M. A. Lee, L. Ratner, C. M. Lane, M. S. Moore, G. Blobel, and M. Bukrinsky. 1998. Viral protein R regulates nuclear import of the HIV-1 pre-integration complex. EMBO J 17:909-917.

369. Popov, S., M. Rexach, L. Ratner, G. Blobel, and M. Bukrinsky. 1998. Viral Protein R Regulates Docking of the HIV-1 Preintegration Complex to the Nuclear Pore Complex. J. Biol. Chem. 273:13347-13352.

370. Pornillos, O., S. L. Alam, D. R. Davis, and W. I. Sundquist. 2002. Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6 protein. Nat. Struct. Biol. 9:812-817.

371. Pornillos, O., S. L. Alam, R. L. Rich, D. G. Myszka, D. R. Davis, and W. I. Sundquist. 2002. Structure and functional interactions of the Tsg101 UEV domain. EMBO J. 21:2397-2406.

372. Pornillos, O., J. E. Garrus, and W. I. Sundquist. 2002. Mechanisms of enveloped RNA virus budding. Trends Cell Biol. 12:569-579.

373. Pornillos, O., D. S. Higginson, K. M. Stray, R. D. Fisher, J. E. Garrus, M. Payne, G. P. He, H. E. Wang, S. G. Morham, and W. I. Sundquist. 2003. HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein. The J. Cell Biol. 162:425-434.

374. Prats, A. C., C. Roy, P. A. Wang, M. Erard, V. Housset, C. Gabus, C. Paoletti, and J. L. Darlix. 1990. cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA. J. Virol. 64:774-783.

245 375. Prats, A. C., L. Sarih, C. Gabus, S. Litvak, G. Keith, and J.-L. Darlix. 1988. Small finger protein of avian and murine retorviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO J. 7:1777-1783.

376. Puffer, B. A., L. J. Parent, J. W. Wills, and R. C. Montelaro. 1997. Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein. J. Virol. 71:6541-6546.

377. Putterman, D., R. B. Pepinsky, and V. M. Vogt. 1990. Ubiquitin in avian leukosis virus particles. Virology 176:633-637.

378. Quigley, J. P., D. B. Rifkin, and E. Reich. 1971. Phospholipid composition of Rous sarcoma virus, host cell membranes and other enveloped RNA viruses. Virology 46:106-116.

379. Rabson,A.B. and B.J.Graves. 1997. Synthesis and processing of viral RNA, p. 205-262. In J.M.Coffin, S.H.Hughes, and H.E.Varmus (eds.), Retroviruses. Cold Spring Harbor Laboratory Press, Plainview, NY.

380. Raiborg, C., K. G. Bache, D. J. Gillooly, I. H. Madshus, E. Stang, and H. Stenmark. 2002. Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat Cell Biol 4:394-398.

381. Ramboarina, S., S. Druillennec, N. Morellet, S. Bouaziz, and B. P. Roques. 2004. Target Specificity of Human Immunodeficiency Virus Type 1 NCp7 Requires an Intact Conformation of Its CCHC N-Terminal Zinc Finger. J. Virol. 78:6682-6687.

382. Rand, R. P. and V. A. Parsegian. 1989. Hydration forces between phospholipid bilayers. Biochimica et Biophysica Acta (BBA) - Reviews on Biomembranes 988:351-376.

383. Rangasamy, D. and V. G. Wilson. 2000. Bovine Papillomavirus E1 Protein Is Sumoylated by the Host Cell Ubc9 Protein. J. Biol. Chem. 275:30487-30495.

384. Rangasamy, D., K. Woytek, S. A. Khan, and V. G. Wilson. 2000. SUMO-1 Modification of Bovine Papillomavirus E1 Protein Is Required for Intranuclear Accumulation. J. Biol. Chem. 275:37999-38004.

385. Raposo, G., M. Moore, D. Innes, R. Leijendekker, A. Leigh-Brown, P. Benaroch, and H. Geuze. 2002. Human Macrophages Accumulate HIV-1 Particles in MHC II Compartments. Traffic 3:718-729.

386. Raymond, C. K., I. Howald-Stevenson, C. A. Vater, and T. H. Stevens. 1992. Morphological classification of the yeast vacuolar protein sorting mutants: Evidence for a prevacuolar compartment in class E vps mutants. Mol. Biol. Cell 3:1389-1402.

246 387. Reed, S. I. 2003. Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nat. Rev. Mol. Cell Biol. 4:855-864.

