MECHANISMS OF CCR5 AGONIST/ANTAGONIST INHIBITION OF HIV-1
ENTRY AND IN VITRO SELECTION OF VIRUS RESISTANT TO
MARAVIROC
by
ANNETTE NICOLE RATCLIFF
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
January, 2013
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Annette Nicole Ratcliff ______
Doctor of Philosophy candidate for the ______degree*.
Jonathan Karn, Ph.D. (signed)______(chair of the committee)
David McDonald, Ph.D. ______
Scott Sieg, Ph.D. ______
John C. Tilton, M.D. ______
Eric J. Arts, Ph.D. ______(dissertation advisor)
October 9, 2012 (date) ______
*We also certify that written approval has been obtained for any proprietary matieral contained therein.
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TABLE OF CONTENTS
TABLE OF CONTENTS ...... 1
LIST OF TABLES ...... 6
LIST OF FIGURES ...... 7
ACKNOWLEDGEMENT ...... 10
LIST OF ABBREVIATIONS ...... 11
ABSTRACT ...... 13
1. CHAPTER 1: INTRODUCTION ...... 15
1.1 HIV and AIDS ...... 16
1.2 HIV-1 Classification and Diversity ...... 16
1.3 HIV-1 Genome and Proteins ...... 18
1.4 HIV Virion ...... 22
1.5 HIV Replication Cycle ...... 23
1.5.1 Viral Entry and Cellular Tropism ...... 23
1.5.2 Reverse Transcription ...... 26
1.5.3 Nuclear Import and Integration ...... 28
1.5.4 RNA Transcription and Export ...... 29
1.5.5 Assembly and Packaging ...... 31
1.5.6 Viral Budding, Release, and Maturation...... 32
1.6 Antiretrovirals and Drug Resistance ...... 33
1.7 Molecular Aspects of HIV Entry ...... 36
1.7.1 Envelope Structure ...... 36
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1.7.2 CD4 and Interactions with gp120 ...... 40
1.7.3 Chemokine Receptors and Interactions with gp120 ...... 41
1.7.4 Gp41 Mediated Membrane Fusion ...... 44
1.8 Entry Inhibitors and Mechanisms of Resistance ...... 46
1.8.1 Attachment Inhibitors ...... 46
1.8.2 Fusion Inhibitors ...... 50
1.8.3 Inhibitors of gp120-CXCR4 Interaction ...... 51
1.8.4 Inhibitors of gp120-CCR5 Interaction ...... 52
1.8.4.1 Chemokine Analog Inhibitors ...... 53
1.8.4.2 Small Molecule Antagonists ...... 54
1.8.4.3 Resistance to CCR5 Inhibitors ...... 56
1.9 Summary of Thesis Work ...... 82
2 CHAPTER 2: MECHANISTIC STUDIES OF HIV-1 ENTRY INHIBITORS
REVEALS DIFFERENTIAL SENSITIVITY TO PSC-RANTES INHIBITION
INVOLVES COMPETITIVE CCR5 BINDING ...... 84
2.1 Preface ...... 85
2.2 Abstract ...... 86
2.3 Introduction ...... 88
2.4 Materials and Methods ...... 91
2.4.1 Cells, Viruses and Inhibitors ...... 91
2.4.2 Plasmids ...... 93
2.4.3 Reverse Transcriptase Activity Assay ...... 94
2.4.4 Multiple-Cycle Drug Susceptibility Assays...... 94
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2.4.5 Single-Cycle Drug Susceptibility Assays ...... 95
2.4.6 Flow Cytometry ...... 95
2.5 Results ...... 97
2.5.1 Dichotomous Sensitivity to PSC-RANTES in Multiple and Single-
Cycle Assays ...... 97
2.5.2 Inhibition of HIV-1 Replication with a Downregulation-Defective
Mutant of CCR5 ...... 98
2.5.3 Prolonged Incubation with PSC-RANTES Recapitulates Multiple-
Cycle Assays ...... 100
2.5.4 PSC-RANTES Inhibits HIV Entry by Competitive CCR5 Binding
...... 102
2.5.5 Kinetics of PSC-RANTES Inhibition of HIV Entry ...... 103
2.6 Discussion ...... 112
3 CHAPTER 3: HIV-1 RESISTANCE TO MARAVIROC CONFERRED BY A CD4
BINDING SITE MUTATION IN THE ENVELOPE GLYCOPROTEIN GP120 ..117
3.1 Preface ...... 118
3.2 Abstract ...... 119
3.3 Introduction ...... 120
3.4 Materials and Methods ...... 124
3.4.1 Cells, Viruses and Inhibitors ...... 124
3.4.2 Resistant Virus Isolation ...... 124
3.4.3 Multiple-Cycle Drug Susceptibility Assays...... 125
3.4.4 Construction of Gp120 Chimeric Viruses...... 125
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3.4.5 Oligonucleotide Ligation Assay ...... 126
3.4.6 Site-Directed Mutagenesis PCR Method ...... 126
3.4.7 In Vitro Fitness Assays ...... 127
3.4.8 Structural Modeling ...... 128
3.5 Results ...... 129
3.5.1 HIV-1 Primary Isolates Demonstrate Variable Sensitivity to
Maraviroc ...... 129
3.5.2 Maraviroc Resistant Mutant Generated in Cell Culture ...... 129
3.5.3 MVC.21 Cross Resistant to another CCR5 Antagonist ...... 131
3.5.4 Multiple Mutations Throughout Gp120 in Resistant Virus ...... 132
3.5.5 Sensitivity of Selected A74 HIV-1 Clones to MVC Inhibition ...135
3.5.6 Relative Fitness of MVC-Resistant and Sensitive HIV-1 Clones
...... 136
3.5.7 K425 is Primary Resistance Mutation ...... 137
3.5.8 Shift in IC50 Versus the MPI Effect Denotes MVC Resistance .137
3.5.9 Model of K425 in Gp120 Structure Suggests Role in CD4 Binding
Affinity ...... 138
3.6 Discussion ...... 153
4 CHAPTER 4: ENHANCED CD4 BINDING AFFINITY NOVEL MECHANISM
OF HIV-1 RESISTANCE TO MARAVIROC ...... 158
4.1 Preface...... 159
4.2 Abstract ...... 160
4.3 Introduction ...... 161
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4.4 Materials and Methods ...... 163
4.4.1 Cells, Viruses and Inhibitors ...... 163
4.4.2 Structural Modeling ...... 164
4.4.3 Drug Susceptibility Assays ...... 165
4.4.4 sCD4 Activation and Inhibition Assays ...... 165
4.4.5 CD4/CCR5 Receptor Affinity Assays ...... 166
4.4.6 Kinetic Fusion Assay ...... 167
4.5 Results ...... 168
4.5.1 Differential Maraviroc Resistance Profiles ...... 168
4.5.2 Recombinant Human Soluble CD4 Enhances Infection of
A74.MVC.21.132...... 170
4.5.3 Efficient Infection of Cells with Low Levels of Surface CD4 ....172
4.5.4 Maraviroc Resistant Virus Demonstrates Increased Sensitivity to
CD4 Mimetic ...... 173
4.5.5 Maraviroc Resistant Virus Exhibits Faster Entry Kinetics ...... 174
4.6 Discussion ...... 182
5 CHAPTER 5: GENERAL DISCUSSION AND FUTURE DIRECTIONS ...... 187
5.1 Competitive Inhibition of HIV Entry Via CCR5 ...... 189
5.2 Envelope Sequence Context Dependency of K425 Mutation...... 191
5.3 Potential of K425 Envelope to Elicit Neutralizing Antibody Response ...... 193
COPYRIGHT RELEASES ...... 195
BIBLIOGRAPHY ...... 198
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LIST OF TABLES
TABLE 1: List of FDA Approved HIV Antiretrovirals ...... 73
TABLE 2: HIV-1 Entry Inhibitors ...... 78
TABLE 3: Summary of MVC Sensitivity in U87.CD4.CCR5 and PBMC Cells ...... 143
TABLE 4: Summary of Los Alamos HIV Database Gp120 Sequence Identity at Codon
425...... 149
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LIST OF FIGURES
FIGURE 1: HIV-1 Genome...... 66
FIGURE 2: HIV-1 Virion ...... 67
FIGURE 3: HIV Replication Cycle ...... 68
FIGURE 4: Model of HIV Entry ...... 69
FIGURE 5: Model of Reverse Transcription ...... 70
FIGURE 6: Nuclear Import and Integration ...... 71
FIGURE 7: HIV mRNA Transcription and Splicing ...... 72
FIGURE 8: Structure of Gp120 ...... 74
FIGURE 9: Structure of CD4 and Role in Immune Activation and HIV Entry...... 75
FIGURE 10: Model of CCR5 Structure and Antagonist Binding Site ...... 76
FIGURE 11: Model of Interactions between CCR5 and HIV Gp120 ...... 77
FIGURE 12: Chemical Structure of Small Molecule CCR5 Antagonists ...... 79
FIGURE 13: Mechanisms of Resistance to Entry Inhibitors ...... 80
FIGURE 14: V3 Loop Mutations Associated with CCR5 Antagonist Resistance ...... 81
FIGURE 15: PSC-RANTES and Maraviroc Sensitivity in Multiple- And Single-Cycle .
