VARIATIONS IN THE V3 CROWN OF HIV-1 ENVELOPE IMPACT AFFINITY FOR

CCR5 AND AFFECT ENTRY AND REPLICATIVE FITNESS

By

MICHAEL ANDREW LOBRITZ

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Dissertation advisor: Eric J. Arts, Ph.D.

Department of Molecular Biology and Microbiology

CASE WESTERN RESERVE UNIVERSITY

August 2007 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

______

candidate for the Ph.D. degree *.

(signed)______(chair of the committee)

______

______

______

______

______

(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents

Chapter 1:

Introduction...... 16

1.A. HIV and AIDS...... 17

1.B. Retroviruses: Structure, Organization, and Replication...... 20

1.B.1. HIV-1 Genome...... 20

1.B.2. HIV-1 Particle...... 23

1.B.3. HIV-1 Replication...... 26

1.B.3.a. Transmission and Replication Dynamics...... 26

1.B.3.a. Host Cell Tropism and Replication...... 26

1.B.3.b. HIV-1 Envelope and Host Cell Entry...... 29

1.B.3.c. Reverse Transcription...... 33

1.B.3.d. Nuclear Translocation and Integration...... 36

1.B.3.e. Transcription and RNA Export...... 39

1.B.3.f. Assembly, Budding and Maturation...... 42

1.B.3.g. HIV-1 Accessory Proteins...... 43

1.D. Antiretroviral Therapy...... 44

1.E. HIV-1 Receptors and Implications of Tropism...... 47

1.F. Interaction of HIV-1 Envelope and CCR5...... 50

1.G. Inhibition of HIV-1 Entry...... 55

1.G.1. Attachment Inhibitors...... 55

1.G.2. Fusion Inhibitors and Mechanisms of Resistance...... 57

1.G.3. Coreceptor Inhibitors and Mechansims of Resistance...... 59

2 1.G.3.a. Chemokine Analogs...... 59

1.G.3.b. Small Molecule Antagonists...... 61

1.G.3.c. Models of Resistance to Coreceptor Inhibitors...... 62

1.H. Implication of HIV-1 Entry Efficiency...... 68

1.I. Hypotheses...... 70

Chapter 2: Natural Variation in the V3 Crown of Human Immunodeficiency Virus...... 72

Type 1 Affects Replicative Fitness and Entry Inhibitor Sensitivity

2.1. Preface...... 73

2.2. Abstract...... 74

2.3. Introduction...... 75

2.4. Materials and Methods...... 78

2.4.1. Cells and Viruses...... 78

2.4.2. Western Detection of Viral Proteins...... 80

2.4.3. Single-cycle Infection Assays...... 80

2.4.4. Entry Inhibitor Sensitivity...... 81

2.4.5. Replicative Fitness from Competition Assays...... 82

2.5. Results...... 84

2.5.1. Natural Variation at HIV-1 V3 Positions 318 and 319...... 84

2.5.2. Replicative Fitness of V3 Mutant Chimeric Viruses in PBMC.....94

2.5.3. Replication of V3 Mutant Chimeric Viruses in...... 106

Macrophage Cultures

2.5.4. Antiretroviral Susceptibility Conferred by V3 Polymorphisms...108

2.5.5. Comparing Replicative Fitness with Sensitivity to...... 116

3 Entry Inhibitors

2.6. Discussion...... 119

Chapter 3. Differential Sensitivity to Inhibition of Human Immunodeficiency...... 128

Virus Type 1 to PSC-RANTES Involves Competitive CCR5 Binding

3.1. Preface...... 129

3.2. Abstract...... 130

3.3. Introduction...... 131

3.4. Materials and Methods...... 135

3.4.1. Reagents...... 135

3.4.2. Plasmids...... 135

3.4.3. Cells...... 135

3.4.4. Viruses and Pseudoviruses...... 136

3.4.5. Multiple-cycle Infection Assays...... 137

3.4.6. Single-cycle Infection Assays...... 138

3.4.7. Flow Cytometry...... 139

3.4.8. Time-of-addition Assay...... 140

3.5. Results...... 141

3.5.1. Dichotomous Sensitivity to PSC-RANTES in Multiple- and...... 141

Single-Cycle Assays

3.5.2. Dichotomous Multiple- and Single-Cycle Sensitivity...... 145

Differences are Specific to PSC-RANTES

3.5.3. Prolonged Incubation with PSC-RANTES Recapitulates...... 146

Multiple-Cycle Assays

4 3.5.4. Inhibition of HIV-1 Replication by PSC-RANTES with a...... 150

Downregulation-defective Variant of CCR5

3.5.5. Multiplicity of Infection Effects on PSC-RANTES IC50 Value..153

3.5.6. Multiplicity of Infection Effects in Wild Type and M7-CCR5...160

3.5.7. Kinetics of PSC-RANTES Inhibition of Entry...... 160

3.6. Discussion...... 166

Chapter 4. Intrinsic Sensitivity of Human Immunodeficiency Virus Type 1...... 171

Isolates to PSC-RANTES is Mediated by Differential CCR5 Affinity

4.1. Preface...... 172

4.2. Abstract...... 173

4.3. Introduction...... 174

4.4. Materials and Methods...... 177

4.4.1. Reagents...... 177

4.4.2. Plasmids...... 177

4.4.3. Cell Lines...... 177

4.4.4. Pseudovirus Production and Single-Round Entry Assays...... 178

4.4.5. Incorporation of Cleaved gp120 into Virions...... 178

4.4.6. Infection of 293 Cells with Inducible Levels of CCR5...... 179

and CD4

4.4.7. Flow Cytometry...... 180

4.4.8. Kinetic Entry Assay...... 181

4.5. Results...... 182

4.5.1. Neutralization of V3 Crown Mutants by sCD4...... 182

5 4.5.2. Effects of CD4 and CCR5 Expression Levels on Infectivity...... 185

of V3 Crown Polymorphisms

4.5.3. Infectivity Impact of Dominant Negative Hetero-...... 191

oligomerization on V3 Crown Polymorphisms

4.5.4. V3 Crown Polymorphisms Impact on Fusion Kinetics...... 195

4.6. Discussion...... 199

Chapter 5. General Discussion...... 206

5.1. V3 Diversity and Sequence Context...... 208

5.2. Intrinsic Viral Sensitivity and Response to Entry Inhibitor Therapy...... 208

5.3. Evolution of CCR5 Inhibitor Resistance...... 210

5.4. Envelope Function and HIV-1 Pathogenesis...... 210

Bibliography...... 212

6 List of Tables

Table 1. Approved therapeutics for HIV infection...... 46

Table 2. Investigational entry inhibitors for HIV infection...... 56

Table 3. V3 (318/319) amino acid frequency by subtype...... 85

Table 4. Replication of V3 Mutant Chimeric Viruses in Macrophage Cultures...... 107

Table 5. Sensitivity of V3 mutant chimeric viruses to antiretroviral compounds...... 109

(IC50 values)

Table 6. Sensitivity of Full Length Primary Isolate Viruses to Antiretroviral...... 110

Compounds (IC50 values)

Table 7. IC50 Values for Multiple- and Single-cycle Assays...... 144

Table 8. IC50 Values in U87-CD4/M7-CCR5 cells...... 154

7 List of Figures

Figure 1. Landmarks in HIV-1 Infection and Progression to AIDS...... 19

Figure 2. HIV-1 Genomic Organization...... 22

Figure 3. HIV-1 Virion Structure...... 25

Figure 4. HIV-1 Replication Scheme...... 28

Figure 5. HIV-1 Entry...... 31

Figure 6. Reverse Ttranscription...... 35

Figure 7. Integration...... 38

Figure 8. Transcription of Viral RNAs...... 41

Figure 9. Model of Ternary Complex Formation...... 53

Figure 10. Models of Resistance to HIV-1 Entry Inhibitors...... 65

Figure 11. Generation of V3 Mutant Chimeric Viruses...... 88

Figure 12. Analysis of Envelope Incorporation by V3 Mutant Chimeric Viruses...... 91

Figure 13. Coreceptor Tropism and Pseudovirus Infection Efficiency...... 93

Figure 14. Replicative Fitness of V3 Polymorphisms in Three Envelope Contexts...... 96

Figure 15. Total Relative Fitness Values of Chimeric Viruses...... 99

Figure 16. Methods for Determining Replicative Fitness...... 103

Figure 17. Fitness Differences Among NL4-3-V3A1-92RW009 Chimeric Viruses...... 105

Figure 18. Sensitivity of 318/319 Polymorphisms to Entry Inhibitors...... 112

Figure 19. Drug Sensitivity Curves for NL4-3-gp120B5-91-US056 viruses...... 115

Figure 20. Correlation of Entry Inhibitor Sensitivity and Total Relative Fitness...... 118

Figure 21. PSC-RANTES Sensitivity in Multiple- and Single-cycle Assays...... 143

Figure 22. Prolonged Incubation of Cultures with PSC-RANTES...... 148

8 Figure 23. Characterization of U87-CD4/M7-CCR5 cells...... 152

Figure 24. Effects of Virus Expansion on PSC-RANTES IC50 Value...... 157

Figure 25. MOI Effect on PSC-RANTES IC50 Value...... 159

Figure 26. MOI Effects on PSC-RANTES Sensitivity in wt- and M7-CCR5...... 162

Figure 27. Kinetics of PSC-RANTES-mediated Inhibition...... 165

Figure 28. Neutralization of Chimeric Viruses by sCD4...... 184

Figure 29. An Inducible System for Expression of CD4 and CCR5...... 187

Figure 30. Effects of Variable CD4 and CCR5 Expression on Viral Infectivity...... 190

Figure 31. Impact of Dominant Negative Hetero-oligomerization on HIV-1 entry...... 194

Figure 32. Kinetic Analysis of Fusion for V3 Crown Polymorphisms...... 198

9 Acknowledgment

Foremost I would like to thank friends and family who have supported me in my progress throughout my graduate studies. Thanks especially to Kara Lassen, who has been both a critic and friend during the last year of this work.

Members of the Arts laboratory past and present, too numerous to mention in limited space, have been incomprehensibly important in my development as a scientist and as a technician in the laboratory. Specific thanks go to Andre Marozsan, for leaving behind so many projects to pursue, and to Ryan Troyer and Awet Abraha, for teaching me everything I know about the technical aspects of virology.

Lastly, I would like to express gratitude to my committee members, who have always been encouraging of my potential, and especially to my advisor, Eric Arts. Thanks Eric for giving me the opportunity to work in the best environment imaginable for graduate studies, for offering up insights into experimental problems and results, and for the constant influx of new, high quality music.

10 List of Abbreviations

AIDS Acquired immune deficiency syndrome

CA Capsid

CCL5 C-C chemokine ligand 5 (RANTES)

CCR5 C-C Chemokine Receptor 5

CD4 Cluster of differentiation 4

ECL-2 Second extracellular loop

ENF Enfuvirtide, T-20, Fuzeon

FI Fusion inhibitor gp120 envelope glycoprotein 120 gp41 envelope glycoprotein 41

HAART Highly active antiretroviral therapy

HIV-1 Human immunodeficiency virus type 1

HIV-2 Human immunodeficiency virus type 2

HR1, 2 Heptad repeat region 1, 2

HTA Heteroduplex tracking assay

IN Integrase

IC50 50% inhibitory concentration

IU Infectious units

LTNP Long term non-progressor

LTR Long terminal repeat

Luc Luciferase

MA Matrix

11 MAb Monoclonal antibody min Minutes

MIP-1α/β Macrophage inflammatory protein-1 α/β (CCL3/CCL4)

MOI Multiplicity of infection

Nef Negative factor

NNRTI Non-nucleoside reverse transcriptase inhibitor

NRTI Nucleoside reverse transcriptase inhibitor

NVP Nevirapine

ORF Open reading frame

PI Protease inhibitor

PIC Preintegration complex

ponA Ponasterone A

pTEFb Positive transcriptional elongation factor b

R5 CCR5-tropic virus

RANTES Regulated upon activation normal T cell expressed and secreted

Rev Regulator of expression of virion proteins

RLU Relative light units

RRE Rev response element

RT Reverse transcriptase

RTC Reverse transcription complex

sCD4 Soluble CD4

SDF-1 Stromal derived factor-1

SP1, 2 Spacer peptide 1, 2

12 TAR Trasactivation response element

Tat Trasactivator

TCID50 50% Tissue culture infection dose

TM Transmembrane unit

Vif Viral infectivity factor

VLP Virus-like particle

Vpr Viral protein R

Vpu Viral protein U

X4 CXCR4-tropic virus

13 Variations in the V3 Crown of HIV-1 Envelope Impact Affinity for CCR5 and

Affect Entry and Replicative Fitness.

Abstract

by

MICHAEL ANDREW LOBRITZ

Entry inhibitors represent a new class of antiretroviral agents for the treatment of infection with HIV-1. The viral envelope glycoprotein, which mediates interaction with host cell receptors and membrane fusion, is an extremely heterogeneous protein.

Consequently, diverse HIV-1 isolates exhibit a broad range of susceptibility to inhibition by agents that target envelope glycoprotein function. The V3 loop is an important functional domain of the envelope glycoprotein which mediates interaction with the HIV-

1 coreceptor, either CCR5 or CXCR4. Amino acid variation within this region has important consequences for the efficiency of the host cell entry process. Polymorphisms at two positions in the V3 crown which negatively impact viral entry efficiency and replication capacity also result in increased susceptibility to entry inhibitors. Conversely, polymorphisms that decrease susceptibility to drugs also confer higher overall replicative fitness. A major mechanism involved in sensitivity to one coreceptor inhibitor, PSC-

RANTES, is competitive binding between the drug and the HIV-1 envelope glycoprotein for CCR5. Correlation between entry inhibitor sensitivity and replicative fitness was most consistent for competitive inhibitors of coreceptor usage. Polymorphisms in the V3 crown appear to alter both entry inhibitor sensitivity and entry efficiency by modulating the affinity relationship between the viral envelope glycoprotein and the host cell

14 coreceptor, specifically affecting the interaction between the V3 crown and the second extracellular loop of CCR5. Higher affinity for coreceptor resulted in faster fusion kinetics and decreased entry inhibitor sensitivity. Increases in coreceptor affinity may be a general adaptive mechanism for all viruses in response to entry inhibitor therapy.

15 Chapter 1

INTRODUCTION

16 1. INTRODUCTION

1.A. HIV and AIDS.

The human disease later known as the acquired immune deficiency syndrome

(AIDS) was first recognized in the United States in 1981 by the presentation of five

homosexual men with atypical pneumonia caused by the agent Pneumocystis carinii.

Simultaneously, an outbreak of rare cancer incidence later identified as Kaposi’s Sarcoma

was reported in homosexual population in New York City. These observations were the

first descriptions of opportunistic infection of individuals suffering from severe

immunodeficiency secondary to infection with the human immunodeficiency virus type 1

(HIV-1). The immunodeficiency was specifically caused by the selective depletion of the

CD4+ T cell subset of the immune system. Our formal understanding of the disease

process of AIDS has matured significantly over the past 25 years, but can be generally

characterized by three major landmarks of infection (14) (Figure 1): 1) acute infection:

initial acquisition of HIV-1 is marked by high level viremia and an acute loss of CD4+ T

cells. 2) Asymptomatic viremia: induction of the adaptive immune response results in a

steady-state level of viremia and chronic high level virus production with minimal

clinical manifestation of disease. This stage can variably last from 2-15 years with a mean of 10 years. 3) Disease progression: onset of AIDS is marked by a sharp decline in

CD4+ T cells, increased viremia, onset of opportunistic infections, and ultimately, death.

17 Figure 1. Landmarks in HIV-1 Infection and Progression to AIDS. Infection with

HIV-1 can be divided into three phases. 1) During acute infection, an acute viremia occurs within weeks after primary infection (red line) while CD4+ T cells rapidly decline from the normal count of 1000 cell/mm3 (blue line). 2) During the asymptomatic phase, immunological control of viremia results in a chronic viral load set point. This is accompanied by a rebound in CD4+ T cell count which is maintained below normal

physiological levels. The asymptomatic phase can last for months to years. 3) During

progression to AIDS, more rapid decline of CD4+ T cells is evident along with a rise in

viremia. Onset to AIDS is marked by the occurrence of opportunistic infection,

continued chronic symptoms, and ultimately, death.

18

19 1.B. Retroviruses: Structure, Organization, and Replication.

Isolation of HIV-1, the causative agent of AIDS, was reported concurrently in

1983 by two separate groups (13, 69). HIV-1 is classified in the family Retroviridae, as

it must reverse transcribe a single-stranded RNA genome into DNA to achieve productive infection of a host cell. The Retroviridae are subclassified into 7 separate

genera that are named α through ε, as well as the lentiviruses and spumaviruses. Within

Retroviridae, HIV-1 is subclassified into the genus lentivirus, based upon its hallmark

pathogenic criteria, i.e. its long period of clinical latency prior to disease progression.

1.B.1. HIV-1 Genome

HIV-1 has a capped, polyadenlyated positive sense RNA genome of approximately 9.3 kilobase pairs coding for 9 gene products in 3 overlapping reading frames (Figure 2). The essential structural and enzymatic retroviral genes gag, pol, and env lie between two promoter repeat sequences (5’ LTR and 3’ LTR). The HIV-1 genome also encodes 6 auxiliary proteins (Tat, Rev, Vif, Vpr, Vpu, and Nef) which are involved in regulating synthesis and processing of viral RNAs, as well as other replicative functions. Transcription of the full length proviral DNA produces mRNA that functions as both genomic RNA for nascent particles as well as message to produce the proteins of new virions. The full length genomic RNA contains well-defined functional domains: the R region, which corresponds to the +1 site of transcription and the site of capping, a 5’ untranslated leader sequence which contains a packaging signal (ψ), open reading frames, a 3’ untranslated region, and a 3’ poly (A) tail. The 5’ untranslated region contains multiple landmarks crucial for viral replication: the TAR loop (required

20 Figure 2. HIV-1 Genomic Organization. The prototypical HXB2 HIV-1 genome encodes nine genes in three overlapping reading frames. Numbers listed on each box indicate the nucleotide number for the beginning and end of each gene.

21

22 for efficient transcription), the polyadenylation signal, the primer binding site (the initiation point for reverse transcription), the major splice donor, the dimerization initiation sequence (essential for genomic RNA dimerization), and the core packaging sequence.

1.B.2. HIV-1 Particle

Electron micrographs indicate the HIV-1 particle is approximately 120 nm in diameter. The particle is enveloped by host cell membrane derived from the budding process, and this membrane is studded with both host cell proteins (28) and HIV-1 envelope glycoproteins (Figure 3). Most evidence suggests that HIV-1 virions contain between 9 and 12 envelope spikes per virion (216, 219, 220). The Gag-derived p17 protein (Matrix, MA) surrounds the inner leaflet of the membrane and remains associated due to post-translational myristylation (81). Virions contain each 2 copies of the RNA genome, which dimerize near their 5’ end. The RNA is coated by 1000-5000 copies of the Nucleocapsid protein (NC), which is responsible for importation of genomic RNA into the virion during assembly (26). Stoichiometric amounts of other Gag molecules are present since they are derived from the same precursor polyprotein (26). The Capsid molecule (p24, CA) forms the lattice-like framework of the conical virus core, which contains the RNA genomes as well as the viral enzymes reverse transcriptase and integrase (26).

23 Figure 3. HIV-1 Virion Structure. HIV-1 is an enveloped virus. Virus membrane is indicated in blue, and is studded by the envelope glycoprotein, made up of the gp120 surface unit (SU) and the gp41 transmembrane unit (TM). The viral matrix protein (p17,

MA) studs the inner leaflet of the viral membrane (yellow). The two copes of viral genomic RNA (red) are encased by the core, which is composed of interlocking units of capsid protein (blue). Functional enzymes reverse transcriptase and integrase are associated with the RNA genome within the viral core (green).

24

25 1.B.3. HIV-1 Replication

1.B.3.a. Transmission and Replication Dynamics. HIV-1 is principally

transmitted through sexual contact or through exchange of blood products (14). The principal targets of infection in vivo are CD4+ T cells and macrophages. HIV-1 replicates

continually throughout the course of infection in untreated individuals, both within

lymphoid tissues and in the peripheral blood (145). HIV-1 replicates preferentially in

activated CD4+ T cells, and these cells generally die within days after infection.

Estimates suggest that the steady-state population size of productively infected CD4+ T cells and macrophages ranges around 2.5 x 107 to approximately 108 total cells at any

moment in time (72). Total body production of virus is estimated at 109 to 1010 virions per day (82, 144). A small subset of memory CD4+ T (1 in 106 resting CD4+ T cells)

cells are in a state of reversible non-productive infection and do not release virus. This

subset of quiescent T cells is referred to as the latent HIV-1 reservoir, and it represents

the major obstacle preventing eradication of HIV-1 from infected individuals (102).

1.B.3.a. Host Cell Tropism and Replication. The replication of HIV-1 is

restricted to a small subset of human cells. The principal targets of HIV-1 replication are

cells of immune function, specifically CD4+ T lymphocytes. Monocyte-derived

macrophages are known as a secondary specific target of viral replication. The cell-

specific tropism of HIV-1 is principally regulated by the expression of receptors that are

required for binding and entry of HIV-1 virions (Figure 4). Entry of HIV-1 into host

cells is a membrane fusion process that results in release of the virus core into the host

cell cytoplasm. The viral core dissolves and the viral RNA is converted into a double

26 Figure 4. HIV-1 Replication Scheme. HIV-1 replication is divided into two major

phases: 1) entry and integration in a susceptible cell, and 2) production, release, and

dissemination of new virions from the infected cell. Infection of a cell is initiated by receptor binding by the envelope glycoprotein. Fusion of viral and cellular membranes results in release of the viral core into the host cell cytoplasm. The RNA genome is reverse transcribed into double stranded DNA, and the viral complex migrates through the host cell cytoplasm and traverses the nuclear envelope. The viral DNA is integrated

into the host cell genome and becomes a stable provirus. Transcription of viral RNAs occurs via cooperation of viral and host cell factors. Full length transcripts are spliced and exported. Some full length transcripts are maintained to ultimately become new virus genomes, while others are translated into essential virion proteins. Envelope glycoproteins are produced through the secretory pathway on free ribosomes and stud the outer membrane of the host cell. Gag monomers multimerize at the host cell inner membrane and pinch off to release new virions. The viral protease cleaves the Gag-pol polyprotein to mature the virion into its infectious form.

27

28 stranded DNA duplex. The viral preintegration complex migrates into the host cell

nucleus and the viral genome is stably integrated randomly into the host chromosome.

From this point, progeny virions are produced and released. Viral RNAs are transcribed,

spliced, and exported from the nucleus to serve as mRNA for viral proteins and to serve

as new genomes. Gag polyproteins assemble at the host cell plasma membrane, which is studded by envelope trimers. New virions pinch off from the cytoplasm and are matured into infectious particles to begin a new cycle of replication.

1.B.3.b. HIV-1 Envelope and Host Cell Entry. HIV-1 can productively

infect cells that express two receptors: the principal receptor CD4 (41), and an auxiliary

co-receptor which derives from the chemokine receptor family, typically either CCR5 or

CXCR4 (16) (Figure 5). HIV-1 enters cells through a pH-independent membrane fusion

event (184) which results in release of the core particle into the cytoplasm in the absence

of a cellular endocytic process. The principal virus protein involved in entry is the

envelope glycoprotein. The envelope gene encodes a protein that measures 160 kDa

when fully glycosylated and is divided into two regions, the surface unit gp120 and the

transmembrane region gp41 (213). The gene is translated as a full length gp160

precursor protein and undergoes secondary structure formation and glycosylation in the

endoplasmic reticulum (51). Gp160 monomers then oligomerize into trimers (58) via

noncovalent interactions between gp41 subunits. Processing of the protein in the golgi

apparatus results in cleavage of the 160 kDa protein by members of the furin family of

endoproteases into the gp120 and gp41 subunits (135). The gp120 surface unit remains

29 Figure 5. HIV-1 Entry. Virus entry into host cells is mediated by the envelope glycoprotein. The major events of the entry process are divided into receptor binding and membrane fusion events. The functional unit of envelope is a trimer composed of three gp41 molecules (blue bars) and three gp120 molecules (blue spheres) which are associated by non-covalent interactions. 1. Virus entry is initiated by the attachment event, binding of gp120 binding host cell CD4. 2. Binding to CD4 results in reconfiguration of the gp120 molecule. The bridging sheet is formed and gp120 is primed for interaction with a coreceptor molecule. 3. CD4-bound gp120 interacts with a coreceptor, either CCR5 or CXCR4. The stoichiometry of the ternary complex is 1:1:1

(gp120 : CD4 : Coreceptor), though the number of trimer subunits and number of trimers required to initiated membrane fusion is unclear. 4. After interaction with the coreceptor, a hydrophobic gp41-derived peptide (the fusion peptide) is inserted into the host cell membrane. This event anchors the virus to the host cell, and the gp41 molecule acts as a bridge between the viral and cellular membranes (gp120 molecules have been removed for clarity). Structural rearrangements within the gp41 molecule are mediated by two triple stranded coiled-coils, the C-terminal (red) and N-terminal (blue) heptad repeat domains. 5. Metastable prefusion intermediates occur during the reconfiguration of the gp41 domains. 6. The C- and N-terminal heptad repeat regions pack into one another forming a stable six-helix bundle. This results in a close approximation of viral and host cell membrane and the formation of a fusion pore. The initial fusion pore enlarges as the membrane lipids mix, ultimately leading to a fusion pore of critical size for release of the viral core into the cytoplasm.

30

31 noncovalently associated with its gp41 partner (35). The envelope is transported to the

cell surface and is incorporated into virions budding from the plasma membrane.

When interacting with a new target cell, the gp120 unit initially makes contact

with the N-terminus of CD4 (128) through interactions with a conserved binding site

(218) (Figure 5B). Interaction with CD4 results in considerable reconfiguration of the

gp120 molecule which results in exposure of a highly conserved coreceptor binding site

(17, 98, 164, 172, 194) (Figure 5C). The coreceptor binding site interacts with the N-

terminus of a coreceptor, either CCR5 or CXCR4 (Figure 5C). Further coreceptor

interactions are mediated by amino acids at the crown of the V3 loop. Ultimately,

conformational changes in the envelope glycoprotein expose the fusion peptide, a

sequence of 15 hydrophobic amino acids located on the amino terminus of gp41, which

insert into and destabilize the host cell membrane (67, 73). At this stage, gp41 is an

integral component common to the viral envelope and host membrane (Figure 5D).

However, further structural transitions in gp41 are necessary to provide the significant

change in free energy required to drive membrane fusion (129, 137, 208). Prior to fusion,

gp41 folds back on itself forming a hairpin structure, the function of which is to bring into close proximity the fusion peptide associated cellular membrane and the integral gp41 associated viral membrane (Figure 5E). This process is mediated by two helical regions in the ectodomain of gp41 termed HR1 and HR2 (29, 45, 68). The N-terminal heptad repeat region, or HR1, and the C-terminal heptad repeat region, or HR2, form a stable 6-helix bundle when the antiparallel HR2 domain binds on the outer grooves of the triple-stranded coiled coil HR1 domain, resulting in the localization of the fusion peptide and transmembrane domains at the same end of the molecule (30, 207) (Figure 5F). This

32 re-orientation and release of free energy drives the fusion of the viral and host cell membranes.

1.B.3.c. Reverse Transcription. A hallmark feature of the biology of

HIV-1 and other retroviruses is the reverse transcription of a single-stranded RNA genome into a double-stranded DNA duplex (Figure 6). The major processes of reverse transcription occur after entry into the host cell cytoplasm (71). The viral and host factors involved in the process of reverse transcription are collectively referred to as the

reverse transcription complex (RTC). Reverse transcription initiates from a virus

producer cell-derived tRNAlys3 primer bound to a conserved region of the HIV-1 genome

called the primer binding site (PBS) (Figure 6A) (71). Reverse transcriptase is a

heterodimer composed of a p51 subunit and a p66 subunit, which includes an RNaseH

domain (115). RT initially extends DNA from this primer towards the 5’ end of the RNA

genome, producing an early DNA marker of reverse transcription called minus strand

strong-stop DNA (Figure 6B). RNaseH digestion removes the region of homology on the

RNA up to the primer binding site (Figure 6C). The first of two strand transfers occurs,

in which the R region found on the –sssDNA fragment aligns with the 3’ R region

(Figure 6D). DNA polymerization and RNaseH digestion continue up to a genomic

element called the polypyrimidine tract (PPT), a pyrimidine rich region which promotes

polymerase stalling (Figure 6E). The undigested PPT acts as a primer for synthesis of

plus-strand strong stop DNA (+sssDNA) (Figure 6F). Extension from this primer covers

the PBS of the aminoacyl-tRNA primer, and RNase H digests the rest of the primer tRNA

(Figure 6G). The second strand transfer occurs due to the new homology domains

33 Figure 6. Reverse Transcription. Primer tRNA is packaged in the virus from the host cell. Primer tRNA is bound to an RNA motif referred to as the primer binding site (PBS), and reverse transcriptase initiates from this point to generate the first DNA fragment, minus strand strong-stop DNA (-sssDNA). RNase H digests the RNA template and the first strand transfer event is mediated by homology of the R region. DNA synthesis and

RNase H digestion proceed to an RNA motif referred to as the polypyrimidine tract

(PPT), which is relatively resistant to RNase H digestion. The PPT RNA acts as primer for plus-strand strong-stop DNA (+sssDNA). RNase H digests the primer tRNA, which initiates the second strand transfer event, mediated by homology of the PBS region. Bi- directional DNA synthesis results in a double stranded DNA product with direct repeats

at the 5’ and 3’ ends (the long terminal repeats). Primer tRNA is indicated in red; viral

RNA is indicated in black; minus-strand DNA is indicated in blue; plus-strand DNA is

indicated in green.

34 35 provided by the PBS sites (Figure 6H). DNA synthesis continues to form a duplex DNA genome (Figure 6I).

1.B.3.d. Nuclear Translocation and Integration. Upon completion of reverse transcription, the unit changes its nomenclature from the RTC to the pre- integration complex (PIC). Unlike many other retroviruses, HIV-1 can infect cells which are terminally differentiated, such as macrophages (186). This has consequences for host cell tropism, as HIV-1 does not require mitotic breakdown of the nuclear envelope to gain access to the host cell chromosomes. Many viral proteins associated with the PIC have been found to contain nuclear localization sequences, including Vpr, IN, MA, and a transient product of reverse transcription referred to as the central DNA flap (186).

Whether these proteins function in a redundant manner to mediate nuclear import is not known, and in fact little is known about the process of active nuclear import of the PIC.

It is further unclear which viral factors remain associated with the PIC after crossing the nuclear envelope.

It is clear, however, that the viral integrase protein must remain associated with the DNA, as it catalyzes the integration event (186). The viral cDNA produced from reverse transcription is initially blunt-ended (Figure 7A). The viral integrase protein trims 2 nucleotides from the 3’ end of each strand, exposing a conserved CA dinucleotide which defines the ends of an integrated provirus (Figure 7B). Integrase catalyzes two simultaneous transesterification reactions. The 3’ hydroxyl group from the viral cDNA attacks a pair of phosphodiester bonds in the host cell DNA (Figure 7B). The distance between the two insertion sites are unique to each retrovirus, and for HIV-1, the targeted

36 Figure 7. Integration. After completion of reverse transcription, the single stranded

RNA genome has been converted into a double stranded DNA genome. (1) The integrase

enzyme processes the 3’ end of each DNA strand to reveal a conserved CA dinucleotide

repeat. (2) The free 3’ hydroxyl groups attach to the host cell chromosome, catalyzed by the integrase enzyme. The transesterification sites are separated by 5 nucleotides in the host cell genome, and any sequence will support HIV-1 integration. (3) The 3’ ends of the virus genome are ligated into the host cell chromosome, leaving a two nucleotide overhang from the virus sequence and a 5 nucleotide gap in the host cell chromosome. (4)

Host cell enzymes cleave the viral sequence remainder and fill the gap to produce a 5 nucleotide direct repeat flanking the stable provirus.

37

38 bonds are separated by 5 nucleotides. The 3’ ends of the viral DNA are joined to the host cell DNA, and the junctions are repaired by host cell enzymes (Figure 7C and D).

1.B.3.e. Transcription and RNA Export. HIV-1 uses its 5’ LTR as its promoter. The integrated provirus uses host transcriptional machinery to produce copies of its own RNA. In the early phases post-integration, host transcription factors activate transcription from the HIV-1 LTR promoter. There is, however, a well characterized elongation defect in the efficiency of transcription from the HIV-1 promoter (92). The elongation defect is not absolute, however, and this inefficient transcription process leads to accumulation of an HIV-1 encoded transactivator protein, Tat, which is essential to efficient production of full length HIV-1 RNAs from the 5’ LTR. Tat binds to an RNA regulatory element produced by secondary structure of the LTR RNA called the TAR element (Trans-activation response element), and in doing so recruits the human positive transcriptional elongation factor b (pTEFb) complex, composed of cyclin T1 and CDK9

(Figure 8). CDK9 mediates hyperphosphorylation of the carboxy-terminal domain of

RNA polymerase II, which increases the elongation processivity (92). Efficient transcription of the HIV-1 genome results in further synthesis of Tat protein and entry into a transcriptional positive feedback loop.

HIV-1 proteins are translated from splice variants which all derive from the full length genomic RNA. With alternative splicing, HIV-1 can produce 47 separate mRNA species (103). The HIV-1 proteins can be separated into three classes based on their splicing characteristics. The multiply spliced mRNA variants encode the regulatory proteins Tat, Rev, and Nef. The singly spliced mRNA species encode the Envelope, Vif,

39 Figure 8. Transcription of Viral RNAs. Transcription of full-length viral RNA

proceeds through cooperation of viral proteins and host cell machinery. The HIV-1 Tat

protein recruits the cyclin T1/CDK9 complex (pTEFb) to an HIV-1 RNA structural

element (the TAR loop). pTEFb mediates phosphorylation of RNA pol II, which increases its processivity and leads to production of full length HIV-1 RNAs. The RNAS

are capped, polyadenylated, and exported into the cytoplasm to serve as mRNAs for viral

proteins or as genomic RNAs for budding virions.

40

41 Vpr, and Vpu. Unspliced RNA species (9.2 kb) function both as genomic RNA and as

mRNA for the Gag-Pol and Gag proteins. Fully spliced transcripts encoding Tat, Rev,

and Nef freely exit the nucleus and are translated, but the singly- and unspliced mRNA species are retained within the nucleus as pre-mRNAs by the interaction of splicing factors (153). The Rev protein facilitates nuclear export of incompletely spliced HIV-1 transcripts. Rev contains both a nuclear export sequence and a nuclear import sequence, and cycles back and forth from the nucleus (153). After translation, Rev translocates

back into the nucleus through interaction with importin-β and binds to a 351 bp RNA

secondary structural motif in the 3’ of env-containing transcripts referred to as the Rev

response element (RRE) (153). Rev interacts with the RNA export machinery CRM1 and

facilitates export of incompletely spliced transcripts to the cytosol.

.

1.B.3.f. Assembly, Budding, and Maturation. Depending upon the cell

type, virus assembly takes place at the plasma membrane or in endosomal compartments

(133). Pr55-Gag contains three essential functional domains involved in assembly: the membrane-binding domain (M), the Gag-Gag interaction domain (I), and the late domain

(L) (133). The M domain is located at the N-terminus in the Matrix (MA) region and consists of a myristic acid, which directs the Gag precursor proteins to the plasma membrane. The I domain, responsible for Gag multimerization, is located within the

Nucleocapsid (NC) and Capsid (CA) domains. The L domain is located in p6 at the polyprotein C-teriminus, and is involved in budding. Assembly and budding is a complex process which can be described by 3 major steps: initiation of Gag

42 multimerization, incorporation of viral RNA, and association of the virus precursor

protein budding domains within the plasma membrane (133).

HIV-1 virions are initially released as immature particles containing a spherical core (114). The viral enzyme Protease (PR) cleaves the precursor proteins in an ordered process. Protease is a 99 amino acid product of the pol gene, and is active as a homodimer (114). Protease initially cleaves itself out from the Gag-Pol polyprotein and then at a site between spacer peptide 1 (SP1) and the Nucleocapsid protein. Next protease cleaves at the Matrix/Capsid junction and SP2/p6 junction, and lastly between

SP1 and Capsid. This last cleavage event is though to induce a step in the virus replication cycle termed maturation, in which the core condenses into a conical shell.

Maturation is a necessary step for virion infectivity, and the mature core is primed for disassembly in a newly infected cell.