388. Reggiori, F. and H. R. Pelham. 2001. Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting. EMBO J. 20:5176- 5186.

389. Reicin, A. S., S. Paik, R. D. Berkowitz, J. Luban, I. Lowy, and S. P. Goff. 1995. Linker insertion mutations in the human immunodeficiency virus type 1 gag gene: effects on virion particle assembly, release and infectivity. J. Virol. 69:642-650.

390. Reil, H., A. A. Bukovsky, H. R. Gelderblom, and H. G. Gottlinger. 1998. Efficient HIV-1 replication can occur in the absence of the viral matrix protein. EMBO J. 17:2699-2708.

391. Rein, A., M. R. McClure, N. R. Rice, R. B. Luftig, and A. M. Schultz. 1986. Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus. Proc. Natl. Acad. Sci. 83:7246-7250.

392. Rey, O., J. Canon, and P. Krogstad. 1996. HIV-1 Gag protein associates with F-actin present in microfilaments. Virology 220:530-534.

393. Rhee, S. S. and E. Hunter. 1987. Myristylation is required for intracellular transport but not for assembly of D-type retrovirus capsids. J. Virol. 61:1045- 1053.

394. Rhee, S. S. and E. Hunter. 1990. Structural role of the matrix protein of type D retroviruses in Gag polyprotein stability and capsid assembly. J. Virol. 64:4383-4389.

395. Richardson, J. H., L. A. Child, and A. M. Lever. 1993. Packaging of human immunodeficiency virus type 1 RNA requires cis-acting sequences outside the 5' leader region. J. Virol. 67:3997-4005.

396. Rocca, A., C. Lamaze, A. Subtil, and A. Dautry-Varsat. 2001. Involvement of the ubiquitin/proteasome system in sorting of the interleukin 2 receptor B chain to late endocytic compartments. Mol. Biol. Cell 12:1293-1301.

397. Rodriguez, M. S., C. Dargemont, and R. T. Hay. 2001. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J. Biol. Chem. 276:12654-12659.

398. Roe, T., T. C. Reynolds, G. Yu, and P. O. Brown. 1993. Integration of murine leukemia virus DNA depends on mitosis. EMBO J. 12:2099-2108.

399. Rogan, S. and S. Heaphy. 2000. The vaccinia virus E3L protein interacts with SUMO-1 and ribosomal protein L23a in a yeast two hybrid assay. Virus Genes

247 21:193-195.

400. Roth, A. F. and N. G. Davis. 2000. Ubiquitination of the PEST-like endocytosis signal of the yeast α-factor receptor. J. Biol. Chem. 275:8143- 8153.

401. Roth, M. G., R. V. Srinivas, and R. W. Compans. 1983. Basolateral maturation of retroviruses in polarized epithelial cells. J. Virol. 45:1065-1073.

402. Rothenberg, E., D. Smotkin, D. Baltimore, and R. A. Weinberg. 1977. In vitro synthesis of infectious DNA of murine leukaemia virus. Nature 269:122- 126.

403. Rotin, D., O. Staub, and R. Haguenauer-Tsapis. 2000. Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. J Membr. Biol 176:1-17.

404. Rous, P. 1911. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J. Exp. Med. 13:397-411.

405. Rousso, I., M. B. Mixon, B. K. Chen, and P. S. Kim. 2000. Palmitoylation of the HIV-1 envelope glycoprotein is critical for viral infectivity. Proc. Natl. Acad. Sci. 97:13523-13525.

406. Rubin, H. 1965. Genetic control of cellular susceptibility to pseudotypes of Rous sarcoma virus. Virology 26:270-6.:270-276.

407. Sachdev, S., L. Bruhn, H. Sieber, A. Pichler, F. Melchior, and R. Grosschedl. 2001. PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies. Genes Dev. 15:3088-3103.

408. Sakalian, M., J. W. Wills, and V. M. Vogt. 1994. Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants. J. Virol. 68:5969-5981.

409. Sakurai, A., J. Yasuda, H. Takano, Y. Tanaka, M. Hatakeyama, and H. Shida. 2004. Regulation of human T-cell leukemia virus type 1 (HTLV-1) budding by ubiquitin ligase Nedd4. Microbes. Infect. 6:150-156.

410. Sampson, D. A., M. Wang, and M. J. Matunis. 2001. The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J. Biol. Chem. 276:21664-21669.