Assays ...... 105
FIGURE 16: Characterization of U87.CD4.M7-CCR5 Cells ...... 106
FIGURE 17: Sensitivity of V3 Chimeric Viruses to Antiretrovirals in Single- And
Multiple- Replication Cycle Assays ...... 107
FIGURE 18: Prolonged Incubation of Cells with PSC-RANTES ...... 108
FIGURE 19: Effect of MOI and Virus Expansion on PSC-RANTES and Maraviroc
IC50 Values ...... 109
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FIGURE 20: Effect of MOI on Inhibition by PSC-RANTES in the Absence of
Receptor Downregulation ...... 110
FIGURE 21: Kinetics of Entry Inhibitor-Mediated Inhibition ...... 111
FIGURE 22: Sensitivity of HIV-1 Primary Isolates To Maraviroc ...... 140
FIGURE 23: Prolonged Tissue Culture Passage to Select for MVC Resistance ...... 141
FIGURE 24: MVC Resistance and Cross Resistance to TAK-779 after Prolonged
Culture with MVC Inhibitor ...... 142
FIGURE 25: Generation and Characterization of Gp120 Chimeric Virus ...... 144
FIGURE 26: Frequency of Gp120 Mutations in PC.21 and MVC.21 Derived Clones ....145
FIGURE 27: Change of Codon 425 Mutation Frequency during Passage Control and
MVC Selection...... 146
FIGURE 28: Oligonucleotide Ligation Assay Determines Change in Gp120 Mutation
Frequency during MVC Selection ...... 147
FIGURE 29: Sensitivity of Gp120 Chimeric Virus to Maraviroc Inhibition ...... 148
FIGURE 30: Replicative Fitness of HIV-1 Gp120 Chimeric Virus Clones Derived from
MVC Selected and Passage Control Experiments ...... 150
FIGURE 31: Effect of the N425K Mutation on MVC Resistance ...... 151
FIGURE 32: Modeling of K425 in Gp120 HIV-1YU-2 Virus Structure Suggests Role in
CD4 Binding Affinity ...... 152
FIGURE 33: Maraviroc Resistance Mutations of R3 and A74.MVC.21 Viruses...... 175
FIGURE 34: Maraviroc Resistance Profiles in Single and Multiple Cycle Assays ...... 176
FIGURE 35: Pre-Exposure to sCD4 Enhances Infection by A74.MVC.21.132 ...... 177
FIGURE 36: Enhancement of Viral Infection and Inhibition by sCD4 is Concentration
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Dependent ...... 178
FIGURE 37: A74.MVC.21.132 Infects Cells Expressing Low Surface Density CD4 .....179
FIGURE 38: Sensitivity to Attachment Inhibitor BMS-806 ...... 180
FIGURE 39: Maraviroc Resistant Virus Demonstrates Enhanced Fusion Kinetics ...... 181
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ACKNOWLEDGEMENT
Foremost, I would like to thank my family for their unwavering love and support throughout the years. Thank you Mom, Dad, Mamaw, and yes, even Travis. Your encouragement and faith have been a constant source of strength and confidence for me and I will always appreciate all that you have done to help me achieve my goals. To my friends, for their constant source of entertainment and laughter, a special thank you for keeping me smiling.
To members of the Arts lab, both past and present, thank you for all of your help and support. I would like to give particular thanks to Ken Henry, Denis Tebit, and Rick
Gibson for being instrumental in helping me develop my technical skills and for being so patient and always willing to help. I owe a particular debt of gratitude to Mastooreh
Chamanian for being my partner in crime so to speak. You are a wonderful friend and comrade and I feel very lucky to have shared my graduate experiences with you.
Lastly, I would like to express appreciation to all of my committee members for generously giving of their time and constantly challenging me to reach my full potential.
I would like to particularly thank my advisor, Eric Arts, for giving me the freedom to pursue my interests but guiding those pursuits. I have learned so much and I am grateful for the opportunity to have worked with such wonderful people.
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LIST OF ABBREVIATIONS
5-FOA: 5-fluoroorotic acid
Ab: antibody
AIDS: Acquired Immune Deficiency Syndrome
CCR5: C-C chemokine receptor type 5
CD4: cluster of differentiation 4
DMEM: Dubelco’s modified Eagle Medium
DNA: deoxyribonucleic acid
ECL: extracellular loop
FBS: fetal bovine serum gp120: glycoprotein 120 gp41: glycoprotein 41
HIV-1: human immunodeficiency virus type 1
IC50: half maximal inhibitory concentration
Ig: immunoglobulin
IU: infectious unit
mg: milligram
MHC: major histocompatibility complex
mL: milliliter
mM: millimolar
MOI: multiplicity of infection
MPI: maximal percent inhibition
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MVC: maraviroc nM: nanomolar
NNRTI: non-nucleoside reverse transcriptase inhibitor
NRTI: nucleoside reverse transcriptase inhibitor
Nt: N-terminus
OBT: optimized background therapy
OLA: oligonucleotide ligation assay
ORF: open reading frame
PBMC: peripheral blood mononuclear cell
PBS: phosphate buffered saline
PDB: Protein Data Bank (http://www.rcsb.org/pdb/home/home.do)
PHA: phytohaemagglutinin
PR: protease
RLU: relative light unit
RNA: ribonucleic acid
RNaseH: ribonuclease H
RT: reverse transcriptase
sCD4: soluble CD4
T20: enfuvirtide
µL: microliter
µM: micromolar
URA3: yeast gene that encodes orotidine 5-phosphate decarboxylase
VVC: vicriviroc
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Mechanisms of CCR5 Agonist/Antagonist Inhibition of HIV-1 Entry and In Vitro
Selection of Virus Resistant to Maraviroc
Abstract
by
ANNETTE NICOLE RATCLIFF
Entry inhibitors represent a new class of antiretrovirals for the treatment of HIV-1 infection. These inhibitors target interactions of the viral envelope glycoprotein with host cell receptors as well as fusion of the viral and host cell membranes. Extensive development of inhibitors that target CCR5, a cellular coreceptor required for viral entry, has resulted in molecules with potent antiviral activity. However, diverse HIV-1 isolates exhibit a wide variation in intrinsic sensitivity to these inhibitors due to factors including the heterogeneity of the envelope glycoprotein, affinity of the envelope for cellular receptors, and rate of entry. The studies presented here demonstrate competitive binding between the envelope glycoprotein and inhibitor for CCR5 is a major mechanism involved in sensitivity to PSC-RANTES. Although downregulation of coreceptor expression from the cell surface was instigated by PSC-RANTES binding, this effect was of short duration and competitive binding modulated viral sensitivity and resistance over time. In addition, in vitro selection of viruses resistant to the CCR5 antagonist maraviroc highlighted a novel mechanism of HIV-1 resistance to this type of inhibitor. Unlike previous studies, resistance was unrelated to changes in the V3 region of the glycoprotein. Rather, resistant virus harbored mutations in the CD4 binding site of the
13
envelope glycoprotein, demonstrated enhanced affinity for binding to CD4, increased
replicative fitness capacity, and increased rates of viral entry. These findings contribute
to the broader knowledge of inhibition of HIV-1 entry, receptor affinity relationships, and adaptive mechanisms of HIV-1 virus in response to treatment with CCR5 entry inhibitors.