1.B.3.g. HIV-1 Accessory Proteins. As a complex retrovirus, HIV-1 encodes 6 genes in addition to the canonical gag, pol, and env. Moving from 5’ to 3’,

HIV-1 encodes the Vif protein (for Viral Infectivity Factor), Vpr (Viral Protein R), Vpu

(Viral Protein U), Tat (Transactivator), Rev (Regulator of Expression of Viral proteins), and Nef (Negative Factor) (Figure 2). The Vif protein is essential for viral replication, and is principally involved in overcoming the effects of an endogenous host restriction factor, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G

(APOBEC3G) (39). APOBEC3G is a cytoplasmic protein associated with mRNA processing bodies which potently restricts HIV-1 replication in human T cells. The Vif protein binds to APOBEC3G protein and targets it for proteasomal degradation in an

43 infected cell. In the absence of Vif, APOBEC3G is specifically incorporated into nascent

virus particles, and exerts its antiviral effect on the next round of replication (39). The

Vpr protein, while not essential for viral replication, has been shown to carry two major

functions for the virus. First, Vpr is specifically incorporated into virions and serves as a

partner in the nuclear targeting of the pre-integration complex by virtue of its nuclear

localization sequence. Further, Vpr has been shown to be involved in cell cycle arrest, and it is believed that this function is important for maintenance of high level transcription and translation of viral products (105). The Vpu protein is also non- essential for viral replication. Vpu functions to modulate particle release and is involved in CD4 downregulation (24). The Tat protein is essential for viral replication and is principally involved in the efficient transcription of the integrated provirus (92). The Rev protein is essential for HIV-1 replication, and it functions to export unspliced and incompletely spliced HIV-1 RNAs into the cytoplasm (153). The Nef protein is not essential in HIV-1 replication, but viruses that lack Nef have a well characterized replication defect. The Nef protein appears to be important in HIV-1 pathogenesis, as

Nef deletions have been associated with long term nonprogressor status in patients (148).

Nef functions to downregulate cell surface CD4 molecules, thereby preventing superinfection of a cell. Nef protein further results in the downregulation of MHC class I molecules, which impairs recognition of virally infected cells by the host immune surveillance, and is believed to be a factor involved in viral persistence (148).

1.D. Antiretroviral Therapy.

44 The major approach to the medical management of HIV infection is the treatment

of patients with antiviral drugs. The enzymatic processes of the HIV-1 replication cycle

present unique approaches for targeted disruption by pharmacological agents. Due to the

high rates of virus production and the mutation rate of the virus (119), treatment of HIV-1 infection is accomplished by administration of 3 agents in combination, referred to as

highly active antiretroviral therapy (HAART). Sustained treatment of patients with three

active drugs results in suppression of viral replication in the peripheral blood to below the

limit of detection of sensitive clinical assays (< 50 RNA copies/ml). Continued virologic

suppression has led to dramatic increases in the life expectancy of HIV-infected

individuals and in time to diagnosis with AIDS, and decreases in HIV-associated

morbidity and opportunistic infection (Bartlett and Gallant). To date, 24 individual drugs

have been approved by the United States Food and Drug Administration for the treatment

of HIV infection. These drugs are distributed into 4 major classes: 1) Nucleoside-analog

reverse transcriptase inhibitors (NRTI), 2) Non-nucleoside reverse transcriptase inhibitors

(NNRTI), 3) Protease inhibitors (PI), and 4) Fusion inhibitors (Table 1).

Nucleoside analog reverse transcriptase inhibitors are functional mimics of

endogenous nucleosides and are incororated by HIV-1 reverse transcriptase into nascent

DNA strands (66, 131). Incorporation of drug into the growing DNA strand results in

chain termination. The inability to complete reverse transcription and integration results

in inhibition of the viral replication cycle. Non-nucleoside analog reverse transcriptase

inhibitors also inhibit the reverse transcriptase enzyme, but these drugs bind to the

enzyme in an allosteric site for their inhibitory activity (42). Again, disruption of the

reverse transcription process is sufficient to block the viral replication cycle. Protease

45 Table 1. FDA approved therapeutics for the treatment of HIV infection

Brand Name Generic Name Class Manufacturer Approval Date

Emtriva emtricitabine, FTC NRTI Gilead Sciences 07/02/03

Epivir Lamivudine, 3TC NRTI GlaxoSmithKline 11/17/95

Hivid Zalcitabine, ddC NRTI Hoffmann-LaRoche 06/19/92

Retrovir Zidovudine, AZT NRTI GlaxoSmithKline 03/19/87

Videx Didanosine, ddI NRTI Bristol-Myers Squibb 10/09/91

Viread Tenofovir, TDF NRTI Gilead Sciences 10/26/01

Zerit Stavudine, d4T NRTI Bristol-Myers Squibb 06/24/94

Ziagen Abacavir, ABC NRTI GlaxoSmithKline 12/17/98

Rescriptor Delavirdine, DLV NNRTI Pfizer 04/04/97

Sustiva Efavirenz, EVF NNRTI Bristol-Myers Squibb 09/17/98

Viramune Nevirapine, NVP NNRTI Boehringer Ingelheim 06/21/96

Agenerase Amprenavir, APV PI GlaxoSmithKline 04/15/99

Aptivus Tipranavir, TPV PI Boehringer Ingelheim 06/22/05

Crixivan Indinavir, IDV PI Merck 03/13/96

Invirase Saquinavir, SQV PI Hoffmann-LaRoche 12/06/95

Lexiva Fosamprenavir, APV PI GlaxoSmithKline 10/20/03

Norvir Ritonavir, RTV PI Abbott Laboratories 03/01/96

Prezista Darunavir PI Tibotec, Inc. 06/23/06

Reyataz Atazanavir, ATV PI Bristol-Myers Squibb 06/20/03

Viracept Nelfinavir, NFV PI Agouron 03/14/97

Fuzeon Enfuvirtide, T-20 FI Trimeris 03/13/03

NRTI – nucleoside reverse transcriptase inhibitor

NNRTI – nonnucleoside reverse transcriptase inhibitor

PI – protease inhibitor

46 inhibitors block the maturation step of HIV-1 replication. Nascent virions from an

infected cell incorporate protease inhibitors and are unable to mature their core, thus

rendering the virions non-infectious (8). Fusion inhibitors represent the newest class of

antiretroviral therapies, and enfuvirtide is the first drug to exploit inhibition of the entry

process (101).

1.E. HIV-1 Receptors and Implications of Tropism.

The concept of inhibiting HIV-1 replication by preventing the virus from entering the host cell has been contemplated since the first identification of the major receptor,

CD4 (41, 128). Early approaches suggested the use of monoclonal antibodies to CD4 to block binding by virus. This approach was limited due to the importance of CD4 in basic immunologic functions. The next major advancement in inhibition of HIV-1 entry was the development of a soluble form of CD4 (sCD4) (44, 64, 88, 180, 191), which could inhibit HIV-1 replication in vitro and in vivo (173, 205), as well as a soluble CD4- immunoglobulin fusion protein (192). However it was soon realized that distinct differences existed between the neutralization sensitivity of laboratory strains of HIV-1, which were originally screened, and primary HIV-1 isolates, and that these differences were based on affinity and association rates for CD4 of the envelope glycoprotein quaternary structure (40, 90). In some cases, treatment with sCD4 resulted in enhancement of infection (4). Ultimately it was observed that therapeutic administration of sCD4 had no effect on viremia or disease (34, 84); however, the sCD4 molecule

provided a tool for greater understanding of the process of HIV-1 entry.

The existence of a second HIV-1 receptor was postulated due to a particular

characteristic of virus growth in culture. All HIV-1 isolates were known to replicate in

47 primary CD4+ T cell cultures. However, viruses then broke down into two subsets: some

viruses propagated well in macrophage cultures, but did not grow in T cell lines (M-

tropic isolates), while a different subset grew well on T cell lines, but not in macrophages

(T-tropic). It was further noticed that T-tropic viruses induced the formation of

multinucleated giant cells in culture, or syncytia. Thus, viruses were characterized as T-

tropic or syncytium inducing (SI), or alternatively as M-tropic or non-syncytium inducing

(NSI). The difference in cell culture tropism was hypothesized to be due to the existence

of two separate coreceptors, which were alternatively expressed on either cell subset, but that were both expressed on primary CD4+ T cells.

Discovery of the coreceptors that mediate HIV-1 entry was facilitated by studies

showing that replication of the virus could be blocked by then unknown, leukocyte

derived, soluble suppressor factors (27). The soluble factors derived from CD8+ T cells were identified as the C-C chemokines RANTES (CCL5), MIP-1α (CCL3), and MIP-1β

(CCL4) (33). Chemokines are small paracrine signaling molecules that are principally involved in the inflammatory response. There are currently four main classes of chemokines, and their nomenclature is based on the number and orientation of N-terminal cysteine motifs (136). C chemokines have a single cysteine residue. C-C chemokines, C-

X-C chemokines, and C-X3-C chemokines each have two cysteine residues, separated by

0, 1, or 3 other resides, respectively. Only the C-C chemokines and C-X-C chemokines, are major factors in HIV-1 infection.

In 1996 the “fusin” cofactor was identified by expression of a cDNA library derived from T-tropic virus-permissive cells against a nonpermissive cell line (63). This receptor was later identified as C-X-C chemokine receptor 4 (CXCR4), and its ligands

48 [stromal derived factor-1 α/β (SDF-1α/β, CXCL12)] can inhibit HIV-1 repication in vitro

(23, 140). Shortly thereafter, C-C chemokine receptor 5 (CCR5) was identified as the

major entry cofactor of M-tropic, NSI HIV-1 isolates (3, 31, 46, 55, 80). The chemokine

receptors are members of the seven transmembrane G protein-coupled receptor

superfamily. They are defined by their coupling to the pertussis toxin-sensitive Gi class of G proteins, expression in leukocytes, and chemotactic signaling function, and are primarily involved in leukocyte activation and directional migration. The chemokine system is highly redundant, with each receptor capable of being bound by multiple ligands, and each ligand binding promiscuously to multiple receptors. This same promiscuity has been investigated for the HIV-1 envelope, and it was revealed that the chemokine receptors CCR2b, CCR3, CCR7, CCR8, STRL33/BONZO, and gpr15/BOB can mediate infection of cells by some viruses (53, 60, 61, 76, 111). Use of these alternative coreceptors appears limited to expression on transfected cell lines, and most evidence suggest that the receptors CCR5 and CXCR4 are the most relevant receptors in vivo. Currently, viruses that utilize CCR5 as an entry cofactor are referred to as R5 viruses, while viruses that utilize CXCR4 are referred to as X4 viruses (15). Rare viruses that can utilize either CCR5 or CXCR4 as an entry cofactor are referred to as dual tropic, or R5X4.

CCR5-tropism is characteristic of viral isolates that persist during asymptomatic disease, and are further thought to be the principal subset of virus responsible for new infections. Over the course of HIV infection, a switch to primarily CXCR4-tropic or dual tropic isolates is generally associated with a rapid depletion of CD4+ T cells and progression to AIDS (36, 174, 187). It was at this time known that a subset of individuals

49 at high risk for infection with HIV-1 remained seronegative despite multiple

opportunities for virus transmission. Genetic analysis of these cohorts revealed that a

subset of these individuals were homozygous for a 32-bp deletion in the CCR5 open

reading frame, and that their CD4+ T cells were resistant to infection by R5 viruses ex

vivo (43, 87, 113, 130, 171, 221). This deletion (Δ32) results in a truncated receptor that

is not expressed on the cell surface. The Δ32 allele is present in the Caucasian population,

with as many as 20% of Caucasians heterozygous for the mutation (Δ32/wt) and 1%

homozygous (Δ32/Δ32) (43). While individuals homozygous for the Δ32 allele are

highly resistant to acquisition of HIV-1 infection (transmission of X4 viruses in Δ32/Δ32 individuals has been reported), heterozygous individuals typically have a more protracted course of infection and experience longer time intervals before progression to AIDS.

Single nucleotide polymorphisms within the promotor region of CCR5 have also been associated with differences in disease progression rates. Specifically, individuals who are

–2459A/A have been shown to progress to AIDS more rapidly than individuals homozygous for the guanine allele (-2459G/G) (6, 125, 127, 141). Remarkably, individuals carrying these receptor polymorphisms lack any discernable biological phenotype other than resistance to HIV infection or delayed progression to AIDS, which indicated the potential value of targeting entry through the CCR5 coreceptor as a viable pharmacological intervention.

1.F. Interaction of HIV-1 Envelope and CCR5.

The envelope glycoprotein, when unligated with CD4, is not in a conformation that can interact with the coreceptor. A subset of HIV-2 viruses and SIV isolates can

50 bind to coreceptor independently of CD4, which has led to the suggestion that the chemokine receptors are the ancestral receptors of the early lineage viruses that gave rise to modern primate immunodeficiency viruses. Use of CD4 is thought to be an adaptation to hide the conserved coreceptor binding site from recognition by the host humoral response. When gp120 binds CD4, structural rearrangements in the envelope glycoprotein lead to exposure of the V3 loop and the formation of the bridging sheet.

The V3 loop is a highly heterogeneous, loop-like structure within gp120. The V3 loop is the immunodominant domain of the HIV-1 envelope, though it is not exposed on most primary isolates and is therefore not considered a major neutralization determinant (75).

The V3 loop is topographically divided into three subsections (Figure 9): 1) The base, formed by a disulfide bond and in close approximation with the gp120 core; 2) the stem, which is a flexible linker region between the base and the tip; 3) the crown, which contains a helix-turn-helix motif defined by a G-P-G-X sequence, and believed to be a critical interacting face with the extracellular loops of CCR5. V3 is the critical determinant of coreceptor tropism. As few as two amino acid changes in the V3 loop can alter the coreceptor specificity from CCR5 and CXCR4 (75). Furthermore, changes in

the V3 have been specifically associated with changes in susceptibility to entry inhibitors

(75).

The current model of gp120-CD4-CCR5 ternary complex formation favors

multiple interactions between gp120 and CCR5 (Figure 9). Studies using deletions and

chimeric receptors (using parts of human CCR2b or murine CCR5, which do not mediate

HIV-1 entry) suggest that the N-terminus of CCR5 is of significant importance for HIV-1

entry (17, 52, 146). Site directed mutagenesis of the V3 region indicate that two domains

51 Figure 9. Model of Ternary Complex Formation. The model is based on the crystal structure of a V3 loop-containing structure of gp120, bound to a soluble fragment of CD4

(Huang et al, 2005). The gp120 core (gray) is bound to the 1st Ig-like domain of CD4

(yellow). This interaction stabilizes the core molecule bridging sheet domain, which is composed of a series of antiparallel β-sheets (arrow). The V3 loop extends outward from the gp120 core inner domain (red). The helix-turn-helix motif in the V3 crown is thought to mediate interaction with the second extracelluar loop of CCR5 (blue). The CCR5 N- terminus is thought to contact both the bridging sheet domain as well as the V3 base.

Adapted from Huang et al.

52

53 in gp120 were essential to the interaction with CCR5. The V3 tip, or crown, is suggested

to interact with the extracellular loops of CCR5, specifically extracellular loop 2 (37).

Consistent with this, monoclonal antibodies that recognize CCR5 ECL2 potently inhibit

HIV-1 entry. Post-translational modification of CCR5 plays a significant role in

coreceptor activity. The N-terminus undergoes both O-glycosylation and tyrosine

sulfation. Inhibition of tyrosine sulfation pathways decreases CCR5 binding by gp120

and HIV-1 entry (62). Mutagenesis or deletion of the intracellular carboxy-terminal

domain of CCR5, which mediates ligand-induced endocytosis, reveals that this is not an

essential function of viral entry. Similarly, deletion or mutagenesis of the second

intracellular loop DRY signaling motif indicates that intracellular signaling is dispensible

for productive viral entry.

Seven transmembrane G protein-coupled receptors exist in multiple allosteric

states due to their function in transmitting information from a single ligand binding site to

other, topographically distinct sites, which include sites of G protein association, oligomerization, and signal transduction scaffolding protein binding sites (32). As such,

CCR5 exists in multiple antigenic configurations (108). Distinct conformational states of

CCR5 in either active or inactive states have been observed using the binding properties

of various antibodies (22, 108). These conformational states exist transiently, and the

stability of each conformation depends upon the lipid environment, the activation state of

the receptor, and the ligand that is bound. Environmental stimuli can stabilize the receptor in particular antigenic configurations. Stabilization may potentially occur through receptor association with scaffolding proteins involved in signal transduction.

Some reports suggest that CCR5 dimerizes through interactions in the first

54 transmembrane region, and that the dimerized form of the molecule is non-permissive for

HIV-1 entry (77). Dimerization of CCR5 has been suggested to be ligand-dependent, though some studies have demonstrated ligand-independent oligomerization of CCR5

(89). The role of CCR5 conformational heterogeneity in HIV-1 entry and coreceptor interaction remains unclear at this time. It remains unknown whether HIV-1 envelope can interact with all, or only a subset of the available CCR5 conformational isomers.

1.G. Inhibition of HIV-1 Entry.

The major enzymatic processes of HIV-1 replication are reverse transcription, integration, and protease maturation. Each represents a unique, virus specific drug target the interruption of which leads to failure of the replication cycle. Uncovering the major mechanisms of HIV-1 host cell entry revealed a series of critical processes that could be targeted for disruption pharmacologically. HIV-1 entry inhibitors fall into three major classes based on the specific entry process that they target: 1) attachment inhibitors, which block the interaction between HIV-1 envelope and CD4, 2) coreceptor inhibitors, which block the interaction between HIV-1 envelope and CCR5 or CXCR4, and 3) fusion inhibitors, which prevent the virus from mixing its membrane with the host cell membrane and releasing the viral core into the cytoplasm (Table 2).

1.G.1. Attachment Inhibitors.

The attachment inhibitor BMS-378806 inhibits both R5- and X4-tropic HIV-1 isolates (112). This compound binds to a pocket on gp120 important for binding CD4 and alters the conformation of the protein such that it cannot recognize CD4 (83). The

55 Table 2. Investigational Entry Inhibitors

Compound Mechanism Status Manufacturer

T-20 Fusion inhibitor Approved Trimeris

T-1249 Fusion inhibitor Discontinued Trimeris

C-34 Fusion inhibitor ------

5-Helix Fusion inhibitor ------

Maraviroc CCR5 antagonist Phase III complete Pfizer

Vicriviroc (SCH-D) CCR5 antagonist Phase IIb Schering-Plough

SCH-C CCR5 antagonist -- Schering-Plough

AD101 CCR5 antagonist -- Schering-Plough

Aplaviroc CCR5 antagonist Discontinued GlaxoSmithKline

TAK-652 CCR5 antagonist Phase I Takeda

TAK-779 CCR5 antagonist -- Takeda

INCB 9471 CCR5 antagonist Phase IIa Incyte

PRO-140 Humanized anti-CCR5 monoconal antibody Phase IIa Progenics

PSC-RANTES Chemokine analog (Microbicide) Gryphon

BMS-378806 Attachment inhibitor Phase IIa Bristol-Myers Squibb

PRO-542 CD4-Ig fusion Discontinued Progenics

TNX-355 Anti-CD4 monoclonal antibody Phase IIa Biogen Idec; Tanox

56 inhibitor binds with significantly higher affinity than CD4, and is an excellent candidate

for therapeutic use. TNX-355 is a humanized anti-CD4 monoclonal antibody that binds to CD4 and inhibits HIV-1 envelope docking, but does not inhibit CD4 function in immunological contexts.

1.G.2. Fusion Inhibitors and Mechanisms of Resistance.

The crystal structure of the gp41 ectodomain (30) and of the ectodomain partnered with an inhibitory peptide (C34) (118) revealed that the fusion active conformation of gp41 was a 6-helix bundle in which three N-helices form an interior, trimeric coiled-coil onto which three antiparallel C-helices pack. Enveloped viruses use a

generally conserved mechanism to initiate the fusion event between the viral and host cell

membrane, and this 6-helix bundle formation has been well studied in the paramyxoviruses family of viruses (51, 91). The essential basis of peptide fusion

inhibitors is that two homologous domains in the viral gp41 protein must interact with

each other to promote fusion, and that mimicry of one of these domains by a heterologous

protein can bind and disrupt the intramolecular interactions of the virus protein. Alpha-

helical peptides homologous to the leucine zipper domain of gp41 had significant

antiviral activity against HIV-1, and this activity depended upon their ordered solution structure (211). Rational design ultimately produced a molecule (T-20, enfuvirtide) with

potent antiviral activity in vivo (93, 101), and many other peptide mimics have been

described (Table 2).

Resistance to early alpha-helical inhibitors was mediated by mutations in the N-

terminal heptad repeat region of gp41 (163), which further suggested the specificity of

57 binding of these peptides to the virus. Monotherapy with enfuvirtide resulted in viral load rebounds after 14 days of therapy, and resistance determinants mapped to the HR-1 domain (G36D, I37T, V38A, V38M, N42T, N42D, N43K) (206). Mutations that confer resistance to enfuvirtide also result in reduced replication capacity of the virus (116), and replicative fitness and enfuvirtide susceptibility were inversely correlated. This is presumably due to adaptations in the gp41 domain that reduce enfuvirtide binding, but consequently reduce the efficiency of 6-helix bundle formation and overall fusion rates

(160). These mutations did not confer cross resistance to other types of entry inhibitors

(attachment inhibitors or coreceptor inhibitors) (158) but did sensitize viruses to neutralization by monoclonal antibodies that target the gp41 domain, presumably by prolonging the exposure of fusion intermediates that are specifically sensitive to these antibodies (160). Adaptation to enfuvirtide has even resulted in viruses that require enfuvirtide for fusion (11).

Though it is clear that gp41 resistance mutations, taken out of the context of the envelope under selection, result in decreased fusion efficiency and reduced viral fitness, the function of envelope context seems to modulate the overall effect of these mutation in vivo (100). Studies of baseline susceptibility to enfuvirtide suggested that large variations in intrinsic susceptibility existed in diverse HIV-1 isolates, and that these variations mapped to regions outside the enfuvirtide binding site (47). Sequences associated with the V3 loop were correlated with intrinsic enfuvirtide susceptibility, suggesting that interactions with the coreceptor were important determinants of susceptibility of a drug that inhibits virus fusion. A seminal observation in the understanding of entry inhibitor susceptibility was made in 2002 by the discovery that

58 efficiency of the fusion process was the principal modulator of intrinsic enfuvirtide susceptibility (159). Mutations in the coreceptor binding site that reduced gp120 affinity for CCR5 resulted in viruses with reduced fusion kinetics (19, 161). The engagement of

CD4 by gp120 initiates a process of structural rearrangement in the envelope glycoprotein resulting in fusion. The completion of this process requires engagement of the coreceptor molecule, but enfuvirtide susceptibility is limited to the time between CD4 engagement and 6-helix bundle formation. Thus the efficiency of this process affects the amount of time that the enfuvirtide has to bind gp41 and prevent fusion. Any change in the system that decreases the rate of fusion (e.g. reducing the levels of coreceptor expression) also increases the susceptibility of a virus to inhibition by enfuvirtide.

Consistent with this, ENF is synergistic with compounds that inhibit CD4 or coreceptor engagement (138, 193, 217).

1.F.3. Coreceptor Inhibitors and Mechanisms of Resistance.

1.f.3.a. Chemokine Analogs. Discovery of the chemokine coreceptors was facilitated by the observation that native chemokines can inhibit HIV-1 replication (33). Thus the first attempt to generate a coreceptor based entry inhibitor was modulation of the endogenous chemokine RANTES (CCL5) to produce a more potent version (179). Modulation of the N-terminus by deletion of the N-terminal methionine residue resulted in increased potency of the chemokine RANTES (179). Addition of an aminooxypentane moiety to the N-terminus of either RANTES or MIP-1αP resulted in further increases in potency and increases in affinity for CCR5 (179, 190). Modulation of the N-terminus of the chemokine was specifically chosen because it appears that chemokines interact selectively with specific receptors by a “message” and “address”

59 system. The core domain of the chemokine is specifically responsible for determining

receptor specificity (the address), while the N-terminus of the chemokine is involved in

elicitation of downstream signal transduction cascades (the message) (20). An HIV-1

inhibitory molecule should ideally not induce signal transduction cascades or the ensuing

lymphocyte activation or chemotaxis. Modifications made to generate met-RANTES and

AOP-RANTES resulted in abrogation of chemotaxis signals but not calcium flux (165,

179).

One major principal for the development of chemokine-like inhibitors is that

chemokines induce ligand-mediated internalization of their receptor through a clathrin-

dependant mechanism. Chemokine receptors, when bound by ligand and in their

signaling-active state, activate G protein-coupled receptor kinases (GRKs). GRKs

phosphorylate the C-terminal cytoplasmic domain of the chemokine receptor. The

phosphorylated form of the receptor is recognized by β-arrestin, which recruits

scaffolding proteins and clathrin precursor proteins to the receptor. Clathrin-coated pits

form invaginations and the receptor is internalized. CCR5 is specifically endocytosed

and moved to a recycling endosomal compartment (178). The contribution of receptor

downregulation in inhibition of HIV-1 entry by chemokines was first observed for

inhibition of X4-tropic HIV-1 isolates by SDF-1 (5). Analysis of a series of RANTES analogs revealed that their potency in blocking HIV-1 entry correlated not with their binding affinity, but with their ability to induce ligand-mediated internalization of CCR5

(143). Consistent with this, mutagenesis of the CCR5 C-terminus, abrogating its recognition and phosphorylation by GRKs, resulted in a mutant that is not internalized

when bound by ligand. Use of this mutant significantly reduced the potency of AOP-

60 MIP-1αP (25). Beyond simple receptor internalization, it has been found that chemokine

analogs result in prolonged sequestration of cell surface receptor (117) The mechanisms

behind prolonged sequestration of CCR5 have been explored but have not been fully

elucidated. Further iterative design of the RANTES N-terminus resulted in a third

generation compound, PSC-RANTES, with even higher potency than AOP-RANTES

(74). PSC-RANTES has been shown to block vaginal transmission in the SHIV-

Macaque model system (107) and is currently in development as a microbicide to prevent

sexual transmission of HIV-1.

A major problem associated with chemokine analogs is their residual capacity to

signal through CCR5 and through cell surface proteoglycans (165, 203). This residual

activity has been associated with stimulation of viral replication by enhancement of viral

integration (124) and through enhancement of virus entry (70). AOP-RANTES has been

shown to activate mitogen activated protein kinases and pertussis-toxin sensitive signals

(124, 166). High concentrations of chemokine analogs clearly have a stimulatory effect

on T cells, and are formally considered partial agonists of CCR5. This residual signaling activity may limit the effectiveness and potential use of chemokine analogs in vivo.

1.F.3.b. Small Molecule CCR5 Antagonists. Small molecule

CCR5 antagonists bind to hydrophobic pockets within the transmembrane helices of

CCR5 (between helices 1, 2, 3, and 7) (56, 199). This site does not overlap the binding sites of either CCR5 agonists or HIV-1 envelope, but instead induces and stabilizes a receptor conformation that is not recognized by either. Thus these molecules are considered allosteric inhibitors. Ideally, a small molecule inhibitor of CCR5 would

61 block binding by HIV-1 envelope but continue to bind native chemokines and facilitate

signal transduction. Most small molecule inhibitors, however, are pure antagonists of the

receptor. Oral administration of small molecule antagonists has been demonstrated to

inhibit viral replication in macaque models (201) and to preventi vaginal transmission

(202). Thus far, two CCR5 antagonists have been shown to inhibit virus replication in

humans (54). The compound Maraviroc has completed phase III efficacy trials and will

likely be approved for therapeutic use in 2007.

1.F.3.c. Models of Resistance to Coreceptor Inhibitors. The

major concern in the therapeutic administration of coreceptor inhibitors is that reisistance

will manifest by a change in coreceptor tropism from CCR5 to CXCR4, or that an

outgrowth of an X4-tropic virus subset will come to dominate the intrapatient virus population. The mechanisms and consequences of coreceptor switching are poorly understood at this time. It is unclear what factors drive this switch, or why switching is

generally limited to late phases of disease progression. Indeed, it is not clear at this time whether a switch from R5 to X4 tropism is a cause or consequence of disease progression.

In the course of preclinical evaluation of CCR5 inhibitors, only one study has identified change in coreceptor tropism as the major mode of resistance (134). On the other hand, resistance to a variety of small molecule CCR5 inhibitors has been generated by passage of inhibitor-susceptible virus isolates in sequential dose escalations of drug. These propagations have been performed in PBMC cultures, which express CCR5 and CXCR4, as well as a variety of other chemokine receptors that have been previously implicated as potential HIV-1 coreceptors. In these experiments, inhibitor-resistant viruses continue to

62 require CCR5 for entry and cannot utilize either CXCR4 or any alternative coreceptors (9,

122, 195, 210). Furthermore, evaluation of coreceptor tropism of viruses derived from patients who failed maraviroc therapy indicate that tropism changes occurred only when

X4 tropic viruses were pre-existing in the patient quasispecies (209). Thus it appears that, though outgrowth of pre-existing X4-tropic viruses in the quasispecies remains a problem for the therapeutic administration of CCR5 antagonists, de novo mutations conferring altered coreceptor usage do not appear to be the favored pathway for resistance in vitro or

in vivo.

If coreceptor switching is not the main mechanism of resistance to small molecule

coreceptor inhibitors, then it is important to understand by what mechanism resistance does arise. Two models of resistance have been proposed: competitive and noncompetitive resistance (Fig. 10A). In the competitive resistance model, gp120 interacts with CCR5 with a given affinity. In the presence of an inhibitor (blue), the high affinity interaction between receptor and inhibitor blocks the lower affinity interaction between the receptor and gp120. Adaptations in gp120 increase the affinity relationship between envelope and coreceptor, and this change in receptor affinity is sufficient for gp120 to compete inhibitor off of the receptor. Experimental evaluation of this mechanism of resistance would appear as a shift in concentration of drug required to inhibit viral replication by 50% in culture (IC50 value), and complete inhibition of virus

entry can be achieved by increasing the concentration of inhibitor (Fig. 10A). Many

questions about this potential form or resistance can be raised: 1) what are the baseline

affinities of each contact point between gp120 and coreceptor? 2) How great are the

variations among HIV-1 isolated in these baseline affinities? 3) What types of change in

63 Figure 10. Models of Resistance to HIV-1 Entry Inhibitors. Two models for

resistance to HIV-1 coreceptor inhibitors have been proposed. (A) The competitive

model may be the most relevant for inhibitors that block HIV-1 envelope binding by

steric hindrance (chemokines, antibodies to CCR5). In this model, an inhibitor bound

receptor is not accessible by HIV-1 envelope proteins with low overall affinities for

receptor, whereas envelope proteins with mutations that confer high affinity can compete

off the inhibitor for interaction with the receptor. This resistance mechanism is

manifested in entry inhibition assays by a fold change in IC50 value (sensitive virus is

depicted by blue diamonds, while a 100-fold resistant variant is depicted by black

diamonds). In this model, increasing inhibitor concentrations result in successful

inhibition (100%) of entry. (B) The noncompetitive model has been proposed for

resistance to small molecule allosteric inhibitors of CCR5 (such as TAK-779). In this

model, envelopes that are sensitive to inhibition cannot recognize the configuration of the

inhibitor bound receptor. Inhibitor-resistant variants can recognize the inhibitor-bound

receptor complex. Entry inhibition assays result in a plateau effect, and the height of this

plateau is indicative of the efficiency with which the HIV-1 envelope utilizes the

inhibitor-bound form of CCR5 relative to the inhibitor-free receptor (a virus that cannot use the inhibitor-bound complex is indicated in blue diamonds, while a virus that uses the inhibitor-bound form of CCR5 with 90% efficiency as compared to the inhibitor-free receptor is indicated in back diamonds).

64

65 the gp160 sequence would result in greater coreceptor affinity? 4) How great a change in

affinity is required to result in differential inhibition by a given drug? 5) Will gp120

adapt to different CCR5 inhibitors by a similar pathway, or by alternative mutations for

each drug (i.e. will cross resistance be a problem)? 6) What are the consequences of affinity changes on overall viral replication? 7) What are the consequences of affinity changes on viral dynamics in vivo and disease progression? Much work is still required

to substantially address these issues.

The second model of resistance to coreceptor inhibitors is noncompetitive

resistance (Fig. 10B). In this scenario, a small molecule inhibitor binds CCR5 in an

allosteric site, which induces and/or stabilizes a receptor configuration that is not

recognized by HIV-1 envelope (or native chemokines). Due to the lack of recognition, receptor docking is blocked and the HIV-1 entry process cannot proceed. Adaptation to this type of inhibition is mediated by changes in gp120 that allow for recognition of the

CCR5 configuration induced by inhibitor binding in the allosteric site. The overall affinity for the inhibitor-free receptor may not be relevant in this circumstance, and the envelope can complex with the inhibitor-bound configuration of CCR5. Experimental evaluation of noncompetitive resistance is manifested by a plateau effect on HIV-1 entry as inhibitor concentration increases (Fig. 10B). In this case, the overall extent of inhibition never reaches 100% independent of the concentration of inhibitor. Evaluation of inhibition curves would reveal that the EC50 for an inhibitor resistant virus (defined as

the half-plateau height, or the concentration at which the inhibitor achieves 50% of its

maximal effect) is equal to the IC50 of the inhibitor against the inhibitor-sensitive variant of the virus. The plateau effect is caused by differences in the efficiency of usage of the

66 inhibitor-free and inhibitor-bound form of the receptor. Thus a completely sensitive virus uses the inhibitor-bound form of CCR5 with 0% efficiency (Fig. 7B, blue line). A virus that uses the inhibitor-bound form of CCR5 with equal efficiency as the inhibitor-free form of CCR5 would not indicate any level of inhibition. Consequently, any virus adaptation that would cause an increase in the efficiency of use of the inhibitor-bound receptor over the efficiency of use of the inhibitor-free receptor would yield a negative inhibitory value, or an apparent enhancement of infection in the presence of saturating levels of inhibitor.

The relevance of these models is currently under intensive investigation.

Genotypic and phenotypic analysis of the passage variants that ultimately gave rise to resistance to AD101 indicate that resistance arose via stepwise accumulation of mutations in the V3 region of gp120 (97). A single mutation was selected for in V3 after 4-6 passages in the presence of AD101 that resulted in modest resistance (3-fold) through an apparent competitive mechanism. This variant had increased capacity to utilize low levels of CCR5 (195) and is hypothesized to have greater overall CCR5 affinity. Upon continued passage, three more mutations arose in the V3 which led to complete insensitivity to AD101. Inhibition studies of virus clones bearing these 4 mutations resulted in a plateau effect which indicated the virus was using the inhibitor-bound CCR5 complex with approximately 90% efficiency of the inhibitor-free form of CCR5, though cell type differences in plateau height did exist (155). Ultimately, the changes required in gp120 to allow for recognition of inhibitor-bound forms of CCR5 are unclear, and the alterations in the geometry of the envelope – receptor complex remain a mystery.

Adaptation to the small molecule SCH-D did not involve changes in the V3 region,

67 suggesting that resistance can arise through alterations in envelope geometry that are not restricted to specific sites within the envelope glycoprotein (122).

1.H. Implications of HIV-1 Entry Efficiency.

HIV-1 envelope-mediated entry is a highly cooperative process. The efficiency of this process can be modulated by factors specific to the host cell, including receptor density on the surface, as well as expression of receptors in antigenically permissive conformations. Entry efficiencies are further modulated by the HIV-1 envelope both through quantitative factors such as receptor affinity as well as through qualitative factors

of receptor interaction (e.g. envelope geometry and receptor-envelope stoichiometry).

Several approaches have been taken to assess cooperativity in HIV-1 envelope

mediated membrane fusion. The earliest study evaluated the effect of varying CCR5

expression levels on virus infection, and estimated that 6 CCR5 molecules were involved

in a stable entry complex (96). By titrating out functional envelope trimers using a

dominant negative gp160 variant, it has been suggested that HIV-1 entry can be achieved

by a single trimer (214), and further that two subunits of the trimer are sufficient for entry

(215). This suggests that two CD4 molecules and two CCR5 molecules would be involved in this process. However, previous studies have implicated a role for at least four CD4 interactions in entry (104), and similar dominant negative gp160 approaches have suggested the involvement of between 4 and 5 trimers (78).