411. Sandefur, S., R. M. Smith, V. Varthakavi, and P. Spearman. 2000. Mapping and characterization of the N-terminal I domain of human immunodeficiency virus type 1 Pr55(Gag). J. Virol. 74:7238-7249.

248 412. Sandefur, S., V. Varthakavi, and P. Spearman. 1998. The I domain is required for efficient plasma membrane binding of human immunodeficiency virus type 1 Pr55Gag. J. Virol. 72:2723-2732.

413. Sandrin, V., D. Muriaux, J. L. Darlix, and F. L. Cosset. 2004. Intracellular Trafficking of Gag and Env Proteins and Their Interactions Modulate Pseudotyping of Retroviruses. J. Virol. 78:7153-7164.

414. Sasaki, H., M. Nakamura, T. Ohno, Y. Matsuda, Y. Yuda, and Y. Nonomura. 1995. Myosin-actin interaction plays an important role in human immunodeficiency virus type 1 release from host cells. Proc. Natl. Acad. Sci. 92:2026-2030.

415. Sattentau, Q. J., J. P. Moore, F. Vignaux, F. Traincard, and P. Poignard. 1993. Conformational changes induced in the envelope glycoproteins of the human and simian immunodeficiency viruses by soluble receptor binding. J. Virol. 67:7383-7393.

416. Sayah, D. M. and J. Luban. 2004. Selection for Loss of Ref1 Activity in Human Cells Releases Human Immunodeficiency Virus Type 1 from Cyclophilin A Dependence during Infection. J. Virol. 78:12066-12070.

417. Scarlata, S. and C. Carter. 2003. Role of HIV-1 Gag domains in viral assembly. Biochimica et Biophysica Acta (BBA) - Biomembranes 1614:62-72.

418. Schaefer-Klein, J., I. Givol, E. V. Barsov, J. M. Whitcomb, M. VanBrocklin, D. N. Foster, M. J. Federspiel, and S. H. Hughes. 1998. The EV-O-derived cell line DF-1 supports the efficient replication of avian leukosis- sarcoma viruses and vectors. Virology 248:305-311.

419. Scheifele, L. Z., R. A. Garbitt, J. D. Rhoads, and L. J. Parent. 2002. Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein. Proc. Natl. Acad. Sci. 99:3944-3949.

420. Scheifele, L. Z., J. D. Rhoads, and L. J. Parent. 2003. Specificity of Plasma Membrane Targeting by the Rous Sarcoma Virus Gag Protein. J. Virol. 77:470- 480.

421. Scheuring, S., R. A. Rohricht, B. Schoning-Burkhardt, A. Beyer, S. Muller, H. F. Abts, and K. Kohrer. 2001. Mammalian Cells Express Two VPS4 Proteins Both of Which are Involved in Intracellular Protein Trafficking. J. Mol. Biol. 312:469-480.

422. Schmidt, D. and S. Muller. 2002. Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity. PROC. NATL. ACAD. SCI. 99:2872-2877.

423. Schnell, J. D. and L. Hicke. 2003. Non-traditional functions of ubiquitin and

249 ubiquitin-binding proteins. J. Biol. Chem. 278:35857-35860.

424. Schroder, A. R., P. Shinn, H. Chen, C. Berry, J. R. Ecker, and F. Bushman. 2002. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110:521-529.

425. Schubert, U., D. E. Ott, E. N. Chertova, R. Welker, U. Tessmer, M. F. Princiotta, J. R. Bennink, H. G. Krausslich, and J. W. Yewdell. 2000. Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Proc. Natl. Acad. Sci. 97:13057-13062.

426. Schultz, A. M. and A. Rein. 1989. Unmyristylated Moloney murine leukemia virus Pr65gag is excluded from virus assembly and maturation events. J. Virol. 63:2370-2373.

427. Schwartz, D. C. and M. Hochstrasser. 2003. A superfamily of protein tags: ubiquitin, SUMO and related modifiers. Trends Biochem. Sci. 28:321-328.

428. Schwartz, D. E., R. Tizard, and W. Gilbert. 1983. Nucleotide sequence of Rous sarcoma virus. Cell 32:853-869.

429. Seeler, J. S. and A. Dejean. 2003. Nuclear and unclear functions of SUMO. Nat. Rev. Mol. Cell Biol. 4:690-699.

430. Serhan, F., M. Penaud, C. Petit, T. Leste-Lasserre, S. Trajcevski, D. Klatzmann, G. Duisit, P. Sonigo, and P. Moullier. 2004. Early detection of a two-long-terminal-repeat junction molecule in the cytoplasm of recombinant murine leukemia virus-infected cells. J. Virol. 78:6190-6199.