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CHAPTER 1
INTRODUCTION
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1.1 HIV and AIDS
Human immunodeficiency virus (HIV), the causative agent of Acquired Immune
Deficiency Syndrome, afflicts an estimated 34 million people worldwide with approximately 25 million fatalities since its identification nearly 30 years ago. The disease, later known as AIDS, was first described in the United States in 1981 in homosexual men suffering from a rare opportunistic infection Pneumocystis carnii pneumonia [28]. Later reports also identified men with Kaposi’s sarcoma, a rare skin cancer normally associated with patients experiencing some form of immune system dysfunction [27]. Massive depletion of CD4+ T lymphocytes was found to be the cause of immune dysfunction in AIDS patients, eventually leading to the simultaneous discovery of the HIV retrovirus in 1983 by two independent research groups [14,70].
HIV infection follows a distinct clinical pattern beginning with primary infection via blood or mucosal routes. Acute infection is associated with increasing HIV viremia and rapid CD4+ T cell depletion, particularly in the gut mucosa [221]. This initial period is followed by an asymptomatic stage in which T cell counts continue to decline albeit at a slower rate than in acute infection. As the body mounts an immune response, a period of clinical latency ensues in which viral loads decline. This stage can last several years.
However, without clinical intervention, immune cell depletion will result in opportunistic infections, progression to AIDS, and ultimately death.
1.2 HIV-1 Classification and Diversity
HIV is classified in the genus Lentivirus, part of the Retroviridae family of viruses.
Lentiviruses fall into five subgroups based on what host species they infect. These five
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categories are primates, sheep and goats, cattle, horses, and felines. The distinguishing
characteristic of primate lentiviruses is their usage of CD4 as a receptor for viral entry
[90,192]. HIV is among the primate lentiviruses as well as several simian
immunodeficiency viruses (SIVs) isolated from different host species.
HIV is considered a complex retrovirus in that it not only encodes the structural
and enzymatic proteins required for viral replication, but encodes additional regulatory
and accessory proteins with a variety of functions. There are two types of HIV: HIV-1
and HIV-2. HIV-1 is the predominant type found worldwide while HIV-2 is limited to
parts of West Africa [36]. It is believed these two genetically distinct virus types
represent separate zoonotic transmissions of retroviruses from non-human primates to
humans. HIV-1 is more closely related to the SIVcpz virus indigenous to chimpanzees
than to HIV-2 [72] while HIV-2 is phylogenetically related to SIVsmm found in sooty
mangabey monkeys [73]. The generic term “HIV” is usually used to refer to HIV type 1.
HIV-1 is further classified into four groups with the “main” group M responsible for the majority of infections. The three other groups O, N, and P are localized in Africa.
Group M is subcategorized into genetically distinct subtypes A, B, C, D, F, G, H, J, K and circulating recombinant forms or CRFs. The majority of subtypes can be found in sub-saharan Africa, largely attributed to the fact this region was the epicenter of the zoonotic jump from non-human primates to humans. HIV subtype distributions in other
regions of the world are less diverse with subtype B dominating infections in North
America and Western Europe.
HIV-1 displays a remarkable level of genetic diversity. In an individual, the diversity of the viral swarm, or quasispecies, is initially limited due to a transmission
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bottleneck, with only a small subset of virus productively establishing an infection [99].
However, once an infection is established the diversity of the viral population expands as
a result of a high error rate of the reverse transcriptase enzyme as well as the rapid rate of
virion production. Major selective forces influencing the quasispecies can include the
host immune response, competition among viruses within the quasispecies, and the
presence of antiretrovirals. As the outer exposed element of the virion, the envelope is
the primary target for the host immune system. In response, the HIV env gene contains
the most diverse regions in the genome. The constant mutation of envelope partially
explains the inability of the immune system to effectively neutralize the virus.
1.3 HIV-1 Genome and Proteins
The single-stranded, positive sense RNA genome contains three open reading
frames (ORFs) and produces a total of fifteen proteins (Fig. 1). Like other retroviruses,
the genome is organized into three major genes: gag, pol and env. These genes encode
the major structural and enzymatic proteins of the virus while accessory genes tat, rev,
vif, vpr, vpu, and nef enhance replication and infectivity.
The capsid (CA), nucleocapsid (NC), and matrix (MA) structural proteins are
produced as a 55 kDa gag polyprotein precursor which is cleaved by the virally encoded protease (PR) enzyme during virion maturation. The gag precursor associates with the
cellular membrane and is sufficient to produce non-infectious and immature virus-like
particles. A ribosomal frame-shift site at the C-terminus of gag results in the ribosome shifting to the pol reading frame approximately 5% of the time [94]. This results in a
gag-pol precursor which includes not only the gag proteins but also the protease,
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integrase, and reverse transcriptase enzymes of the pol gene. An auto-catalytic cleavage process releases protease which proceeds to cleave CA, NC, and MA on gag and gag-pol polyproteins during maturation [206].
As previously mentioned, the proteins encoded on the pol gene are transcribed as part of a gag-pol polyprotein precursor. Cleavage of the protein products releases the major enzymes required for HIV replication. In order from 5’ to 3’ on the genome they are protease, reverse transcriptase, and integrase. The protease enzyme cleaves individual protein products from the gag and gag-pol polyprotein precursors during virion maturation. Reverse transcriptase is a heterodimer of a 51 kDa and 66 kDa subunits. The
51 kDa subunit is a product of protease cleavage of the C-terminal end of the 66 kDa subunit. The major function of reverse transcriptase is to convert the ssRNA genome of
HIV into a dsDNA genome that can be integrated into the host cell DNA. This integration process is performed by the 32 kDa integrase enzyme.
The HIV env gene encodes two glycoproteins which form the envelope spike on the virion surface. The envelope proteins are translated from spliced mRNA on endoplasmic reticulum associated ribosomes as a polyprotein precursor, gp160. The gp160 precursor is glycosylated and oligomerizes into trimer complexes before being transported to the Golgi complex for proteolytic cleavage by the cellular protease furin.
Furin process the gp160 precursor into the transmembrane (gp41) and surface (gp120) glycoproteins [83]. Following cleavage, gp120 and gp41 associate through noncovalent interactions and the envelope complex is transported to the cell surface where it can be incorporated into budding virions.
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The regulatory proteins tat and rev are each encoded by two exons with full
transcripts produced during mRNA splicing. Tat stands for trans-activator of transcription and functions to enhance viral mRNA transcription after proviral DNA integration into cellular chromosome [9,205]. The rev (regulator of expression of viral
proteins) protein aids in the nuclear export of unspliced and partially-spliced mRNA transcripts of viral genes which would otherwise be retained in the nucleus by cellular factors [64,231].
The HIV accessory proteins, although considered non-essential for replication in
some tissue culture lines, do perform important roles in infection by either promoting
virus protein production or counteracting cellular defense mechanisms meant to restrict
viral replication. The specific functions of these proteins have been studied extensively
in the last two decades with the hope to understand their role in HIV infection and
pathogenesis.
The vpr gene encodes a 14 kDa protein which is incorporated in viral particles.
Multiple functions have been reported for the vpr protein including (a) influencing the
mutation rate during reverse transcription [135], (b) facilitating nuclear import of viral
DNA as part of the preintegration complex (PIC) [168], (c) transactivation of the HIV
LTR promoter as well as stimulation of gene expression via cellular promoters
[33,38,178,183], (d) inducing cell cycle arrest [97], and (e) regulating apoptosis [151].