There are several implications to the multiple receptor stoichiometry of HIV-1 entry. One assumption is that receptor density will play a significant role in entry efficiency, and furthermore, affinity relationships between envelope and CD4 as well as

68 envelope and coreceptor will have implications on the rate and efficiency of entry. A

stoichiometry requiring the aggregation of multiple CCR5 molecules to form a stable

entry complex will be significantly inhibited in environments with limiting amounts of

CCR5, such as in the presence of inhibitor. This prolongation of the entry step may allow

for greater efficacy of various virus deactivation processes, such as endocytosis or

complement mediated lysis.

The efficiency of the HIV-1 entry process is thought to have implications for

overall viral replicative fitness (123, 157). HIV-1 exists as a genetically variable

population, or quasispecies (50, 168) as a consequence of a high mutation rate (3.4 x 10-5 per base pair) (119) and rapid turnover. Fitness is a complex factor within an infected host. In a simple model, intrinsic rates of viral replication determine the relative proportion of an HIV-1 clone in the overall population. Although low fitness variants can contribute to the genetic pool in the quasispecies theory, the most fit variants will dominate the population due to their competitive replicative advantage. Host selection is a major determinant of fitness in vivo. However, replicative fitness, also termed replication capacity (a measure of the growth kinetics of a virus) has important implications on viral fitness and pathogenesis. For example, viruses derived from long term nonprogressor cohorts have been shown to have lower replicative fitness than viruses from individuals with normal disease progression (156). Viruses lacking the nef gene also have a replication defect in vitro, and these viruses are frequently associated with less severe disease symptoms. Efficiency of virus entry may be an important determinant of viral replicative fitness, and consequently of viral pathogenesis.

69 Variations in the efficiency of the entry process may have an important role in the

context of inhibiting HIV-1 entry in vivo. Many studies have looked at the baseline

susceptibility of primary isolate HIV-1 viruses to inhibition by a broad range of entry

inhibitors. As was seen with enfuvirtide, a wide range of susceptibility (>1000 fold)

exists among primary HIV-1 isolates to molecules that inhibit at the level of the

coreceptor (47, 99, 170, 185, 189, 197). The clinical relevance of such variations is

unclear at this time. Furthermore, the mechanisms involved in these variations are not

clear. The intrapatient population of viruses must infect cells in the presence of chemokine inhibitors, and some studies have suggested that viruses grow resistant to inhibition by chemokines over the course of HIV-1 infection in a single patient (95). One possible consequence of this hypothesis is that virus variants found at acute infection may be more susceptible to entry inhibitors that variants found late in infection. Variations in coreceptor affinity may be involved in determining sensitivity to coreceptor inhibitors.

1.I. Hypotheses.

Clearly many problems remain in our understanding of HIV-1 entry. It has become essential to answer many of these questions as inhibitors of the HIV-1 entry process begin widespread use in the treatment of HIV infection. Much emphasis has been placed on the understanding of noncompetitive forms of resistance to coreceptor antagonists, but significantly less has been placed on understanding the mechanisms involved in and clinical relevance of competitive resistance to entry inhibitors. We wish to understand how natural variations in the HIV-1 envelope glycoprotein impact sensitivity to entry inhibitors, and what effect these variations have on overall viral

70 replication capacity. Furthermore, due to the high degree of diversity in the HIV-1 envelope glycoprotein, we suspect that the context in which mutations or polymorphisms arise is crucial in determining the overall impact of those changes. We suspect that many inhibitors select for competitive resistance and that adaptations through the competitive model will impact on entry inhibitor sensitivity and viral replicative fitness. Our approach to these questions is to identify key amino acids in a functional domain of HIV-

1 envelope (V3) which are associated with variable susceptibility to entry inhibitors. We analyze the mechanisms associated with this phenotype by generating virus clones containing identified polymorphisms and assessing viral replication, entry inhibitor susceptibilities, growth characteristics, fusion kinetics, and receptor affinities.

We hypothesize that increases in coreceptor affinity result in faster virus fusion rates, a byproduct of which is decreased susceptibility to entry inhibitors. We wish to evaluate whether competitive resistance mutations will confer a type of “cross resistance” to all classes of entry inhibitors. We wish to quantify affinity changes between HIV-1 envelope and CCR5 and understand how changes of specific magnitudes relate to sensitivity to entry inhibitors and changes in replicative fitness. We hypothesize that a major determinant of gp120-CCR5 interaction, the gp120 V3 region, may have an important contribution to overall replication capacity and entry inhibitor susceptibility.

The answers to these questions will lead to a greater understanding of responses to antiretroviral therapy targeting HIV-1 entry, to further rational design of subsequent inhibitors and therapeutic strategies, and to a greater understanding of viral dynamics and the disease processes of HIV-1 infection.

71 Chapter 2

NATURAL VARIATION IN THE V3 CROWN OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 AFFECTS ENTRY INHIBITOR SENSITIVITY AND REPLICATIVE FITNESS

Authors: Michael A. Lobritz1,2, Andre J. Marozsan2, Ryan M. Troyer2, and Eric J.

Arts1,2

1Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA 44106

2Division of Infectious Diseases, Department of Medicine, Case Western Reserve University

72 2.1. PREFACE

Prior to the initiation of this study, a previous graduate student (Vincent Torre

M.Sc.) performed a screen of primary HIV-1 isolates for sensitivity to the chemokine analog AOP-RANTES. He observed a 31-fold difference between 14 separate isolates to inhibition and observed a correlation between sequences in the V3 crown and AOP-

RANTES susceptibility. Another graduate student (Andre Marozsan, Ph.D.) initiated a project to study the relevance of these V3 crown polymorphisms and generated three of the chimeric viruses used in the study.

73 2.2. ABSTRACT

The HIV-1 envelope is a highly flexible genetic element. Natural variation in the

HIV-1 envelope glycoprotein may affect both sensitivity to entry inhibitors and viral

replicative fitness. Differences in replicative fitness and sensitivity to entry inhibitors

may be further defined by the V3 crown due to its role in engagement of CCR5. In

particular, two positions in the crown (318 and 319) appear to be important in

determining intrinsic susceptibility to multiple entry inhibitors. We evaluated a series of natural polymorphisms at positions 318 and 319 in three distinct CCR5-tropic envelope genetic backgrounds to address the role of these polymorphisms in replicative fitness and sensitivity to RANTES analogs, as well as other entry inhibitors. Change at position 319 to each of the three major consensus amino acids (A, T, and R) resulted in variation in sensitivity to entry inhibitors and altered replicative fitness, but the effects of any one amino acid depended on envelope context. Change of the nearly invariant tyrosine at position 318 to a rare arginine resulted in increased sensitivity to entry inhibitors and decreased replicative fitness independent of envelope context. Polymorphisms in the V3 crown that showed increased susceptibility to entry inhibitors also exhibited decreased replication capacity. Sensitivity to PSC-RANTES correlated most significantly with replicative fitness. Viruses containing V3 crown polymorphisms with the lowest replicative fitness and highest entry inhibitor sensitivity also replicated poorly in

environments with limiting CCR5 density (e.g. primary macrophages), suggesting that

differences in coreceptor affinity may underlie both of these phenotypic characteristics.

74 2.3. INTRODUCTION

The HIV-1 envelope glycoprotein mediates entry of virus into host cells through

sequential interaction with CD4, a coreceptor (either CCR5 or CXCR4), and subsequent

membrane fusion. Entry inhibitors can disrupt this process by preventing any one of

these critical events (16, 59, 213). Primary HIV-1 isolates display a wide range of

susceptibilities to entry inhibitors, with 50% inhibitory concentrations (IC50) varying by

as much as 1000-fold. This is in significant contrast to inhibitors of reverse transcription

and protease cleavage, which exhibit modest differences in intrinsic sensitivity across

diverse HIV-1 isolates. Large susceptibility variations in primary HIV-1 isolates have

been documented for the chemokine derivative AOP-RANTES (189), the fusion inhibitor

enfuvirtide (ENF, T-20) (99, 159, 161), and many small molecule coreceptor antagonists

[TAK-779 (159, 170), maraviroc (54), SCH-C (170), SCH-D (vicriviroc) (185), and

AMD-3100 (99)]. Due to the high degree of diversity among HIV-1 env genes of the

same or different HIV-1 subtypes, it is difficult to identify specific sequence variations

that may be associated with variable sensitivity to entry inhibitors.

Evaluation of intrinsic sensitivity differences to T-20 and TAK-779 revealed that

kinetic factors of fusion were largely responsible for variations in IC50 (159). Sensitivity to T-20 mapped to the V3 loop of env (47), but mutations in the bridging sheet are also sufficient to modulate intrinsic susceptibility to these inhibitors (159, 161). Multiple factors are involved in the efficiency of host cell entry. Upon CD4 binding, structural rearrangements within the envelope occur which reveal the coreceptor binding site. The current model of ternary complex formation favors multiple interaction sites between

75 HIV-1 envelope and CCR5. The bridging sheet and V3 stem interact with the CCR5 N-

terminus, while the V3 crown interacts with the second extracellular loop of CCR5. The hypervariable V3 loop must evolve by balancing attempts to escape host humoral response with the need to engage the CCR5 coreceptor for host cell entry. The affinity relationship between CCR5 and envelope, which can be modulated by the density of

CCR5 on the cell surface, may be important in influencing the efficiency of entry. Some views hold that the major rate-limiting process in host cell entry is the formation of 6- helix bundles (149), but other data suggests that ternary complex formation is the major rate-limiting step of the entry process (132). The affinity relationship between CCR5 and

V3 may be important in influencing the efficiency of entry through either of these pathways.

Mechanisms involved in variable susceptibility to chemokines such as CCL5

(RANTES) or their derivatives have not been evaluated. These inhibitors differ from small molecule CCR5 antagonists in their ability to occupy surface receptor as well as trigger internalization of CCR5 (143). We have previously determined the sensitivity of a panel of primary HIV-1 isolates from all subtypes to the CCL5 analog AOP-RANTES

(189). We found a >30-fold difference in intrinsic susceptibility and suggested that this variability in AOP-RANTES sensitivity may be related to sequence differences in the V3 crown, specifically at positions 318 and 319 (HXB2 numbering). Similar variations at position 319 were observed when comparing intrinsic sensitivities of a different panel of primary isolate viruses to inhibition by TAK-779 and SCH-C (170). Further underscoring the potential relevance of these sites in intrinsic entry inhibitor sensitivity, treatment of HIV-infected hu-PBL-SCID mice with the CCL5 analog NNY-RANTES

76 selected for mutation at position 318 in the challenge virus, suggesting a potential role of this polymorphism in escape (134).

In this study, we assessed the effect of natural polymorphisms at position 318 and

319 in the V3 crown on entry inhibitor sensitivity and overall replicative fitness.

Previous studies have suggested that replicative fitness of primary HIV-1 isolates is associated with the env gene and overall efficiency of host cell entry (12, 123, 157).

Given the central role of the V3 region in coreceptor interactions, it is possible that variation within this region may have a profound impact on the replicative fitness of the whole virus. To assess this, we inserted a series of observed polymorphisms into position

318 and 319 in three distinct envelope backgrounds: the C2-V3 region of a subtype A primary isolate (A1-92RW009), the gp120 region of a subtype B primary isolate (B5-

91US056), and the gp160 region of the Yu-2 strain. These envelopes were inserted into the NL4-3 backbone, and the viruses were tested for sensitivity to a variety of entry inhibitors and other antiretroviral agents. It was determined that polymorphisms at these sites can have a dramatic impact on entry inhibitor sensitivity in a context-dependent and

–independent manner. Competitive replication assays further showed that these polymorphisms can significantly impact replicative fitness. In general, entry inhibitor sensitivity in each background correlated with replication capacity, suggesting involvement of a common factor, such as the affinity/avidity of HIV-1 envelope and

CCR5.

77 2.4. MATERIALS AND METHODS

2.4.1. Cells and Viruses. U87 human glioma cells expressing CD4 and either

CCR5 or CXCR4 were obtained through the AIDS Research and Reference Reagent

Program. These cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM,

Mediatech, Inc., Herndon, VA) supplemented with 15% fetal bovine serum (FBS,

Mediatech, Inc.), penicillin (100 U/ml), streptomycin (100 μg/ml), geneticin (G418, 300

μg/ml), and puromycin (1 μg/ml). 293T cells were grown in DMEM containing 10%

FBS and penicillin/streptomycin. Peripheral blood mononuclear cells (PBMC) were

extracted from HIV-seronegative donors by Ficoll-Hypaque density gradient

centrifugation of heparin-treated venous blood. PBMC were maintained in RPMI 1640

(Mediatech, Inc.) supplemented with 10% FBS and penicillin/streptomycin, and

stimulated with phytohemagglutinin (PHA, 2 μg/ml, Sigma-Aldrich, St. Louis, MO) and

interleukin-2 (IL-2, 1 ng/ml, BD Pharmingen, San Diego, CA) for 3 days prior to

infection. CD4+ T cells were isolated from PHA-blasted PBMC cultures by negative

selection using the Miltenyi CD4+ T cell magnetic isolation kit (Miltenyi Biotec,

Bergisch Gladbach, Germany) and maintained in RPMI supplemented with 10% FBS and

IL-2. Monocytes were isolated from unstimulated PBMC by positive selection for CD14

positive cells (Miltenyi Biotec), and macrophages were matured by adherence to plastic and growth in the presence of GM-CSF for 7 days. Macrophages were maintained in

o RPMI supplemented with 10% new human serum (Cellgro) at 37 C in 5% CO2.

78 Primary HIV-1 isolates were obtained from the AIDS Research and Reference Reagent

Program. For all strains used, the letter and number before the dash indicate the subtype of the virus envelope and the laboratory strain number, which precedes the year of isolation, country of origin, and repository strain number (e.g. A1-92RW009 denotes a subtype A virus isolated in Rwanda in 1992). NL4-3-envelope chimeric viruses were generated using a yeast recombination/gap repair technique (120). Briefly, the C2-V3 region of the subtype A primary isolate A1-92RW009 was PCR amplified using the forward primer E80-RECOM and the reverse primer E105-RECOM and cotransformed into yeast with the Sac II linearized vector pREC-envΔV3/URA3. Recombined plasmids were selected on leucine dropout media containing FOA. The EcoRI – XhoI fragment, which includes the entire HXB2-derived coding region of gp160 as well as the first and second exons of tat and rev, was shuttled into the infectious molecular clone pNL4-3.

Cloning of the gp120 region of the subtype B primary isolate B5-91US056 has been previously described (120, 123). Cloning of the Yu-2 gp160 was performed essentially as in Marozsan et al (120). Site directed mutagenesis was performed on the envelope genes using the QuikChange XL site directed mutagenesis kit (Stratagene, La Jolla, CA).

Infectious chimeric virus was produced by transfecting 293T cells using the Effectene transfection reagent (Qiagen, Valencia, CA). Supernatant containing virus was collected

2 d post-transfection, clarified by centrifugation at 2500 rpm for 15 min, and purified through a 0.45 μm filter (Millipore, Billerica, MA). Viruses were subsequently passaged through U87-CD4/CCR5 cells to remove debris produced in transfection. Sequence analysis of the C2-V3 region was performed in triplicate on virus stocks to confirm that reversion or mutation did not occur. Viral titers were calculated using a limiting dilution

79 + TCID50 method on PHA and IL-2 activated PBMC, CD4 T cells, macrophages, or U87-

CD4/CCR5 cells as previously described (12).

2.4.2. Western Detection of Viral Proteins. Viral incorporation of gp120 was assessed by Western blot. Equal TCID50 values of virus stock were first clarified of cell

debris by retaining the supernatant after centrifugation at 3,000 rpm for 10 min and then

pelleting the virus in supernatant by centrifugation at 38,000 rpm for 45 min. Virus

pellets were lysed with SDS lysis buffer which was then separated on 10% PAGE and

transferred to nitrocellulose. Blots were blocked with 5% gelatin solution and probed for

gp41 with the chessie 8 antibody obtained through the AIDS Research and Reference

Reagent Program. The presence of gp120 was assessed using the B13 antibody (a kind

gift of Dr. George Lewis, The Institute of Human Virology, Baltimore, MD, and Bruce

Chesebro, NIAID, Hamilton, MT) (2). Envelope detection was controlled by p24

quantification, assessed using the 24-4 MAb, obtained through the AIDS Research and

Reference Reagent Program. Primary blots were incubated with HRP-conjugated goat-

anti-mouse secondary antibody (Pierce Biotechnology, Inc., Piscataway, NJ).

2.4.3. Single-cycle Infection Assays. Envelope expression vectors were

generated from the pREC-env shuttle vectors used in the yeast recombination cloning

step. Generation of envelope expression vectors was previously described (120).

Plasmids were digested with Xba I and religated to align the poly-A signal sequence with

the 3’ end of the envelope open reading frame. Envelope pseudotypes were generated by

cotransfection of 293T cells with the 1 μg of the luciferase-encoding pseudotyping vector

80 NLLuc.AM (155) and 1 μg of envelope expression vector. Cells were washed after 24 h, and pseudoviruses were collected after a subsequent 48 h. Relative particle numbers were determined by limiting dilution reverse transcriptase assay (121). Limiting dilution infectivity assays were performed to determine a linear range for each pseudovirus for infection of U87-CD4/CCR5 cells. Cells were incubated with 1:5 dilutions of pseudovirus for 48 h, washed with PBS, and lysed with 100 μl Glo Lysis Buffer

(Promega, Madison, WI). Samples were assessed for luciferase activity on the Bio-Rad

Lumimark-Plus (Bio-Rad).

2.4.4. Entry Inhibitor Sensitivity. Most drug sensitivity assays (excluding

sensitivity to 2D7) were performed in multiple cycle replication assays on U87-

CD4/CCR5 cells. Cells were added to 96-well plates (104 cells/well) and allowed to

adhere overnight. Cells were incubated with serial 10-fold dilutions of drug

(CCL3/CCL4/CCL5 [100nM to 0.1 pM], PSC-RANTES [10 nM to 0.1 pM], T-20 [10

μM to 0.01 nM], TAK-779 [10 μM to 0.1 nM], 3TC [10 μM to 0.1 nM], nevirapine [10

μM to 0.1 nM], ritonavir [10μg/ml to 0.1 ng/ml], 118-D-24 [100 μM to 1 nM], and 2D7

[25 μg/ml to 0.25 μg/ml] ) for 1 h prior to the addition of virus (MOI = 0.005). Cells

were incubated with virus for 24 h, washed with PBS, and fresh medium that contained

the appropriate concentration of drug was replaced. Cell-free supernatant samples were

taken each at day 4-8 post-infection and monitored for virus production by radioactive

reverse transcriptase (RT) assay protocol previously described (189). Plots of RT activity

versus drug concentration were constructed and analyzed to determine the 50% inhibitory

concentration (IC50) for each drug. For the monoclonal antibody 2D7, cells were

81 incubated with serial 10-fold dilutions of antibody for 1 h prior to the addition of

pseudovirus. Cells were incubated for 48 h, washed with PBS, lysed, and luciferase

activity determined. Plots of luciferase activity versus drug concentration were used to determine IC50 values for each pseudovirus.

2.4.5. Replicative Fitness from Competition Assays. The replicative fitness of each chimeric virus was determined using a dual infection competition assay. Chimeric viruses containing V3 crown polymorphisms were competed against a panel of CCR5- tropic primary isolate viruses (B2-92BR017, Yu-2, B5-91US056, C5-97ZA003, A1-

92RW009, and B10-92BR003). Virus was added alone or in pairs to PHA/IL-2

stimulated PBMC at a multiplicity of infection (MOI) of 0.0005 IU/cell to 5 x 105 cells

per well in a 48-well plate (100 IU/well). Fresh IL-2 was added 4 days post-infection.

All assays were performed using one donor from a single blood draw in duplicate. Cell-

free supernatants were assessed for reverse transcriptase activity at days 4, 8, and 12

postinfection. Cells and cell-free supernatant were harvested 12 days post-infection and

stored at –80o C for subsequent analysis. Proviral DNA was extracted from infected

PBMC using the QIAamp DNA blood kit (Qiagen). Proviral DNA was PCR amplified

over the C2-V3 region as previously described (12). For competitions that include

viruses with identical C2-V3 regions, PCR amplification was performed over the vif gene

region. Nested PCR products from the competitions were analyzed by heteroduplex

tracking assay. A radioactive DNA probe was generated by PCR amplification from a

plasmid template containing the C2-V3 region of a heterologous virus. The forward primer was labeled with 2 μCi of [γ-32P] ATP using T4 polynucleotide kinase (Invitrogen,

82 Carlsbad, CA). Nested PCR reactions from competition DNA were annealed to probe by mixing PCR product with radioactive probe and denaturing at 95oC for 3 min and annealing at 37oC for 5 min. Reactions contained DNA annealing buffer (100 mM NaCl,

10 mM Tris-HCl [pH 7.8], and 2 mM EDTA), 10 μl of unlabeled PCR-amplified DNA from the competition, and approximately 400 counts per minute (cpm) of probe per reaction. The DNA heteroduplexes were resolved on tris-borate-EDTA buffer-containing

10% nondenaturing polyacrylamide gels for 2 h at 200 V. Gels were dried and exposed to X-ray film (Eastman Kodak Co., Rochester, N.Y.). Quantification of heteroduplex bands was performed on a Bio-Rad phosphorimager using QuantityOne software (Bio-

Rad, Hercules, CA). Replicative fitness of each chimeric virus was estimated from the relative abundance of each virus quantified from the heteroduplex tracking assay relative to the probe binding of the monoinfection. Production of each virus in the dual infection

(f0) was divided by the initial proportion in the inoculum (i0) and is referred to as the relative fitness of that virus (w = f0/i0). The fitness difference (WD) between the two viruses in competition is representative of the relative fitness of the more fit virus (wM) versus that of the less fit virus (wL): WD = wM/wL.

83 2.5. RESULTS

2.5.1. Natural Variation at HIV-1 V3 Positions 318 and 319.

Multiple studies have demonstrated a potential role for positions 318 and 319

(HXB2 numbering) in the V3 region in affecting sensitivity to entry inhibitors (134, 170,

189). Previous work described a 30-fold variation in sensitivity to the CCL5 analog

AOP-RANTES in a panel of diverse HIV-1 isolates (n=14). Sequence analysis of the V3

region suggested a possible association between AOP-RANTES sensitivity and the

identity of the amino acid at position 319 being either a threonine or an alanine (189). A more recent study on the CCR5 monoclonal antibody PA14 showed a distinct pattern of sensitivity to the CCR5 antagonists SCH-C and TAK-779. This sensitivity was mapped to the V3 crown, and included the polymorphisms alanine, threonine, and arginine at position 319 (170). Furthermore, treatment of hu-PBL-SCID mice infected with the

HIV-1 strain 242 with NNY-RANTES resulted in mutation in the V3 crown position 318

(H→R), which may have been associated with escape from drug pressure (134). We analyzed the frequency of the amino acids alanine, threonine, and arginine at position 319, and tyrosine and arginine at position 318 from 31,408 HIV-1 V3 sequences from all subtypes deposited in the Los Alamos database (Table 3). Alanine, threonine, and arginine are the major consensus amino acids at position 319, but their frequency varies by subtype. Alanine is the consensus amino acid for subtypes A, C, F, G, and H, while threonine is the consensus amino acid for subtypes B and D. In subtype A, the alanine polymorphism is present in the large majority of sequences (84%) while the threonine is relatively rare (7%). In subtype B, both threonine (53%) and alanine (42%) are present in

84 85 the majority of sequences. The 319 consensus amino acid for subtype CRF01_AE is the

more atypical arginine, and alanine and threonine are present at very low abundance.

Position 318, on the other hand, is very well conserved across subtypes. The consensus

amino acid for all subtypes is tyrosine, and few variations at this site have been recorded.

Arginine at position 318 is present in conjunction with either alanine or threonine at

position 319 in very low frequencies across subtypes.

To assess polymorphisms at position 318 and 319 in a systematic fashion, it was essential that we use envelope chimeric viruses. To rule out replicative fitness effects caused by other regions of the HIV-1 genome, we used NL4-3 as a neutral backbone for all chimeric viruses. We generated replication competent chimeric viruses with three different envelope genes in the NL4-3 backbone: 1) an envelope chimeric virus containing the C2-V3 region of a subtype A primary isolate, A1-92RW009, in the HXB2 envelope background, 2) the gp120 coding region of a subtype B primary isolate, B5-

91US056, with the gp41 derived from HXB2, and 3) the gp160 region of the Yu-2 strain

(Fig. 11A). The HXB2 env in the NL4-3 backbone is preferred over the complete NL4-3 sequence because HXB2 lacks the G36D mutation present in the NL4-3 gp41, which

confers reduced susceptibility to T-20. Site directed mutagenesis was performed on these

envelopes to introduce the relevant natural polymorphisms at positions 318 and 319 (Fig.

11A). The wild type sequence for A1-92RW009 and B5-91US056 is Y318A319, and the wild type Yu-2 sequence is Y318T319 (Fig 11A). These positions are four amino acids

downstream of the conserved GPGX tip of the V3 crown (Fig. 1B).

Infectious chimeric virus was produced and analyzed to quantify envelope

3.5 incorporation. Equal TCID50 values of viral supernatants [10 (3162) infectious units]

86 Figure 11. Generation of V3 Mutant Chimeric Viruses. (A) Three different chimeric

viruses were generated: NL4-3-V3A1-92RW009 contains the V3 region of the primary,

CCR5-tropic isolate A1-92RW009 in the NL4-3 background. NL4-3-gp120B5-91US056 contains the gp120 region derived from the primary, CCR5-tropic isolate B5-91US056 in the NL4-3 background, and NL4-3-gp160Yu-2 is an NL4-3 construct with the full-length

envelope coding sequence of the CCR5-tropic isolate Yu-2. Site-directed mutagenesis

was performed at positions 318 and 319 to change the wild type amino acids (A1-

92RW009 – YA; B5-91US056 – YA; Yu-2 – YT) into the relevant polymorphisms

(318Y/319A, 318Y/319T, 318Y/319R, 318R/319A, 318R/319T). Sequence of the 34

amino acid V3 loop is shown, and the crown sequence is boxed. The amino acids at 318

and 319 are indicated in bold. Wild type virus sequences are indicated in bold. (B) The

X-ray crystal structure of the V3 loop derived from Huang et al (86) is presented as a

ribbon and a space filling model. The crown GPGR of the subtype B JR-FL strain is

depicted in this structure (purple). In JR-FL, position 319 is a threonine. Since a tyrosine

or arginine at position 318 and an alanine or threonine at position 319 are natural

polymorphisms in both subtype A and B, energy minimizations were performed with the

T319A substitutions in the JR-FL structure and using the GROMOS96 algorithm within

the DeepView/Swiss-PdbViewer v3.7 (assuming a uniform dielectric constant inside the

protein). The alanine was easily accommodated without perturbing the minimal energy

predicted by the model.

87

88 were pelleted and analyzed by Western blot to detect HIV-1 gp120 and p24. All chimeric

viruses incorporated similar amounts of gp120 with respect to p24 antigen content (Fig.

12A). Processing of the gp160 precursor into gp120 and gp41 occurred with similar

efficiencies in all envelopes (Fig. 12A). Coreceptor tropism for each of the V3 mutant envelopes was determined using a luciferase-based envelope pseudotype assay.

Pseudoviruses were produced bearing each mutant envelope, and equivalent particle numbers were used to infect U87 cells expressing either CD4 alone, CD4 and CXCR4, or

CD4 and CCR5. All envelopes effectively mediated infection of U87-CD4/CCR5 cells, but not cells expressing CXCR4 or no coreceptor (Fig. 13A-C).

Using luciferase-expressing pseudoviruses, luciferase activity is proportional to the number of particles that infect the cells (155). Thus efficiency of virus entry can be roughly assessed from luciferase activity. The A to T change at position 319 in the V3A1-

92RW009 envelope modestly increased entry efficiency, while the A to R change modestly decreased efficiency. The Y to R change at position 318 resulted in a 100-fold decrease in luciferase activity in conjunction with either an A or T at position 319 of the V3A1-

92RW009 envelope (Fig. 13A). In the B5-91US056 envelope, change between A and T had

no effect on entry efficiency, while in the Yu-2 gp160 context, change from the wild type

T to alanine at 319 resulted in a 10-fold increase in entry efficiency (Fig. 13B and C).

However, insertion of arginine at position 318 or 319 resulted in significant decreases in

entry efficiency (greater than 100-fold) in both subtype B envelope contexts (Fig. 13B

and C). In the Yu-2 context, insertion of arginine at either position 318 or 319 severely

attenuated transduction of U87-CD4/CCR5 cells by pseudovirus, with >1000-fold

decreases in luciferase activity detected (Fig. 13C).

89 Figure 12. Analysis of Envelope Incorporation by V3 Mutant Chimeric Viruses. To ensure equivalent incorporation of the envelope protein in the chimeric viruses, equal

TCID50 of replication competent virus was pelleted at 38,000 rpm and subjected to

Western blot. (A) 103.5 infectious units of each virus were pelleted and resuspended in

100 μl of SDS lysis buffer. 10 μl of this sample, or 316 infectious units, were diluted serially 1:5 for each NL4-3-V3A1-92RW009 chimeric virus and were probed for gp120 (B13

MAb), gp41 (Chessie 8 MAb), and p24 (24-4 MAb). (B) 316 infectious units of each

NL4-3-gp120B5-91US056 and NL4-3-gp160Yu-2 V3 crown mutant chimeric virus were probed for p24 and gp120 content.

90

91 Figure 13. Coreceptor Tropism and Pseudovirus Infection Efficiency. Equivalent particle numbers of pseudovirus, as determined by limiting dilution reverse transcriptase assay, were used to infect U87-CD4, U87-CD4/CXCR4, or U87-CD4/CCR5 cells. Cells were incubated with pseudovirus for 48 h, the cells lysed, and luciferase activity was determined. Pseudoviruses produced without envelope served as negative control for infection, while treatment of cells with 100 μM 3TC served as a specificity control for luciferase activity. Values are presented as relative light units, and error bars represent standard deviations from two independent experiments performed in triplicate.

92

93 2.5.2. Replicative Fitness of V3 Mutant Chimeric Viruses in PBMC.

Competitive replicative fitness of a virus in ex vivo culture assays describes the relative ability of one virus versus another to expand in a cell population. Previous studies have suggested that the entry process is an important determinant of replicative fitness of the wild type primary HIV-1 isolates (12, 123, 157). We anticipated that polymorphisms within the V3 region, which mediated significant differences in transduction of U87-CD4/CCR5 by pseudoviruses, would have a significant impact on replicative fitness. Thus, we assessed viral replicative fitness in PHA/IL-2 activated

PBMC cultures by competing each V3 mutant chimeric virus against each of six primary

HIV-1 reference isolates: B2-92BR017, Yu-2, B5-91US056, C5-97ZA003, A1-

92RW009, and B10-92BR003 (Fig. 14). This set of reference HIV-1 isolates were selected based on having a range of relative fitness values common to wild type primary

HIV-1 isolates (Fig. 14A) (12). A subset of V3 mutant chimeric viruses was employed in pairwise competitions to confirm the relevance of comparison to a reference panel (Fig.

16 and 17). Relative virus production was monitored at 12 days post-infection using heteroduplex tracking assay (see Materials and Methods or Fig. 16 relating to pairwise competitions) (7, 12).

Insertion of the A1-92RW009 V3 region into the NL4-3 backbone increased the fitness of this chimeric virus over the parental A1-92RW009 primary isolate (Fig. 14B).

Comparison of A1-92RW009 to NL4-3-V3A1-92RW009(YA) showed an approximate 5-fold increase in replicative efficiency by the chimeric virus. This increased fitnesss in the

NL4-3 backbone may be related to optimization of this laboratory strain through in vitro selection. We consistently observe increases in replicative fitness of the NL4-3/env

94 Figure 14. Replicative Fitness of V3 Polymorphisms in Three Envelope Contexts.

Each chimeric virus was competed in pairwise dual infection in PHA/IL-2 activated

PBMCs against a panel of CCR5-tropic primary isolate viruses (B2-92BR017, Yu-2, B5-

91US056, C5-97ZA003, A1-92RW009, and B10-92BR003). Relative fitness values

were calculated as described in materials and methods. Plots are of fitness difference

values and are indicative of fold-difference replication capacity. Fitness differences are

plotted on logarithmic scale, and bars that fall above the midline (WD = 1) indicate

competitions in which the chimeric virus was the winner, while bars falling below the

midline indicate competitions in which the primary reference isolate was the winner.

Bars that lie near the midline (WD = 1) indicate competitions in which both viruses were

of nearly equivalent replicative fitness. All competitions were performed in duplicate.

(A) Fitness order of full length viruses used in the reference panel. (B) Fitness

difference values of NL4-3-V3A1-92RW009 chimeric viruses with V3 polymorphisms against the reference panel. (C) Fitness difference values of NL4-3-gp120B5-91US056 chimeric viruses with V3 polymorphisms against the reference panel. (D) Fitness difference values of NL4-3-gp160Yu-2 chimeric viruses with V3 polymorphsims against

the reference panel.

95

96 chimeric virus over the parental primary HIV-1 isolate. Considering the slight increase in

fitness of the chimeric viruses versus the parental strain, the fitness impact of 318 and

319 substitutions were always compared among themselves rather than directly to their

parental primary isolate. Change from alanine to threonine at position 319 resulted in a

modest increase in replicative fitness consistent with the pseudovirus data (Compare Fig.

13A and Fig. 14B). The A→R change at position 319 of NL4-3-V3A1-92RW009(YA) resulted in a significant but still modest decrease in fitness compared to the wild type chimeric virus. However, insertion of arginine at position 318 significantly reduced replicative fitness. A threonine as opposed to alanine at position 319 provided some compensation for the decreased fitness conferred by R318 in the NL4-3-V3A1-92RW009(YA)

virus. A total relative fitness value was derived for the addition of the relative fitness

values derived from competitions against the six reference strains (Fig. 15). This

collective analysis revealed a fitness order for the V3 polymorphisms in NL4-3-V3A1-

92RW009 context: Y318T319 > YA > YR > RT > RA. However, all of these chimeric viruses

had fitness values in the range of wild type, CCR5-tropic primary HIV-1 isolates

circulating in the infected population.

In the subtype A population, alanine is significantly more frequent than threonine

at position 319 (84% versus 7%, Table 3), while in the subtype B population, alanine and

threonine are present at nearly equal frequency (42% versus 53%, Table 3). Change

between A and T at position 319 in both subtype B contexts had modest fitness impact.

The wild type NL4-3-gp120B5-91US056(YA) and NL4-3-gp120B5-91US056(YT) variants had

nearly equivalent fitness, and the wild type YA was slightly more fit that the parental

primary B5-91US056 isolate (Fig. 14C). However, the wild type NL4-3-gp160Yu-2(YT)

97 Figure 15. Total Relative Fitness Values of Chimeric Viruses. Total relative fitness was calculated by adding the relative fitness value (w) of the chimeric virus from each competition with a reference panel virus.

98

99 virus was significantly less fit than the NL4-3-gp160Yu-2(YA), but both chimeric viruses

could out-compete the parental Yu-2 virus (Fig. 14D). Insertion of arginine into position

V3 position 318 or 319 of NL4-3-gp120B5-91US056 or NL4-3-gp160Yu-2 yielded viruses

with significantly reduced replication capacities as seen by their poor ability to compete against the six reference HIV-1 isolates (Fig. 14C and D). Evaluation of total relative fitness values revealed that natural variations at sites 318-319 had a greater impact on

replicative fitness in the context of the B5-91US056 or Yu-2 envelope than in the A1-

92RW009 C2-V3 context (Fig. 15). This decrease in the fitness impact in the NL4-3-

V3A1-92RW009 chimeric viruses may again be related to having more of the HXB2 env gene associated with the A1-92RW009 V3 sequence.

To determine overall replication capacity of each virus, we competed each chimeric virus against a panel of reference viruses. To confirm the relevance of this type of comparison, we competed the NL4-3-V3A1-92RW009 V3 mutant chimeric viruses against

each other in pairwise competition. All of the V3 mutant NL4-3-V3A1-92RW009 chimeric

viruses contained one of three synonymous mutation vif sequence tags [i.e. vifA (wild

type NL4-3 sequence), vifB, or vifC, Fig. S1]. Each tag is a region of altered nucleotide

sequence in vif that codes for the same amino acid sequence. These sequence tags were

used to distinguish and quantify two NL4-3-V3A1-92RW009 chimeric viruses by

heteroduplex tracking assay in a dual infection (Fig. 16). Competition between two

viruses that are isogenic but differ only in their vif sequence tag showed no fitness

difference, indicating that the tag sequences did not affect replicative fitness (Fig. 17). A

transitive relationship was evident in this fitness matrix such that fitness order of YT >

YA > YR > RA > RT (Fig. 17) also resulted in the YT variant having greater fitness than

100 Fig. 16. Methods for Determining Replicative Fitness. (A) To distinguish between

the single amino acid polymorphic chimeric viruses in competition, genetic tags were

incorporated into the NL4-3 genome by introduction of synonymous mutations in the

accessory gene vif. The silent mutations permit differentiation of probe binding on

heteroduplex tracking assay without altering the amino acid sequence. (B) Proviral DNA

of vif-tagged chimeric viruses from dual infections [e.g. NL4-3-V3A1-92RW009(YT) vifA vs.