431. Shank, P. R., S. H. Hughes, H. J. Kung, J. E. Majors, N. Quintrell, R. V. Guntaka, J. M. Bishop, and H. E. Varmus. 1978. Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA. Cell 15:1383-1395.

432. Shank, P. R. and H. E. Varmus. 1978. Virus-specific DNA in the cytoplasm of avian sarcoma virus-infected cells is a precursor to covalently closed circular viral DNA in the nucleus. J. Virol. 25:104

433. Shehu-Xhilaga, M., H. G. Kraeusslich, S. Pettit, R. Swanstrom, J. Y. Lee, J. A. Marshall, S. M. Crowe, and J. Mak. 2001. Proteolytic Processing of the P2/Nucleocapsid Cleavage Site Is Critical for Human Immunodeficiency Virus Type 1 RNA Dimer Maturation. J. Virol. 75:9156-9164.

434. Shehu-Xhilaga, M., S. Ablan, D. G. Demirov, C. Chen, R. C. Montelaro, and E. O. Freed. 2004. Late Domain-Dependent Inhibition of Equine Infectious Anemia Virus Budding. J. Virol. 78:724-732.

435. Shields, A., W. N. Witte, E. Rothenberg, and D. Baltimore. 1978. High

250 frequency of aberrant expression of Moloney murine leukemia virus in clonal infections. Cell 14:601-609.

436. Shih, S. C., D. J. Katzmann, J. D. Schnell, M. Sutanto, S. D. Emr, and L. Hicke. 2002. Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis. Nat Cell Biol 4:389-393.

437. Shimada, M., H. Hosaka, H. Takaku, J. S. Smith, M. J. Roth, S. Inouye, and M. Inouye. 1994. Specificity of priming reaction of HIV-1 reverse transcriptase, 2'-OH or 3'-OH. J. Biol. Chem. 269:3925-3927.

438. Shoemaker, C., S. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore. 1980. Structure of a cloned circular Moloney murine leukemia virus DNA molecule containing an inverted segment: implications for retrovirus integration. Proc. Natl. Acad. Sci. 77:3932-3936.

439. Shoemaker, C., J. Hoffman, S. P. Goff, and D. Baltimore. 1981. Intramolecular integration within Moloney murine leukemia virus DNA. J. Virol. 40:164-172.

440. Skehel, J. J. and D. C. Wiley. 2000. Receptor Binding and Membrane Fusion in Virus Entry: The Influenza Hemagglutinin. Annu. Rev. Biochem. 69:531- 569.

441. Smith, J. G., W. Mothes, S. C. Blacklow, and J. M. Cunningham. 2004. The Mature Avian Leukosis Virus Subgroup A Envelope Glycoprotein Is Metastable, and Refolding Induced by the Synergistic Effects of Receptor Binding and Low pH Is Coupled to Infection. J. Virol. 78:1403-1410.

442. Sokolskaja, E., D. M. Sayah, and J. Luban. 2004. Target Cell Cyclophilin A Modulates Human Immunodeficiency Virus Type 1 Infectivity. J. Virol. 78:12800-12808.

443. Sonstegard, T. S. and P. B. Hackett. 1996. Autogenous regulation of RNA translation and packaging by Rous sarcoma virus Pr76gag. J. Virol. 70:6642- 6652.

444. Spence, J., R. R. Gali, G. Dittmar, F. Sherman, M. Karin, and D. Finley. 2000. Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell 102:67-76.

445. Spengler, M. L., K. Kurapatwinski, A. R. Black, and J. Azizkhan-Clifford. 2002. SUMO-1 Modification of Human Cytomegalovirus IE1/IE72. J. Virol. 76:2990-2996.

446. Srinivasakumar, N., M.-L. Hammarskjold, and D. Rekosh. 1995. Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol

251 precursor incorporation. J. Virol. 69:6106-6114.

447. Staub, O., S. Dho, P. Henry, J. Correa, T. Ishikawa, J. McGlade, and D. Rotin. 1996. WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome. EMBO J. 15:2371-2380.

448. Staub, O., I. Gautschi, T. Ishikawa, K. Breitschopf, A. Ciechanover, L. Schild, and D. Rotin. 1997. Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. EMBO J. 16:6325-6336.