Vpr has been implicated in HIV pathogenicity and it is required for infection of primary
macrophages [141]. The role vpr plays in pathogenicity may be inferred from the
observation that a higher frequency of vpr mutations are found in viral strains from long-
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term nonprogressors [130], individuals infected with HIV but who do not exhibit immunosuppression even in the absence of therapy.
Another accessory protein, vif or virus infectivity factor, promotes viral infectivity by counteracting the antiviral activity of the cellular DNA cytosine deaminase
APOBEC3G [200]. APOBEC3G is a cellular antiviral factor that converts cytosines to uracils during minus-strand DNA synthesis. In the absence of vif, APOBEC3G can be incorporated into virus particles [240]. During reverse transcription, cytosine deamination results in guanine to adenine hypermutations in viral DNA and may lead to degradation of the viral genome by uracil DNA glycosidases. However, the vif protein targets APOBEC3G by inducing ubiquitination and proteasomal degradation [142].
Two major functions of the vpu protein have been described: (a) degradation of newly synthesized CD4 [237] and (b) enhancement of virus particle release. Degradation of newly synthesized CD4 occurs through the ubiquitin-proteasome pathway. Vpu binding directly to the cytosolic end of CD4 in the endoplasmic reticulum (ER) recruits cellular factors to the vpu-CD4 complex and leads ultimately to proteosomal degradation of CD4 [196]. This function serves an important role during replication since the envelope precursor gp160 becomes associated with CD4 in the ER and is prevented from transport to the cell surface [237]. Vpu degradation of CD4 frees the envelope gp160 to continue its transportation pathway. The role vpu plays in enhancing virus particle release has long been known, however only recently was the mechanism by which vpu promotes particle release identified. Retention of virus particles at the cell membrane was observed for viruses lacking a functional vpu gene [102]. A cellular protein, named
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tetherin for its ability to retain immature virus particles at the cell surface, was identified
as a target of the vpu protein [154].
The nef gene partially overlaps the 3’ LTR and produces a 27-kDa protein. Nef is a membrane-associated protein produced early in mRNA transcription of viral genes. A number of functions have been attributed to nef including (a) downregulation of surface
CD4, MHC-I, MHC-II, CD3, and CD28, (b) regulate T cell activation pathways, and (c) enhance viral infectivity. Nef association with the cytoplasmic tail of CD4 promotes rapid internalization of the receptor from the cell surface through clathrin-coated pits
[74]. Within the endosome, nef promotes CD4 lysosomal degradation by linking CD4 with membrane transport adaptors such as COP I which target the endosome to the lysosome [167]. In addition to promoting CD4 downregulation, nef has also been implicated in downregulation of MHC-1 and MHC-II molecules, as well as the CD3 T-
cell receptor complex and CD28 costimulatory molecule from the cell surface
[15,21,208,211]. This function of nef serves to limit antigen presentation and cytotoxic T
lymphocyte recognition of infected T cells, thereby evading host immune responses.
1.4 HIV Virion
The mature HIV-1 virion is approximately 100-150nm in diameter and consists of
two copies of positive single strand RNA genome (Fig. 2) [77]. These RNA strands are encapsulated along with reverse transcriptase, protease and integrase inside a canonical shaped capsid structure. This capsid structure is surrounded by a lipid bilayer composed of host cellular membrane lipids taken during viral budding. Matrix proteins help stabilize viral envelope spikes embedded in the membrane.
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1.5 HIV Replication Cycle
The HIV-1 replication cycle initiates with binding of the envelope to a CD4+ host
cell (Fig. 3). A chemokine receptor, either CCR5 or CXCR4, is required as a coreceptor
for entry. Upon fusion with the host cell membrane the viral core is released into the
cytoplasm where it degrades, releasing two copies of the ssRNA genome and viral
proteins. Reverse transcription of the viral RNA into double-stranded DNA is followed by integration into the host cell DNA. Cellular machinery transcribes and translates the genome to produce viral protein products. Viral assembly occurs at the plasma
membrane followed by budding and maturation to produce an infectious virion. A single
replication cycle takes approximately 24 hours to complete. This leads to a rapid
replication rate in infected individuals with millions of new virions produced each day.
1.5.1 Viral Entry and Cellular Tropism
Not long after the discovery of HIV, it was determined that CD4 was a required
receptor for viral infection [43]. However, it was also noted that viruses exhibited
differential infection of CD4+ cell types with some capable of infecting primary
macrophages and others replicating in T cell lines. To reflect these preferences, viruses
were classified as macrophage tropic (M-tropic) or T cell line tropic (T-tropic). These
observations, in addition to evidence that human CD4 expression alone in murine cells
was insufficient to support HIV infection, suggested a secondary receptor, or coreceptor,
was required for viral entry into human cells and that more than one receptor may fulfill
this role [133].
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Identification of the first coreceptor capable of mediating HIV entry occurred in
1996 with a functional cDNA library clone to identify a cofactor that would permit fusion
of murine cells expressing human CD4 with cells expressing HIV envelope of a T-tropic
virus [62]. The result of this study was the identification of a G-protein coupled receptor,
then termed “fusin”. At the time, no functional activity had been described for this
receptor, although the cDNA had been previously cloned and named LESTR [128].
However, sequence homology suggested LESTR/fusin was related to the chemokine
receptor for the chemoattractant molecule interleukin-8. It was later determined to
belong to the CXC subfamily of chemokine receptors and is now known as CXCR4.
The discovery of a chemokine receptor as the cofactor required for T-tropic viral entry led the way for the identification of the cofactor required for M-tropic virus entry
later that same year. Since the mid-1980s it had been known that CD8+ cytotoxic T cells secreted proteins capable of interfering with HIV infection at an early stage in the viral lifecycle. However, the identity of these inhibitory factors was not determined until 1995 when Cocchi et. al identified RANTES, MIP-1α, and MIP-1β as the M-tropic virus inhibitory factors produced by CD8+ T cells [37]. These proteins were all chemokine ligands of the CC subfamily, implicating a CC chemokine receptor as the potential cofactor required for M-tropic virus entry. Two independent research groups described a
CC chemokine receptor with specificity for these three ligands, designated CC CKR5
[39,189]. Not long after these studies, multiple groups reported the CC CKR5 receptor, now known as CCR5, was the cofactor required for entry of M-tropic strains [2,46,50].
Based on these findings and the description of viruses capable of replicating in cells
expressing either coreceptor, viruses are now classified as R5, X4, or R5X4/dual tropic.
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The process of HIV entry into a target cell is divided into three main events: 1)
attachment to a host cell, 2) binding to a seven transmembrane chemokine coreceptor,
and 3) fusion of the viral and cellular membrane (Fig. 4). The HIV envelope complex mediates interactions with the cellular receptors and membrane. The envelope is comprised of an outer surface glycoprotein gp120 and a transmembrane glycoprotein gp41. These glycoproteins associate via noncovalent interactions to form heterodimeric subunits. A trimer of these heterodimeric subunits comprises an envelope spike on the surface of the virion and functions to mediate entry into host cells.
HIV entry initiates with the attachment of the envelope glycoprotein gp120 to the cellular receptor CD4 [43,133]. Engagement with CD4 induces a structural rearrangement in gp120 exposing a coreceptor binding site. CCR5 and CXCR4 are the two major coreceptors used by viruses for entry. Although over fourteen different seven transmembrane receptors including CCR1, CCR2b, CCR3, CCR8, CCR9,
BONZO/CXCR6, Bob/GPR15 have been shown to support HIV infection in CD4+ cell lines, only CCR5 and CXCR4 have been shown to act as coreceptors in vivo
[34,119,199]. Coreceptor binding initiates further conformational changes in the envelope, permitting insertion of gp41 fusion peptide into the cellular membrane.
Through intermediate gp41 structural rearrangements, a fusion pore develops as a result of membrane mixing between the viral and cellular membranes. The capsid core structure, containing the viral genome, passes through the fusion pore and into the cytoplasm of the cell.