NL4-3-V3A1-92RW009(YA) vifB] was PCR amplified using the external and nested PCR set

within the vif gene, producing a 290 bp amplicon that begins 50 nucleotides upstream of

the vif genetic tag. A radiolabeled probe was generated by PCR amplification from

pNL4-3vifA or vifB, using a common downstream primer and an upstream primer that

overlaps the vif genetic tag. Heteroduplex tracking analysis of the bulk amplicon was

resolved on 8% nondenaturing polyacrylamide gels, permitting quantification of two

heteroduplex species. In the example autoradiograph, lane 1 shows the migration of a

vifA probe, lanes 2 and 3 show a heteroduplex product per lane resulting from an NL4-3-

V3A1-92RW009(YT) chimeric virus (vifA) and an NL4-3-V3A1-92RW009(YA) chimeric virus

(vifB) monoinfections, respectively. Lanes 4 and 5 are replicates showing two heteroduplex products per lane resulting from a competition between the NL4-3-V3A1-

92RW009(YT) chimeric (vifA) and NL4-3-V3A1-92RW009(YA) chimeric (vifB) viruses.

Phosphor-imaging analysis was used to quantify the production of each virus in dual

infection relative to the virus production in the monoinfections. (C) Panel C provides an

example of the fitness determination for the NL4-3-V3A1-92RW009(YT) chimeric virus

relative to the NL4-3-V3A1-92RW009(YA) chimeric virus . The vifA, vifB, and vifC versions

of the NL4-3-V3A1-92RW009(YT) chimeric virus were competed in duplicate against the

101 vifA, vifB, and vifC versions of NL4-3-V3A1-92RW009(YA) chimeric virus. Relative fitness

values were derived from the mean of these 12 NL4-3-V3A1-92RW009(YT) vs. NL4-3-V3A1-

92RW009(YA) competitions. These mean values are reported in Fig. S2. Previous

experiments have involved multiple competitions between the vifA, vifB, and vifC versions of wild type NL4-3 and found that the synonymous vif substitutions did not affect replicative fitness (data not shown).

102

103 Fig. 17. Fitness Differences Among NL4-3-V3A1-92RW009 Chimeric Viruses. The fitness difference (WD) for the virus indicated on the Y axis is plotted versus all other V3 polymorphsims. Bars that lie above the midline (WD = 1) indicate competitions in which the virus on the Y axis is the winner, and bars that lie below the midline indicate competitions in which the virus bearing the polymorphisms indicated on the bar is the winner. Competitions performed between viruses with the same V3 318/319 polymorphisms but different vif signature sequences indicate that synonymous change in the vif sequences did impact replicative fitness of the virus. Comparison of these viruses resulted in a pure transitive relationship among fitness values, and the fitness differences recapitulated comparison to the reference panel of primary isolates.

104

105 the YA variant in direct competition, and consequently YT > RA, YT > RT, YT > RA

(Fig. S2). Fitness differences using pairwise comparison were identical to those found by

comparison to the reference panel (Compare Fig. 17 and Fig. 15). Thus all other chimeric viruses were competed solely against the reference panel to determine a replicative fitness value.

2.5.3. Replication of V3 Mutant Chimeric Viruses in Macrophage Cultures.

The levels of CD4 and CCR5 expressed on the surface of macrophages is significantly lower than the levels of each receptor on CD4+ T cells (49, 109, 110, 200).

It is commonly observed that viruses with expanded tropism for macrophages have

evolved higher affinity for one or more receptors (57, 154, 188). Thus we examined if

the chimeric viruses with natural V3 polymorphisms displayed different replication

kinetics in macrophage cultures, and if the ability to infect macrophages correlated with

replicative fitness in PBMCs. Limiting dilution TCID50 assays were performed side-by- side in macrophage cultures and in CD4+ T cell cultures from the same donor and blood

draw. In the subtype B contexts, the V3 polymorphisms with an arginine at position 318

or 319 that induced the lowest repicative fitness in PBMC cultures failed to grow to

detectable levels in macrophage cultures (Table 4). The NL4-3-V3A1-92RW009 viruses

experienced more significant growth in macrophage cultures relative to CD4+ T cell cultures as compared to the other chimeric viruses. The 318 and 319 polymorphisms containing arginine did replicate detectably in macrophages, but with slower kinetics than the YA and YT polymorphisms. Enhanced macrophage tropism in these viruses may again be a complementation effect with the HXB2 envelope fragment.

106

107 2.5.4. Antiretroviral Susceptibility Conferred by V3 Polymorphisms.

Replication competent chimeric viruses harboring the various V3 polymorphisms

were employed in drug susceptibility assays using a broad range of antiretroviral

compounds in U87-CD4/CCR5 cell cultures. Virus production was measured by reverse

transcriptase activity in the supernatant. Changes at position 318 and 319 mediated

differential sensitivity to entry inhibitors in all envelope contexts (Table 5 and Fig. 18).

Variable inhibition of the V3 mutant chimeric viruses was observed with (i) native β-

chemokines and their analogs (CCL4, CCL5, PSC-RANTES), (ii) monoclonal CCR5

antibodies (2D7), (iii) noncompetitive allosteric inhibitors of CCR5 (TAK-779), and (iv)

inhibitors of virus fusion (ENF) (Fig. 18A-C and Table 5). No differences in IC50 values were observed for the V3 mutant chimeric viruses with compounds that inhibit reverse transcription (3TC, nevirapine), protease cleavage (ritonavir), or integrase function (118-

D-24) (Table 5). Drug sensitivity assays performed with the primary isolate viruses A1-

92RW009, B5-91US056, and Yu-2 indicate that the envelope chimeric viruses recapitulate the intrinsic sensitivity of the parental viruses to PSC-RANTES, ENF, TAK-

779, and 3TC (Table 6).

In the NL4-3-V3A1-92RW009 virus context, change at position 319 among T, A, and

R polymorphisms had only modest effects with most entry inhibitors (Fig. 18, A-C).

Arginine at 319 in the NL4-3-V3A1-92RW009 viruses resulted in increased susceptibility exclusively to ENF (Fig. 18B). Arginine at position 318 increased sensitivity to nearly all entry inhibitors in conjunction with both alanine and threonine at position 319 (Table

5, Fig. 18 A-C). Only the NL4-3-V3A1-92RW009 viruses containing arginine at position 318

108

109

110 Figure 18. Sensitivity of 318/319 Polymorphisms to Entry Inhibitors. U87-

CD4/CCR5 cells were incubated with 10-fold dilutions of PSC-RANTES (a), ENF (B),

TAK-779 (C), or 3TC (D) and then exposed to virus for 24 h. After washing out input

virus, supernatant samples were analyzed for reverse transcriptase activity at 4, 6, and 8

days post-infection. Drug susceptibility curves (Fig. 19) were plotted to determine IC50 values using the Probit algorithm. IC50 values and standard deviations for each respective

drug and virus are plotted in the panels. Asterisks indicate cutoff for significance of p <

0.01.

111

112 were susceptible to inhibition by CCL5 and CCL4, but no viruses were inhibited by

CCL3 (Table 5).

The 318/319 V3 polymorphisms in the subtype B env contexts had similar

impacts on sensitivity to entry inhibitors as observed with NL4-3-V3A1-92RW009 viruses.

In the NL4-3-gp120B5-91US056 virus context, the T and A polymorphisms at position 319

did not significantly alter sensitivity to most entry inhibitors (Fig. 18 A-C, Fig. 19A-D).

Arginine at position 319 in this envelope context resulted in increased susceptibility to

PSC-RANTES, ENF, and 2D7 (Fig. 18A and B, Table 5, Fig. 19A, B, and D). This

variant did not exhibit increased susceptibility to TAK-779 (Fig. 18C, Fig. 19C).

Arginine at position 318 in conjunction with either alanine or threonine at position 319

significantly increased sensitivity to all entry inhibitors (Fig. 18 A-C, Fig. 19 A-D, Table

5). The susceptibility of NL4-3-gp120B5-91US056(YT) differed from NL4-3-gp120B5-

91US056(RT) by 50-fold to PSC-RANTES, and by 100-fold for ENF and TAK-779. As

with the NL4-3-V3A1-92RW009, arginine at position 318 in the NL4-3-gp120B5-91US056 context resulted in susceptibility to CCL5 and CCL4 (Table 5).

V3 polymorphisms in the NL4-3-gp160Yu-2 context had dramatic effects on

sensitivity to entry inhibitors. A change of the wild type 319T to alanine yielded a NL4-

3-gp160Yu-2 virus with reduced susceptibility to PSC-RANTES, ENF, and 2D7 (Fig. 18A,

B, and C; Table 5). Insertion of arginine at either position 318 or 319 resulted in NL4-3-

gp160Yu-2 viruses with exquisite sensitivity to inhibition by PSC-RANTES, ENF, and

TAK-779 (Fig. 18 A-C). As described above, the NL4-3-gp160Yu-2 viruses with the V3

polymorphisms were equally sensitive to a protease inhibitor (ritonavir), nucleoside RT

113 Fig. 19. Drug Sensitivity Curves for NL4-3-gp120B5-91US056 Viruses. Relative

supernatant RT activity from 8 days post-infection was plotted versus drug concentration

to determine IC50 values. Values are derived from a single experiment performed in triplicate on U87-CD4/CCR5 cells. Error bars represent standard deviation of each value.

Curves were used to determine IC50 values.

114

115 inhibitor (3TC), non-nucleoside RT inhibitor (nevirapine), and integrase inhibitor (118-

D-24) (Table 5 and Fig. 18D).

2.5.5. Comparing Replicative Fitness with Sensitivity to Entry Inhibitors.

The data described above suggests that replicative fitness in PBMCs conferred by variation in the V3 crown is directly related to the efficiency of entry by pseudotyped virus in U87-CD4/CCR5 cells and inversely related to sensitivity to entry inhibitors.

Using regression analysis, we observed a highly significant correlation between sensitivity to PSC-RANTES inhibition and total relative fitness [r = 0.93, p < 0.000014,

Pearson product-moment correlation (PPMC)] (Figure 20). The relationships between total relative fitness and IC50 values with ENF (r = 0.73, p < 0.0028, PPMC) and 2D7 (r =

0.64, p < 0.028, PPMC) were weaker but still significant (Figure 20). In contrast, there was no significant correlation between IC50 values to TAK-779 (r = 0.40, p < 0.14) or

3TC (r = 0.19, p < 0.51) and total relative fitness of these V3 mutant chimeric viruses

(Figure 20). These results suggest that reduced susceptibility to entry inhibitors (as assessed by increasing IC50 value) may be associated with substitutions that increase replicative fitness.

116 Figure 20. Correlation of Entry Inhibitor Sensitivity and Total Relative Fitness.

Coefficients were determined by Pearson product-moment correlation.

117

118 2.6. DISCUSSION

In this study, we have addressed the effects of natural polymorphisms in the V3

crown on HIV-1 replication capacity and sensitivity to various entry inhibitors. Changes

at position 318 and 319 in the V3 crown, immediately downstream of the GPGX motif,

have been implicated in variable entry inhibitor susceptibility in three separate analyses

(134, 170, 189). We evaluated the three major consensus amino acids at position 319 (A,

T, and R). The frequency of each polymorphism varies significantly by subtype (Table

3). We further evaluated two amino acids at position 318 (Y and R). Tyrosine at

position 318 is highly conserved across all subtypes, but arginine arises at low frequency

in subtypes A, B, C, and D (Table 3). In this study, polymorphisms at these sites were

inserted alone or in combination into three distinct envelope contexts. Clonal, chimeric viruses expressing the mutant envelopes in the background of the laboratory strain NL4-3 were assessed in multiple replication cycle drug sensitivity assays and growth competition assays.

Variation at position 319, which is polymorphic across HIV-1 subtypes, affected replication and entry inhibitor sensitivity in a context-dependent manner. On the other hand, change at position 318, where tyrosine is highly conserved across subtypes, resulted in increased entry inhibitor sensitivity and decreased replicative fitness independent of envelope background. The V3 region in env codes for an exposed and immunogenic loop-like structure that provides vital functions in mediating co-receptor binding and virus entry. Recent X-ray crystallography data on gp120 suggests that residues 318 and 319 are in a cavity just below the GPGX crown of the V3 loop (Fig.

119 11B) (86). These amino acids may influence the flexibility of the region or possibly the angle of the crown relative to the base. Consequently, substitutions at positions 318 and

319 may affect the strength of the interaction between the V3 crown and the second extracellular loop of CCR5 or the positioning of the crown relative to the receptor.

Modulation of this interaction may then have a dramatic impact on viral replication and sensitivity to drugs that target entry, as the overall efficiency of this process has been altered.

Change of the amino acid at position 319 from one natural polymorphism to another resulted in altered susceptibility to entry inhibitors and modulation of replicative fitness in all three envelope contexts (Fig. 15 and 18). However, the effect of any one amino acid change varied depending on the envelope background in which it occurred.

Threonine at position 319 in the NL4-3-V3A1-92RW009 context resulted in an increase in replication capacity over the wild type alanine (Fig. 15), even though it is rare in the subtype A population (Table 3). Surprisingly, threonine in the subtype B background, where it is the more common amino acid (53% T versus 42% A) had the opposite effect.

The wild type Yu-2 sequence is Y318T319. However, change from the wild type T to A at position 319 caused an increase in replicative fitness (Fig. 15) and increased resistance to

PSC-RANTES, ENF, and 2D7 (Fig. 18 A and B, Table 5). On the other hand, we observed that a single amino acid change could result in different effects within the context of the same HIV-1 subtype. Change between T and A in the NL4-3-gp120B5-

91US056 context resulted in no significant changes in replicative fitness (Figure 15) or entry inhibitor sensitivity (Fig. 18). Change to the amino acid arginine at position 319 resulted in decreased replication capacity and increased entry inhibitor sensitivity in all three

120 envelopes (Fig. 15 and 18). However, envelope sequence context did impact on the

magnitude of this change. For example, insertion of arginine at position 319 in the NL4-

3-V3A1-92RW009 context resulted in only modest reduction in replication capacity, whereas

this change dramatically reduced the efficiency of entry and replicative fitness in the

subtype B background (Fig. 15). In general, the existence of 3 major consensus amino

acids at position 319 in all subtypes suggests flexibility at this position (Table 3).

The 318 position is highly conserved across all HIV-1 isolates. Change of the

nearly invariable tyrosine to an arginine resulted in increased sensitivity to entry

inhibitors and decreased replicative fitness in all envelope contexts (Fig. 15 and 18). The magnitude of the effect, however, was again dependent upon the envelope context. In the subtype A V3 background, the fitness of both the R318A319 and R318T319 variant was

within the normal range of primary HIV-1 isolates (Fig. 14B). On the other hand,

insertion of arginine into position 318 of the Yu-2 context resulted in viruses which

barely replicated in culture (Fig. 14D). The reduced impact of the 318R polymorphism in

the subtype A V3 sequence may be a consequence of the chimeric nature of this envelope.

A substitution to 318R was selected in an HIV-1 isolate during treatment of

infected hu-PBL-SCID mice with the chemokine analog NNY-RANTES (134). This

result provided the rationale for our analysis of this polymorphism. In this case, the wild

type 242 molecular clone contains an arginine at position 318, but the stock virus used in

the study was found to contain a histidine. During the course of treatment, the arginine

re-emerged in NNY-RANTES treated but not control-treated animals. The re-

emergence of arginine at position 318 may have been due to outgrowth of the wild type

242 virus in the presence of NNY-RANTES, or may have been associated with

121 coreceptor switching. In the sequence contexts we analyzed, however, arginine at position 318 was associated with low replicative fitness and high intrinsic susceptibility to PSC-RANTES as well as endogenous chemokines. Furthermore, this change was not sufficient to change tropism of the virus. The 242 sequence context may contain other polymorphisms that are linked to and support replication of a 318R containing virus.

However, outgrowth of the histidine containing variant in tissue culture passage that produced the stock virus suggests that, in the passage environment, the arginine was significantly less fit than the histidine.

We determined ex vivo replicative fitness by competing HIV-1 strains in human

CD8-depleted PBMCs. The relative replicative fitness values, derived from competitions with a set of primary CCR5-tropic reference strains, closely matched efficiency of host cell entry derived from single round entry assays using envelope pseudotyped viruses

(Compare Fig. 13 and Fig. 15). As an example, we found that in the Yu-2 context, arginine-containing envelopes resulted in a 1000-fold infectivity reduction in single-cycle infections as compared to the wild type pseudoviruses (Fig. 13C). The same envelopes, when used in replication-competent, chimeric viruses and employed in competition assays showed a similary dramatic decrease in replicative fitness (Fig. 14D and Fig. 15).

Given the isogenic background of each virus, entry efficiency was the major determinant of relative replicative fitness, and we conclude that the fitness differences we observed in this analysis were specifically due to changes at position 318 and 319.

Adaptations in the V3 loop have been shown to increase envelope fusogenicity

(150). These changes can be associated with an increase in affinity for the receptor, but the affinity relationship is not an exclusive determinant of entry efficiency (151).

122 Previous studies have also suggested that not all polymorphisms that alter coreceptor affinity result in decreased fusion kinetics (161). Natural polymorphisms at positions

318/319 result in changes in entry inhibitor sensitivity and entry efficiency. Viruses bearing 318/319 polymorphisms that conferred high level susceptibility to entry inhibitors had relatively low replicative fitness in PBMCs and could not efficiently infect macrophage cultures when compared to more robust replication in CD4+ T cells derived

from the same donor (Table 4). This data suggests that these viruses are sensitive to the

cell surface density of CCR5 (or CD4). We hypothesize that these V3 crown

polymorphisms are specifically affecting coreceptor affinity and fusion kinetics.

Preliminary data exploring this hypothesis suggests that the differences in replicative

fitness between the NL4-3-V3A1-92RW009(YA) virus and the full length A1-92RW009 virus

may be due to differences in CD4 affinity, as the C2-V3 chimeric virus, but not the

primary isolate, is sensitive to neutralization by soluble CD4.

We observed a general correlation between increasing replicative fitness and

decreasing sensitivity to most entry inhibitors. This observation extended to a chemokine

analog (PSC-RANTES), anti-CCR5 antibody (2D7), and a fusion inhibitor (enfuvirtide)

(Fig. 20). This broad increase in sensitivity to entry inhibitors was surprising considering

these were modest changes which occurred in a variable region of the V3 crown.

However, previous reports have suggested a correlation between sensitivity to

mechanistically distinct entry inhibitors (94, 162, 167). Even though the overall fitness

was altered by these V3 polymorphisms, we observed equivalent sensitivity to other

inhibitors blocking the steps of reverse transcription, DNA integration, and proteolytic

processing. The strongest and most significant correlate of fitness was sensitivity to

123 PSC-RANTES (r = 0.93) followed by T-20 (r = 0.73) and 2D7 (r = 0.64). Although there

was a trend for increased sensitivity to TAK-779 and decreasing replicative fitness, this

relationship was not significant. Correlation between PSC-RANTES sensitivity and

replicative fitness with these V3 polymorphisms was somewhat surprising considering

the dual inhibitory mechanism of PSC-RANTES (143). However, we hypothesize that differences in susceptibility to PSC-RANTES are more closely related to competitive

CCR5 binding between drug and virus than to receptor downregulation. Unlike allosteric inhibitors, it is possible that the competitive inhibitor bound form of CCR5 is maintained in a conformation that is recognized by HIV-1 envelope, but sterically hindered.

Increased CCR5 affinity conferred by polymorphisms in the V3 loop may increase the ability of envelope to compete with PSC-RANTES for CCR5 docking, which would be reflected in higher IC50 values. Correlation between fitness and 2D7 was less significant

than PSC-RANTES, even though it also acts as a competitive inhibitor of CCR5. PSC-

RANTES, but not 2D7, mediates CCR5 internalization, and thus competition between

virus envelope and drug occurs in two different environments for these two drugs.

Envelope competes with 2D7 in an environment with high CCR5 expression, while

envelope competes with PSC-RANTES in an environment of low CCR5 expression. We

may anticipate a stronger relationship between sensitivity to 2D7 and replicative fitness if

drug sensitivity assays are performed in a cell type with lower expression levels of CCR5.

However, we also had fewer data points on the V3 mutant chimeric viruses to test this

relationship. Future experiments are exploring the relationship between levels of CCR5

on the cell surface versus inhibition by competitive inhibitors (e.g. 2D7) of viruses with

varying fitness.

124 We also observed a significant relationship between replicative fitness of V3

crown polymorphisms and sensitivity to enfuvirtide. This finding was predicted by

previous studies indicating that mutations in the gp120 bridging sheet which affected

CCR5 binding and decreased entry efficiency also resulted in increased ENF sensitivity

(19, 159, 161). HIV-1 is susceptible to inhibition by ENF in a window that begins with

CD4 binding and ends when the gp41 C- and N-terminal heptad repeat regions rearrange

into six-helix bundles. This kinetic window and the association rate of the inhibitor are

major determinants of fusion inhibitor susceptibility (159, 182). Coreceptor affinities or

association rates appear to play an important role in the size of the inhibitory window for

fusion inhibitors (132, 149), and polymorphisms in the V3 crown are likely mediating

kinetic fusion differences. Furthermore, delayed fusion kinetics conferred by V3 crown

polymorphisms would certainly affect the rate of viral replication in culture, thus

accounting for correlations between ENF sensitivity and replicative fitness.

Although TAK-779 inhibits HIV-1 envelope binding to CCR5 as do PSC-

RANTES and 2D7, there was only a trend relating TAK-779 inhibition and replicative

fitness. We hypothesize that viruses with greater replicative fitness and entry efficiency

related to higher affinity for CCR5 can compete with PSC-RANTES and 2D7 for

occupancy of CCR5. In the case of small allosteric molecules such as TAK-779, the

inhibitor-bound CCR5 is functionally masked from viral envelope regardless of its coreceptor affinity (18, 175). Differential sensitivity of these mutant V3 chimeric viruses to allosteric compounds may then be more related to the ability of the virus to scavenge drug-free receptor at sub-saturating concentrations. Furthermore, affinity-mediated differences in TAK-779 may be more closely related to interaction between the envelope

125 and the CCR5 N-terminus, and we hypothesize that the V3 crown polymorphisms are specifically affecting interaction between envelope and the CCR5 second extracellular loop. However, these inhibitors do not bind CCR5 to form a dead-end complex, but have defined dissociation rates. Thus, relative affinity of these viruses for CCR5, which do impact replicative fitness, could account for the weak correlation with TAK-779 susceptibility.

Studies examining the emergence of HIV-1 resistance to small molecule CCR5 antagonists suggest that resistance mutations emerge throughout the env gene by evolutionary pathways which may be dependent on sequence context (1, 9, 97, 122, 210).

Early in this research, it was clear that primary HIV-1 isolates displayed a wide range of sensitivities to entry inhibitors and even to native β-chemokines (47, 99, 170, 185, 189,

197). We have been unsuccessful in our attempts to select for resistance to AOP-

RANTES and PSC-RANTES utilizing protocols similar to those successfully employed for generating AD101, Maraviroc, and SCH-D, and TAK-652 resistance in vitro (97, 122,

195, 210). Resistance to small molecule allosteric inhibitors appears to be mediated by usage of both drug-bound and drug-free forms of CCR5 for entry (155, 210), but intermediates in this pathway seemed to adapt to the inhibitor initially by increasing coreceptor affinity (155, 195). In this study, instead of selecting for resistance, we identified key residues in the V3 loop associated with intrinsic sensitivity to PSC-

RANTES (> 50-fold variation in IC50 values). The term “resistance” has not been applied to HIV-1 isolates harboring polymorphisms which confer reduced sensitivity to entry inhibitors. Reduced susceptibility to entry inhibitors may emerge during disease progression or through circulation of HIV-1 through the human population as an

126 adaptation to innate and acquired host factors (95, 181, 196), providing a framework for variations in sensitivity to entry inhibitors. Changes in the V3 crown that reduced susceptibility to entry inhibitors also increased entry efficiency and fitness, possibly by altering the affinity of the crown for CCR5. Though affinity differences may be irrelevant for complete insensitivity to CCR5 antagonists, treatment with these drugs may initially select for strains that resist drug inhibition by increasing affinity for CCR5.

While resistance to most antiretroviral drugs results in decreased fitness, these partially resistant variants may have higher replicative fitness in vivo. Increases in CCR5 affinity as an adaptation to entry inhibitor therapy may be an important step for all viruses in the evolution of resistance to coreceptor antagonists and may affect the course of disease progression.

127 Chapter 3.

DIFFERENTIAL SENSITIVITY TO INHIBITION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 TO PSC-RANTES INVOLVES COMPETITIVE CCR5 BINDING

Authors: Michael A. Lobritz1,2, Andre J. Marozsan2, and Eric J. Arts1,2

1Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA 44106

2Division of Infectious Diseases, Department of Medicine, Case Western Reserve University

128 3.1. PREFACE

Having observed a strong correlation between replicative fitness and sensitivity to inhibition by PSC-RANTES, we sought to understand the mechanism of inhibition of this compound. Chemokine analogs are known to initiate ligand-dependent internalization of

CCR5, which functionally removes coreceptor from the cell surface. We wished to evaluate alternative inhibitory mechanisms by PSC-RANTES from the perspective of receptor binding.

129 3.2. ABSTRACT

The chemokine analog PSC-RANTES inhibits replication of HIV-1 by blocking viral envelope interaction with the CCR5 coreceptor. The potency of PSC-RANTES is generally ascribed to a mechanism involving downregulation and retention of cell surface

CCR5. This model suggests that most viruses should have similar sensitivities to inhibition. We have evaluated the mechanism of inhibition of PSC-RANTES using 2

HIV-1 clones which differ by two amino acids in the V3 crown. These viruses differed in sensitivity to inhibition by PSC-RANTES by 10-fold in multiple cycle replication assays, but exhibited no sensitivity difference in single-cycle infection assays. Using prolonged drug exposure assays and a CCR5 mutant that is not downregulated, we found that intrinsic sensitivity differences between these two viruses are related to competitive

CCR5 binding by PSC-RANTES rather than receptor sequestration. We find that potent inhibitory activity by PSC-RANTES is possible in the absence of cell surface downregulation, and that resistance to PSC-RANTES is based on a model of competitive inhibition.

130 3.3. INTRODUCTION

The entry process of the human immunodeficiency virus type 1 (HIV-1) involves the sequential interaction of the viral envelope glycoprotein (gp160) with two separate human receptors. The gp120 surface unit interacts first with human CD4, which induces conformational changes in gp120 and allows it to interact with a chemokine coreceptor, either CCR5 or CXCR4. Significant effort toward pharmacological interruption of this process has yielded a wide variety of molecules that can be globally considered entry inhibitors, but which operate through significantly divergent mechanisms. The first entry inhibitor to be approved for therapeutic administration was enfuvirtide (T-20, Fuzeon), which blocks the virus-host cell membrane fusion process (101). Recent advancements have been largely aimed at targeting coreceptor dependent steps of HIV-1 entry. To this end, a variety of pharmacologic approaches have been taken which include small molecule antagonists (TAK-779, AD101, SCH-C, SCH-D, Maraviroc, Aplaviroc), monoclonal antibodies (PA-12, PA-14), and analogs of native chemokines (met-

RANTES, AOP-RANTES, NNY-RANTES, PSC-RANTES, AOP-MIP-1αP).

Discovery of the corecptor CCR5 was aided by the observation that the endogenous ligands of CCR5 (MIP-1α, MIP-1β, and RANTES) can inhibit HIV-1 replication in vitro, albeit with low potency (33). Modification of the N-terminus of these molecules results in derivatives with enhanced potency over the native chemokine (179).

Several generations of iterative design have been applied to the chemokine RANTES

(Met-RANTES, NNY-RANTES, AOP-RANTES) to generate the high potency inhibitor

PSC-RANTES [N-nonanoyl, des-Ser1[L-thioproline2, L-cyclohexylglycine3]-

131 RANTES(2-68)] (74). PSC-RANTES shows potent activity against HIV-1 in vitro (74,

143) and in the SHIV-macaque vaginal challenge model (106). Like previous

generations, PSC-RANTES is a result of modification of the RANTES N-terminus,

which is thought to be principally involved in receptor activation and signal transduction,

while the intact chemokine core is involved in receptor selectivity and binding (20, 21).

Studies conducted with AOP-RANTES indicated that despite differences in signal

transduction pathways, the derivative resulted in more robust and long-lasting

internalization of CCR5 through a clathrin-dependent endocytic process (178), fundamentally removing the coreceptor from the cell surface. It further appears that

RANTES analogues prolong the internalization phase before the receptor is ultimately

recycled to the cell surface (117). Studies with PSC-RANTES indicate that it in fact

causes internalization of CCR5 with enhanced potency over AOP-RANTES, and thus

internalization of receptor has been considered the dominant mechanism of inhibition of

HIV-1 entry (74, 143). Early studies suggested that met-RANTES and AOP-RANTES

bound CCR5 with higher affinity than the native chemokine (179), but later studies with

PSC-RANTES did not recapitulate this result (74).

A common feature of entry inhibitors is that the concentration of drug required to

inhibit 50% of viral replication in culture (IC50) varies over a wide range (10-1000 fold)

when comparing a group of primary isolate viruses that have never been exposed to drug

(47, 99, 170, 185). This variation in “intrinsic sensitivity” to inhibition is not observed

for nucleoside reverse transcriptase inhibitiors (NRTIs), non-nucleoside reverse

transcriptase inhibitors (NNRTIs), or protease inhibitors (PIs). Screening of a panel of 14

primary isolate viruses to inhibition by AOP-RANTES demonstrated a 31-fold variation

132 in sensitivity to inhibition (189). Insertion of single nucleotide polymorphisms at positions 318 and 319 in the V3 loop of envelope was sufficient to augment PSC-

RANTES sensitivity by as much as 50-fold in three separate envelope genetic backgrounds (Lobritz et al). Furthermore, sensitivity to inhibition by PSC-RANTES correlated closely with viral replicative fitness. These results suggest that the inhibitory activities of AOP- and PSC-RANTES are consistent with other entry inhibitors in as much as a wide variation is seen in “intrinsic sensitivity” to inhibition. The proposed mechanism of inhibition by RANTES analogues (i.e. receptor sequestration) does not sufficiently describe a mechanism by which differential sensitivity to the inhibitor might be observed. In other words, if receptor downregulation is the principal mechanism of inhibition, then variable sensitivity to inhibition should not be observed.

The cell biological processes associated with the effects of RANTES analogs on

CCR5 downregulation and cell signaling have been carefully evaluated. However, a significant virological analysis of PSC-RANTES mediated inhibition of HIV-1 replication has not been performed. To address the apparent discordance between the mechanism of inhibition of PSC-RANTES and the observed variations in sensitivity, we have evaluated intrinsic sensitivity to PSC-RANTES sensitivity using two virus clones which have been shown to have a 10-fold difference in sensitivity to inhibition by PSC-

RANTES in multiple-cycle replication assays. The viruses differ solely by two amino acid polymorphisms at positions 318 and 319 (HXB2 numbering) in the V3 crown.

Inhibition by PSC-RANTES was studied in multiple replication cycle tissue culture assays, as well as in single-cycle replication assays using replication competent and replication defective viruses. We have evaluated the effect of prolonged culture of cells

133 with PSC-RANTES prior to infection. We have further studied the inhibitory effects of

PSC-RANTES in multiple- and single-cycle assays using a mutant CCR5 (M7-CCR5) that is not competent for ligand-induced receptor downregulation. We have evaluated the effect of input virus titer on sensitivity to inhibition by PSC-RANTES in the wild type

and M7-CCR5 context in multiple- and single-cycle assays. Lastly, we have used time-

of-addition experiments to assess the kinetics of PSC-RANTES mediated inhibition.

Overall, we have found that the inhibitory effects of PSC-RANTES early after drug exposure is mediated primarily by receptor sequestration events, but that in extended replication systems, competitive receptor binding is the principal mechanism of inhibition.

Further, we have determined that receptor sequestration is not necessary for potent

inhibition of HIV-1 replication by PSC-RANTES, and that resistance to PSC-RANTES is

consistent with a competitive model of inhibition.

134 3.3. MATERIALS AND METHODS

3.4.1. Reagents. PSC-RANTES was a provided by Dr. Oliver Hartley. T-20,

TAK-779, 3TC, Nevirapine, and Saquinavir were obtained from the AIDS Research and

Reference Reagent Program. Stock solutions of inhibitors (excluding PSC-RANTES and

T-20) were dissolved in DMSO and filter-sterilized.

3.4.2. Plamsids. The pBABE-M7-CCR5 construct was a kind gift of Dr.

Nathaniel Landau (25). This vector was used to construct U87-CD4/M7-CCR5 cells by

lentiviral transduction of U87-CD4 cells. Cells stably expressing M7-CCR5 were selected under puromycin and sorted for CCR5 positive cells by reactivity with the 2D7 monoclonal antibody. The pseudotyping vector pNL4-3.E- was constructed by digestion

of pNL4-3 with Nhe I, blunting, and religation. HIV-1 envelope pseudotyped particles produced with this vector lack the envelope gene and are restricted to a single cycle of

replication. Due to a lack of a reporter gene in this background, infection must be scored

by a reporter gene in trans. The pseudotyping vector NLLuc.AM was a kind gift of Dr.

John Moore (155). Pseudoviruses produced with this vector are also restricted to a single round of replication, but insertion of the luciferase gene in the env position allows scoring of viral replication by production of luciferase enzyme.

3.4.3. Cells. U87-CD4/CCR5 (obtained from the AIDS Research and

Reference Reagent Program) and U87-CD4/M7-CCR5 cells were maintained in

Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 15% fetal bovine

135 serum (FBS), penicillin and streptomycin (pen/strep), G418 (300 μg/ml), and puromycin

(1 μg/ml). U87-CD4/CCR5/Luc cells were generated by transducing U87-CD4/CCR5

cells with VSV-G pseudotyped particles of HR’-Luc, which encodes the firefly luciferase

protein under control of the HIV-1 LTR promoter. Briefly, lentiviral vector was

generated by cotransfection of 293T cells with pHR’-Luc, pΔR8.9I (encoding the HIV-1

gag-pol under control of the CMV promoter) and pMD.G (encoding the VSV-G

envelope). Lentiviral particles were collected and used to transduce U87-CD4/CCR5

cells. 293T cells were maintained in DMEM supplemented with 10% FBS and pen/strep.

Purified CD4+ T cells were derived from peripheral blood mononuclear cells (PBMC)

from HIV-1 seronegative donors and maintained in RPMI supplemented with 10% FBS, pen/strep, and interleukin-2 (IL-2, 1 ng/mL). PBMC were isolated by Ficoll-hypaque density gradient centrifugation. Total PBMC were activated by phytohemagglutinin (2

μg/mL, Sigma-Aldrich, St. Louis, MO) and IL-2 for 3 days. Stimulated cells were used to isolate CD4+ T cells by negative selection using the Miltenyi CD4+ T cell isolation kit.

The purity of the CD4+ cell population was determined to be > 95% by flow cytometry.

3.4.4. Viruses and Pseudoviruses. The NL4-3/A1-92RW009 V3 chimeric

viruses have been previously described (Lobritz et al). Replication competent chimeric

viruses were produced by transfection of 293T cells with 2 μg of each infectious

molecular clone with the Effectene transfection system. Cells were washed after 24 h

and virus recovered after 72 h. The 293T transfection supernatant was assessed for

reverse transcriptase activity as previously described (189) and virus was propagated on

U87-CD4/CCR5 cells for enrichment of the total virus population and clarification of

136 debris from transfection. Supernatants from the propagation were pooled and frozen in aliquots for subsequent assays. Infectious titer was determined by limiting dilution

+ TCID50 measurements on U87-CD4/CCR5 cells and on activiated CD4 T cells as previously described. To confirm that reversion of mutation of the V3 region of each virus did not occur, DNA was extracted after the limiting dilution TCID50 (10 days in propagation) and the C2-V3 region was PCR amplified from proviral DNA as previously described and sequenced.