449. Stewart, L., G. Schatz, and V. M. Vogt. 1990. Properties of avian retrovirus particles defective in viral protease. J. Virol. 64:5076-5092.

450. Stoltzfus, C. M., K. Dimock, S. Horikami, and T. A. Ficht. 1983. Stabilities of avian sarcoma virus RNAs: comparison of subgenomic and genomic species with cellular mRNAs. J. Gen. Virol. 64 (Pt 10):2191-2202.

451. Strack, B., A. Calistri, M. A. Accola, G. Palu, and H. G. Gottlinger. 2000. A role for ubiquitin ligase recruitment in retrovirus release. Proc. Natl. Acad. Sci. 97:13063-13068.

452. Strack, B., A. Calistri, S. Craig, E. Popova, and H. G. Gottlinger. 2003. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114:689-699.

453. Stromberg, K., N. E. Hurley, N. L. Davis, R. R. Rueckert, and E. Fleissner. 1974. Structural studies of avian myeloblastosis virus: comparison of polypeptides in virion and core component by dodecyl sulfate- polyacrylamide gel electrophoresis. J. Virol. 13:513-528.

454. Strous, G. J., P. van Kerkhof, R. Govers, A. Ciechanover, and A. L. Schwartz. 1996. The ubiquitin conjugation system is required for ligand- induced endocytosis and degradation of the growth hormone receptor. EMBO J. 15:3806-3812.

455. Stuchell, M. D., J. E. Garrus, B. Muller, K. M. Stray, S. Ghaffarian, R. McKinnon, H. G. Krausslich, S. G. Morham, and W. I. Sundquist. 2004. The Human Endosomal Sorting Complex Required for Transport (ESCRT-I) and Its Role in HIV-1 Budding. J. Biol. Chem. 279:36059-36071.

456. Swanson, K. A., R. S. Kang, S. D. Stamenova, L. Hicke, and I. Radhakrishnan. 2003. Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation. EMBO J. 22:4597-4606.

457. Swanstrom,R. and J.W.Wills. 1997. Synthesis, assembly, and processing of viral proteins, p. 263-334. In J.M.Coffin, S.H.Hughes, and H.E.Varmus (eds.), Retroviruses. Cold Spring Harbor Laboratory Press, Plainview, NY.

252 458. Takahashi, K. i., S. Baba, Y. Koyanagi, N. Yamamoto, H. Takaku, and G. Kawai. 2001. Two Basic Regions of NCp7 Are Sufficient for Conformational Conversion of HIV-1 Dimerization Initiation Site from Kissing-loop Dimer to Extended-duplex Dimer. J. Biol. Chem. 276:31274-31278.

459. Tan, K., J. h. Liu, J. h. Wang, S. Shen, and M. Lu. 1997. Atomic structure of a thermostable subdomain of HIV-1 gp41. Proc. Natl. Acad. Sci. 94:12303- 12308.

460. Tanaka, N., K. Kaneko, H. Asao, H. Kasai, Y. Endo, T. Fujita, T. Takeshita, and K. Sugamura. 1999. Possible Involvement of a Novel STAM- associated Molecule "AMSH" in Intracellular Signal Transduction Mediated by Cytokines. J. Biol. Chem. 274:19129-19135.

461. Tanese, N. and S. P. Goff. 1988. Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities. Proc. Natl. Acad. Sci 85:1777-1781.

462. Tang, C., Y. Ndassa, and M. F. Summers. 2002. Structure of the N-terminal 283-residue fragment of the immature HIV-1 Gag polyprotein. Nat. Struct. Biol. 9:537-543.

463. Tang, S., T. Murakami, B. E. Agresta, S. Campbell, E. O. Freed, and J. G. Levin. 2001. Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants That Exhibit Aberrant Core Morphology and Are Blocked in Initiation of Reverse Transcription in Infected Cells. J. Virol. 75:9357-9366.

464. Tang, Y., U. Winkler, E. O. Freed, T. A. Torrey, W. Kim, H. Li, S. P. Goff, and H. C. Morse. 1999. Cellular motor protein KIF-4 associates with retroviral Gag. J. Virol. 73:10508-10513.

465. Tanzi, G. O., A. J. Piefer, and P. Bates. 2003. Equine Infectious Anemia Virus Utilizes Host Vesicular Protein Sorting Machinery during Particle Release. J. Virol. 77:8440-8447.