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1.5.2 Reverse Transcription
The defining characteristic of retroviruses is their ability to convert their single-
stranded RNA genomes into double-stranded DNA through the process of reverse
transcription (Fig. 5). The reverse transcriptase (RT) enzyme is a virally encoded DNA
polymerase with both RNA-dependent and DNA-dependent activities. RT also has an associated ribonuclease H (RNaseH) activity for degradation of RNA in RNA-DNA
duplexes.
Reverse transcription occurs in the cytosol of cells and requires an RNA primer to
initiate polymerization from the ssRNA genome template. In the case of HIV-1,
tRNALys3 serves as this primer and binds to an 18 nucleotide region known as the primer binding site (PBS) near the 5’ end of the genome. It has been shown that tRNALys3 is
incorporated into virions and that some primer extension occurs prior to viral entry [129].
Once the tRNA binds to the PBS site, polymerization of minus (-) strand DNA proceeds
from the 3’-OH of the tRNA towards the 5’ end of the genome. The RNase H domain of
the RT enzyme is responsible for digesting the (+) RNA genome once hybridized with
the newly synthesized (-) DNA strand. Digestion of the genomic RNA frees this short
ssDNA, known as minus strand strong stop DNA, which is then able to hybridize with
the 3’ end of the genome via a short repeat (R) region of homology found at both the 5’
and 3’ ends of the genome. DNA synthesis proceeds along the remaining length of RNA
genome with RNase H digesting the genomic RNA along the way. RNase H leaves a
region of RNA in the env coding region known as the polypurine tract (PPT). The PPT
serves as a primer for positive-strand DNA synthesis which proceeds in a 5’ to 3’ manner
using the minus-strand DNA as template. Once the 3’ end of the minus-strand DNA is
26
reached, the PPT region and the tRNALys3 are digested by RNaseH activity. A second
strand transfer event occurs where the positive-strand DNA binds to the minus-strand
DNA at the PBS region which is now present in both DNA. Second strand DNA synthesis completes through bidirectional synthesis by the circular binding of the long minus-strand DNA to the shorter positive-strand DNA at the U5, R, and U3 regions. The double-stranded DNA product of reverse transcription contains identical regions at the 5’ and 3’ ends termed the long terminal repeats (LTR). These regions are essential for proviral mRNA transcription and contain the enhancer, promoter, transcription initiation, transcription termination and polyadenylation signals.
An interesting caveat in retroviral replication is the ability to produce recombinant genomes during reverse transcription. Since the virus particle contains two copies of ssRNA, it is possible for heterologous genomes to be packaged into the same virion during dual infection of a single host cell by two or more genetically distinct viral strains.
The strand transfer events that occur as part of reverse transcription, as well as stalling of
the RT complex on an RNA template, promote intermolecular jumps between the ssRNA
genomes and lead to a recombinant dsDNA product. Progeny virions produced following
such a recombination event may contain unique sequence combinations reflecting the
heritage of the parental viral strains. Preferential recombination sites, often termed
hotspots, are typically located in conserved regions of the genome. However, the site of
recombination can introduce nonsynonymous mutations in a gene coding region and
result in a sequence unique to the recombinant genome. Recombination, in conjunction
with the high mutation rate of RT (3 X 10-5 mutations per replication cycle [136]), is a
major mechanism for viral evolution and the generation of drug resistant viral strains.
27
1.5.3 Nuclear Import and Integration
Following reverse transcription, the newly synthesized viral dsDNA must be
imported into the cell nucleus for integration into the host chromosome. This is
accomplished through the formation of the pre-integration complex (PIC), a complex of host and viral proteins that actively transport the dsDNA into the cell nucleus. The PIC consists of viral dsDNA, the integrase (IN) enzyme, the viral vpr protein, matrix protein, and cellular factors. Among the cellular factors identified are coactivators of transcription such as lens epithelium–derived growth factor (LEDGF/p75) as well as
nuclear import proteins importin-α, transportin-3 (TNPO3), and transportin-SR2 (TRN-
SR2). Due to the size of the PIC complex, it cannot passively transfer from the cell
cytoplasm into the nucleus but must be actively carried into the nucleus through a cellular
nuclear import pathway (Fig. 6A). Nuclear localization signals found in the matrix, vpr, and integrase recruit nuclear importin proteins to the PIC. The directionality of nuclear transport relies on a differential concentration gradient between RanGTP and RanGDP
with RanGTPase hydrolyzing RanGTP to RanGDP in the cytoplasm. After translocation
through the nuclear pore complex, association of importin-α to RanGTP release the PIC.
This active nuclear transport process permits HIV infection of non-dividing cells such as
macrophages.
Once inside the nucleus, the PIC is targeted to the cellular DNA for integration.
Although integration site selection is not fully understood, HIV integrase targets
transcriptionally active regions of chromatin DNA. Site selection specificity has been detected for LEDGF/p75-modulated genes [201]. LEDGF/p75 is a survival protein that
28
acts as a transcriptional coactivator of anti-apoptotic proteins and is thought to function
as a tether for integrase, targeting viral DNA incorporation to LEDGF-modulated genes
[134].
The process of integration initiates with a reaction known as 3’-end processing
(Fig. 6B). The integrase enzyme cleaves off two nucleotides from the 3’ ends of both
strands of linear viral DNA. Integrase also cleaves the cellular DNA in a staggered
manner, resulting in non-blunt ends. The 3’ recessed ends of the viral DNA bind with the
cleaved cellular DNA in a strand transfer event. Integration is completed when the gaps
between the integrated viral DNA and cellular DNA are filled in by cellular repair
enzymes. Once integrated, the viral DNA is referred to as “proviral” DNA.
1.5.4 RNA Transcription and Export
Following successful integration into the host chromosome, the proviral DNA is
used as a template for the synthesis of viral RNAs which encode the necessary structural,
enzymatic, and regulatory proteins for the production of progeny virus. Cellular machinery are utilized for the transcription of viral RNAs, RNA-splicing, and nuclear export.
Basal transcription activity from the HIV LTR promoter is very low. However, the tat protein is produced from a small number of RNA transcripts generated early in cells [98]. Tat potentiates a positive feedback mechanism promoting high level HIV mRNA transcription. Once present, tat binds to a transactivation response (TAR) element, a cis-acting RNA element that forms a stable stem-loop structure located
downstream of the transcription initiation site [149]. Tat-TAR interaction promotes
29
recruitment of a multicomponent kinase complex termed P-TEFb or positive- transcriptional elongation factor b [249]. P-TEFb is a cyclin dependent kinase which phosphorylates the C-terminal domain of RNA polymerase II, relieving the polymerase
from negative elongation factors and promoting processive mRNA elongation (Fig. 7A).
The kinase subunit of P-TEFb is cyclin dependent kinase 9 (CDK9) which is regulated by
interactions with a cyclin regulatory subunit [228]. Tat binding to the TAR RNA element
recruits the Cyclin T1 subunit, which in turn recruits the CDK9 kinase. The presence of
tat increases processive transcription elongation of the proviral DNA by as much as two
logarithms [44,65].
Three major groups of RNA transcription products are produced from the proviral
DNA: 1) unspliced RNAs, 2) partially spliced mRNAs, and 3) multiply spliced mRNAs
(Fig. 7B). The unspliced RNAs serve dual roles as templates for the translation of the
gag and gag-pol polyprotein precursors as well as serving as genomic RNA for packaging
in progeny virions. Partially spliced mRNAs encode the env, vif, vpu and vpr proteins
while short, multiply spliced mRNAs encode the rev, tat, and nef proteins. Differential
splicing patterns can result in as many as thirty or more unique mRNA transcripts being
produced [171].