Vectors for expressing the NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) envelopes have been previously described (Lobritz et al). Envelope pseudotyped particles with no reporter gene were generated by cotransfection of 293T cells with an envelope expression vector and the HIV-1 pseudotyping vector pNL4-3.E-. Envelope pseudotyped viruses with a genomic copy of firefly luciferase were generated by cotransfection of 293T cells with an envelope expression vector and the pseudotyping vector pNLLuc.AM (155). Cells were transfected with 2 μg total plasmid with the

Effectene transfection reagent (Qiagen), washed after 24 h, and pseudovirus-containing supernatant was recovered after 72 h. Cellular debris was removed from the supernatant by low speed centrifugation (2000 rpm x 10 min). Quantification of pseudovirus particles was performed by limiting dilution reverse transcriptase assay.

3.4.5. Multiple-cycle infection assays. Sensitivity to HIV-1 inhibitors was assessed using assays that involve multiple cycles of HIV-1 replication in either U87-

CD4/CCR5 cells or in purified CD4+ T lymphocytes. Cells were plated to 96-well plates

(2 x 104 U87-CD4/CCR5 cells/well or 2 x 105 CD4+ T cells/well). Drugs were added to

137 wells in serial 10-fold dilutions (PSC-RANTES: 100 nM – 100 fM; T-20: 10 μM - 10 pM;

TAK-779: 10 μM – 10 pM; 3TC: 10 μM – 10 pM; Nevirapine 10μM – 10 pM) and

incubated for 1 h at 37oC. Virus was added to the cells at a multiplicity of infection of

0.005 unless otherwise specified, and the cells and virus were incubated for 24 h at 37oC.

Input virus was removed and the cells washed with PBS. Fresh medium containing the

appropriate concentration of drug was added back to the cells. Virus production into the

supernatant was monitored by radioactive reverse transcriptase assay. Virus production was plotted versus drug concentration to determine the 50% inhibitory concentration

(IC50) for each virus and drug.

3.4.6. Single-cycle infection assays. Single-cycle virus infection assays were

performed using three strategies. In a conventional assay, replication-defective particles containing a genomic copy of the firefly luciferase gene (NLLuc.AM) were pseudotyped

with different HIV-1 envelopes and used to infect U87-CD4/CCR5 cells. Virion particle

numbers were assessed by limiting dilution reverse transcriptase activity (121), and

infectivity of the stock pseudoviruses was assessed by limiting dilution infection of U87-

CD4/CCR5 cells. Drug sensitivity assays were performed using a volume of pseudovirus

in the linear range of infection. The second single-cycle infection assay was performed

using replication-defective psuedovirus particles that lack any form of reporter gene

within the HIV-1 genome (NL4-3.E-). Using this pseudovirus, infection was scored by

addition of virions to U87-CD4/CCR5 cells containing an integrated copy of the firefly

luciferase gene under control of the HIV-1 LTR in a tat deficient background (U87-

CD4/CCR5/Luc). In this assay system, integration of the pseudovirus genome into the

138 host cell chromosome results in production of Tat protein, which can activate transcription of its own genome (cis activation) or the LTR which drives production of the luciferase reporter gene (trans activation). Infectivity of pseudovirus stocks was

assessed by limiting dilution infection, and drug sensitivity assays were performed using

a volume of pseudovirus within the linear range of detection. A background level of

luciferase activity is detectable in uninfected cells due to constitutive activity of the LTR,

and this value was used as a background subtraction. The third assay for single-cycle

infection was through infection of U87-CD4/CCR5/Luc reporter cells with replication

competent HIV-1 virions. The purpose of this assay was to use virus that had been

prepared from infection rather than transfection as it has been shown that these types of

virus preparations differ in the proportion of fully processed envelope on the virions (79).

Equivalent TCID50 values of virus were used to infect U87-CD4/CCR5/Luc cells. After

8 hours of exposure to virus, the supernatant was removed, the cells washed, and medium

was replaced with the addition of 10 μg/mL saquinavir, a protease inhibitor. This

inhibitor prevents maturation of virions produced from the first round of infection, and

renders them non-infectious. Production of Tat protein from the integrated virus

stimulates transcription of luciferase reporter from the LTR in trans, and this luciferase

production is indicative only of the virus that has successfully entered the cell prior to the

wash step and addition of saquinavir, functionally assessing single-cycle replication.

3.4.7. Flow cytometry. FACS analysis of CCR5 expression was performed on

U87-CD4/CCR5 and U87-CD4/M7-CCR5 cells. Cells were pelleted at 2000 rpm for 5

min, washed with PBS, and washed in FACS staining buffer (PBS, 5% FBS, 1% BSA,

139 0.1% sodium azide). Cells were again pelleted (2000 rpm x 5 min) and resuspended in

FACS staining buffer in addition to antibodies: CCR5 was detected with phycoerythrin-

(PE-) conjugated anti-CCR5 (clone 2D7 or clone 3A9, Beckton Dickinson) or with

isotype controls (PE-conjugated IgG2a κ). All antibodies were incubated at 12.8 μg/ml at

room temperature for 30 min. Cells were diluted in an additional 150 μl FACS staining

buffer and pelleted again (2000 rpm x 5 min) and resuspended in 400 μl FACS staining

buffer for analysis. For internalization assays, cells were treated with PSC-RANTES (10 nM - 1 pM) for 2 h prior to detection of CCR5. Cells were analyzed on a FACScalibur flow cytometer (Beckton Dickinson).

3.4.8. Time-of-addition assay. HIV-1 pseudoviruses bearing either NL4-3-

V3A1-92RW009(YA) or NL4-3-V3A1-92RW009(RT) envelope were spinonculated onto U87-

CD4/CCR5 cells. 2.5 x 106 cells were spin-infected with 5 mL of pseudovirus-containing supernatant for 90 min at 2500 x g at 4oC. The cells were washed twice with cold phosphate buffered saline (PBS) to remove unbound virions. Cells were resuspended in cold medium and split into 96-well plates (50 μl/well). Virus-cell mixes were synchronized for entry by addition of 130 μl of 37oC medium, and then the appropriate

concentration of inhibitor was added to each well in 20 μl of medium at fixed-time

intervals after addition of warm medium, which is defined as time = 0 for

o synchronization of viral replication. Cells were incubated for 48 h at 37 C and 5% CO2.

Cells were then treated with lysis buffer and luciferase activity was determined.

140 3.5. RESULTS

3.5.1. Dichotomous Sensitivity to PSC-RANTES in Multiple- and Single-

cycle assays. PSC-RANTES, as well as other chemokine analogs, has been described as

an inhibitor of HIV-1 by functioning principally through downregulation of CCR5 from

the cell surface (143). The virologic impact of this mechanism suggests that, when

comparing a variety of HIV-1 isolates, all viruses should be inhibited to a similar degree

by the same concentration of PSC-RANTES. In contrast to this, we have observed a >

30-fold difference in sensitivity to inhibition by AOP-RANTES against a panel (n = 14)

of diverse primary HIV-1 isolates. Polymorphisms in the V3 region, specifically at

positions 318 and 319 in V3 (HXB2 numbering), can alter the sensitivity level of the

virus by as much as 50-fold. We generated two viruses [NL4-3-V3A1-92RW009(YA) and

NL4-3-V3A1-92RW009(RT)] that are completely isogenic except for two amino acid polymorphisms at position 318 and 319 in the V3 crown. NL4-3-V3A1-92RW009(YA) was less sensitive to inhibition by PSC-RANTES than was NL4-3-V3A1-92RW009(RT) by a

factor of 10 in multiple-cycle replication assays using U87-CD4/CCR5 cells (Figure 21A,

Table 7). U87-CD4/CCR5 cells are a human glioma cell line transfected to express both

HIV-1 receptors. The cell surface levels of CCR5 on transfected cell lines do not

accurately mimic either the frequency or density of receptor expression on natural target

cells, CD4+ T cells (109). We therefore performed multiple replication cycle drug

+ sensitivity assays in purified CD4 T cells with NL4-3-V3A1-92RW009(YA) and NL4-3-

V3A1-92RW009(RT). These results recapitulated the 10-fold difference in sensitivity to

inhibition by PSC-RANTES (Figure 21A, Table 7).

141 Figure 21. PSC-RANTES Sensitivity in Multiple- and Single-cycle Assays. (A) The

sensitivity of the V3 loop chimeric viruses was assessed in a series of multiple-cycle and

single-cycle assays. IC50 values were determined using replication competent chimeric viruses in multiple-cycle assays in U87-CD4/CCR5 cells and in purified primary CD4+ T

lymphocytes, and virus production was measured by assessing reverse transcriptase

activity in cell-free supernatants. Single-cycle assays were performed in three ways: (1)

replication competent viruses were used to infect U87-CD4/CCR5 cells with an

integrated LTR-luciferase reporter construct (LUC in trans). Infections were treated with

1 μM saquinavir to prevent maturation of nascent virions and subsequent rounds of

infection. (2) env pseudotypes lacking a genomic reporter gene were used to infect U87-

CD4/CCR5/luc cells (LUC in trans). (3) env pseudotyped viruses containing a genomic

copy of the firefly luciferase reporter gene were used to infect U87-CD4/CCR5 cells

(LUC in cis). Infection was scored by intracellular luciferase activity. Bars indicate fold

difference in IC50 value between NL4-3-V3A1-92RW009(YA) (set to 1) and NL4-3-V3A1-

92RW009(RT). Error bars indicate standard deviation of the IC50 value calculation derived

from two independent experiments performed in triplicate. (B) Comparison of

sensitivity differences in multiple- and single-cycle assays for various antiretroviral

agents. Bars indicate the fold-change in IC50 value of the NL4-3-V3A1-92RW009(RT)

variant as compared to the NL4-3-V3A1-92RW009(YA) variant (set to 1, indicated by the

dashed line). Fold difference IC50 in the multiple-cycle assay is indicated by the gray

bars, and single-cycle differences are indicated by the white bars. Error bars indicate the

standard deviation of the IC50 value calculation, which is derived from two independent

experiments performed in triplicate.

142

143

144 We next assessed sensitivity of these two viruses to inhibition by PSC-RANTES using single-cycle infection assays. Single-cycle replication drug sensitivity assays were

performed in three separate manners: (i) infection of U87-CD4/CCR5 cells with an

envelope-deleted, replication defective pseudovirus which contains a genomic luciferase

gene (in cis), and assessment of intracellular luciferase activity; (ii) infection of U87-

CD4/CCR5.Luc cells with an envelope-deleted, replication defective pseudovirus and

assessment of the luciferase production from an integrated LTR-luciferase reporter in trans; and (iii) infection of U87-CD4/CCR5/Luc cells with a replication competent virus and assessment of luciferase produced by an integrated LTR-luciferase reporter construct

(in trans). Unexpectedly, all three mechanisms of assessing sensitivity to PSC-RANTES by single-cycle replication showed no difference between NL4-3-V3A1-92RW009(YA) and

NL4-3-V3A1-92RW009(RT) in their sensitivity to inhibition by PSC-RANTES (Figure 21A,

Table 7).

3.5.2. Dichotomous Multiple- and Single-cycle Sensitivity Differences are

Specific to PSC-RANTES. Having observed dichotomous PSC-RANTES sensitivity

differences when comparing multiple- and single-cycle assays, we postulated that this

assay-dependent difference may be a consequence of cell surface CCR5 downregulation.

We thus compared multiple- and single-cycle assays using entry inhibitors that are not

known to affect steady-state levels of CCR5 on the surface. We further evaluated assay-

dependent differences for two reverse transcriptase inhibitors. Multiple-cycle and single-

cycle drug sensitivity assays were performed with both viruses using ENF, TAK-779,

3TC, and nevirapine. The IC50 values obtained by single-cycle assays differed from the

145 multiple-cycle assays for ENF and TAK-779; however, the viruses still differed by nearly

100-fold for both drugs (Figure 21B, Table 7). This differs from the effect observed with

PSC-RANTES, where a 10-fold difference observed in multiple-cycle assays diminishes

to no difference in single-cycle (Figure 21B). The viruses did not differ from each other

in IC50 value for 3TC or nevirapine in either multiple-cycle or single-cycle replication

assays (Fig. 21B).

3.5.3. Prolonged Incubation with PSC-RANTES Recapitulates Multiple-

cycle Assays. Since entry inhibitors that are not known to alter the steady-state

concentration of CCR5 on the cell surface did not abrogate the sensitivity differences

observed between the two virus clones when comparing single- and multiple-cycle assays,

we hypothesized that competitive receptor binding by PSC-RANTES was important in driving differential susceptibility to inhibition by this drug, but that the effect of CCR5

downregulation, which occurs rapidly after drug exposure, may be masking this behavior

in short course single-cycle replication assays. To address this, we manipulated our cell

culture system by performing single-cycle replication assays over a range of PSC-

RANTES concentrations after the prolonged culture of target cells with PSC-RANTES.

Previous studies have shown that cells in the continued presence of PSC-RANTES begin

returning significant proportions of CCR5 to the cell surface after 4-6 days of drug

exposure (143). In a multiple replication cycle assay, virus replication occurs over 6 days

after an initial inoculum of 20 infectious units (MOI of 0.005 in 20,000 cells). Assuming

a 36-hour replication cycle, 6 days is sufficient for the completion of 4 complete

replication cycles (Figure 22A). Further assuming exponential growth of virus in culture,

146 Figure 22. Prolonged Incubation of Cultures with PSC-RANTES. Sensitivity to

inhibition by PSC-RANTES of NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) was assessed in single-cycle after prolonged incubation of cells with PSC-RANTES. (A)

Multiple-cycle assays allow for 4 complete replication cycles over a 6 day period. (B)

Single-cycle assays are competed in 48 h. (C) U87-CD4/CCR5 cells were exposed to

10-fold dilutions of PSC-RANTES and for 5 days. After 5 days cells were exposed to virus for 48 hours and infection was scored by luciferase activity. (D) Parallel cultures were established in which one set was treated with PSC-RANTES over ten fold concentrations as in (C), but the second set of cultures were left untreated. Both sets of cultures were incubated for 5 days. After 5 days, the supernatant from the PSC-

RANTES treated cells was transferred to the cells which had been untreated for 5 days.

The cells which had been in the presence of PSC-RANTES were given fresh medium containing freshly diluted PSC-RANTES at the appropriate concentration. Both sets of parallel cultures were incubated for 1 h and exposed to virus for 48 h. Infection was scored by luciferase activity. Plots indicate fold difference in IC50 between NL4-3-V3A1-

92RW009(YA) (gray bars, set to 1) and NL4-3-V3A1-92RW009(RT) (white bars). Black lines

indicate periods of exposure to PSC-RANTES. White boxes indicate cycles of viral

replication. Error bars indicate standard deviation of the IC50 value calculation from 2

independent experiments performed in triplicate.

147

148 4 replication cycles is sufficient for infection of all susceptible cells in culture when

starting from an MOI of 0.001 (20 cells – 200 cells – 2000 cells – 20,000 cells). U87-

CD4/CCR5 cells were incubated with concentrations of PSC-RANTES for 5 days, and then exposed to virus (Figure 22C). Luciferase was quantified 48 h post-infection, and it was determined that in this context, a 10-fold variation in sensitivity to PSC-RANTES was observed between NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) (Figure

22C), similar to the multiple-cycle replication results (Figure 22A), but not to single-

cycle experiments performed with infection after 1 hour of PSC-RANTES exposure

(Figure 22B). A second prolonged incubation experiment was performed using parallel cultures of U87-CD4/CCR5/LUC cells (Figure 22D). In one set of cultures, cells were incubated with 10-fold concentrations of PSC-RANTES for 5 days, while a parallel set of cells were incubated in medium alone. After 5 days, the medium containing PSC-

RANTES was transferred to PSC-RANTES-naïve cells. Following medium transfer, the

cells which had been in the presence of PSC-RANTES for 5 days were provided with

fresh medium containing fresh PSC-RANTES at the appropriate concentration. All

cultures were incubated for 1 hour and then exposed to virus for single-cycle replication

assays. Differential sensitivity to PSC-RANTES between NL4-3-V3A1-92RW009(YA) and

NL4-3-V3A1-92RW009(RT) in the cultures that were in the presence of PSC-RANTES for 5

days and then were provided with fresh PSC-RANTES (Figure 22D). No difference was

observed between the two viruses in sensitivity to PSC-RANTES in the cultures that

received 5 day-old PSC-RANTES for 1 hour prior to single-cycle replication assays. The

potency of the 5 day-old PSC-RANTES was reduced by 10-fold as compared to fresh

PSC-RANTES.

149 3.5.4. Inhibition of HIV-1 Replication with a Downregulation-defective

Variant of CCR5. By performing single-cycle replication assays in cell cultures with extended exposure to PSC-RANTES, we intended to infect cells after steady-state CCR5 density has returned to pre-drug levels. Endocytosis of CCR5 is achieved through phosphorylation of its C-terminal domain by G protein coupled receptor kinases (GRKs) and recognition of the phophorylated receptor by β-arrestin (25). A mutant of CCR5 in which the potential serine phosphorylation residues on the C-terminal domain have been converted to alanine (M7-CCR5) has been previously characterized (25). To assess the inhibitory activity of PSC-RANTES in the absence of receptor downregulation, we generated stable U87-CD4/M7-CCR5 cells. These cells expressed CCR5 similar levels of CCR5 as U87-CD4/wt-CCR5 cells as assessed by flow cytometry, and supported wild type levels of HIV-1 replication (data not shown). We assessed the effect of PSC-

RANTES on downregulation by flow cytometry. We used two specific antibodies to

CCR5, clone 2D7 and clone 3A9. The epitope for clone 2D7 resides in the second extracellular loop of CCR5 and has been reported to overlap the PSC-RANTES binding site (155). The epitope for clone 3A9 resides on the N-terminus, and binding of this antibody is not inhibited by PSC-RANTES binding (155). After treatment of cells with

PSC-RANTES for 2 hours, cells were stained with the appropriate antibody in the continued presence of PSC-RANTES. Flow cytometry with MAb clone 3A9 indicates that the wild type receptor was efficienty downregulated from the cell surface, while the

M7-CCR5 variant was not (Fig. 23B). Staining by clone 2D7 was inhibited by PSC-

RANTES in both the wild type and M7-CCR5 system, suggesting that PSC-RANTES can efficiently bind M7-CCR5 and inhibit antibody binding sterically (Fig. 23A). PSC-

150 Figure 23. Characterization of U87-CD4/M7-CCR5 Cells. (A) Detection of cell surface CCR5 after treatment of cells with 10-fold dilutions of PSC-RANTES for 2 h by

CCR5 MAb 2D7. Detection of M7-CCR5 is indicated by black diamonds and wt-CCR5 is indicated by white squares. (B) Detection of cell surface CCR5 after treatment of cells with 10-fold dilutions of PSC-RANTES for 2 h by CCR5 MAb 3A9. (C) Fold difference in sensitivity NL4-3-V3A1-92RW009(YA) (gray, IC50 value set to 1) and NL4-3-

V3A1-92RW009(RT) (white) using wt- and M7-CCR5 variants in multiple- and single-cycle.

Error bars represent standard deviation from the IC50 calculations from two independent

experiments performed in triplicate.

151

152 RANTES sensitivity assays performed in U87-CD4/M7-CCR5 cells revealed an

approximate 10-fold difference between NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-

92RW009(RT) in both multiple- and single-cycle replication assays (Fig. 23C, Table 8).

The IC50 values obtained in multiple-cycle replication did not differ significantly from

results in cells expressing wild type CCR5. This is in contrast to the single-cycle

replication assay, in which the IC50 value for each virus was significantly higher, though

still different by a factor of 10 (Table 8). The IC50 values obtained by drug sensitivity

assays with T-20, TAK-779, 3TC, and Nevirapine did not differ significantly from those

found in cells expressing wild type CCR5 (Table 8). Thus PSC-RANTES can potently

inhibit HIV-1 replication in the absence of receptor downregulation, though a large

discrepancy exists between the potency of the drug in single round assays and after

multiple cycles of replication.

3.5.5. Multiplicity of Infection Effects on PSC-RANTES IC50 Value. When treating cells expressing M7-CCR5 with PSC-RANTES, downregulation of cell surface

CCR5 no longer contributes to the inhibitory effect. Thus inhibition in this context is exclusively related to occlusion of gp120 binding to CCR5 by PSC-RANTES occupancy.

We hypothesized that RANTES bound to cell surface-expressed CCR5 may inhibit HIV-

1 entry through a competitive mechanism. In this model, the inhibitory potential of PSC-

RANTES in an exclusively competitive mechanism will be dependent upon the quantity of virus which is CD4-bound and primed for coreceptor interaction. Thus we assessed sensitivity to PSC-RANTES in U87-CD4/M7-CCR5 cells at a multiplicity of infection of

0.1, 0.01, 0.001, and 0.0001 infectious units/cell using a single virus [NL4-3-V3A1-

153

154 92RW009(YA)]. Supernatant reverse transcriptase activity was assessed on days 3, 4, 5, 6,

and 10 postinfection. Figure 24 (A-D) indicates that supernatant RT activity accumulated

over the 10 days at all multiplicities of infection. The earliest day at which supernatant

RT activity could be detected varied by the input titer (Day 3 for MOI = 0.1, Day 4 for

MOI = 0.01, and Day 5 for MOI = 0.001 and 0.0001) (Figure 24). Day 10 RT activities

were similar across all multiplicities of infection (Figure 24). Correction of the absolute

RT activities at each drug concentration to the RT activity of the drug-free control

infections on each day allows determination of the IC50 value for each dilution for each

day (Figure 24E-H). Within each multiplicity of infection, the IC50 value increased each

day as virus grew out in the culture. This increase was approximately 5-fold between

each measurement. Day 10 IC50 values indicate the maximum IC50 value for each virus before death of the cells in culture and a decrease in virus production (data not shown).

Thus as more virus is present in the culture, the inhibitory activity of PSC-RANTES is reduced. Importantly, PSC-RANTES mediated complete inhibition of viral replication in the context of M7-CCR5 in all but the highest multiplicity of infection (Figure 24).

To assess the effect of input multiplicity of infection on PSC-RANTES IC50 value, we compared the IC50 values at each multiplicity of infection from a single sampling day

(Fig. 25). At day 3, only cultures receiving the highest MOI of input virus (0.1) had

produced detectable virus into the supernatant (Figure 25A). On day 4, cultures receiving

an MOI of 0.1 and 0.01 were producing virus, and the measured IC50 value differed by

10-fold (Figure 25B). For days 5, 6, and 10, all cultures were producing virus, but the

recorded IC50 values differed by 1000-fold (Fig. 25C-E). Thus we conclude that when

receptor downregulation is not present, measured IC50 values for a single virus type in

155 Figure 24. Effect of virus expansion in culture on PSC-RANTES IC50 value. U87-

CD4/M7-CCR5 cells were infected with four 10-fold dilutions of the same NL4-3-V3A1-

92RW009(YA) virus stock over a range of PSC-RANTES concentrations. Supernatant RT

activity was determined on days 3, 4, 5, 6, and 10 post-infection. Absolute reverse

transcriptase activities in each culture are depicted in panels A-D. RT activity corrected

to the no drug control is depicted in panels E-H. Error bars were removed to decrease complexity, but standard deviations were within 10% of the average value.

156

157 Figure 25. MOI Effect on PSC-RANTES IC50 Value. Relative IC50 values for each multiplicity of infection were plotted as sampled on a single day. Error bars were removed to decrease complexity, but standard deviations were within 10% of the average value.

158

159 multiple cycle replication is highly dependent upon the input titer of virus and on the

sampling day.

3.5.6. Multiplicity of Infection Effects in Wild Type and M7-CCR5. We

evaluated the effect of input virus multiplicity of infection on PSC-RANTES sensitivity

in the wild type and M7-CCR5 context in multiple-cycle and single-cycle assays. Similar

to the effect seen in multiple replication cycle assays using M7-CCR5, MOI has an effect

on IC50 curves using a single virus [NL4-3-V3A1-92RW009(YA)] over a 1000-fold dilution of virus input using M7-CCR5 in single-cycle (Figure 26C and D). Higher MOI inputs resulted in higher IC50 values. Again, the potency of inhibition in single-cycle was

significantly reduced when compared to the multiple-cycle assays. In single-cycle assays

using wt-CCR5, the measured IC50 value was independent of MOI over 100-fold dilution of input titer (Figure 26B). However, in multiple-cycle replication using wt-CCR5, the

IC50 value decreased as input titer decreased as was observed in cells expressing M7-

CCR5 (Figure 26A). In this assay, no viral replication was detected above 10 nM PSC-

RANTES for any MOI in wt-CCR5 (Figure 26A). This suggests that PSC-RANTES

treatment at or above 10 nM resulted in cell surface CCR5 downregulation below a

threshold level required to seed the cultures with the inoculum virus.

3.5.7. Kinetics of PSC-RANTES Inhibition of Entry. We next wished to assess the kinetics of inhibition by PSC-RANTES as compared to other entry inhibitors.

We performed time-of-addition experiments using virus synchronized for entry. U87-

CD4/M7-CCR5 cells were spinoculated with virus at a temperature that is not permissive

160 Figure 26. MOI Effects on PSC-RANTES Sensitivity in wt- and M7-CCR5. The effect of varying input virus titer on sensitivity to PSC-RANTES was assessed in both multiple- and single-cycle assays using U87-CD4 cells expressing either wt-CCR5 or

M7-CCR5. Virus was added to cells over a range of 10-fold dilutions, from a multiplicity of infection (MOI) of 0.1 infectious units/cell (IU/cell) to 0.0001 IU/cell.

IC50 curves from the multiple-cycle assays are all derived from the supernatant RT activities corrected to the no drug control from day 7 post-infection. Single-cycle assay

IC50 curves are based on luciferase activity corrected to the no drug control.

161

162 for cell fusion (4oC). Cells were then sequentially treated with completely inhibitory

concentrations of either PSC-RANTES (10 nM), TAK-779 (10 μM), or T-20 (10 μM)

after synchronization of viral entry by the addition of 37oC medium. The inhibitory

activity of each drug was assessed over 2 hours for its ability to prevent entry and

luciferase production by HIV-1 pseudoviruses of a single envelope type [NL4-3-V3A1-

92RW009(YA)]. The T1/2 values obtained for PSC-RANTES was similar to that of TAK-

779 (Figure 27). Both drugs lost their inhibitor activity in cultures earlier than did T-20,

as would be expected when comparing inhibitors of coreceptor binding against an

inhibitor of membrane fusion (Figure 27). Using this system, both T-20 and TAK-779

inhibited entry by 100 % at T = 0. PSC-RANTES, however, was only able to inhibit

86% of HIV-1 entry at T = 0 (Figure 27). This effect may be due to the requirement of some contribution from receptor downregulation to achieve full inhibitory capacity. On the other hand, this modest breakthrough from inhibition may be due to the amount of virus that is spin-inoculated onto the cell surface, which we are not able to measure with a great degree of accuracy.

163 Figure 27. Kinetics of PSC-RANTES-mediated Inhibition. U87-CD4/M7-CCR5 cells were spinoculated with an env pseudotype of NL4-3-V3A1-92RW009 at 2500 x g for 90 min at 4oC. Cells were washed twice with cold PBS to remove unbound virus, and split into 96 well plates. Fully inhibitory concentrations of entry inhibitors (PSC-RANTES =

10 nM, TAK-779 = 10 μM, ENF = 10 μM) were added sequentially after synchronization of virus entry with warm drug-free medium. Cells were incubated for 48 h, lysed, and infection scored by luciferase activity. Plots represent the % infection compared to the luciferase activity from the addition of drug at 120 min post-synchronization.

164

165 3.6. DISCUSSION

In this study, we have addressed the virologic aspects of inhibition of HIV-1 entry

by PSC-RANTES. A great deal of information about the regulation of CCR5 expression in the presence of CCL5 analogs has been determined (117, 124, 165, 169, 178, 203). To date, study of the pharmacological activity of PSC-RANTES and other chemokine analogs has been largely focused on receptor downregulation (74, 143, 169). However, we have observed as much as 50-fold variation in sensitivity to PSC-RANTES by viruses that differ in sequence only in the V3 crown. Furthermore, we found a strong correlation between susceptibility to PSC-RANTES and viral replicative fitness. Pharmacolgical models which do not evaluate the binding mechanism of PSC-RANTES to CCR5 are insufficient to explain the underlying difference between these viruses and why they exhibit large susceptibility differences. Thus we sought to understand more fully the inhibitory effects of PSC-RANTES caused by steric occupancy of CCR5 and determined that inhibition by PSC-RANTES is mediated by a competitive process.

Viruses that exhibited 10-fold differences in sensitivity to PSC-RANTES in multiple replication cycle assays in transformed cell lines and primary cells did not exhibit this difference in single-cycle assays. In an attempt to exclude artifacts from the single-cycle assay system, we utilized three distinct single-cycle assays, employing

pseudovirus as well as replication-competent virus, and using markers of infection

derived from both the cell and the virus. We found no difference in IC50 value between

these two viruses in any single-cycle assay system (Figure 21A). A major limitation to

single-cycle assays is duration of drug and virus exposure. In multiple-cycle replication

166 assays, we utilize a multiplicity of infection of 0.001 infectious units per cell in 20,000

cells. Carried over 6 days, this allows for infection of all cells in culture after four rounds

of replication (Figure 22A). Neither this spreading effect nor the effect of CCR5

recycling to the cell surface is taken into account by single-cycle assays. Through the manipulation of our tissue culture system, we attempted to tease out the underlying differences between the single- and multiple-cycle assay systems for PSC-RANTES

(Figure 22A-D). We anticipated that extended exposure to PSC-RANTES would result

in cell surface expression levels of CCR5 that would reach a steady-state level (143).

After extended exposure to drug, we observed in single-cycle infection a 10-fold

variation in sensitivity to PSC-RANTES between our two model viruses. By transferring

5-day-old PSC-RANTES from one cell culture into a new cell culture, we also confirmed

that PSC-RANTES has a long activity half-life in tissue culture. The potency of

inhibition by 5 day old PSC-RANTES was only reduced by 10-fold over freshly added

PSC-RANTES.

Using the M7-CCR5 variant, we were able to evaluate the pharmacology of PSC-

RANTES in the absence of receptor downregulation. A small degree of receptor

downregulation did occur at high PSC-RANTES concentrations (Figure 23A and B), but

this was significantly reduced over the wild type receptor. In this system, we observed

that inhibition by PSC-RANTES was consistent with a competitive mechanism. In the

absence of receptor downregulation, we observed a 10-fold difference in IC50 values between our two model viruses in both multiple- and single-cycle assays (Figure 23C), again suggesting that the major difference between multiple- and single-cycle assays with

PSC-RANTES is the presence of receptor sequestration. Furthermore, increasing

167 substrate concentration (that is, the virus) resulted in decreases in the overall potency of

the inhibitor (Figure 24 and 25). This increase in IC50 value occurred when virus input

was increased at the onset (by multiplicity of infection, Figure 25) or when the number of infected cells producing virus into the supernatant increased (as when culture outgrowth

occurred, Figure 24). These data show that the inhibitory effects of a competitive

inhibitor are extremely fluid, and that factors such as input titer, cell number, and day of

sampling are extremely important when attempting to determine an IC50 value.

Presumably, CCR5 density on the cell surface will also play a distinct role in modulating

IC50 values. This results in added complexity when attempting to assess the comparative

IC50 values of two different viruses. Thus it is extremely important that the virus stocks

being used are carefully evaluated for titer and that assays are performed rigorously. It

also becomes clear that absolute IC50 values for a virus using a competitive inhibitor are constantly fluctuating, and only relative data can be accurately reported.

In the absence of CCR5 downregulation, we observed a distinct difference between the concentration of PSC-RANTES necessary for inhibition of 100% of HIV-1 entry and the concentration of PSC-RANTES necessary to prevent accumulation of quantifiable reverse transcriptase activity in the supernatant (Figure 23C, Figure 26).

This finding was apparent when we performed drug sensitivity assays using dilutions of input virus in multiple-cycle versus single-cycle assays. One important consideration is the sensitivity of an assay that measures the activity of the firefly luciferase enzyme versus one that measures the activity of reverse transcriptase. We would suggest that the luciferase activity used in single-cycle assays is significantly more sensitive than is RT activity. Thus we are able to detect a small amount of virus seeding in the cultures by

168 luciferase activity. This seeding may be insufficient to expand in the culture in the

presence of inhibitor to a level detectable by RT activity, which appears to be less

sensitive at the lower end of the linear range of the assay. Thus the IC50 value is further

dependent upon the sensitivity of the assay used to score infection.

What are the properties of the PSC-RANTES interaction with CCR5 that are

relevant for antiviral activity? One consideration is the relative binding affinity of PSC-

RANTES for CCR5 when compared to RANTES. Recent studies demonstrated only

small differences between PSC-RANTES, AOP-RANTES, and RANTES in their ability

to displace 125I-MIP-1α or MIP-1β (74). In contrast to this, Townson et al used 125I-

murine MIP-1α displacement studies of both AOP-RANTES and AOP-MIP-1αP

indicated that each aminooxypentane derivate had a higher CCR5 binding affinity than

their respective native chemokines (190). Early characterization of AOP-RANTES

affinity was in accordance with an enhanced binding affinity for AOP-RANTES versus

RANTES (179). Moreover, displacement assays suggested a single high-affinity binding site for AOP-RANTES, whereas RANTES showed a biphasic displacement curve, suggestive of two binding sites. Antibody-based binding assays have suggested a two component displacement curve for PSC-RANTES (Lobritz et al, work in progress).

PSC-RANTES may then be interacting with CCR5 in a qualitatively different manner

than RANTES. At this time the qualitative and quantitative aspects of PSC-RANTES

binding to CCR5 are unclear.

What are the differences between the NL4-3-V3A1-92RW009(YA) virus and the

NL4-3-V3A1-92RW009(RT) variant that results in differential sensitivity to inhibition by

PSC-RANTES? These two viruses have significantly different replicative fitness as

169 assessed by entry efficiency and viral replicative fitness (Lobritz et al). One possibility is

that the R318T319 V3 region has a lower affinity for CCR5 than the Y318A319 variant.

There can be multiple consequences for this affinity difference in the context of PSC-

RANTES inhibition in a competitive model. First, a higher affinity for the CCR5 extracellular loop 2 may result in an increased ability to compete with PSC-RANTES for

CCR5 binding. Second, enhanced affinity for CCR5 may confer upon the wild type virus an increased capacity to scavange low levels of cell surface CCR5 that would be limiting in the presence of PSC-RANTES. We favor a model in which the PSC-RANTES-bound form of CCR5 remains in a configuration that is recognized by the HIV-1 envelope glycoprotein. Thus in coreceptor limiting environments, caused by downregulation of

CCR5 by PSC-RANTES, the ability to scavenge receptor and compete with drug for binding of residual receptor may be largely a consequence of V3 loop affinities and receptor association rates, and this may be a reflection of viral replication capacity.

170 Chapter 4

INTRINSIC SENSITIVITY OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 ISOLATES TO PSC-RANTES IS MEDIATED BY DIFFERENTIAL CCR5 AFFINITY

Authors: Michael A. Lobritz1,2, Sandra Nguyen3, Andre J. Marozsan2, Benhur Lee3, and

Eric J. Arts1,2

1Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA 44106

2Division of Infectious Diseases, Department of Medicine, Case Western Reserve University

3Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California 90095

171 4.1. PREFACE

We have established that naturally occurring polymorphisms in the V3 crown are sufficient to modulate sensitivity of viruses to a wide array of entry inhibitors. Mutations in this region also cause changes in entry efficiency and impact overall viral replication capacity in a manner that is inversely proportional to sensitivity to entry inhibitors, which suggested that a similar biological process was involved in both of these phenotypes.

Furthermore, we have established that viruses bearing V3 crown polymorphisms are differentially susceptible to PSC-RANTES in the competitive model of inhibition. These findings have suggested that the V3-CCR5 affinity and the rates of virus fusion have been specifically altered by V3 crown polymorphisms. In this chapter, experiments were performed to map virus-host interactions that have been affected by V3 crown changes and to specifically test hypotheses about receptor affinity and fusion kinetics.

172 4.2. ABSTRACT

Natural variation in the V3 crown of HIV-1 results in dramatic differences in

entry inhibitor sensitivity and overall entry efficiency. The mechanism underlying these

differences is unclear, but likely involves the interaction of V3 with CCR5 and the rates

of the overall fusion process. We evaluated a series of viruses containing natural

polymorphisms in the V3 crown positions 318 and 319. Infection of cells with inducible

levels of CD4 and CCR5 indicate that V3 crown variants with high replicative fitness and

reduced susceptibility to entry inhibitors have an increased capacity to scavenge low

levels of CCR5. No correlation was found between sensitivity to CCR5 inhibitors and

CD4 usage. Furthermore, these variants were more resistant to the effects of hetero-

oligomerization with dominant negative envelopes. Faster fusion kinetics was associated

with increased use of low level CCR5 and resistance to dominant negative hetero-

oligomerization. Taken together, our data suggest that natural variation in the V3 crown modulate coreceptor affinity and fusion kinetics, and these parameters specifically modulate entry inhibitor susceptibility and replicative fitness.