466. Telesnitsky,A. and S.Goff. 1997. Reverse transcriptase and the generation of retroviral DNA, p. 121-160. In J.M.Coffin, S.H.Hughes, and H.E.Varmus (eds.), Retroviruses. Cold Spring Harbor Laboratory Press, Plainview, NY.

467. Temin, H. M. and H. Rubin. 1958. Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology 6:669-688.

468. Terrell, J., S. Shih, R. Dunn, and L. Hicke. 1998. A function for monoubiquitination in the internalization of a G protein-coupled receptor. Mol. Cell 1:193-202.

469. Thali, M., A. A. Bukovski, E. Kondo, B. Rosenwirth, C. T. Walsh, J. G.

253 Sodroski, and H. G. Gottlinger. 1994. Functional association of cyclophilin A with HIV-1 virions. Nature 372:363-365.

470. Timmins, J., G. Schoehn, S. Ricard-Blum, S. Scianimanico, T. Vernet, R. W. H. Ruigrok, and W. Weissenhorn. 2003. Ebola Virus Matrix Protein VP40 Interaction with Human Cellular Factors Tsg101 and Nedd4. J. Mol. Biol. 326:493-502.

471. Toh, H., R. Kikuno, H. Hayashida, T. Miyata, W. Kugimiya, S. Inouye, S. Yuki, and K. Saigo. 1985. Close structural resemblance between putative polymerase of a Drosophila transposable genetic element 17.6 and pol gene product of Moloney murine leukaemia virus. EMBO J. 4:1267-1272.

472. Tounekti, N., M. Mougel, C. Roy, R. Marquet, J. L. Darlix, J. Paoletti, B. Ehresmann, and C. Ehresmann. 1992. Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA. J. Mol. Biol. 223:205-220.

473. Tritel, M. and M. D. Resh. 2000. Kinetic analysis of human immunodeficiency virus type 1 assembly reveals the presence of sequential intermediates. J. Virol. 74:5845-5855.

474. Trono, D. 1992. Partial reverse transcripts in virions from human immunodeficiency and murine leukemia virus. J. Virol. 66:4893-4900.

475. Urbanowski, J. L. and R. C. Piper. 2001. Ubiquitin Sorts Proteins into the Intralumenal Degradative Compartment of the Late-Endosome/Vacuole. Traffic 2:622-630.

476. Vana, M. L., Y. Tang, A. Chen, G. Medina, C. Carter, and J. Leis. 2004. Role of Nedd4 and Ubiquitination of Rous Sarcoma Virus Gag in Budding of Virus-Like Particles from Cells. J. Virol. 78:13943-13953.

477. Verderame, M. F., T. D. Nelle, and J. W. Wills. 1996. The membrane-binding domain of the Rous sarcoma virus Gag protein. J. Virol. 70:2664-2668.

478. VerPlank, L., F. Bouamr, T. J. LaGrassa, B. Agresta, A. Kikonyogo, J. Leis, and C. A. Carter. 2001. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc. Natl. Acad. Sci. 98:7724-7729.

479. Vincent, O., L. Rainbow, J. Tilburn, H. N. Arst, Jr., and M. A. Penalva. 2003. YPXL/I is a protein interaction motif recognized by aspergillus PalA and its human homologue, AIP1/Alix. Mol. Cell Biol 23:1647-1655.

480. Vodicka, M. A., D. M. Koepp, P. A. Silver, and M. Emerman. 1998. HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes Dev. 12:175-185.

254 481. Vogt, P. K. and R. Ishizaki. 1965. Reciprocal patterns of genetic resistance to avian tumor viruses in two lines of chickens. Virology 26:664-672.

482. Vogt,V.M. 1997. Retroviral virions and genomes, J.M.Coffin, S.H.Hughes, and H.Varmus (eds.), Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

483. Vogt, V. M. and M. N. Simon. 1999. Mass Determination of Rous Sarcoma Virus Virions by Scanning Transmission Electron Microscopy. J. Virol. 73:7050-7055.

484. von der Helm, K. 1977. Cleavage of Rous sarcoma viral polypeptide precursor into internal structural proteins in vitro involves viral protein p15. Proc. Natl. Acad. Sci. 74:911-915.

485. von Schwedler, U., J. Song, C. Aiken, and D. Trono. 1993. Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells. J. Virol. 67:4945-4955.