Most cellular mRNAs are fully spliced prior to leaving the nucleus. However,
HIV replication requires that partially and unspliced RNAs are transported out of the
nucleus to the cytoplasm. In order to overcome this dilemma, the rev protein (regulator of expression of viral proteins) binds to unspliced viral RNAs and shuttles them out of the nucleus. Rev binds to a cis-acting RNA element known as the rev-responsive element (RRE) located in the env gene. The RRE is a series of highly structured stem-
30
loops found in all unspliced and partially spliced viral RNAs. Multiple rev proteins bind to the RRE and form a complex capable of interacting with nuclear export machinery.
Rev is able to shuttle the unspliced or partially spliced RNAs to the cytoplasm and return to the nucleus by way of its nuclear localization signal. Once in the cytoplasm, viral protein synthesis from mRNA transcripts ensues through normal cellular translational machinery.
1.5.5 Assembly and Packaging
Production of gag and gag-pol polyproteins promotes assembly of new virus particles at the plasma membrane. The matrix (MA) domain of gag, which is located at the N-terminus of the p55 precursor protein, is cotranslationally modified by the addition of myristic acid. Mutational studies suggest that a positively charged face of MA interacts with negatively charged phospholipids of the plasma membrane, stabilizing membrane binding [68].
In addition to associating with the plasma membrane, gag must also participate in gag-gag and gag-RNA interactions. The gag-gag interaction is thought to involve
multiple regions of the precursor including portions of capsid (CA), the p2 peptide, and
nucleocapsid (NC). In tandem, interactions between NC and a cis-acting element located
5’ of the gag initiation site known as the major packaging signal, or ψ-site, mediate the
incorporation of genomic RNA into virus particles. Although regions outside of the ψ- site have been implicated in viral RNA packaging [29], the ψ-site is the region primarily responsible for gag association as well as dimerization of two ssRNA genomes [67,120].
31
The envelope precursor protein, gp160, is synthesized in the ER and cotranslationally translocated into the ER lumen by means of an N-terminal signal peptide comprised of approximately the first thirty amino acids of the polyprotein. In the lumen, gp160 folds, disulfide bonds are formed, the protein is glycosylated, and oligimerizes to form trimers. After gp160 is properly folded and trimerized, it is transported to the Golgi complex where the polyprotein is cleaved by cellular proteases into the gp120 and gp41 subunits. The gp41 subunit is anchored to membrane and noncovalently associates with gp120. The envelope complex is trafficked to the plasma membrane through a regulated secretory pathway. Mutational studies indicate that the
MA domain of gag promotes envelope incorporation into budding virions through interaction with the cytosolic tail of gp41 [19].
1.5.6 Viral Budding, Release, and Maturation
Accumulation of gag and associated RNA at the plasma membrane enhances the final stage of virion assembly known as budding. A proline rich domain at the C- terminus of p55 gag known as p6 encodes a small protein that promotes viral particle release from the plasma membrane [79]. Multiple studies have indicated a connection between the endosomal sorting pathway and the viral budding process. The observation that p6 interacts with a component of the ESCRT-I complex (endosomal sorting complex required for transport) further points to the importance of the endosomal sorting pathway in virion release [76]. In addition to interacting with cellular factors to promote budding, p6 has also been shown to direct the incorporation of the vpr protein into new virions
[164].
32
A cellular defense mechanism to prevent release and maturation of budded virus particles involves a cellular factor, tetherin, which physically tethers budding virus particles to the cell membrane. These nascent particles are subject to internalization through endocytic pathways. While it has been known for some time that the accessory protein vpu promotes release of virus particles [153], it has only recently been
demonstrated that vpu does so through antagonization of tetherin [102,154].
Shortly after virus release from the membrane, the protease enzyme cleaves the
gag and gag-pol precursors into the mature proteins encoded by gag and pol. Once
released, the structural proteins induce rearrangements in the virion morphology in a
process known as maturation. Mature virions contain a conical shaped core
encapsulating the RNA genome. Immature virions are not capable of establishing
productive infections and thus maturation is a requirement for infectious particle
production.
1.6 Antiretrovirals and Drug Resistance
Shortly after the discovery of HIV as the etiologic agent causing severe immune
deficiency in AIDS patients, a campaign ensued to develop therapeutic interventions to
slow disease progression and viral proliferation. Today, multiple antiretroviral
compounds targeting a variety of host and viral proteins have been developed and are in
clinical use. Antiretrovirals are broadly classified based on the phase of the viral
replication cycle that they target. Currently there are six classes of HIV antiretrovirals:
1) nucleoside reverse transcriptase inhibitors (NRTI), 2) nonnucleoside reverse
transcriptase inhibitors (NNRTI), 3) protease inhibitors (PI), 4) integrase inhibitors (II),
33
5) fusion inhibitors (FI), and 6) chemokine receptor antagonists (CRA). According to the
Food and Drug Administration (FDA), there are thirty-five individual or combination drugs approved for therapeutic administration for HIV infection in the United States
(Table 1).
The first antiretroviral for the treatment of HIV infection was approved for use in
1987 by the FDA. Zidovudine, commonly known as AZT, is a nucleoside analogue that,
when incorporated into a DNA strand during reverse transcription, causes premature
termination of DNA synthesis. AZT is one of a number of nucleoside analogs that target
the virally encoded reverse transcriptase enzyme to prevent proviral DNA synthesis and
incorporation into the host cell genome. DNA synthesis requires a 3’ hydroxyl (-OH)
group for addition of the subsequent nucleotide to the elongating DNA strand.
Nucleoside inhibitors are prodrugs that require phosphorylation by cellular proteases
prior to incorporation in growing DNA or RNA strands. Members of this class of drugs
do not possess the appropriate 3’-OH group necessary for addition of the next nucleotide.
By this mechanism, nucleoside inhibitors prematurely terminate viral DNA synthesis and
inhibit HIV replication.
In contrast to the NRTIs, the nonnucleoside reverse transcriptase inhibitors are
noncompetitive inhibitors. These compounds bind an allosteric site on the p66 subunit of
the enzyme and inhibit polymerase activity by altering the conformation of the active site.
Protease inhibitors prevent the maturation of virus particles into mature infectious
virions by competitively binding the viral protease enzyme active site and inhibiting
cleavage of gag and gag-pol polyproteins. Immature virions are not capable of infecting
a new cell.
34
An obligate step in HIV replication is the integration of viral dsDNA into the host cell genome. Raltegravir, marketed as Isentress by Merck & Co., is the only inhibitor clinically available to date that specifically targets the activity of the integrase enzyme.
It does so by preventing the DNA strand transfer event catalyzed by integrase during viral
DNA integration into chromosome. Raltegravir interferes with covalent interactions between integrase and the phosphodiester backbone of DNA.
The newest class of HIV antiretrovirals includes inhibitors that target the process of HIV entry into cells. Collectively these are known as entry inhibitors, however they are more specifically classified based on which step of entry they target. Currently there are two entry inhibitors available for use in patients, a fusion inhibitor and a CCR5 small molecule antagonist.
Historically, the reverse transcriptase inhibitors were the first to be approved for the treatment of HIV infection. Unfortunately, the rapid mutation rate of the virus meant that drug resistance developed quickly during monotherapy and often conferred cross resistance to inhibitors in the same drug class. This outcome was greatly reduced in 1996 with the implementation of Highly Active Antiretroviral Therapy (HAART). HAART generally consists of three or more antiretrovirals from at least two different drug classes.
Typically initial regimens consist of two reverse transcriptase inhibitors and a protease inhibitor. However, increased mortality as a result of HAART in combination with emergence of multi-drug resistant viral strains has driven demand for novel antiretrovirals targeting viral and/or cellular processes required for HIV replication. As the newest class of HIV drugs, entry inhibitors contribute to the arsenal of antiretrovirals available to improve clinical outcomes for patients.
35
1.7 Molecular Aspects of HIV Entry
The following sections will outline the current knowledge on the specific
interactions of the HIV envelope and cellular receptors as well as discuss pharmacologic
intervention strategies targeting these interactions and viral resistance mechanisms that
arise as a result.