173 4.3. INTRODUCTION

The HIV-1 envelope glycoprotein gp160 mediates entry of virus into host cells by

binding to two separate receptors on the host cell membrane, CD4 and a coreceptor

(CCR5 or CXCR4). The entry process is highly cooperative and must be completed in an

ordered manner for productive fusion to take place. The cooperativity of this process

suggests that variations in envelope glycoprotein affinities for each receptor and entry complex stoichiometry can have drastic impacts on the kinetic processes of entry. These

differences may impact on the efficiency of virus entry and have implications for

replicative fitness and sensitivity to entry inhibitors.

Evaluation of baseline receptor affinities of the HIV-1 envelope glycoprotein have

suggested that small variations exist between viruses in their affinity for CD4 (44, 64, 88,

180, 191, 192). Of notable exception is that the association rate for CD4 differs

significantly between primary isolate HIV-1 envelope glycoproteins and envelopes from

laboratory adapted strains (40, 90, 137). Extended propagation of primary isolate viruses

in tissue culture, even in primary cells, results in viruses with increased susceptibility to

neutralization by soluble CD4, a surrogate marker for CD4 association rate (154). It is

hypothesized that host selection factors, specifically antibody-based selection, maintain

primary isolate virus envelope in a quaternary structure configuration that is resistant to such selection factors, but which limits CD4 association rates (154).

On the other hand, the affinity of the HIV-1 envelope glycoprotein for CCR5 has been shown to be extremely variable among HIV-1 isolates (17, 52, 80, 152).

Measurement of coreceptor affinity for the HIV-1 envelope is confounded by the

174 apparent multiple interactions between envelope and CCR5. The current model of

coreceptor binding suggests that the CD4-ligated form of envelope interacts with CCR5

at the N-terminus with the bridging sheet domain of gp120 as well as the base of the V3

loop, while the V3 crown interacts with the second extracellular loop of CCR5 (37, 38,

85, 86, 147, 164, 194, 204). Individual affinities at these contact points are difficult to

ascertain.

The functional consequences of altered receptor affinity has been evaluated in the

context of sensitivity to enfuvirtide (159). Mutations in the gp120 bridging sheet which reduced affinity for CCR5 resulted in decreased fusion kinetics and a consequent increase in sensitivity to enfuvirtide (19, 159, 161). Presumably, changes in the V3 loop sequence may have similar results (47). Adaptations in the V3 loop can specifically alter

coreceptor affinity, and this change has been associated with variable susceptibility to

entry inhibitors (149, 150).

Multiple studies have shown that diverse HIV-1 viruses have differential

susceptibility to inhibitors that target the entry process. We have observed a > 50 fold

variation in susceptibility to PSC-RANTES and other entry inhibitors caused by natural

amino acid variation at positions 318 and 319 in the V3 crown (Lobritz et al).

Polymorphisms that increased entry inhibitor sensitivity also resulted in decreased entry

efficiency and viral replication capacity. In this study, we examined the functional

impact of V3 crown mutations which are known to cause variations in entry inhibitor

susceptibility and entry efficiency. We evaluated relative CD4 and CCR5 affinity by

neutralization with sCD4 and infection of cell lines with a 25-fold dynamic range in CD4

expression and 15-fold dynamic range in CCR5 expression. We further evaluated these

175 V3 mutations by assessing the impact of dominant negative envelope hetero- oligomerization on infectivity. Lastly, we assessed overall replication kinetics of these

V3 mutations and determined that reduced CCR5 affinity results in decreased fusion kinetics and increased susceptibility to coreceptor inhibitors.

176 4.4. MATERIALS AND METHODS

4.4.1. Reagents. Minocycline (Sigma Aldrich, St. Louis, MO) was dissolved in

DMSO to generate a stock concentration of 1 mg/ml. Ponasterone A (Invitrogen,

Carlsbad, CA) was dissolved in 100% ethanol to generate a stock of 1 mM. Enfuvirtide,

C34, and TAK-779 were acquired from the AIDS Research and Reference Reagent

Program.

4.4.2. Plasmids. The wild-type and dominant negative HIV-1 envelopes were

expressed from the vector pREC-envΔleu (120). Dominant negative envelope mutants

were generated by site-directed mutagenesis using the QuikChange XL site-directed

mutagenesis kit (Stratagene, La Jolla, CA). Cleavage site mutants were generated by

changing the primary cleavage site REKR (amino acids 508 – 511 in the gp120 coding

region) to SEKS. Mutations were confirmed by sequencing the gp120-gp41 junction.

All mutations and polymorphisms in the envelope gene are designated by the wild type

amino acid represented by the single letter code, the residue number, and the substituted

amino acid. Residue numbering is based on the prototypical HXB2 envelope sequence

nomenclature.

4.4.3. Cell Lines. 293T cells were maintained in Dulbecco’s Modified Eagle’s

Medium (DMEM, Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine

serum (FBS, Mediatech, Inc.) and penicillin (100 U/ml) and streptomycin (100 μg/ml)

(pen/strep). U87-CD4/CCR5 cells were maintained in DMEM supplemented with 15%

177 FBS, pen/strep, G418 (300 μg/ml) and puromycin (1 μg/ml). 293 inducible cells were maintained in DMEM supplemented with 10% FBS, pen/strep, and blasticidin (50 μg/ml).

o All cells were maintained at 37 C and 5% CO2.

4.4.4. Pseudovirus Production and Single-round Entry Assays. Pseudoviruses were produced by cotransfection of 293T cells with 1 μg of luciferase-encoding pseudotyping vector (NLLuc.AM) and 1 μg of envelope expression vector. Cells were washed after 24 h, and pseudovirus was collected after an additional 48 h. Infectivity of pseudovirus stocks was assessed by limiting dilution infection of U87-CD4/CCR5 cells and assessment of intracellular luciferase activity 48 h post-infection. For soluble CD4 neutralization assays, envelope pseudotyped viruses were incubated with 10-fold concentrations of sCD4 (Progenics pharmaceuticals) for 90 minutes and then added to

U87-CD4/CCR5 cells. In some experiments, mixtures of wild type and dominant negative envelopes were cotransfected into 293T cells to generate virion stocks containing variable frequencies of dominant negative heterooligomeric envelope trimers.

In these experiments, the total amount of envelope expression vector was always 1 μg; however, the ratios of wild type and dominant negative envelopes varied. In order to maintain accuracy of plasmid addition, plasmids were diluted so that all volumes for pipetting were between 5 and 25 μl. Dominant negative envelopes were cotransfected with wild type envelopes at a frequency of 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 0.9, and 1.0.

4.4.5. Incorporation of Cleaved gp120 into Virions. Analysis of gp120 content in virions containing various frequencies of dominant negative cleavage mutant

178 envelope was assessed by Western blot. Equivalent particle numbers of virus were pelleted and reusepended in SDS lysis buffer. Samples were separated by SDS-PAGE and blotted to nitrocellulose. Total envelope protein was detected by the B13 monoclonal antibody clone. Blots were incubated with an HRP-conjugated secondary antibody and revealed with the ECL pico detection kit (Amersham).

4.4.6. Infection of 293 Cells with Inducible Levels of CCR5 and CD4. 293 inducible cells were generated by selection of 4 vector stable cells. CCR5 expression is controlled by a two vector ecdysone-inducible promoter. pVgRXR encodes the VgEcR fusion protein under control of the CMV promoter, and the RXR open reading frame under control of the RSV 5’ long terminal repeat. pIND-CCR5 encodes CCR5 under control of the minimal heat shock promoter with inducible control provided by five repeats of the glucocorticoid receptor DNA binding domains (5X E/GRE). Addition of the ecdysone derivative ponasterone A (the inducer) results in recruitment of a transcriptional coactivator to the 5X E/GRE element and activation of transcription of the

CCR5 ORF. CD4 expression is inducibly regulated by the TREx expression system

(Invitrogen). Cells contain pcDNA5-TO-CD4 and transcription of the CD4 ORF is controlled by the addition of the tetracycline analog minocycline. Single cell clones were isolated to generate cell populations with consistent levels of induction upon stimulation of CD4 and CCR5 expression.

293 inducible cells were plated at a density of 20,000 cells per well in a 48-well plate and allowed to adhere overnight. Cells were induced in a matrix pattern to express

CD4 and CCR5. Minocycline was added to cells in 2-fold dilutions over 8 separate

179 dilutions (20 ng/ml – 0.15625 ng/ml) to induce CD4 expression. Ponasterone A was

added in 2-fold dilutions over 8 separate dilutions from a final concentration of 2 μM to

0.015625 μM to induce CCR5 expression. This matrix results in 64 unique CD4 and

CCR5 induction environments. Each drug concentration was induced in triplicate. Cells were induced for 24 hours prior to infection. Cells were then exposed to pseudovirus for

48 h, washed with PBS, and lysed with Glo lysis buffer (Promega, Inc.). Luciferase activity was assessed using the Lumimark Plus instrument (Bio-Rad, Hercules, CA).

4.4.7. Flow Cytometry. CD4 and CCR5 expression levels after induction with minocycline (CD4) and Ponasterone A (CCR5) were assessed by flow cytometry. 106 cells were plated in a 6 well plate and induced overnight with 2-fold dilutions of minocycline or ponasterone A. Cells were removed from their wells by treatment with 3 mM EDTA/PBS for 5 min at 37oC. Cells were pelleted (2000 rpm for 5 min) and washed

with FACS staining buffer (PBS, 5% FBS, 1% BSA, 0.1% sodium azide). Cells were

pelleted again (2000 rpm for 5 min) and resuspended in FACS staining buffer in addition to antibodies: phycoerythrin- (PE-) conjugated anti-CCR5 (clone 2D7, BD Pharmingen),

fluoroisothiocyante- (FITC-) conjugated anti-CD4 (BD Pharmingen), or with isotype

controls (PE-conjugated IgG2AΚ or FITC-conjugated IgG1, BD Pharmingen). All

antibodies were incubated at 12.8 μg/ml at room temperature for 30 min. Cells were

diluted in an additional 150 μl FACS staining buffer and pelleted again (2000 rpm for 5

min) and resuspended in 400 μl FACS staining buffer for analysis. Cells were analyzed

on a FACScalibur flow cytometer with CellQuest software (BD Biosciences).

180 4.4.8. Kinetic Entry Assay. The kinetics of entry for different HIV-1 envelopes

was assessed by measuring the rate of desensitization of synchronized pseudoviruses to

various entry inhibitors. HIV-1 pseudoviruses bearing heterologous envelopes were

spinoculated onto 106 U87-CD4/CCR5 cells (90 min, 2500 x g, 4oC). After spinoculation,

cells were washed twice with cold PBS to remove unbound virions. The cell/virus

mixture was resuspended in cold medium and divided into a 96-well plate (50 μl/well).

Entry of pseudoviruses into target cells was synchronized by the addition of 130 μl of

37oC medium. HIV-1 entry inhibitors were added to each well at 100% inhibitory

concentrations at fixed-time intervals after synchronization of entry. The time of addition of warm medium was defined as T = 0 for synchronization of virus entry. Cells were

o incubated for 48 h at 37 C and 5% CO2, lysed, and luciferase activity was determined.

181 4.5. RESULTS

4.5.1. Neutralization of V3 Crown Mutants by sCD4. We have previously

assessed a series of V3 crown polymorphisms for their sensitivity to entry inhibitors and

overall impact on viral replicative fitness. Change at position 318 from a conserved

tyrosine to a rare arginine in three separate envelope contexts resulted in increased

susceptibility to PSC-RANTES, ENF, TAK-779, and MAb 2D7 without impacting on

sensitivity to inhibitors that target reverse transcription, integration, or protease cleavage.

This same change resulted in 20 – 100 fold decreases in replicative fitness versus the wild type tyrosine. Furthermore, change from the wild type tyrosine at position 319 in the Yu-

2 context to a threonine resulted in decreased susceptibility to entry inhibitors and higher replicative fitness and entry efficiency. To better understand the mechanisms involved in the efficiency of host cell entry conferred by these polymorphisms, we evaluated the affinity changes conferred by this mutation to the major HIV-1 receptors, CD4 and CCR5.

Neutralization by soluble CD4 (sCD4) has been suggested as a surrogate measure of the association rate of HIV-1 gp120 for the surface bound receptor. Viruses were exposed to sCD4 for 90 min at 37oC and then added to cells. Infections were scored after

48 hours by determining luciferase activity. NL4-3-V3A1-92RW009 chimeric viruses

containing either a tyrosine or arginine at position 318 were equivalently neutralized by

sCD4 with an IC50 of 7 and 4 μg/ml, respectively (Figure 28A). Neither the tyrosine nor

the arginine in the NL4-3-gp120B5-91US056 context were neutralized to any extent by the concentrations of sCD4 that were evaluated (up to 25 μg/ml) (Figure 28B). The NL4-3- gp160Yu-2 variants containing either alanine or threonine at position 319 were partially

182 Figure 28. Neutralization of Chimeric Viruses by sCD4. Envelope pseudotypes were incubated with 10-fold concentrations of sCD4 for 90 min at 37oC. Complexes were added to U87-CD4/CCR5 cells, and luciferase activity was determined after 48 h. Plots indicate luciferase activity for each virus relative to the sCD4-free control.

183

184 neutralized by sCD4 at concentrations of 22 and 18 μg/ml, respectively (Figure 28C).

These data indicated that the NL4-3-V3A1-92RW009 viruses have the highest CD4

association rate or affinity, followed by the Yu-2 variants, and that the B5-91US056

variants have the lowest affinity of the three. Furthermore, changes in the V3 crown did

not impact CD4 association rates or affinities.

4.5.2. Effects of CD4 and CCR5 Expression Levels on Infectivity of V3

Crown Polymorphisms. To better asses the impact of CD4 and CCR5 affinity of these

V3 crown mutations in their contexts, we evaluated the infectivity of envelope

pseudotypes in a cell line with inducible levels of CD4 and CCR5. The line is based on

293 cells containing a CD4 expression cassette with a tetracycline inducible operon, and

a CCR5 expression cassette which is regulated by the ecdysone derivative ponasterone A

(Figure 29A). Expression of both receptors can be independently regulated by the dose

of each drug in the supernatant. Addition of the tetracycline derivative minocycline over a concentration range of 150 pg/ml to 10 ng/ml resulted in a 22-fold difference in receptor expression level as measured by the mean fluorescence intensity (MFI) of the

cell population by flow cytometry (100 relative light units (RLU) at 150 pg/ml to 2200

RLU at 10 ng/ml) (Figure 29B). Minocycline concentration had no effect on CCR5

induction or on cell viability (data not shown). Addition of ponasterone A over a

concentration range from 63 nM to 2 μM resulted in a 13.3-fold induction of CCR5

expression as measured by the MFI of the cell population by flow cytometry (300 RLU at

63 nM to 4000 RLU at 2 μM) (Fig. 29C). Ponasterone A had no effect on CD4

185 Figure 29. An Inducible System for Expression of CD4 and CCR5. (A) Minocycline addition controls expression of CD4 and ponasterone A addition controls expression of

CCR5. (B) Dynamic range of CD4 induction was assessed by flow cytometry. Data points represent cell population mean fluorescence intensity after 24 h induction with the indicated concentration of minocycline. Basal expression was detected at 100 relative light units (RLU) and maximal expression was detected at 2200 RLU with 10 ng/ml minocycline (22-fold induction). (C) Dynamic range of CCR5 expression was assessed by flow cytometry. Basal expression was detected at 300 RLU and maximal expression was detected at 4000 RLU (13.3-fold induction).

186

187 expression levels or on cell viability. Neither minocycline at 20 ng/ml nor ponasterone A

at 4 μM had any effect on HIV-1 infectivity of U87-CD4/CCR5 cells (data not shown).

The impact of CD4 and CCR5 expression levels on infectivity of V3 crown polymorphisms in the A1-92RW009 V3, B5-91US056 gp120, and Yu-2 gp160 contexts was assessed by infection of CD4/CCR5 inducible cells by envelope pseudotypes. 293 cells were induced for 24 hours in a matrix of 8 concentrations of minocycline (20 ng/ml to 0.15625 ng/ml) and 8 concentrations of ponasterone A (2 μM to 0.015625 μM) and then exposed to virus. After an additional 48 h, cells were lysed and the luciferase activity of each infection determined. Luciferase activity of each minocycline/ponasterone A concentration was plotted as a function of the luciferase activity detected at the highest minocycline/ponasterone A concentration (20 ng/ml and 2

μM). Reduction of CCR5 levels impacted the infectivity of all viruses, but to varying extents (Figure 30). At the lowest CCR5 expression level, NL4-3-V3A1-92RW009(YA) was able to infect at a level of 31 to 20% of the maximal expression level depending on the

CD4 expression level (Figure 30A), while NL4-3-V3A1-92RW009(RT) was only able to

infect from 10 to 8% of maximal infection, dependent upon CD4 expression level (Figure

30B). The overall rate of infecitivity loss was faster in for NL4-3-V3A1-92RW009(RT) as compared to NL4-3-V3A1-92RW009(YA) as measured by the concentration of ponasterone

A required for 50% of maximal infection (EC50). The EC50 for NL4-3-V3A1-92RW009(YA) was 250 nM ponasterone A (Figure 30A), while the EC50 for NL4-3-V3A1-92RW009(YA) was 500 nM ponasterone A (Figure 30B). Change at position 318 in the NL4-3-gp120B5-

91US056 context had a similar effect. NL4-3-gp120B5-91US056(YA) infected at a range of 40

to 4% of maximum in the low CCR5 environment (Figure 30C), while NL4-3-gp120B5-

188 Figure 30. Effects of Variable CD4 and CCR5 Expression on Viral Infectivity. 293 cells with inducible expression of CD4 and CCR5 were induced over a range of 8 CD4 expression levels and 8 CCR5 expression levels for a total of 64 unique receptor expression environments. After 24 h of induction, cells were exposed to env pseudotyped viruses and luciferase activity was determined after another 48 h. Plots of each virus indicate luciferase activity in a single receptor environment as a percent of the luciferase activity at the highest drug concentration (20 ng/ml minocycline, 2 μM ponasterone A).

The X-axis indicates the concentration of minocycline (in ng/ml) and the z-axis indicates the concentration of ponasterone A (in μM). Infections at each drug concentration were performed in triplicate.

189

190 91US056(RT) infected from 17 to 2% of maximum in the low CCR5 environments (Figure

30D). The EC50 concentration for NL4-3-gp120B5-91US056(YA) was 125 nM (Figure 30C),

while the EC50 for NL4-3-gp120B5-91US056(RT) was 250 nM (Figure 30D). Similar results

were observed comparing NL4-3-gp160Yu-2(YA) to NL4-3-gp160Yu-2(YT) (Figure 30E

and F).

The effect of CD4 expression levels on viruses that were neutralized by sCD4 was

minimal compared to the effect on NL4-3-gp120B5-91US056 variants, which were not

successfully neutralized by soluble CD4 (Figure 30). This suggests that the NL4-3-

gp160Yu-2 and NL4-3-gp160A1-92RW009 viruses had a significantly increased capacity to

scavenge low levels of CD4. The lowest concentration of CD4 resulted in approximately

30% reductions in infectivity in these two virus contexts (Figure 30A and B, E and F),

whereas a similar reduction in CD4 levels resulted in a 95% reduction in infectivity in the

primary B5-91US056 context (Figure 30C and D).

4.5.3. Infectivity Impact of Dominant Negative Hetero-oligomerization on V3

Crown Mutations. The functional unit of entry for HIV-1 is a trimer. Fusion competent

envelopes are composed of three fully cleaved gp160 subunits. Trimerization of gp160

molecules occurs soon after they are translated (58). Cleavage of gp160 into gp120 and

gp41 occurs after the trimerization event (183). When two different species of envelope glycoproteins are expressed in the same cell, heterotrimers can form. In fact, heterotrimer formation can occur with an efficiency that is close to random mixing of monomers. To assess the affinity relationship of envelopes containing V3 crown polymorphisms and CCR5, we assessed the infectivity of virus stocks containing

191 different ratios of wild type and dominant negative envelope mutants. This approach has

been utilized to assess trimer and subunit stoichiomery of HIV-1, MLV, ASLV-A, and influenza HA entry complexes (214, 215). We utilized a defective envelope variant that

exerts a dominant negative entry effect, that is, incorporation of a single mutant envelope

monomer into a trimer causes the entire trimer to be defective. The R508S/R511S

envelope variant is completely defective for proteolytic cleavage of the gp160 envelope

precursor protein due to two mutations in the furin recognition sequence (65, 126). This

mutant is efficiently incorporated into wild type trimers and exported to the cell surface

(58, 177, 183, 212). Furthermore, this mutant does exert a dominant negative effect on

trimers that incorporate a single monomer (78). We cotransfected 293T cells with

experimentally controlled ratios of wild type and dominant negative envelope expression

vectors. We assumed that expressed protein levels were directly related to the quantity of

transfected plasmid. Thus we can model the frequency of trimer species in a population

of virions from the perspective of the frequency of the dominant negative mutant in the

cotransfection experiment (fM) (Figure 31A) (214). In a population of virions produced

by transfection, the frequency of trimers containing exclusively dominant negative

3 mutants is equal to fM (Figure 31A, blue line). This proportion increases as the

frequency of the mutant increases in the transfection, rising to 100% as fM = 1.0.

2 Heterotrimers containing two mutant subunits can be expressed as 3fM (1-fM) (Figure

31A, red line), and heterotrimers containing one mutant subunit can be expressed as

2 3fM(1-fM) (Figure 31A, yellow line). Trimers composed exclusively of wild type subuits

3 can then be expressed as (1-fM) (Figure 31A, green line) .

192 Figure 31. Impact of Dominant Negative Hetero-oligomerization on HIV-1 Entry.

(A) Predicted frequencies of trimer species in env pseudotyped virus populations with

varying frequencies of transfected dominant negative env monomers. The green line

indicates the frequency of homotrimers with exclusively wild type env monomers relative

to the frequency of dominant negative env monomers. These wild type homotrimers are

predicted to be the only functional trimer subset. (B) Western blot of env pseudotyped

virus populations with increasing frequencies of dominant negative (R508S/R511S) env

monomers. A portion of uncleaved gp160 is present even in the absence of dominant

negative env monomers, but the gp120 is titrated away as the mutant frequency increases.

(C) The stoichiometry model proposed by Yang et al suggests that relative infectivity (RI)

of a virus population bearing mutant env monomers at frequency fM will be equal to (1-

3T fM) where T = the number of trimers involved in the entry process. Hypothetical data for T = 1-4 are presented (colored lines), along with infectivity data derived from dominant negative hetero-oligomerization of NL4-3-V3A1-92RW009(YA) (black diamonds)

and NL4-3-V3A1-92RW009(RT) (white squares). Error bars represent standard deviations

from two independent assays performed in triplicate.

193

194 Virus stocks containing various frequencies of dominant negative envelope

monomers were generated in the context of the NL4-3-V3A1-92RW009(YA) and the NL4-3-

V3A1-92RW009(RT) wild type envelope. Stock viruses were pelleted and evaluated by

western blot to confirm incorporation of the dominant negative variant. Increasing the

amount of the dominant negative envelope in the stock resulted in titration of functional

gp120 from the virion populations (Figure 31B).

The model proposed by Sodroski and colleagues suggests that, as only wild type

homotrimers can functionally mediate infection, then the relative infectivity of a virus

stock [RI(%)] must be related to the frequency of wild type homotrimers expressed on the

3T virion surface. Thus RI (%) = (1-fM) x 100%, where T = the critical threshold of

trimers involved in mediated virus entry (Figure 31C). We performed infectivity assays

of each virus stock on U87-CD4/CCR5 cells and quantified luciferase activity. We

observed a close fit between the NL4-3-V3A1-92RW009(YA) virus and the model that

supports T = 1 infection levels, or single trimer stoichiometry involved in the entry

complex (Figure 31C). On the other hand, we observed a close fit between NL4-3-V3A1-

92RW009(RT) and the T = 2 model, suggestive of two trimers involved in the formation of the entry complex for this virus (Figure 31C).

4.5.4. V3 Crown Polymorphisms Impact on Fusion Kinetics. To assess the impact of V3 crown polymorphisms on fusion kinetics, we utilized an assay that measures the rates of desensitization to the fusion inhibitors enfuvirtide and C34.

Envelope pseudotyped viruses were spinoculated onto cells at 4oC and washed to remove

unbound particles. Entry of all particles was synchronized by the addition of warm

195 medium, and 100% inhibitory concentrations of ENF or C34 were added to cultures at

time intervals after the addition of warm medium. Cells were then incubated for 48 hours,

lysed, and luciferase activity determined. The time required for half-maximal

desensitization to ENF [a surrogate marker for the formation of 6-helix-bundles, a critical

endpoint of fusion (t1/2)] for NL4-3-gp160Yu-2(YA) and NL4-3-V3A1-92RW009(YA) were

both rapid (t1/2 = 28 and 25 minutes, respectively) (Figure 32A). NL4-3-V3A1-92RW009(RT) fused with significantly delayed kinetics (t1/2 = 49 minutes) compared to NL4-3-V3A1-

92RW009(YA) (24 minute lag time) (Figure 32A). The slowest overall fusion kinetics were observed for NL4-3-V3B5-91US056(YA), with a t1/2 of 58 minutes (Figure 32A).

Similar results were obtained when evaluating fusion kinetics with C34 instead of

ENF. Half-maximal fusion for NL4-3-gp160Yu-2(YA) and NL4-3-V3A1-92RW009(YA) was again rapid (t1/2 = 25 and 30 minutes, respectively) (Figure 32B). Change of tyrosine to

arginine in the NL4-3-V3A1-92RW009 context again resulted in delayed fusion kinetics (t1/2 =

44 minutes) with a lag time of 14 minutes (Figure 32B). Again, the primary B5-

91US056 envelope mediated very slow fusion kinetics (t1/2 = 70 minutes).

196 Figure 32. Kinetic Analysis of Fusion for V3 Crown Polymorphisms. U87-

CD4/CCR5 cells were spinoculated with an env pseudotype virus at 2500 x g for 90 min at 4oC. Cells were washed twice with cold PBS to remove unbound virus, and split into

96 well plates. Fully inhibitory concentrations of entry inhibitors (ENF = 10 μM, C34 =

10 μM) were added sequentially after synchronization of virus entry with warm drug-free medium. Cells were incubated for 48 h, lysed, and infection scored by luciferase activity.

Plots represent the % infection compared to the luciferase activity from the addition of drug at 180 min post-synchronization for ENF (A) or C34 (B).

197

198 4.6. DISCUSSION

We have previously determined that variations at positions 318 and 319 have a significant impact in viral replication and entry inhibitor sensitivity. In this study, we evaluated the mechanisms contributing to these effects. We evaluated relative CD4 and

CCR5 affinities of viruses containing V3 crown polymorphisms by assessing neutralization sensitivity to sCD4 and infection of cells expressing variable levels of each receptor. Furthermore, we assessed viral receptor affinity by titrating out the amount of functional gp120 expressed on the virion surface. Lastly, we directly measured virus fusion kinetics using time-of-addition assays with fusion inhibitors. We found that polymorphisms in the V3 crown that reduce entry inhibitor sensitivity and increase replicative fitness do so by increasing apparent affinity with CCR5 and enhancing fusion kinetics. These viruses are more able to scavenge low levels of CCR5 coreceptor for infection, more resistant to hetero-oligomerization with dominant negative variants of

HIV-1 envelope, and fuse with host cell membranes with faster overall kinetics.

The envelope glycoprotein functions as a trimer, but early biochemical studies of envelope function relied on soluble, monomeric gp120 molecules. The solution structure of these monomers was found to poorly recapitulate the quaternary structure of the function envelope trimer unit. Thus functional studies of the envelope glycoprotein must be performed in the native trimer context. One approach to the study of trimer biochemistry is to generate secreted forms of envelope that have been stabilized as a trimer by the introduction of various disulfide bonds (48, 142). A second approach to the study of envelope trimer function is to perform experiments using whole virions, though

199 assays involving whole virions are generally laborious. Affinity measurements are

typically calculated by limiting dilution of competitior ligands and determination of the

dissociation constant. However, relative affinity measurements of the native trimer can

be performed by manipulating the functional levels of cell surface receptor in the

infection process (96, 152) or by titrating out the amount of functional envelope

expressed on virion surfaces (215).

We have utilized a novel inducible receptor system for the evaluation of relative

CD4 and CCR5 affinities. The range of CD4 and CCR5 expression levels in this inducible system faithfully recapitulates expression levels found on primary cells (S.

Nguyen and B. Lee, work in progress). Induction of CD4 ranges from 2000 molecules per cell (at basal level) to 50,000 molecules per cell (10 ng/ml minocycline). This represents a 25-fold dynamic range, and is consistent with levels of CD4 measured on primary CD4+ T cells (65,000 molecules per cell) and macrophages (5000 – 15,000

molecules per cell) (109). Induction of CCR5 expression in this system ranges from 800

molecules per cell (basal) to 12,000 molecules per cell (2 μM ponasterone A), or a

dynamic range of 15-fold induction. CD4+ T cells have been shown to express from

1000 to 9000 molecules per cell, depending upon the donor, and macrophages expressing

from 500 to 5000 molecules per cell (109). We observed significant differences in the

ability of various HIV-1 envelopes to utilize low levels of CD4 and CCR5. We assessed

64 unique receptor expression levels for each envelope, producing a 3-dimensional

surface plot that was highly reproducible and unique for each virus assessed.

Comparison of viruses that are isogenic except for V3 crown polymorphisms at

position 318 and 319 revealed that these polymorphisms significantly impacted the

200 ability of the envelope to infect cells at low CCR5 density relative to high CCR5 expression levels. The differences between these viruses are consistent with their

replicative fitness and entry inhibitor susceptibility profiles. That is, viruses with high

replicative fitness and low susceptibility to entry inhibitors exhibit increased capacity to scavenge low level receptor. This finding was consistent in all three envelope contexts that were tested. This suggests that a major factor underlying all these processes is differential affinity for coreceptor. Thus we observe that viruses with low coreceptor affinity have decreased replicative fitness and increased susceptibility to entry inhibitors.

We further observed a significant decrease in the capacity of the primary B5-

91US056 envelope to utilize low level CD4 compared to the Yu-2 or A1-92RW009 V3 chimeric virus. Interestingly, these differences were recapitulated in their sensitivity to neutralization by sCD4. These differences were dependent upon the envelope context, and changes in the V3 crown positions 318 and 319 amino acids had no effect upon CD4 usage. The A1-92RW009 V3 chimeric virus contains most of its envelope derived from the laboratory strain HXB2. Previous studies have indicated that laboratory adapted strains and primary isolates exhibit significant differences in their CD4 affinity profiles

(40, 154). The Yu-2 envelope is also derived from a primary isolate that did not undergo multiple rounds of tissue culture passage. The Yu-2 strain, however, is derived from a cerebrospinal fluid virus sample. Previous studies have suggested that neurotropic viruses achieve this tropism due to increases in their CD4 affinity (57, 188). The major target of infection in the central nervous system are microglial cells, which are monocyte/macrophage lineage cells, and express low levels of CD4 (109, 152, 200).

Importantly, we did not observe an association between replicative fitness, entry inhibitor

201 susceptibility, and CD4 affinity. The NL4-3-gp120B5-91US056(YA) virus exhibited high replicative fitness and relative resistance to entry inhibitors when compared to NL4-3-

V3A1-92RW009(YA) or NL4-3-gp160Yu-2(YA).

V3 crown polymorphisms conferring high replicative fitness and reduced entry inhibitor susceptibility were further resistant to the effects of hetero-oligomerization with dominant negative variants of gp160. The use of dominant negative envelope variants incorporated with wild type monomers was used by Sodroski and colleagues to determine the entry complex stoichiometry of HIV-1, amphotropic murine leukemia virus (A-MLV), avian sarcoma/leucosis virus type A (ASLV-A), and influenza virus (214). They determined that the prototypic HIV-1 R5 strain Yu-2 could enter host cell with a stoichiometry of T = 1 trimer. Subsequent analysis using a similar methodology suggested that Yu-2 mediated entry with two subunits of a single trimer (215). Our data using two viruses with V3 crown mutations suggests that, when utilizing the same mathematical model system, variations would exist in the entry stoichiomety of HIV-1 isolates, and that this difference is based on coreceptor affinity. The NL4-3-V3A1-

92RW009(YA) infectivity data fit closely to the T = 1 stoichiometry model, while the NL4-

3-V3A1-92RW009(RT) envelope fit closely to the T = 2 stoichiometry model. Assuming the accuracy of the mathematical model, our data would suggest that affinity differences in the V3 loop for CCR5 can specifically modulate the stoichiometry of HIV-1 envelope trimers involved in the formation of a functional entry complex. This finding would further suggest mechanisms involved in differential susceptibility to PSC-RANTES and other entry inhibitors as well as differences in replicative fitness observed in these two viruses. The requirement of two versus one trimer for a stable entry complex would

202 suggest the involvement of a higher number of coreceptor molecules. Further limiting

CCR5 accessibility by the introduction of a coreceptor inhibitor would more significantly impact a virus requiring a larger number of CCR5 molecules for entry.

The findings of Yang et al are, however, in significant contrast to previous studies which indicate that entry required between 4 and 6 molecules of CCR5, suggesting the requirement of a minimum of two envelope trimers to be engaged (96). Studies on CD4 requirements for functional entry have also suggested the involvement of multiple gp120 trimers contacting with CD4 to develop sufficient avidity for stable entry complex formation (104). A more recent study has directly challenged the findings of Yang et al.

Moore and colleagues utilized dominant negative hetero-oligomerization with wild type

HIV-1 envelopes and observed differences in the impact of uncleaved trimers on virus infectivity (78). Proposing a different mathematical model, they observed isolate-specific differences in the critical number of trimers involved in the entry complex. A subset of their viruses utilized CXCR4 for entry, however, and they did not observe a direct association between envelope stoichiometry criticality and any other phenotype, such as coreceptor affinity, entry inhibitor sensitivity, or replicative fitness, as has been done in this study. Moore and colleagues have proposed that, assuming an average of 9 functional envelope trimers per virion (219, 220), a minimum of 4 to 5 trimers are required for formation of a stable entry complex (78). Our favored hypothesis is that the titration of functional gp120 molecules from a population of virions is more accurately a measure of receptor affinity. In the absence of differences in CD4 affinity, as we have observed between NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT), we would

203 suggest that differences in infectivity that we observed are directly related to differential affinity for CCR5 conferred by the V3 crown.

We observe now a direct correlation between coreceptor affinity levels and fusion kinetics. Viruses with increased susceptibility to entry inhibitors, decreased replicative fitness, and decreased capacitiy to scavenge low level CCR5 also fused with cells with significantly decreased kinetics. This decrease in entry kinetics is likely the basis for what we term “entry efficiency.” In this model, equivalent virus particles are bound to host cells through interactions with CD4. Ternary complex formation occurs with variable kinetics, as the viral envelope glycoproteins aggregate a critical threshold of ternary complexes to achieve sufficient avidity for the formation of a fusion pore. The major rate limiting step in this model is the formation of ternary complexes by the slow kinetics of coreceptor binding, followed by a rapid reorganization of gp41 into 6-helix bundles and lipid mixing. This model is consistent with previous proposals (132).

Furthermore, comparison of the B5-91US056 envelope would suggest that CD4 affinity is further a determinant of fusion kinetics, and based on kinetic data, this virus should have the lowest replicative fitness of the viruses tested. This is not what we have observed with this isolate, however. This virus demonstrates a high affinity for CCR5, but a low affinity for CD4. We would suggest that the lag in fusion kinetics observed for this virus is due to low CD4 binding, and that the fusion events downstream of CD4 binding for this virus are in fact quite rapid. It is possible that low CD4 affinity results in a low efficiency of spinoculation, as this procedure has been shown to enhance virus infectivity by promoting gp120-CD4 interactions (139). We are currently evaluating the effect of CD4 affinity on fusion kinetics by performing kinetic assays with CD4

204 inhibitors, as well as coreceptor inhibitors and fusion inhibitors, utilizing temperature control of the fusion process.