486. von Schwedler, U. K., T. L. Stemmler, V. Y. Klishko, S. Li, K. H. Albertine, D. R. Davis, and W. I. Sundquist. 1998. Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly. EMBO J. 17:1555-1568.

487. von Schwedler, U. K., K. M. Stray, J. E. Garrus, and W. I. Sundquist. 2003. Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J. Virol. 77:5439-5450.

488. von Schwedler, U. K., M. Stuchell, B. Muller, D. M. Ward, H. Y. Chung, E. Morita, H. E. Wang, T. Davis, G. P. He, D. M. Cimbora, A. Scott, H. G. Krausslich, J. Kaplan, S. G. Morham, and W. I. Sundquist. 2003. The protein network of HIV budding. Cell 114:701-713.

489. Wang, H., K. M. Norris, and L. M. Mansky. 2002. Analysis of bovine leukemia virus Gag membrane targeting and late domain function. J. Virol. 76:8485-8493.

490. Wang, H., N. J. Machesky, and L. M. Mansky. 2004. Both the PPPY and PTAP Motifs Are Involved in Human T-Cell Leukemia Virus Type 1 Particle Release. J. Virol. 78:1503-1512.

491. Weinberg, J. B., T. J. Matthews, B. R. Cullen, and M. H. Malim. 1991. Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes. J. Exp. Med. 174:1477-1482.

492. Weissenhorn, W., A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley. 1997. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387:426- 430.

255 493. Weldon, R. A., Jr., C. R. Erdie, M. G. Oliver, and J. W. Wills. 1990. Incorporation of chimeric Gag protein into retroviral particles. J. Virol. 64:4169-4179.

494. Weldon, R. A., Jr., P. Sarkar, S. M. Brown, and S. K. Weldon. 2003. Mason-Pfizer monkey virus Gag proteins interact with the human sumo conjugating enzyme, hUbc9. Virology 314:62-73.

495. Weldon, R. A., Jr. and J. W. Wills. 1993. Characterization of a small (25- kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release. J. Virol. 67:5550-5561.

496. Werner, S. and B. M. Wohrl. 2000. Asymmetric subunit organization of heterodimeric Rous sarcoma virus reverse transcriptase alphabeta: localization of the polymerase and RNase H active sites in the alpha subunit. J. Virol. 74:3245-3252.

497. Whitcomb, J. M., B. A. Ortiz-Conde, and S. H. Hughes. 1995. Replication of avian leukosis viruses with mutations at the primer binding site: Use of alternative tRNAs as primers. J. Virol. 69:6228-6238.

498. Wilk, T., B. Gowen, and S. D. Fuller. 1999. Actin associates with the nucleocapsid domain of the human immunodeficiency virus Gag polyprotein. J. Virol. 73:1931-1940.

499. Wilk, T., I. Gross, B. E. Gowen, T. Rutten, F. de Haas, R. Welker, H. G. Krausslich, P. Boulanger, and S. D. Fuller. 2001. Organization of immature human immunodeficiency virus type 1. J. Virol. 75:759-771.

500. Wills, J. W., C. E. Cameron, C. B. Wilson, Y. Xiang, R. P. Bennett, and J. Leis. 1994. An assembly domain of the Rous sarcoma virus Gag protein required late in budding. J. Virol. 68:6605-6618.

501. Wills, J. W. and R. C. Craven. 1991. Form, function, and use of retroviral Gag proteins [editorial]. AIDS 5:639-654.

502. Wills, J. W., R. C. Craven, and J. A. Achacoso. 1989. Creation and expression of myristylated forms of Rous sarcoma virus Gag protein in mammalian cells. J. Virol. 63:4331-4343.

503. Wills, J. W., R. C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C. R. Erdie. 1991. Suppression of retroviral MA deletions by the amino-terminal membrane -binding domain of p60src. J. Virol. 65:3804-3812.

504. Wu, X., Y. Li, B. Crise, and S. M. Burgess. 2003. Transcription start regions in the human genome are favored targets for MLV integration. Science 300:1749-1751.

256 505. Wyma, D. J., A. Kotov, and C. Aiken. 2000. Evidence for a stable interaction of gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1 particles. J. Virol. 74:9381-9387.

506. Xiang, Y., C. E. Cameron, J. W. Wills, and J. Leis. 1996. Fine mapping and characterization of the Rous sarcoma virus Pr76gag late assembly domain. J. Virol. 70:5695-5700.