1.7.1 Envelope Structure
The envelope spike consists of a trimer of heterodimeric subunits comprised of a
transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The gp120 and
gp41 glycoproteins associate through noncovalent interactions after proteolytic processing of the gp160 precursor in the endoplasmic reticulum. Although a trimer of subunits is required for envelope function, it is not well understood how many heterodimeric subunits must participate in receptor interactions for entry.
Many insights into gp120 structure/function relationships have been gained with the crystallization of CD4-bound and, more recently, unliganded gp120 core structures.
Although crystallization methods demanded truncation of variable loop regions, valuable information regarding CD4 binding site, bridging sheet formation, and broadly neutralizing antibody epitopes have been discerned from these crystallization studies.
The first crystal structure of a gp120 core resolved in 1998 identified major structural regions of gp120 when liganded with CD4 and monoclonal antibody 17b [109].
Gp120 consists of five conserved regions (C1-C5) with interspersed variable loop regions
36
(V1-V5). The structure provided by Kwong et al lacked the V1-V2 and V3 loop regions
as well as the amino- and carboxy-terminal ends of gp120. Additionally, the gp120 core
used was stripped of carbohydrate groups. Despite these modifications, the gp120 core
retained the ability to bind CD4 as well as other antibodies targeting gp120. The crystal
structure revealed gp120 folded into a heart-shaped configuration composed of 25 β-
strands, 5 α-helices, and 10 loop segments. Portions of the conserved regions of gp120
comprised a “core” structure with the variable regions forming surface-exposed loops
stabilized by cysteine-cysteine disulfide bonds at the base of each loop. Since resolution
of this first structure, several other gp120 structures have been solved in complex with
various ligands and antibodies. Four major regions could be identified in these gp120
structures: the inner domain, the outer domain, the bridging sheet, and the third variable
loop (V3) (Fig. 8A).
The inner domain of gp120 mediates interactions with gp41 and is relatively
conserved among different HIV strains. Within the viral spike, the inner domain is
proximal to the trimer axis and relatively inaccessible. Recent crystallization of a gp120
with intact amino- and carboxy- terminal ends revealed an ordered organization within
the inner domain [161]. A prominent feature is a seven-stranded β-sandwich from which the amino- and carboxy- terminal ends emanate toward the viral membrane. Mutagenic
studies have implicated the terminal ends as well as parts of this β-sandwich in noncovalent interactions with the ectodomain of gp41 [86,243]. Emitting away from the
β-sandwich, toward the cellular membrane, are three topological layers believed to
change conformation during transition from unliganded to the CD4-bound state [63,161].
Alteration of multiple residues within these layers was found to impede the gp120-CD4
37
binding interaction even though these inner domain residues did not directly contact CD4
[63]. Thus, the gp120 core inner domain is believed to mediate interactions with gp41 as
well as promote CD4 binding site formation through conformational changes during
transition from unbound to ligand-bound states.
The outer domain of gp120 is largely exposed and distal to the trimer axis in the
envelope spike. The surface exposed region is densely packed with glycosylated
residues (Fig. 8B). This glycan shield is thought to be the reason for poor
immunogenicity of this region in eliciting humoral immune response in patients.
The bridging sheet is composed of four anti-parallel β-sheets and links the inner and outer domains. The structure of an unliganded SIVmac gp120 suggested the bridging
sheet undergoes significant structural rearrangement as a result of CD4 binding [31],
however the recent resolution of an unliganded HIV gp120 revealed this structure was
intact even in the absence of CD4 [107]. Multiple studies have identified key elements
within the bridging sheet contribute to the formation of the binding site of CD4 as well as
the coreceptor binding site (Fig. 8C) [91,109]. This region is also a target for
neutralization, however it is believed to be shielded by the heavily glycosylated V1/V2
loops from surface exposure prior to receptor engagement [63,107].
The V3 loop of gp120 has long been known to interact with coreceptor. Indeed,
simple substitutions in the tip of the V3 loop can alter viral tropism from CCR5 to
CXCR4 [92]. It is believed that the tip directly interacts with the extracellular loops of
the chemokine coreceptor and, in conjunction with the V3 loop base and bridging sheet,
forms a discontinuous coreceptor binding site. Truncation of the V3 loop severely
impairs entry efficiency.
38
Despite the inability to resolve structures of a native envelope trimer, superimposition of monomeric gp120 subunit atomic structures with density maps derived through cryoelectron microscopic tomography studies of native trimers revealed architectural similarities between the monomeric subunits and those involved in tertiary structure of the envelope complex [123,248]. These data lend confidence that the resolved monomer structures of gp120 reflect conformations seen in the functional envelope spike.
A major impediment to the prevention of HIV infection has been the inability to develop an effective vaccine. Although there are several reasons for this, one major contributing factor is the inability to elicit broadly neutralizing antibodies that are capable of targeting envelopes of diverse virus strains. The structural information just described alludes to reasons for this dilemma. As mentioned, the exposed surface of gp120 is highly glycosylated (Fig. 8B) shielding variable regions which may serve as the best neutralizing epitopes. Although some broadly neutralizing antibodies, such as 2G12, do target and bind N-linked glycan residues on gp120, these antibodies are rarely produced.
The majority of broadly neutralizing antibodies identified (b12, 17b, VRC01, 2F5, 4E10,
HJ16, PG9, PG16) target regions of gp120 that bind CD4 or coreceptor or prevent conformational transitions in gp120 and gp41 required for entry (Fig. 8D) [88]. These regions are largely inaccessible for neutralization in unliganded structures. However, the recent data by Kwon et al. contradicts the long held belief that major structural rearrangements occur in gp120 in the transition from unliganded to CD4-bound states
[107]. Future studies will be needed to reconcile these issues.
39
1.7.2 CD4 and Interactions with gp120
Not long after the discovery that HIV targets lymphocytes was it determined that
the cell surface receptor CD4 played a major role in HIV cellular tropism [43,133]. CD4
is expressed on cells of the immune system, specifically monocytes, macrophages,
dendritic cells, and T cell subsets including naïve, central and effector memory T cells. It can also be found on microglia and macrophages in the central nervous system [96,226].
CD4 plays a major role in helper T cell activation by enhancing specificity of the T cell receptor (TCR) complex interaction with antigen bound to a major histocompatibility complex class II (MHCII) molecule on an antigen-presenting cell (Fig. 9B).
CD4 is a member of the immunoglobulin (Ig) superfamily and consists of a short cytoplasmic tail, a single transmembrane segment, and four sequential extracellular immunoglobin-like domains (D1-D4) [185]. The first Ig-like domain, D1, interacts with both MHCII molecules as part of CD4’s normal immune function as well as the HIV envelope glycoprotein gp120 [109,224]. The putative binding site for gp120 is comprised of residues 25-64 of D1 with 22 amino acids of CD4 interacting with 26 residues of gp120 [109]. D1 is organized into nine strands (AGFCC’C”BED) with the three antiparallel β-strands C-C’-C” and the D strand composing the gp120 binding site (Fig.
9A). Alanine scanning mutagenesis analyses revealed two key residues in CD4 for its
interaction with gp120 [10,143]. Mutation of phenylalanine 43 in the C” strand or
arginine 59 in the D strand to alanine or glycine significantly reduced gp120 binding.
Structurally, F43 of CD4 projects into a conserved hydrophobic cavity on the surface of
gp120 while R59 forms a salt bridge with D368 of gp120 [109]. Interestingly, these same
residues are critical for CD4’s interactions with MHCII molecules [224].
40
According to the work by Kwong et al., the F43 residue accounts for nearly one- quarter of the interatomic interactions necessary for gp120 binding. F43 interacts with gp120 residues E370, I371, N425, M426, W427, G473, and D368. Additional, mostly hydrophobic gp120 residues (W112, V255, T257, F382, Y384, and M475), line the cavity on gp120 into which F43 projects termed the “F43 cavity”. This binding cavity is located at the interface of the inner domain, outer domain, and bridging sheet (Fig.