Taken together, we have found that CCR5 affinity is a unifying determinant of multiple virus entry phenotypes. We would suggest that ternary complex formation between gp120, CD4, and CCR5 is the major rate-limiting step of the entry process and that this association has important implications for virus susceptibility to all therapeutic agents that would target the entry process. Furthermore, the kinetics of ternary complex formation, and specifically the V3 loop, impinge on overall viral replicative fitness, which may have implications for intrapatient virus evolution and disease progression.

205 Chapter 5

GENERAL DISCUSSION

206 5. Discussion

HIV-1 entry is a highly cooperative and complex process. The envelope-receptor

interaction critically determines host cell tropism and is a major factor in disease

progression and virus transmission. Detailing of the major mechanisms involved have

led to a greater understanding of virus transmission, pathogenesis, and furthermore, to the

development of potent inhibitors which may have a great impact on the clinical outcome of infected patients. Central to our understanding of the entry process is the interaction of the HIV-1 envelope with the CCR5 coreceptor, particularly by the V3 loop structure.

The envelope and V3 are subjected to high levels of selection in vivo by host selection factors and endogenous chemokines. Because of this pressure, the V3 loop is an extremely heterogeneous functional element of the envelope glycoprotein. Little is understood about the implications of this diversity, or the effects of envelope context on

the major functional impact of V3, namely, binding to coreceptor.

It is imperative that we understand the ramifications of therapeutic administration

of entry inhibitors. Many questions involving the entry process remain of critical

importance, and these studies have attempted to address several of these: What is the

effect of V3 sequence heterogeneity and envelope sequence context on entry, replication, and entry inhibitor sensitivity? How will viruses adapt to coreceptor inhibitors in vivo?

Is a model of competitive resistance relevant in the evolution of inhibitor resistance?

What are the consequences of increased coreceptor affinity and fitness? Does entry efficiency have consequences for pathogenesis?

207 5.1. V3 Diversity and Sequence Context.

The V3 region of the HIV-1 envelope glycoprotein is a highly heterogeneous element which is subjected to host immunological selection in vivo. Though the amino acid identity at any one site can be fluid, the overall length of the V3 loop is relatively constant at 35 amino acids, suggesting that the the distance between the gp120 core and the V3 tip must remain somewhat constant for optimal geometry of the ternary complex.

We have observed that despite the heterogeneity of this element, there are distinct constraints on amino acid changes and their effects on envelope function. The same amino acid, when inserted into three different contexts, can alternatively enhance viral replication, decrease replication, or have no effect whatsoever. Our data suggest that sequence analysis of HIV-1 envelope glycoproteins will be insufficient to predict protein function in an isolate-specific manner. Furthermore, observation of amino acid polymorphisms is often made in primary isolate viruses. To study the effects of these polymorphisms, a standard practice is to insert them into an easily manipulable laboratory virus background which exists in the form of a plasmid. Our studies have shown that generalization of effects conferred by mutations in envelope cannot be made by performing site directed mutagenesis studies outside of the native context in which the polymorphism is initially discovered.

5.2. Intrinsic Viral Sensitivity and Response to Entry Inhibitor Therapy.

It has been suggested that viruses from early and late infection differ in their susceptibility to the chemokine CCL5 (95). The basic concept behind this observation is that viruses are constantly utilizing CCR5 for host cell entry in the face of continuous

208 host immunological pressure. A consequence of this host immune pressure is the elaboration of chemokines. The constant presence of chemokine receptor ligands provides evolutionary pressure for resistance to these chemokines. Our data suggests that resistance to chemokines comes through increases in coreceptor affinity, and this affinity increase confers a replicative advantage. We have previously observed that HIV-1 quasispecies diversity increases throughout the course of infection, and that increases in replicative fitness occur concomitantly (176, 198). Potentially these increases in replicative fitness are mediated by changes in receptor affinity. A logical extension of this possibility is that HIV-1 infected patients may respond to entry inhibitor therapy differently depending upon whether they are in early or late stages of infection. This is an important question which has implications on the design of optimal drug regimens and timing of the implementation of entry inhibitors. Most new antiretroviral agents are initially utilized as salvage therapy for patients who are in advanced disease, who harbor drug resistant viruses, and who have limited therapeutic options. It may be, however, that

CCR5 inhibitors will have limited efficacy in these patient populations, and that the results that are observed from this early, limited distribution of CCR5 inhibitor therapy will mask the potential benefit that would be experienced by patients who start entry inhibitor therapy earlier in disease progression. It will be important to monitor rates of therapy failure and disease progression in both early and late disease patient populations to determine the optimal timing of administration of drug regimens containing entry inhibitors to maximize virologic response to therapy and to prevent the development of drug resistant viruses.

209 5.3. Evolution of CCR5 Inhibitor Resistance. Generation of CCR5 inhibitor

resistance has been performed for several HIV-1 isolate in in vitro selection models.

From the data accumulated thus far, we speculate that the generation of inhibitor

resistance will be a process unique to each virus and host. Monitoring of inhibitor

resistance to NRTI, NNRTI, and PI in clinical situations generally relies on the fact that

discrete, well characterized mutations in the HIV-1 reverse transcriptase or protease open

reading frames accurately predict inhibitor resistance. Sequence analysis of plasma viruses from a patient and screening of these loci suggests when patients should change therapy and indicates which drugs would be active against the circulating virus. Again we would suggest that sequence analysis alone will not be sufficient to determine susceptibility to CCR5 antagonists. Functional assays of full length patient-derived envelopes will be necessary to determine both coreceptor tropism and inhibitor susceptibility. This unfortunate complexity and expense in susceptibility determination may significantly limit the usefulness of CCR5 inhibitors in clinical settings.

5.4. Envelope Function and HIV-1 Pathogenesis. Recent studies have suggested that the HIV-1 envelope glycoprotein is a major determinant of overall viral replicative fitness (12, 123, 157). It is unclear at this time what aspect of entry underlies this difference in viral replication capacity, though it is suggested that overall avidity for coreceptor may be an important factor (123). We have observed that modest changes in a functional domain of the envelope can have significant impacts on overall viral replicative fitness. However it is unclear at this time whether viral replicative fitness and virulence are related phenotypes for HIV-1 viruses in vivo. Long term nonprogressor

210 (LTNP) patients are HIV-1 infected individuals who maintain high CD4 counts and do

not progress to disease over a long period of time. Previous studies have suggested that

viruses derived from LTNP are significantly less fit in vitro than are viruses from

matched patients who progress normally to disease (156). A further subset of individuals referred to as elite suppressors (ES) maintain viremia below the detection of laboratory

assays in the absence of antiretroviral therapy (10). We are only beginning to understand

the basic virology and immunology involved in suppression of viremia in these patients.

Perhaps these patients harbor viruses with significant entry defects, contributing to an

overall low replicative fitness in vivo. We have developed assays, and studies are now

underway to assess envelope function of viruses derived from LTNP and ES individuals

which may provide insight into virus-specific factors involved in control of viremia.

These experiments are designed to assess the association between entry efficiency, fitness,

and HIV-1 pathogenesis in vivo.

211 Bibliography

1. Aarons, E. J., S. Beddows, T. Willingham, L. Wu, and R. A. Koup. 2001.

Adaptation to blockade of human immunodeficiency virus type 1 entry imposed

by the anti-CCR5 monoclonal antibody 2D7. Virology 287:382-390.

2. Abacioglu, Y. H., T. R. Fouts, J. D. Laman, E. Claassen, S. H. Pincus, J. P.

Moore, C. A. Roby, R. Kamin-Lewis, and G. K. Lewis. 1994. Epitope mapping

and topology of baculovirus-expressed HIV-1 gp160 determined with a panel of

murine monoclonal antibodies. AIDS Res.Hum.Retroviruses 10:371-381.

3. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M.

Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1alpha, MIP-

1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science

272:1955-1958.

4. Allan, J. S., J. Strauss, and D. W. Buck. 1990. Enhancement of SIV infection

with soluble receptor molecules. Science 247:1084-1088.

5. Amara, A., S. L. Gall, O. Schwartz, J. Salamero, M. Montes, P. Loetscher, M.

Baggiolini, J. L. Virelizier, and F. Arenzana-Seisdedos. 1997. HIV coreceptor

downregulation as antiviral principle: SDF-1alpha-dependent internalization of

the chemokine receptor CXCR4 contributes to inhibition of HIV replication.

J.Exp.Med. 186:139-146.

212 6. An, P., M. P. Martin, G. W. Nelson, M. Carrington, M. W. Smith, K. Gong,

D. Vlahov, S. J. O'Brien, and C. A. Winkler. 2000. Influence of CCR5

promoter haplotypes on AIDS progression in African-Americans. AIDS 14:2117-

2122.

7. Arien, K. K., A. Abraha, M. E. Quinones-Mateu, L. Kestens, G. Vanham,

and E. J. Arts. 2005. The replicative fitness of primary human

immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2

isolates. J.Virol. 79:8979-8990.

8. Ashorn, P., T. J. McQuade, S. Thaisrivongs, A. G. Tomasselli, W. G. Tarpley,

and B. Moss. 1990. An inhibitor of the protease blocks maturation of human and

simian immunodeficiency viruses and spread of infection.

Proc.Natl.Acad.Sci.U.S.A 87:7472-7476.

9. Baba, M., H. Miyake, X. Wang, M. Okamoto, and K. Takashima. 2007.

Isolation and characterization of human immunodeficiency virus type 1 resistant

to the small-molecule CCR5 antagonist TAK-652. Antimicrob.Agents Chemother.

51:707-715.

10. Bailey, J. R., K. G. Lassen, H. C. Yang, T. C. Quinn, S. C. Ray, J. N.

Blankson, and R. F. Siliciano. 2006. Neutralizing antibodies do not mediate

suppression of human immunodeficiency virus type 1 in elite suppressors or

213 selection of plasma virus variants in patients on highly active antiretroviral

therapy. J.Virol. 80:4758-4770.

11. Baldwin, C. E., R. W. Sanders, Y. Deng, S. Jurriaans, J. M. Lange, M. Lu,

and B. Berkhout. 2004. Emergence of a drug-dependent human

immunodeficiency virus type 1 variant during therapy with the T20 fusion

inhibitor. J.Virol. 78:12428-12437.

12. Ball, S. C., A. Abraha, K. R. Collins, A. J. Marozsan, H. Baird, M. E.

Quinones-Mateu, A. Penn-Nicholson, M. Murray, N. Richard, M. Lobritz, P.

A. Zimmerman, T. Kawamura, A. Blauvelt, and E. J. Arts. 2003. Comparing

the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates

of subtypes B and C. J.Virol. 77:1021-1038.

13. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J.

Gruest, C. Dauguet, C. Axler-Blin, F. Vezinet-Brun, C. Rouzioux, W.

Rozenbaum, and L. Montagnier. 1983. Isolation of a T-lymphotropic retrovirus

from a patient at risk for acquired immune deficiency syndrome (AIDS). Science

220:868-871.

14. Bartlett, J. G. and J. E. Gallant. 2007. Medical Management of HIV Infection.

Johns Hopkins University Press.

214 15. Berger, E. A., R. W. Doms, E. M. Fenyo, B. T. Korber, D. R. Littman, J. P.

Moore, Q. J. Sattentau, H. Schuitemaker, J. Sodroski, and R. A. Weiss. 1998.

A new classification for HIV-1. Nature 391:240.

16. Berger, E. A., P. M. Murphy, and J. M. Farber. 1999. Chemokine receptors as

HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu.Rev.Immunol.

17:657-700.

17. Bieniasz, P. D., R. A. Fridell, I. Aramori, S. S. Ferguson, M. G. Caron, and B.

R. Cullen. 1997. HIV-1-induced cell fusion is mediated by multiple regions

within both the viral envelope and the CCR-5 co-receptor. EMBO J. 16:2599-

2609.

18. Billick, E., C. Seibert, P. Pugach, T. Ketas, A. Trkola, M. J. Endres, N. J.

Murgolo, E. Coates, G. R. Reyes, B. M. Baroudy, T. P. Sakmar, J. P. Moore,

and S. E. Kuhmann. 2004. The differential sensitivity of human and rhesus

macaque CCR5 to small-molecule inhibitors of human immunodeficiency virus

type 1 entry is explained by a single amino acid difference and suggests a

mechanism of action for these inhibitors. J.Virol. 78:4134-4144.

19. Biscone, M. J., J. L. Miamidian, J. M. Muchiri, S. S. Baik, F. H. Lee, R. W.

Doms, and J. D. Reeves. 2006. Functional impact of HIV coreceptor-binding site

mutations. Virology 351:226-236.

215 20. Blanpain, C., B. J. Doranz, A. Bondue, C. Govaerts, A. De Leener, G. Vassart,

R. W. Doms, A. Proudfoot, and M. Parmentier. 2003. The core domain of

chemokines binds CCR5 extracellular domains while their amino terminus

interacts with the transmembrane helix bundle. J.Biol.Chem. 278:5179-5187.

21. Blanpain, C., B. J. Doranz, J. Vakili, J. Rucker, C. Govaerts, S. S. Baik, O.

Lorthioir, I. Migeotte, F. Libert, F. Baleux, G. Vassart, R. W. Doms, and M.

Parmentier. 1999. Multiple charged and aromatic residues in CCR5 amino-

terminal domain are involved in high affinity binding of both chemokines and

HIV-1 Env protein. J.Biol.Chem. 274:34719-34727.

22. Blanpain, C., J. M. Vanderwinden, J. Cihak, V. Wittamer, E. Le Poul, H.

Issafras, M. Stangassinger, G. Vassart, S. Marullo, D. Schlndorff, M.

Parmentier, and M. Mack. 2002. Multiple active states and oligomerization of

CCR5 revealed by functional properties of monoclonal antibodies. Mol.Biol.Cell

13:723-737.

23. Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark-Lewis, J. Sodroski, and

T. A. Springer. 1996. The lymphocyte chemoattractant SDF-1 is a ligand for

LESTR/fusin and blocks HIV-1 entry. Nature 382:829-833.

24. Bour, S. and K. Strebel. 2003. The HIV-1 Vpu protein: a multifunctional

enhancer of viral particle release. Microbes.Infect. 5:1029-1039.

216 25. Brandt, S. M., R. Mariani, A. U. Holland, T. J. Hope, and N. R. Landau.

2002. Association of chemokine-mediated block to HIV entry with coreceptor

internalization. J.Biol.Chem. 277:17291-17299.

26. 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.

27. Brinchmann, J. E., G. Gaudernack, and F. Vartdal. 1990. CD8+ T cells inhibit

HIV replication in naturally infected CD4+ T cells. Evidence for a soluble

inhibitor. J.Immunol. 144:2961-2966.

28. Cantin, R., S. Methot, and M. J. Tremblay. 2005. Plunder and stowaways:

incorporation of cellular proteins by enveloped viruses. J.Virol. 79:6577-6587.

29. Chambers, P., C. R. Pringle, and A. J. Easton. 1990. Heptad repeat sequences

are located adjacent to hydrophobic regions in several types of virus fusion

glycoproteins. J.Gen.Virol. 71 ( Pt 12):3075-3080.

30. 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.

31. 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.

217 1996. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by

primary HIV-1 isolates. Cell 85:1135-1148.

32. Christopoulos, A. and T. Kenakin. 2002. G protein-coupled receptor allosterism

and complexing. Pharmacol.Rev. 54:323-374.

33. Cocchi, F., A. L. DeVico, A. Garzino-Demo, S. K. Arya, R. C. Gallo, and P.

Lusso. 1995. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the

major HIV-suppressive factors produced by CD8+ T cells. Science 270:1811-

1815.

34. Collier, A. C., R. W. Coombs, D. Katzenstein, M. Holodniy, J. Gibson, J.

Mordenti, A. E. Izu, A. M. Duliege, A. J. Ammann, T. Merigan, and . 1995.

Safety, pharmacokinetics, and antiviral response of CD4-immunoglobulin G by

intravenous bolus in AIDS and AIDS-related complex.

J.Acquir.Immune.Defic.Syndr.Hum.Retrovirol. 10:150-156.

35. Colman, P. M. and M. C. Lawrence. 2003. The structural biology of type I viral

membrane fusion. Nat.Rev.Mol.Cell Biol. 4:309-319.

36. Connor, R. I., K. E. Sheridan, D. Ceradini, S. Choe, and N. R. Landau. 1997.

Change in coreceptor use coreceptor use correlates with disease progression in

HIV-1--infected individuals. J.Exp.Med. 185:621-628.

218 37. Cormier, E. G. and T. Dragic. 2002. The crown and stem of the V3 loop play

distinct roles in human immunodeficiency virus type 1 envelope glycoprotein

interactions with the CCR5 coreceptor. J.Virol. 76:8953-8957.

38. Cormier, E. G., D. N. Tran, L. Yukhayeva, W. C. Olson, and T. Dragic. 2001.

Mapping the determinants of the CCR5 amino-terminal sulfopeptide interaction

with soluble human immunodeficiency virus type 1 gp120-CD4 complexes.

J.Virol. 75:5541-5549.

39. Cullen, B. R. 2006. Role and mechanism of action of the APOBEC3 family of

antiretroviral resistance factors. J.Virol. 80:1067-1076.

40. Daar, E. S., X. L. Li, T. Moudgil, and D. D. Ho. 1990. High concentrations of

recombinant soluble CD4 are required to neutralize primary human

immunodeficiency virus type 1 isolates. Proc.Natl.Acad.Sci.U.S.A 87:6574-6578.

41. Dalgleish, A. G., P. C. Beverley, P. R. Clapham, D. H. Crawford, M. F.

Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essential

component of the receptor for the AIDS retrovirus. Nature 312:763-767.

42. De Clercq, E. 2004. Non-nucleoside reverse transcriptase inhibitors (NNRTIs):

past, present, and future. Chem.Biodivers. 1:44-64.

219 43. Dean, M., M. Carrington, C. Winkler, G. A. Huttley, M. W. Smith, R.

Allikmets, J. J. Goedert, S. P. Buchbinder, E. Vittinghoff, E. Gomperts, S.

Donfield, D. Vlahov, R. Kaslow, A. Saah, C. Rinaldo, R. Detels, and S. J.

O'Brien. 1996. Genetic restriction of HIV-1 infection and progression to AIDS

by a deletion allele of the CKR5 structural gene. Hemophilia Growth and

Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia

Cohort Study, San Francisco City Cohort, ALIVE Study. Science 273:1856-1862.

44. Deen, K. C., J. S. McDougal, R. Inacker, G. Folena-Wasserman, J. Arthos, J.

Rosenberg, P. J. Maddon, R. Axel, and R. W. Sweet. 1988. A soluble form of

CD4 (T4) protein inhibits AIDS virus infection. Nature 331:82-84.

45. Delwart, E. L., G. Mosialos, and T. Gilmore. 1990. Retroviral envelope

glycoproteins contain a "leucine zipper"-like repeat. AIDS Res.Hum.Retroviruses

6:703-706.

46. Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di

Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B. Davis, S. C. Peiper, T. J.

Schall, D. R. Littman, and N. R. Landau. 1996. Identification of a major co-

receptor for primary isolates of HIV-1. Nature 381:661-666.

47. Derdeyn, C. A., J. M. Decker, J. N. Sfakianos, X. Wu, W. A. O'Brien, L.

Ratner, J. C. Kappes, G. M. Shaw, and E. Hunter. 2000. Sensitivity of human

220 immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by

coreceptor specificity defined by the V3 loop of gp120. J.Virol. 74:8358-8367.

48. Dey, A. K., K. B. David, P. J. Klasse, and J. P. Moore. 2007. Specific amino

acids in the N-terminus of the gp41 ectodomain contribute to the stabilization of a

soluble, cleaved gp140 envelope glycoprotein from human immunodeficiency

virus type 1. Virology 360:199-208.

49. Di Marzio, P., J. Tse, and N. R. Landau. 1998. Chemokine receptor regulation

and HIV type 1 tropism in monocyte-macrophages. AIDS Res.Hum.Retroviruses

14:129-138.

50. Domingo, E. and J. J. Holland. 1997. RNA virus mutations and fitness for

survival. Annu.Rev.Microbiol. 51:151-178.

51. Doms, R. W., R. A. Lamb, J. K. Rose, and A. Helenius. 1993. Folding and

assembly of viral membrane proteins. Virology 193:545-562.

52. Doranz, B. J., Z. H. Lu, J. Rucker, T. Y. Zhang, M. Sharron, Y. H. Cen, Z. X.

Wang, H. H. Guo, J. G. Du, M. A. Accavitti, R. W. Doms, and S. C. Peiper.

1997. Two distinct CCR5 domains can mediate coreceptor usage by human

immunodeficiency virus type 1. J.Virol. 71:6305-6314.

221 53. 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.

54. Dorr, P., M. Westby, S. Dobbs, P. Griffin, B. Irvine, M. Macartney, J. Mori,

G. Rickett, C. Smith-Burchnell, C. Napier, R. Webster, D. Armour, D. Price,

B. Stammen, A. Wood, and M. Perros. 2005. Maraviroc (UK-427,857), a potent,

orally bioavailable, and selective small-molecule inhibitor of chemokine receptor

CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.

Antimicrob.Agents Chemother. 49:4721-4732.

55. 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 381:667-673.

56. Dragic, T., A. Trkola, D. A. Thompson, E. G. Cormier, F. A. Kajumo, E.

Maxwell, S. W. Lin, W. Ying, S. O. Smith, T. P. Sakmar, and J. P. Moore.

2000. A binding pocket for a small molecule inhibitor of HIV-1 entry within the

transmembrane helices of CCR5. Proc.Natl.Acad.Sci.U.S.A 97:5639-5644.

57. Dunfee, R. L., E. R. Thomas, P. R. Gorry, J. Wang, J. Taylor, K. Kunstman,

S. M. Wolinsky, and D. Gabuzda. 2006. The HIV Env variant N283 enhances

222 macrophage tropism and is associated with brain infection and dementia.

Proc.Natl.Acad.Sci.U.S.A 103:15160-15165.

58. Earl, P. L., B. Moss, and R. W. Doms. 1991. Folding, interaction with GRP78-

BiP, assembly, and transport of the human immunodeficiency virus type 1

envelope protein. J.Virol. 65:2047-2055.

59. Eckert, D. M. and P. S. Kim. 2001. Mechanisms of viral membrane fusion and

its inhibition. Annu.Rev.Biochem. 70:777-810.

60. Edinger, A. L., T. L. Hoffman, M. Sharron, B. Lee, Y. Yi, W. Choe, D. L.

Kolson, B. Mitrovic, Y. Zhou, D. Faulds, R. G. Collman, J. Hesselgesser, R.

Horuk, and R. W. Doms. 1998. An orphan seven-transmembrane domain

receptor expressed widely in the brain functions as a coreceptor for human

immunodeficiency virus type 1 and simian immunodeficiency virus. J.Virol.

72:7934-7940.

61. Farzan, M., H. Choe, K. Martin, L. Marcon, W. Hofmann, G. Karlsson, Y.

Sun, P. Barrett, N. Marchand, N. Sullivan, N. Gerard, C. Gerard, and J.

Sodroski. 1997. Two orphan seven-transmembrane segment receptors which are

expressed in CD4-positive cells support simian immunodeficiency virus infection.

J.Exp.Med. 186:405-411.

223 62. Farzan, M., T. Mirzabekov, P. Kolchinsky, R. Wyatt, M. Cayabyab, N. P.

Gerard, C. Gerard, J. Sodroski, and H. Choe. 1999. Tyrosine sulfation of the

amino terminus of CCR5 facilitates HIV-1 entry. Cell 96:667-676.

63. 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.

64. Fisher, R. A., J. M. Bertonis, W. Meier, V. A. Johnson, D. S. Costopoulos, T.

Liu, R. Tizard, B. D. Walker, M. S. Hirsch, R. T. Schooley, and . 1988. HIV

infection is blocked in vitro by recombinant soluble CD4. Nature 331:76-78.

65. Freed, E. O., D. J. Myers, and R. Risser. 1989. Mutational analysis of the

cleavage sequence of the human immunodeficiency virus type 1 envelope

glycoprotein precursor gp160. J.Virol. 63:4670-4675.

66. Furman, P. A., J. A. Fyfe, M. H. St Clair, K. Weinhold, J. L. Rideout, G. A.

Freeman, S. N. Lehrman, D. P. Bolognesi, S. Broder, H. Mitsuya, and . 1986.

Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-

triphosphate with human immunodeficiency virus reverse transcriptase.

Proc.Natl.Acad.Sci.U.S.A 83:8333-8337.

67. Gallaher, W. R. 1987. Detection of a fusion peptide sequence in the

transmembrane protein of human immunodeficiency virus. Cell 50:327-328.

224 68. Gallaher, W. R., J. M. Ball, R. F. Garry, M. C. Griffin, and R. C. Montelaro.

1989. A general model for the transmembrane proteins of HIV and other

retroviruses. AIDS Res.Hum.Retroviruses 5:431-440.

69. Gelmann, E. P., M. Popovic, D. Blayney, H. Masur, G. Sidhu, R. E. Stahl,

and R. C. Gallo. 1983. Proviral DNA of a retrovirus, human T-cell leukemia

virus, in two patients with AIDS. Science 220:862-865.

70. Gordon, C. J., M. A. Muesing, A. E. Proudfoot, C. A. Power, J. P. Moore,

and A. Trkola. 1999. Enhancement of human immunodeficiency virus type 1

infection by the CC-chemokine RANTES is independent of the mechanism of

virus-cell fusion. J.Virol. 73:684-694.

71. Gotte, M., X. Li, and M. A. Wainberg. 1999. HIV-1 reverse transcription: a

brief overview focused on structure-function relationships among molecules

involved in initiation of the reaction. Arch.Biochem.Biophys. 365:199-210.

72. Haase, A. T. 1999. Population biology of HIV-1 infection: viral and CD4+ T cell

demographics and dynamics in lymphatic tissues. Annu.Rev.Immunol. 17:625-

656.

73. Harter, C., P. James, T. Bachi, G. Semenza, and J. Brunner. 1989.

Hydrophobic binding of the ectodomain of influenza hemagglutinin to

membranes occurs through the "fusion peptide". J.Biol.Chem. 264:6459-6464.

225 74. Hartley, O., H. Gaertner, J. Wilken, D. Thompson, R. Fish, A. Ramos, C.

Pastore, B. Dufour, F. Cerini, A. Melotti, N. Heveker, L. Picard, M. Alizon, D.

Mosier, S. Kent, and R. Offord. 2004. Medicinal chemistry applied to a

synthetic protein: development of highly potent HIV entry inhibitors.

Proc.Natl.Acad.Sci.U.S.A 101:16460-16465.

75. Hartley, O., P. J. Klasse, Q. J. Sattentau, and J. P. Moore. 2005. V3: HIV's

switch-hitter. AIDS Res.Hum.Retroviruses 21:171-189.

76. He, J., Y. Chen, M. Farzan, H. Choe, A. Ohagen, S. Gartner, J. Busciglio, X.

Yang, W. Hofmann, W. Newman, C. R. Mackay, J. Sodroski, and D.

Gabuzda. 1997. CCR3 and CCR5 are co-receptors for HIV-1 infection of

microglia. Nature 385:645-649.

77. Hernanz-Falcon, P., J. M. Rodriguez-Frade, A. Serrano, D. Juan, A. del Sol,

S. F. Soriano, F. Roncal, L. Gomez, A. Valencia, A. Martinez, and M.

Mellado. 2004. Identification of amino acid residues crucial for chemokine

receptor dimerization. Nat.Immunol. 5:216-223.

78. Herrera, C., P. J. Klasse, C. W. Kibler, E. Michael, J. P. Moore, and S.

Beddows. 2006. Dominant-negative effect of hetero-oligomerization on the

function of the human immunodeficiency virus type 1 envelope glycoprotein

complex. Virology 351:121-132.

226 79. Herrera, C., P. J. Klasse, E. Michael, S. Kake, K. Barnes, C. W. Kibler, L.

Campbell-Gardener, Z. Si, J. Sodroski, J. P. Moore, and S. Beddows. 2005.

The impact of envelope glycoprotein cleavage on the antigenicity, infectivity, and

neutralization sensitivity of Env-pseudotyped human immunodeficiency virus

type 1 particles. Virology 338:154-172.

80. Hill, C. M., H. Deng, D. Unutmaz, V. N. Kewalramani, L. Bastiani, M. K.

Gorny, S. Zolla-Pazner, and D. R. Littman. 1997. Envelope glycoproteins from

human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus

can use human CCR5 as a coreceptor for viral entry and make direct CD4-

dependent interactions with this chemokine receptor. J.Virol. 71:6296-6304.

81. 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.U.S.A 93:3099-3104.

82. Ho, D. D., A. U. Neumann, A. S. Perelson, W. Chen, J. M. Leonard, and M.

Markowitz. 1995. Rapid turnover of plasma virions and CD4 lymphocytes in

HIV-1 infection. Nature 373:123-126.

83. Ho, H. T., L. Fan, B. Nowicka-Sans, B. McAuliffe, C. B. Li, G. Yamanaka, N.

Zhou, H. Fang, I. Dicker, R. Dalterio, Y. F. Gong, T. Wang, Z. Yin, Y. Ueda,

J. Matiskella, J. Kadow, P. Clapham, J. Robinson, R. Colonno, and P. F. Lin.

227 2006. Envelope conformational changes induced by human immunodeficiency

virus type 1 attachment inhibitors prevent CD4 binding and downstream entry

events. J.Virol. 80:4017-4025.

84. Hodges, T. L., J. O. Kahn, L. D. Kaplan, J. E. Groopman, P. A. Volberding,

A. J. Amman, C. J. Arri, L. M. Bouvier, J. Mordenti, A. E. Izu, and . 1991.

Phase 1 study of recombinant human CD4-immunoglobulin G therapy of patients

with AIDS and AIDS-related complex. Antimicrob.Agents Chemother. 35:2580-

2586.

85. Hu, Q., J. O. Trent, G. D. Tomaras, Z. Wang, J. L. Murray, S. M. Conolly, J.

M. Navenot, A. P. Barry, M. L. Greenberg, and S. C. Peiper. 2000.

Identification of ENV determinants in V3 that influence the molecular anatomy of

CCR5 utilization. J.Mol.Biol. 302:359-375.

86. Huang, C. C., M. Tang, M. Y. Zhang, S. Majeed, E. Montabana, R. L.

Stanfield, D. S. Dimitrov, B. Korber, J. Sodroski, I. A. Wilson, R. Wyatt, and

P. D. Kwong. 2005. Structure of a V3-containing HIV-1 gp120 core. Science

310:1025-1028.

87. Huang, Y., W. A. Paxton, S. M. Wolinsky, A. U. Neumann, L. Zhang, T. He,

S. Kang, D. Ceradini, Z. Jin, K. Yazdanbakhsh, K. Kunstman, D. Erickson,

E. Dragon, N. R. Landau, J. Phair, D. D. Ho, and R. A. Koup. 1996. The role

228 of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat.Med.

2:1240-1243.

88. Hussey, R. E., N. E. Richardson, M. Kowalski, N. R. Brown, H. C. Chang, R.

F. Siliciano, T. Dorfman, B. Walker, J. Sodroski, and E. L. Reinherz. 1988. A

soluble CD4 protein selectively inhibits HIV replication and syncytium formation.

Nature 331:78-81.

89. Issafras, H., S. Angers, S. Bulenger, C. Blanpain, M. Parmentier, C. Labbe-

Jullie, M. Bouvier, and S. Marullo. 2002. Constitutive agonist-independent

CCR5 oligomerization and antibody-mediated clustering occurring at

physiological levels of receptors. J.Biol.Chem. 277:34666-34673.

90. Ivey-Hoyle, M., J. S. Culp, M. A. Chaikin, B. D. Hellmig, T. J. Matthews, R.

W. Sweet, and M. Rosenberg. 1991. Envelope glycoproteins from biologically

diverse isolates of immunodeficiency viruses have widely different affinities for

CD4. Proc.Natl.Acad.Sci.U.S.A 88:512-516.

91. Joshi, S. B., R. E. Dutch, and R. A. Lamb. 1998. A core trimer of the

paramyxovirus fusion protein: parallels to influenza virus hemagglutinin and

HIV-1 gp41. Virology 248:20-34.

92. Karn, J. 1999. Tackling Tat. J.Mol.Biol. 293:235-254.

229 93. Kilby, J. M., S. Hopkins, T. M. Venetta, B. DiMassimo, G. A. Cloud, J. Y.

Lee, L. Alldredge, E. Hunter, D. Lambert, D. Bolognesi, T. Matthews, M. R.

Johnson, M. A. Nowak, G. M. Shaw, and M. S. Saag. 1998. Potent suppression

of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated

virus entry. Nat.Med. 4:1302-1307.

94. Koning, F. A., C. Koevoets, T. J. van der Vorst, and H. Schuitemaker. 2005.

Sensitivity of primary R5 HTV-1 to inhibition by RANTES correlates with

sensitivity to small-molecule R5 inhibitors. Antivir.Ther. 10:231-237.

95. Koning, F. A., D. Kwa, B. Boeser-Nunnink, J. Dekker, J. Vingerhoed, H.

Hiemstra, and H. Schuitemaker. 2003. Decreasing sensitivity to RANTES

(regulated on activation, normally T cell-expressed and -secreted) neutralization

of CC chemokine receptor 5-using, non-syncytium-inducing virus variants in the

course of human immunodeficiency virus type 1 infection. J.Infect.Dis. 188:864-

872.

96. Kuhmann, S. E., E. J. Platt, S. L. Kozak, and D. Kabat. 2000. Cooperation of

multiple CCR5 coreceptors is required for infections by human

immunodeficiency virus type 1. J.Virol. 74:7005-7015.

97. Kuhmann, S. E., P. Pugach, K. J. Kunstman, J. Taylor, R. L. Stanfield, A.

Snyder, J. M. Strizki, J. Riley, B. M. Baroudy, I. A. Wilson, B. T. Korber, S.

M. Wolinsky, and J. P. Moore. 2004. Genetic and phenotypic analyses of human

230 immunodeficiency virus type 1 escape from a small-molecule CCR5 inhibitor.

J.Virol. 78:2790-2807.

98. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A.

Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in

complex with the CD4 receptor and a neutralizing human antibody. Nature

393:648-659.

99. Labrosse, B., J. L. Labernardiere, E. Dam, V. Trouplin, K. Skrabal, F.

Clavel, and F. Mammano. 2003. Baseline susceptibility of primary human

immunodeficiency virus type 1 to entry inhibitors. J.Virol. 77:1610-1613.

100. Labrosse, B., L. Morand-Joubert, A. Goubard, S. Rochas, J. L.

Labernardiere, J. Pacanowski, J. L. Meynard, A. J. Hance, F. Clavel, and F.

Mammano. 2006. Role of the envelope genetic context in the development of

enfuvirtide resistance in human immunodeficiency virus type 1-infected patients.

J.Virol. 80:8807-8819.

101. Lalezari, J. P., K. Henry, M. O'Hearn, J. S. Montaner, P. J. Piliero, B.

Trottier, S. Walmsley, C. Cohen, D. R. Kuritzkes, J. J. Eron, Jr., J. Chung, R.

DeMasi, L. Donatacci, C. Drobnes, J. Delehanty, and M. Salgo. 2003.

Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North

and South America. N.Engl.J.Med. 348:2175-2185.

231 102. Lassen, K., Y. Han, Y. Zhou, J. Siliciano, and R. F. Siliciano. 2004. The

multifactorial nature of HIV-1 latency. Trends Mol.Med. 10:525-531.

103. Lassen, K. G., J. R. Bailey, and R. F. Siliciano. 2004. Analysis of human

immunodeficiency virus type 1 transcriptional elongation in resting CD4+ T cells

in vivo. J.Virol. 78:9105-9114.

104. Layne, S. P., M. J. Merges, M. Dembo, J. L. Spouge, and P. L. Nara. 1990.

HIV requires multiple gp120 molecules for CD4-mediated infection. Nature

346:277-279.

105. Le Rouzic, E. and S. Benichou. 2005. The Vpr protein from HIV-1: distinct

roles along the viral life cycle. Retrovirology. 2:11.

106. Lederman, M. M., R. S. Veazey, R. Offord, D. E. Mosier, J. Dufour, M.

Mefford, M. Piatak, Jr., J. D. Lifson, J. R. Salkowitz, B. Rodriguez, A.

Blauvelt, and O. Hartley. 2004. Prevention of vaginal SHIV transmission in

rhesus macaques through inhibition of CCR5. Science 306:485-487.

107. Lederman, M. M., R. S. Veazey, R. Offord, D. E. Mosier, J. Dufour, M.

Mefford, M. Piatak, Jr., J. D. Lifson, J. R. Salkowitz, B. Rodriguez, A.