507. Xiang, Y., T. W. Ridky, N. K. Krishna, and J. Leis. 1997. Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation. J. Virol. 71:2083-2091.

508. Yamashita, M. and M. Emerman. 2004. Capsid Is a Dominant Determinant of Retrovirus Infectivity in Nondividing Cells. J. Virol. 78:5670-5678.

509. Yasuda, J., E. Hunter, M. Nakao, and H. Shida. 2002. Functional involvement of a novel Nedd4-like ubiquitin ligase on retrovirus budding. EMBO Rep 3:636-640.

510. Yasuda, J., M. Nakao, Y. Kawaoka, and H. Shida. 2003. Nedd4 Regulates Egress of Ebola Virus-Like Particles from Host Cells. J. Virol. 77:9987-9992.

511. Yoshimori, T., F. Yamagata, A. Yamamoto, N. Mizushima, Y. Kabeya, A. Nara, I. Miwako, M. Ohashi, M. Ohsumi, and Y. Ohsumi. 2000. The Mouse SKD1, a Homologue of Yeast Vps4p, Is Required for Normal Endosomal Trafficking and Morphology in Mammalian Cells. Mol. Biol. Cell 11:747-763.

512. Young, J. A. T., P. Bates, K. Willert, and H. E. Varmus. 1990. Efficient incorporation of human CD4 protein into avian leukosis virus particles. Science 250:1421-1423.

513. Yu, F., S. M. Joshi, Y. M. Ma, R. L. Kingston, M. N. Simon, and V. M. Vogt. 2001. Characterization of Rous sarcoma virus Gag particles assembled in vitro. J. Virol. 75:2753-2764.

514. Yu, X., X. Yuan, Z. Matsuda, T.-H. Lee, and M. Essex. 1992. The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral enveloped protein into mature virions. J. Virol. 66:4966-4971.

515. Yuan, B., X. Li, and S. P. Goff. 1999. Mutations altering the moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle. EMBO J 18:4700-4710.

516. Yuan, X., X. Yu, T.-H. Lee, and M. Essex. 1993. Mutations in the N-terminal region of human immunodeficiency virus type 1 matrix protein block intracellular transport of the Gag precursor. J. Virol. 67:6387-6394.

517. Zabransky, A., E. Hunter, and M. Sakalian. 2002. Identification of a

257 Minimal HIV-1 Gag Domain Sufficient for Self-Association. Virology 294:141-150.

518. Zaitseva, M., K. Peden, and H. Golding. 2003. HIV coreceptors: role of structure, posttranslational modifications, and internalization in viral-cell fusion and as targets for entry inhibitors. Biochim. Biophys. Acta 1614:51-61.

519. Zheng, Y. H., A. Plemenitas, T. Linnemann, O. T. Fackler, and B. M. Peterlin. 2001. Nef increases infectivity of HIV via lipid rafts. Current Biology 11:875-879.

520. Zhou, W., L. J. Parent, J. W. Wills, and M. D. Resh. 1994. Identification of a membrane-binding domain within the amino- terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J. Virol. 68:2556-2569.

258 VITA

Jared L. Spidel

Education:

1995-1999 B.S. Biochemistry Lebanon Valley College Annville, PA

1999-2005 Ph.D. Microbiology Pennsylvania State University & Immunology Hershey, PA

Publications:

Zhou Z., D. Fisher, J. Spidel, J. Greenfield, B. Patson, A. Fazal, C. Wigal, O. A. Moe, J. D. Madura. Kinetic and docking studies of the interaction of quinones with the quinone reductase active site. Biochemistry. 2003 Feb 25;42(7):1985-94.

Spidel J. L., R. C. Craven, C. B. Wilson, A. Patnaik, H. Wang, L. M. Mansky, J. W. Wills. Lysines close to the Rous sarcoma virus late domain critical for budding. J Virol. 2004 Oct;78(19):10606-16.

Johnson M. C., J. L. Spidel, D. Ako-Adjei, J. W. Wills, V. M. Vogt. The C-terminal half of TSG101 blocks Rous sarcoma virus budding and sequesters Gag into unique non- endosomal structures. J Virol. In press.

Spidel J. L. and J. W. Wills. A potential sumoylation site in Rous sarcoma virus capsid important for replication. In preparation.

Spidel J. L., M. Johnson, V. M. Vogt, J. W. Wills. Analysis of ESCRT protein complexes during Rous sarcoma virus budding. In preparation.