Blauvelt, and O. Hartley. 2004. Prevention of vaginal SHIV transmission in

rhesus macaques through inhibition of CCR5. Science 306:485-487.

232 108. Lee, B., M. Sharron, C. Blanpain, B. J. Doranz, J. Vakili, P. Setoh, E. Berg,

G. Liu, H. R. Guy, S. R. Durell, M. Parmentier, C. N. Chang, K. Price, M.

Tsang, and R. W. Doms. 1999. Epitope mapping of CCR5 reveals multiple

conformational states and distinct but overlapping structures involved in

chemokine and coreceptor function. J.Biol.Chem. 274:9617-9626.

109. Lee, B., M. Sharron, L. J. Montaner, D. Weissman, and R. W. Doms. 1999.

Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets,

dendritic cells, and differentially conditioned monocyte-derived macrophages.

Proc.Natl.Acad.Sci.U.S.A 96:5215-5220.

110. Lewin, S. R., S. Sonza, L. B. Irving, C. F. McDonald, J. Mills, and S. M.

Crowe. 1996. Surface CD4 is critical to in vitro HIV infection of human alveolar

macrophages. AIDS Res.Hum.Retroviruses 12:877-883.

111. Liao, F., G. Alkhatib, K. W. Peden, G. Sharma, E. A. Berger, and J. M.

Farber. 1997. STRL33, A novel chemokine receptor-like protein, functions as a

fusion cofactor for both macrophage-tropic and T cell line-tropic HIV-1.

J.Exp.Med. 185:2015-2023.

112. Lin, P. F., W. Blair, T. Wang, T. Spicer, Q. Guo, N. Zhou, Y. F. Gong, H. G.

Wang, R. Rose, G. Yamanaka, B. Robinson, C. B. Li, R. Fridell, C. Deminie,

G. Demers, Z. Yang, L. Zadjura, N. Meanwell, and R. Colonno. 2003. A small

233 molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4

receptor binding. Proc.Natl.Acad.Sci.U.S.A 100:11013-11018.

113. Liu, R., W. A. Paxton, S. Choe, D. Ceradini, S. R. Martin, R. Horuk, M. E.

MacDonald, H. Stuhlmann, R. A. Koup, and N. R. Landau. 1996.

Homozygous defect in HIV-1 coreceptor accounts for resistance of some

multiply-exposed individuals to HIV-1 infection. Cell 86:367-377.

114. Louis, J. M., I. T. Weber, J. Tozser, G. M. Clore, and A. M. Gronenborn.

2000. HIV-1 protease: maturation, enzyme specificity, and drug resistance.

Adv.Pharmacol. 49:111-146.

115. Lowe, D. M., A. Aitken, C. Bradley, G. K. Darby, B. A. Larder, K. L. Powell,

D. J. Purifoy, M. Tisdale, and D. K. Stammers. 1988. HIV-1 reverse

transcriptase: crystallization and analysis of domain structure by limited

proteolysis. Biochemistry 27:8884-8889.

116. Lu, J., P. Sista, F. Giguel, M. Greenberg, and D. R. Kuritzkes. 2004. Relative

replicative fitness of human immunodeficiency virus type 1 mutants resistant to

enfuvirtide (T-20). J.Virol. 78:4628-4637.

117. Mack, M., B. Luckow, P. J. Nelson, J. Cihak, G. Simmons, P. R. Clapham, N.

Signoret, M. Marsh, M. Stangassinger, F. Borlat, T. N. Wells, D. Schlondorff,

and A. E. Proudfoot. 1998. Aminooxypentane-RANTES induces CCR5

234 internalization but inhibits recycling: a novel inhibitory mechanism of HIV

infectivity. J.Exp.Med. 187:1215-1224.

118. Malashkevich, V. N., D. C. Chan, C. T. Chutkowski, and P. S. Kim. 1998.

Crystal structure of the simian immunodeficiency virus (SIV) gp41 core:

conserved helical interactions underlie the broad inhibitory activity of gp41

peptides. Proc.Natl.Acad.Sci.U.S.A 95:9134-9139.

119. Mansky, L. M. and H. M. Temin. 1995. Lower in vivo mutation rate of human

immunodeficiency virus type 1 than that predicted from the fidelity of purified

reverse transcriptase. J.Virol. 69:5087-5094.

120. Marozsan, A. J. and E. J. Arts. 2003. Development of a yeast-based

recombination cloning/system for the analysis of gene products from diverse

human immunodeficiency virus type 1 isolates. J.Virol.Methods 111:111-120.

121. Marozsan, A. J., E. Fraundorf, A. Abraha, H. Baird, D. Moore, R. Troyer, I.

Nankja, and E. J. Arts. 2004. Relationships between infectious titer, capsid

protein levels, and reverse transcriptase activities of diverse human

immunodeficiency virus type 1 isolates. J.Virol. 78:11130-11141.

122. Marozsan, A. J., S. E. Kuhmann, T. Morgan, C. Herrera, E. Rivera-Troche,

S. Xu, B. M. Baroudy, J. Strizki, and J. P. Moore. 2005. Generation and

235 properties of a human immunodeficiency virus type 1 isolate resistant to the small

molecule CCR5 inhibitor, SCH-417690 (SCH-D). Virology 338:182-199.

123. Marozsan, A. J., D. M. Moore, M. A. Lobritz, E. Fraundorf, A. Abraha, J. D.

Reeves, and E. J. Arts. 2005. Differences in the fitness of two diverse wild-type

human immunodeficiency virus type 1 isolates are related to the efficiency of cell

binding and entry. J.Virol. 79:7121-7134.

124. Marozsan, A. J., V. S. Torre, M. Johnson, S. C. Ball, J. V. Cross, D. J.

Templeton, M. E. Quinones-Mateu, R. E. Offord, and E. J. Arts. 2001.

Mechanisms involved in stimulation of human immunodeficiency virus type 1

replication by aminooxypentane RANTES. J.Virol. 75:8624-8638.

125. Martin, M. P., M. Dean, M. W. Smith, C. Winkler, B. Gerrard, N. L. Michael,

B. Lee, R. W. Doms, J. Margolick, S. Buchbinder, J. J. Goedert, T. R.

O'Brien, M. W. Hilgartner, D. Vlahov, S. J. O'Brien, and M. Carrington.

1998. Genetic acceleration of AIDS progression by a promoter variant of CCR5.

Science 282:1907-1911.

126. McCune, J. M., L. B. Rabin, M. B. Feinberg, M. Lieberman, J. C. Kosek, G.

R. Reyes, and I. L. Weissman. 1988. Endoproteolytic cleavage of gp160 is

required for the activation of human immunodeficiency virus. Cell 53:55-67.

236 127. McDermott, D. H., P. A. Zimmerman, F. Guignard, C. A. Kleeberger, S. F.

Leitman, and P. M. Murphy. 1998. CCR5 promoter polymorphism and HIV-1

disease progression. Multicenter AIDS Cohort Study (MACS). Lancet 352:866-

870.

128. McDougal, J. S., J. K. Nicholson, G. D. Cross, S. P. Cort, M. S. Kennedy, and

A. C. Mawle. 1986. Binding of the human retrovirus HTLV-III/LAV/ARV/HIV

to the CD4 (T4) molecule: conformation dependence, epitope mapping, antibody

inhibition, and potential for idiotypic mimicry. J.Immunol. 137:2937-2944.

129. 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. 151:413-423.

130. Michael, N. L., G. Chang, L. G. Louie, J. R. Mascola, D. Dondero, D. L. Birx,

and H. W. Sheppard. 1997. The role of viral phenotype and CCR-5 gene defects

in HIV-1 transmission and disease progression. Nat.Med. 3:338-340.

131. Mitsuya, H., K. J. Weinhold, P. A. Furman, M. H. St Clair, S. N. Lehrman, R.

C. Gallo, D. Bolognesi, D. W. Barry, and S. Broder. 1985. 3'-Azido-3'-

deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and

cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-

associated virus in vitro. Proc.Natl.Acad.Sci.U.S.A 82:7096-7100.

237 132. Mkrtchyan, S. R., R. M. Markosyan, M. T. Eadon, J. P. Moore, G. B.

Melikyan, and F. S. Cohen. 2005. Ternary complex formation of human

immunodeficiency virus type 1 Env, CD4, and chemokine receptor captured as an

intermediate of membrane fusion. J.Virol. 79:11161-11169.

133. Morita, E. and W. I. Sundquist. 2004. Retrovirus budding. Annu.Rev.Cell

Dev.Biol. 20:395-425.

134. Mosier, D. E., G. R. Picchio, R. J. Gulizia, R. Sabbe, P. Poignard, L. Picard,

R. E. Offord, D. A. Thompson, and J. Wilken. 1999. Highly potent RANTES

analogues either prevent CCR5-using human immunodeficiency virus type 1

infection in vivo or rapidly select for CXCR4-using variants. J.Virol. 73:3544-

3550.

135. Moulard, M. and E. Decroly. 2000. Maturation of HIV envelope glycoprotein

precursors by cellular endoproteases. Biochim.Biophys.Acta 1469:121-132.

136. Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K.

Matsushima, L. H. Miller, J. J. Oppenheim, and C. A. Power. 2000.

International union of pharmacology. XXII. Nomenclature for chemokine

receptors. Pharmacol.Rev. 52:145-176.

238 137. Myszka, D. G., R. W. Sweet, P. Hensley, M. Brigham-Burke, P. D. Kwong, W.

A. Hendrickson, R. Wyatt, J. Sodroski, and M. L. Doyle. 2000. Energetics of

the HIV gp120-CD4 binding reaction. Proc.Natl.Acad.Sci.U.S.A 97:9026-9031.

138. Nagashima, K. A., D. A. Thompson, S. I. Rosenfield, P. J. Maddon, T. Dragic,

and W. C. Olson. 2001. Human immunodeficiency virus type 1 entry inhibitors

PRO 542 and T-20 are potently synergistic in blocking virus-cell and cell-cell

fusion. J.Infect.Dis. 183:1121-1125.

139. O'Doherty, U., W. J. Swiggard, and M. H. Malim. 2000. Human

immunodeficiency virus type 1 spinoculation enhances infection through virus

binding. J.Virol. 74:10074-10080.

140. Oberlin, E., A. Amara, F. Bachelerie, C. Bessia, J. L. Virelizier, F. Arenzana-

Seisdedos, O. Schwartz, J. M. Heard, I. Clark-Lewis, D. F. Legler, M.

Loetscher, M. Baggiolini, and B. Moser. 1996. The CXC chemokine SDF-1 is

the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1.

Nature 382:833-835.

141. Ometto, L., R. Bertorelle, M. Mainardi, M. Zanchetta, S. Tognazzo, O.

Rampon, E. Ruga, L. Chieco-Bianchi, and A. De Rossi. 2001. Polymorphisms

in the CCR5 promoter region influence disease progression in perinatally human

immunodeficiency virus type 1-infected children. J.Infect.Dis. 183:814-818.

239 142. Pancera, M., J. Lebowitz, A. Schon, P. Zhu, E. Freire, P. D. Kwong, K. H.

Roux, J. Sodroski, and R. Wyatt. 2005. Soluble mimetics of human

immunodeficiency virus type 1 viral spikes produced by replacement of the native

trimerization domain with a heterologous trimerization motif: characterization and

ligand binding analysis. J.Virol. 79:9954-9969.

143. Pastore, C., G. R. Picchio, F. Galimi, R. Fish, O. Hartley, R. E. Offord, and D.

E. Mosier. 2003. Two mechanisms for human immunodeficiency virus type 1

inhibition by N-terminal modifications of RANTES. Antimicrob.Agents

Chemother. 47:509-517.

144. Perelson, A. S., A. U. Neumann, M. Markowitz, J. M. Leonard, and D. D. Ho.

1996. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and

viral generation time. Science 271:1582-1586.

145. Piatak, M., Jr., M. S. Saag, L. C. Yang, S. J. Clark, J. C. Kappes, K. C. Luk,

B. H. Hahn, G. M. Shaw, and J. D. Lifson. 1993. High levels of HIV-1 in

plasma during all stages of infection determined by competitive PCR. Science

259:1749-1754.

146. Picard, L., G. Simmons, C. A. Power, A. Meyer, R. A. Weiss, and P. R.

Clapham. 1997. Multiple extracellular domains of CCR-5 contribute to human

immunodeficiency virus type 1 entry and fusion. J.Virol. 71:5003-5011.

240 147. Picard, L., G. Simmons, C. A. Power, A. Meyer, R. A. Weiss, and P. R.

Clapham. 1997. Multiple extracellular domains of CCR-5 contribute to human

immunodeficiency virus type 1 entry and fusion. J.Virol. 71:5003-5011.

148. Piguet, V. and D. Trono. 1999. The Nef protein of primate lentiviruses.

Rev.Med.Virol. 9:111-120.

149. Platt, E. J., J. P. Durnin, and D. Kabat. 2005. Kinetic factors control

efficiencies of cell entry, efficacies of entry inhibitors, and mechanisms of

adaptation of human immunodeficiency virus. J.Virol. 79:4347-4356.

150. Platt, E. J., S. E. Kuhmann, P. P. Rose, and D. Kabat. 2001. Adaptive

mutations in the V3 loop of gp120 enhance fusogenicity of human

immunodeficiency virus type 1 and enable use of a CCR5 coreceptor that lacks

the amino-terminal sulfated region. J.Virol. 75:12266-12278.

151. Platt, E. J., D. M. Shea, P. P. Rose, and D. Kabat. 2005. Variants of human

immunodeficiency virus type 1 that efficiently use CCR5 lacking the tyrosine-

sulfated amino terminus have adaptive mutations in gp120, including loss of a

functional N-glycan. J.Virol. 79:4357-4368.

152. Platt, E. J., K. Wehrly, S. E. Kuhmann, B. Chesebro, and D. Kabat. 1998.

Effects of CCR5 and CD4 cell surface concentrations on infections by

241 macrophagetropic isolates of human immunodeficiency virus type 1. J.Virol.

72:2855-2864.

153. Pollard, V. W. and M. H. Malim. 1998. The HIV-1 Rev protein.

Annu.Rev.Microbiol. 52:491-532.

154. Pugach, P., S. E. Kuhmann, J. Taylor, A. J. Marozsan, A. Snyder, T. Ketas, S.

M. Wolinsky, B. T. Korber, and J. P. Moore. 2004. The prolonged culture of

human immunodeficiency virus type 1 in primary lymphocytes increases its

sensitivity to neutralization by soluble CD4. Virology 321:8-22.

155. Pugach, P., A. J. Marozsan, T. J. Ketas, E. L. Landes, J. P. Moore, and S. E.

Kuhmann. 2007. HIV-1 clones resistant to a small molecule CCR5 inhibitor use

the inhibitor-bound form of CCR5 for entry. Virology 361:212-228.

156. Quinones-Mateu, M. E., S. C. Ball, A. J. Marozsan, V. S. Torre, J. L.

Albright, G. Vanham, G. G. van Der, R. L. Colebunders, and E. J. Arts. 2000.

A dual infection/competition assay shows a correlation between ex vivo human

immunodeficiency virus type 1 fitness and disease progression. J.Virol. 74:9222-

9233.

157. Rangel, H. R., J. Weber, B. Chakraborty, A. Gutierrez, M. L. Marotta, M.

Mirza, P. Kiser, M. A. Martinez, J. A. Este, and M. E. Quinones-Mateu. 2003.

242 Role of the human immunodeficiency virus type 1 envelope gene in viral fitness.

J.Virol. 77:9069-9073.

158. Ray, N., J. E. Harrison, L. A. Blackburn, J. N. Martin, S. G. Deeks, and R. W.

Doms. 2007. Clinical resistance to enfuvirtide does not affect susceptibility of

human immunodeficiency virus type 1 to other classes of entry inhibitors. J.Virol.

81:3240-3250.

159. Reeves, J. D., S. A. Gallo, N. Ahmad, J. L. Miamidian, P. E. Harvey, M.

Sharron, S. Pohlmann, J. N. Sfakianos, C. A. Derdeyn, R. Blumenthal, E.

Hunter, and R. W. Doms. 2002. Sensitivity of HIV-1 to entry inhibitors

correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics.

Proc.Natl.Acad.Sci.U.S.A 99:16249-16254.

160. Reeves, J. D., F. H. Lee, J. L. Miamidian, C. B. Jabara, M. M. Juntilla, and R.

W. Doms. 2005. Enfuvirtide resistance mutations: impact on human

immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus

neutralization. J.Virol. 79:4991-4999.

161. Reeves, J. D., J. L. Miamidian, M. J. Biscone, F. H. Lee, N. Ahmad, T. C.

Pierson, and R. W. Doms. 2004. Impact of mutations in the coreceptor binding

site on human immunodeficiency virus type 1 fusion, infection, and entry

inhibitor sensitivity. J.Virol. 78:5476-5485.

243 162. Repits, J., M. Oberg, J. Esbjornsson, P. Medstrand, A. Karlsson, J. Albert, E.

M. Fenyo, and M. Jansson. 2005. Selection of human immunodeficiency virus

type 1 R5 variants with augmented replicative capacity and reduced sensitivity to

entry inhibitors during severe immunodeficiency. J.Gen.Virol. 86:2859-2869.

163. Rimsky, L. T., D. C. Shugars, and T. J. Matthews. 1998. Determinants of

human immunodeficiency virus type 1 resistance to gp41-derived inhibitory

peptides. J.Virol. 72:986-993.

164. Rizzuto, C. and J. Sodroski. 2000. Fine definition of a conserved CCR5-binding

region on the human immunodeficiency virus type 1 glycoprotein 120. AIDS

Res.Hum.Retroviruses 16:741-749.

165. Rodriguez-Frade, J. M., A. J. Vila-Coro, A. Martin, M. Nieto, F. Sanchez-

Madrid, A. E. Proudfoot, T. N. Wells, A. Martinez, and M. Mellado. 1999.

Similarities and differences in RANTES- and (AOP)-RANTES-triggered Signals:

Implications for Chemotaxis. J.Cell Biol. 144:755-765.

166. Roscic-Mrkic, B., M. Fischer, C. Leemann, A. Manrique, C. J. Gordon, J. P.

Moore, A. E. Proudfoot, and A. Trkola. 2003. RANTES (CCL5) uses the

proteoglycan CD44 as an auxiliary receptor to mediate cellular activation signals

and HIV-1 enhancement. Blood 102:1169-1177.

244 167. Rusert, P., H. Kuster, B. Joos, B. Misselwitz, C. Gujer, C. Leemann, M.

Fischer, G. Stiegler, H. Katinger, W. C. Olson, R. Weber, L. Aceto, H. F.

Gunthard, and A. Trkola. 2005. Virus isolates during acute and chronic human

immunodeficiency virus type 1 infection show distinct patterns of sensitivity to

entry inhibitors. J.Virol. 79:8454-8469.

168. Saag, M. S., B. H. Hahn, J. Gibbons, Y. Li, E. S. Parks, W. P. Parks, and G.

M. Shaw. 1988. Extensive variation of human immunodeficiency virus type-1 in

vivo. Nature 334:440-444.

169. Sabbe, R., G. R. Picchio, C. Pastore, O. Chaloin, O. Hartley, R. Offord, and

D. E. Mosier. 2001. Donor- and ligand-dependent differences in C-C chemokine

receptor 5 reexpression. J.Virol. 75:661-671.

170. Safarian, D., X. Carnec, F. Tsamis, F. Kajumo, and T. Dragic. 2006. An anti-

CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by

inhibiting different stages of human immunodeficiency virus type 1 entry.

Virology 352:477-484.

171. Samson, M., F. Libert, B. J. Doranz, J. Rucker, C. Liesnard, C. M. Farber, S.

Saragosti, C. Lapoumeroulie, J. Cognaux, C. Forceille, G. Muyldermans, C.

Verhofstede, G. Burtonboy, M. Georges, T. Imai, S. Rana, Y. Yi, R. J. Smyth,

R. G. Collman, R. W. Doms, G. Vassart, and M. Parmentier. 1996. Resistance

245 to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5

chemokine receptor gene. Nature 382:722-725.

172. Sattentau, Q. J. and J. P. Moore. 1991. Conformational changes induced in the

human immunodeficiency virus envelope glycoprotein by soluble CD4 binding.

J.Exp.Med. 174:407-415.

173. Schooley, R. T., T. C. Merigan, P. Gaut, M. S. Hirsch, M. Holodniy, T. Flynn,

S. Liu, R. E. Byington, S. Henochowicz, E. Gubish, and . 1990. Recombinant

soluble CD4 therapy in patients with the acquired immunodeficiency syndrome

(AIDS) and AIDS-related complex. A phase I-II escalating dosage trial.

Ann.Intern.Med. 112:247-253.

174. Schuitemaker, H., M. Koot, N. A. Kootstra, M. W. Dercksen, R. E. de Goede,

R. P. van Steenwijk, J. M. Lange, J. K. Schattenkerk, F. Miedema, and M.

Tersmette. 1992. Biological phenotype of human immunodeficiency virus type 1

clones at different stages of infection: progression of disease is associated with a

shift from monocytotropic to T-cell-tropic virus population. J.Virol. 66:1354-

1360.

175. Seibert, C., W. Ying, S. Gavrilov, F. Tsamis, S. E. Kuhmann, A. Palani, J. R.

Tagat, J. W. Clader, S. W. McCombie, B. M. Baroudy, S. O. Smith, T. Dragic,

J. P. Moore, and T. P. Sakmar. 2006. Interaction of small molecule inhibitors of

HIV-1 entry with CCR5. Virology 349:41-54.

246 176. Shankarappa, R., J. B. Margolick, S. J. Gange, A. G. Rodrigo, D. Upchurch,

H. Farzadegan, P. Gupta, C. R. Rinaldo, G. H. Learn, X. He, X. L. Huang,

and J. I. Mullins. 1999. Consistent viral evolutionary changes associated with the

progression of human immunodeficiency virus type 1 infection. J.Virol.

73:10489-10502.

177. Si, Z., N. Phan, E. Kiprilov, and J. Sodroski. 2003. Effects of HIV type 1

envelope glycoprotein proteolytic processing on antigenicity. AIDS

Res.Hum.Retroviruses 19:217-226.

178. Signoret, N., A. Pelchen-Matthews, M. Mack, A. E. Proudfoot, and M. Marsh.

2000. Endocytosis and recycling of the HIV coreceptor CCR5. J.Cell Biol.

151:1281-1294.

179. Simmons, G., P. R. Clapham, L. Picard, R. E. Offord, M. M. Rosenkilde, T.

W. Schwartz, R. Buser, T. N. Wells, and A. E. Proudfoot. 1997. Potent

inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5

antagonist. Science 276:276-279.

180. Smith, D. H., R. A. Byrn, S. A. Marsters, T. Gregory, J. E. Groopman, and D.

J. Capon. 1987. Blocking of HIV-1 infectivity by a soluble, secreted form of the

CD4 antigen. Science 238:1704-1707.

247 181. Stalmeijer, E. H., R. P. Van Rij, B. Boeser-Nunnink, J. A. Visser, M. A.

Naarding, D. Schols, and H. Schuitemaker. 2004. In vivo evolution of X4

human immunodeficiency virus type 1 variants in the natural course of infection

coincides with decreasing sensitivity to CXCR4 antagonists. J.Virol. 78:2722-

2728.

182. Steger, H. K. and M. J. Root. 2006. Kinetic dependence to HIV-1 entry

inhibition. J.Biol.Chem. 281:25813-25821.

183. Stein, B. S. and E. G. Engleman. 1990. Intracellular processing of the gp160

HIV-1 envelope precursor. Endoproteolytic cleavage occurs in a cis or medial

compartment of the Golgi complex. J.Biol.Chem. 265:2640-2649.

184. Stein, B. S., S. D. Gowda, J. D. Lifson, R. C. Penhallow, K. G. Bensch, and E.

G. Engleman. 1987. pH-independent HIV entry into CD4-positive T cells via

virus envelope fusion to the plasma membrane. Cell 49:659-668.

185. Strizki, J. M., S. Xu, N. E. Wagner, L. Wojcik, J. Liu, Y. Hou, M. Endres, A.

Palani, S. Shapiro, J. W. Clader, W. J. Greenlee, J. R. Tagat, S. McCombie,

K. Cox, A. B. Fawzi, C. C. Chou, C. Pugliese-Sivo, L. Davies, M. E. Moreno,

D. D. Ho, A. Trkola, C. A. Stoddart, J. P. Moore, G. R. Reyes, and B. M.

Baroudy. 2001. SCH-C (SCH 351125), an orally bioavailable, small molecule

antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1

infection in vitro and in vivo. Proc.Natl.Acad.Sci.U.S.A 98:12718-12723.

248 186. Suzuki, Y. and R. Craigie. 2007. The road to chromatin - nuclear entry of

retroviruses. Nat.Rev.Microbiol. 5:187-196.

187. Tersmette, M., R. E. de Goede, B. J. Al, I. N. Winkel, R. A. Gruters, H. T.

Cuypers, H. G. Huisman, and F. Miedema. 1988. Differential syncytium-

inducing capacity of human immunodeficiency virus isolates: frequent detection

of syncytium-inducing isolates in patients with acquired immunodeficiency

syndrome (AIDS) and AIDS-related complex. J.Virol. 62:2026-2032.

188. Thomas, E. R., R. L. Dunfee, J. Stanton, D. Bogdan, J. Taylor, K. Kunstman,

J. E. Bell, S. M. Wolinsky, and D. Gabuzda. 2007. Macrophage entry mediated

by HIV Envs from brain and lymphoid tissues is determined by the capacity to

use low CD4 levels and overall efficiency of fusion. Virology 360:105-119.

189. Torre, V. S., A. J. Marozsan, J. L. Albright, K. R. Collins, O. Hartley, R. E.

Offord, M. E. Quinones-Mateu, and E. J. Arts. 2000. Variable sensitivity of

CCR5-tropic human immunodeficiency virus type 1 isolates to inhibition by

RANTES analogs. J.Virol. 74:4868-4876.

190. Townson, J. R., G. J. Graham, N. R. Landau, B. Rasala, and R. J. Nibbs.

2000. Aminooxypentane addition to the chemokine macrophage inflammatory

protein-1alpha P increases receptor affinities and HIV inhibition. J.Biol.Chem.

275:39254-39261.

249 191. Traunecker, A., W. Luke, and K. Karjalainen. 1988. Soluble CD4 molecules

neutralize human immunodeficiency virus type 1. Nature 331:84-86.

192. Traunecker, A., J. Schneider, H. Kiefer, and K. Karjalainen. 1989. Highly

efficient neutralization of HIV with recombinant CD4-immunoglobulin molecules.

Nature 339:68-70.

193. Tremblay, C. L., C. Kollmann, F. Giguel, T. C. Chou, and M. S. Hirsch. 2000.

Strong in vitro synergy between the fusion inhibitor T-20 and the CXCR4 blocker

AMD-3100. J.Acquir.Immune.Defic.Syndr. 25:99-102.

194. Trkola, A., T. Dragic, J. Arthos, J. M. Binley, W. C. Olson, G. P. Allaway, C.

Cheng-Mayer, J. Robinson, P. J. Maddon, and J. P. Moore. 1996. CD4-

dependent, antibody-sensitive interactions between HIV-1 and its co-receptor

CCR-5. Nature 384:184-187.

195. Trkola, A., S. E. Kuhmann, J. M. Strizki, E. Maxwell, T. Ketas, T. Morgan, P.

Pugach, S. Xu, L. Wojcik, J. Tagat, A. Palani, S. Shapiro, J. W. Clader, S.

McCombie, G. R. Reyes, B. M. Baroudy, and J. P. Moore. 2002. HIV-1 escape

from a small molecule, CCR5-specific entry inhibitor does not involve CXCR4

use. Proc.Natl.Acad.Sci.U.S.A 99:395-400.

196. Trkola, A., H. Kuster, C. Leemann, C. Ruprecht, B. Joos, A. Telenti, B.

Hirschel, R. Weber, S. Bonhoeffer, and H. F. Gunthard. 2003. Human

250 immunodeficiency virus type 1 fitness is a determining factor in viral rebound and

set point in chronic infection. J.Virol. 77:13146-13155.

197. Trkola, A., W. A. Paxton, S. P. Monard, J. A. Hoxie, M. A. Siani, D. A.

Thompson, L. Wu, C. R. Mackay, R. Horuk, and J. P. Moore. 1998. Genetic

subtype-independent inhibition of human immunodeficiency virus type 1

replication by CC and CXC chemokines. J.Virol. 72:396-404.

198. Troyer, R. M., K. R. Collins, A. Abraha, E. Fraundorf, D. M. Moore, R. W.

Krizan, Z. Toossi, R. L. Colebunders, M. A. Jensen, J. I. Mullins, G. Vanham,

and E. J. Arts. 2005. Changes in human immunodeficiency virus type 1 fitness

and genetic diversity during disease progression. J.Virol. 79:9006-9018.

199. Tsamis, F., S. Gavrilov, F. Kajumo, C. Seibert, S. Kuhmann, T. Ketas, A.

Trkola, A. Palani, J. W. Clader, J. R. Tagat, S. McCombie, B. Baroudy, J. P.

Moore, T. P. Sakmar, and T. Dragic. 2003. Analysis of the mechanism by

which the small-molecule CCR5 antagonists SCH-351125 and SCH-350581

inhibit human immunodeficiency virus type 1 entry. J.Virol. 77:5201-5208.

200. Tuttle, D. L., J. K. Harrison, C. Anders, J. W. Sleasman, and M. M.

Goodenow. 1998. Expression of CCR5 increases during monocyte differentiation

and directly mediates macrophage susceptibility to infection by human

immunodeficiency virus type 1. J.Virol. 72:4962-4969.

251 201. Veazey, R. S., P. J. Klasse, T. J. Ketas, J. D. Reeves, M. Piatak, Jr., K.

Kunstman, S. E. Kuhmann, P. A. Marx, J. D. Lifson, J. Dufour, M. Mefford,

I. Pandrea, S. M. Wolinsky, R. W. Doms, J. A. DeMartino, S. J. Siciliano, K.

Lyons, M. S. Springer, and J. P. Moore. 2003. Use of a small molecule CCR5

inhibitor in macaques to treat simian immunodeficiency virus infection or prevent

simian-human immunodeficiency virus infection. J.Exp.Med. 198:1551-1562.

202. Veazey, R. S., M. S. Springer, P. A. Marx, J. Dufour, P. J. Klasse, and J. P.

Moore. 2005. Protection of macaques from vaginal SHIV challenge by an orally

delivered CCR5 inhibitor. Nat.Med. 11:1293-1294.

203. Vila-Coro, A. J., M. Mellado, d. A. Martin, A. Martinez, and J. M.

Rodriguez-Frade. 1999. Characterization of RANTES- and Aminooxypentane-

RANTES-Triggered Desensitization Signals Reveals Differences in Recruitment

of the G Protein-Coupled Receptor Complex. J.Immunol. 163:3037-3044.

204. Wang, W. K., T. Dudek, M. Essex, and T. H. Lee. 1999. Hypervariable region

3 residues of HIV type 1 gp120 involved in CCR5 coreceptor utilization:

therapeutic and prophylactic implications. Proc.Natl.Acad.Sci.U.S.A 96:4558-

4562.

205. Watanabe, M., K. A. Reimann, P. A. DeLong, T. Liu, R. A. Fisher, and N. L.

Letvin. 1989. Effect of recombinant soluble CD4 in rhesus monkeys infected

with simian immunodeficiency virus of macaques. Nature 337:267-270.

252 206. Wei, X., J. M. Decker, H. Liu, Z. Zhang, R. B. Arani, J. M. Kilby, M. S. Saag,

X. Wu, G. M. Shaw, and J. C. Kappes. 2002. Emergence of resistant human

immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20)

monotherapy. Antimicrob.Agents Chemother. 46:1896-1905.

207. 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.

208. Weissenhorn, W., S. A. Wharton, L. J. Calder, P. L. Earl, B. Moss, E.

Aliprandis, J. J. Skehel, and D. C. Wiley. 1996. The ectodomain of HIV-1 env

subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of

gp120 and the N-terminal fusion peptide. EMBO J. 15:1507-1514.

209. Westby, M., M. Lewis, J. Whitcomb, M. Youle, A. L. Pozniak, I. T. James, T.

M. Jenkins, M. Perros, and R. E. van der. 2006. Emergence of CXCR4-using

human immunodeficiency virus type 1 (HIV-1) variants in a minority of HIV-1-

infected patients following treatment with the CCR5 antagonist maraviroc is from

a pretreatment CXCR4-using virus reservoir. J.Virol. 80:4909-4920.

210. Westby, M., C. Smith-Burchnell, J. Mori, M. Lewis, M. Mosley, M. Stockdale,

P. Dorr, G. Ciaramella, and M. Perros. 2007. Reduced maximal inhibition in

phenotypic susceptibility assays indicates that viral strains resistant to the CCR5

antagonist maraviroc utilize inhibitor-bound receptor for entry. J.Virol. 81:2359-

2371.

253 211. Wild, C., T. Oas, C. McDanal, D. Bolognesi, and T. Matthews. 1992. A

synthetic peptide inhibitor of human immunodeficiency virus replication:

correlation between solution structure and viral inhibition.

Proc.Natl.Acad.Sci.U.S.A 89:10537-10541.

212. Willey, R. L., J. S. Bonifacino, B. J. Potts, M. A. Martin, and R. D. Klausner.

1988. Biosynthesis, cleavage, and degradation of the human immunodeficiency

virus 1 envelope glycoprotein gp160. Proc.Natl.Acad.Sci.U.S.A 85:9580-9584.

213. Wyatt, R. and J. Sodroski. 1998. The HIV-1 envelope glycoproteins: fusogens,

antigens, and immunogens. Science 280:1884-1888.

214. Yang, X., S. Kurteva, X. Ren, S. Lee, and J. Sodroski. 2005. Stoichiometry of

envelope glycoprotein trimers in the entry of human immunodeficiency virus type

1. J.Virol. 79:12132-12147.

215. Yang, X., S. Kurteva, X. Ren, S. Lee, and J. Sodroski. 2006. Subunit

stoichiometry of human immunodeficiency virus type 1 envelope glycoprotein

trimers during virus entry into host cells. J.Virol. 80:4388-4395.

216. Zanetti, G., J. A. Briggs, K. Grunewald, Q. J. Sattentau, and S. D. Fuller.

2006. Cryo-electron tomographic structure of an immunodeficiency virus

envelope complex in situ. PLoS.Pathog. 2:e83.

254 217. Zhang, X. Q., M. Sorensen, M. Fung, and R. T. Schooley. 2006. Synergistic in

vitro antiretroviral activity of a humanized monoclonal anti-CD4 antibody (TNX-

355) and enfuvirtide (T-20). Antimicrob.Agents Chemother. 50:2231-2233.

218. Zhou, T., L. Xu, B. Dey, A. J. Hessell, D. Van Ryk, S. H. Xiang, X. Yang, M.

Y. Zhang, M. B. Zwick, J. Arthos, D. R. Burton, D. S. Dimitrov, J. Sodroski,

R. Wyatt, G. J. Nabel, and P. D. Kwong. 2007. Structural definition of a

conserved neutralization epitope on HIV-1 gp120. Nature 445:732-737.

219. Zhu, P., E. Chertova, J. Bess, Jr., J. D. Lifson, L. O. Arthur, J. Liu, K. A.

Taylor, and K. H. Roux. 2003. Electron tomography analysis of envelope

glycoprotein trimers on HIV and simian immunodeficiency virus virions.

Proc.Natl.Acad.Sci.U.S.A 100:15812-15817.

220. Zhu, P., J. Liu, J. Bess, Jr., E. Chertova, J. D. Lifson, H. Grise, G. A. Ofek, K.

A. Taylor, and K. H. Roux. 2006. Distribution and three-dimensional structure

of AIDS virus envelope spikes. Nature 441:847-852.

221. Zimmerman, P. A., A. Buckler-White, G. Alkhatib, T. Spalding, J. Kubofcik,

C. Combadiere, D. Weissman, O. Cohen, A. Rubbert, G. Lam, M.

Vaccarezza, P. E. Kennedy, V. Kumaraswami, J. V. Giorgi, R. Detels, J.

Hunter, M. Chopek, E. A. Berger, A. S. Fauci, T. B. Nutman, and P. M.

Murphy. 1997. Inherited resistance to HIV-1 conferred by an inactivating

mutation in CC chemokine receptor 5: studies in populations with contrasting

255 clinical phenotypes, defined racial background, and quantified risk. Mol.Med.

3:23-36.

256