8A,C). Mutation of any of four gp120 residues (T257, D368, E370, or W427) highly
conserved among primate immunodeficiency viruses significantly reduces gp120-CD4
binding efficiency [157].
In addition to the F43 binding interaction, the R59 residue of CD4 forms a second
critical interaction with gp120. R59 forms double hydrogen bonds with the carboxylate
group of gp120 residue D368 [109]. Furthermore, R59 appears sandwiched between
D368 and V430, forming interactions with both residues. Mutation of R59 to alanine or
aspartate has been shown to reduce gp120 binding to CD4 by as much as 10-fold
[10,143].
1.7.3 Chemokine Receptors and Interactions with gp120
CCR5 and CXCR4 are members of a receptor super-family known as G-protein
coupled receptors (GPCR). Despite their diverse functions, members of this receptor
family are thought to share a similar structural conformation consisting of a seven
transmembrane helix bundle with an extracellular N-terminal tail and three extracellular
loops (ECL) (Fig. 10). Chemokine receptors are divided into subfamilies based on the arrangement of cysteine residues in the N-terminus of the chemokine ligands which bind
41
them. There are four subfamilies: C, CC, CXC, and CX3C. The C subfamily of
chemokines contains a single cysteine residue in the N-terminus while the CC subfamily has two adjacent cysteines. The CXC subfamily contains two cysteines, however a non- conserved residue resides between them while the CX3C subfamily has three non- conserved amino acids separating the cysteines. A revision in nomenclature has resulted
in chemokine ligands that were previously named to be renamed such that CCR5 ligands
RANTES, MIP-1α, and MIP-1β, are referred to as CCL5, CCL3, and CCL4, respectively.
Additionally, the CXCR4 ligand SDF-1 is referred to as CXCL12. Despite this effort to
simplify the nomenclature, ligands for these receptors are still often referred to by their historical names in literature.
Upon activation, GPCRs associate with G protein complexes instigating
intracellular signaling events. In the case of CCR5, ligand activation can result in C- terminal receptor phosphorylation, association with β-arrestin, desensitization of the receptor to further signaling, and clathrin-dependent endocytosis. Upon internalization the receptor can be marked for degradation pathways or resensitized and cycled back to the cell surface. The specific details of receptor sorting pathways remain under
investigation. Basal activation of chemokine receptors in the absence of agonist binding
have been described [40]. Chemokine receptors are known to exist in multiple antigenic
forms, both active and inactive. Differential monoclonal antibody binding to CCR5
epitopes reveals multiple functional forms of the receptor exist transiently dependent on
ligand binding, lipid composition, and activation state [17,23,117]. The promiscuous
binding of chemokine ligands to multiple receptors would suggest recognition of specific
42
receptor conformations by individual ligands. It is unclear whether the HIV envelope is
capable of recognizing all of these antigenic forms or only a subset thereof.
Although the structures of CCR5 and CXCR4 have not been determined
experimentally yet, their structures have been computationally predicted using the few
solved crystal structures of other GPCRs, specifically the bovine rhodopsin structure, as a
template (Fig. 10) [159]. In spite of these limitations, mutagenesis studies have
determined key interactions between the glycoprotein gp120 and coreceptors.
Gp120 is believed to interact with CCR5 at two key sites on the coreceptor: the
N-terminal tail (Nt) and the second extracellular loop (ECL2). Specifically, the V3 loop
tip of gp120 interacts with the ECL2 while the V3 loop base and the four-stranded bridging sheet interact specifically with sulfated tyrosine residues on the Nt of CCR5.
There are four sulfonated tyrosine residues at positions 3, 10, 14, and 15 within the Nt of the receptor. Mutation of any of these sulfo-tyrosine residues to alanine has been found to impair viral entry efficiency, albeit to varying degrees [59,174]. The crystallization of gp120 in complex with the Nt of CCR5 and monoclonal antibody 412d provided further evidence for the importance of these sulfo-tyrosine residues in gp120 interactions [91].
The dichotomy between N-terminus and extracellular loop binding appears to have inherent variability amongst viral strains. Receptors with mutations in these regions mediate infection differently dependent on viral strain. For example, a CCR5 mutant receptor in which the Nt of CCR5 is replaced with the Nt of CCR2b mediated entry of
HIVJR-FL but did not efficiently mediate entry of HIV89.6 [184]. The vast diversity of HIV-
1 env coding sequence as well as the flexibility of the glycoprotein structure may explain
this variability. It is reasonable to imagine a model in which gp120-CCR5 interactions
43
fall within a spectrum for a given viral envelope (Fig. 11). Envelopes that depend heavily on Nt interactions would fall on one end of this spectrum while those relying mainly on ECL2 interactions would fall at the other end. Viral envelopes would most likely fall somewhere between these two endpoints with those depending equally upon
both interactions lying in the middle.
An additional consideration for both CD4 and coreceptor binding is the
stoichiometry of envelope subunits and receptors required to trigger entry events. While
some studies have suggested as many as five envelope trimers are required for entry [87],
others have proposed that a single trimer is sufficient to mediate HIV entry [241]. The
number of gp120 subunits within an envelope trimer that must engage CD4 and
coreceptor remains unclear as well. Work utilizing nonfunctional gp120 monomers
incorporated in envelope trimers predicted two functional gp120 subunits are adequate
[242] which conflicts with earlier reports that at least four CD4 receptor interactions are
required [115]. Thus, the issues of trimer number and gp120 subunit stoichiometry
remain largely unresolved. Equally important is the matter of receptor stoichiometry
necessary for entry. If multiple receptors are essential, then receptor cell surface density
would have significant implications for entry efficiency and gp120-receptor affinities.
CD4, CCR5, and CXCR4 densities can vary significantly in T cell lines used for in vitro
infections as well as in lymphocyte subpopulations [118]. In environments where
receptor numbers are limited, as in the presence of entry inhibitors, receptor
stoichiometry requirements could significantly impact entry.
1.7.4 Gp41 Mediated Membrane Fusion
44
The gp41 glycoprotein consists of three regions: a large ectodomain (Ecto) exposed on the outside of the virion as part of the envelope complex, a membrane- spanning domain (MSD) anchoring gp41 in the viral membrane, and a cytoplasmic tail thought to interact with MA in the virion. The gp41 ectodomain contains a hydrophobic fusion peptide at the N-terminal end. This fusion peptide inserts into the cellular membrane, tethering the virus to the cell. The ectodomain also contains two helical repeat regions: N-terminal helical repeat (NHR or HR1) and C-terminal helical repeat
(CHR or HR2). In a functional envelope complex, the HR1 regions of the three gp41 subunits pack together forming a homotrimeric, coiled-coil core structure [230]. During fusion, the CHR regions of the subunits fold around the NHR core structure in an antiparallel fashion to form a stable, six-helix bundle intermediate. Similar intermediate structures have been identified in the fusion events of influenza, Ebola, and Moloney murine leukemia viruses suggesting a common membrane fusion mechanism [25,60,229].
Formation of this intermediate brings the viral and cellular membranes into close proximity allowing creation of a fusion pore and the completion of viral entry.
Some evidence suggests that gp120 binding to CD4 not only induces changes in gp120 but also promotes changes in gp41. An increase in immunoreactivity of gp41 domains has been observed following sCD4 exposure [193]. Whether this increased reactivity is a result of significant gp41 structural changes or simply unmasking of gp41 epitopes as a result of gp120 structural changes remains unclear. It has been suggested that CD4 binding results in exposure of a pre-hairpin intermediate of gp41 in which the fusion peptide is inserted into the cell membrane but that coreceptor engagement is
45
required for six-helix bundle formation. Additional studies are required to clarify these
events.
1.8 Entry Inhibitors and Mechanisms of Resistance
As the first step in HIV infection, the process of viral entry into host cells has
long been considered an attractive target for intervention therapy. The extensive use of
other antiretroviral targets in HAART therapy, while greatly impacting mortality rates
among infected individuals, also heightened the need for novel drug classes targeting
alternative viral processes. The emergence of drug resistant viral strains in treatment
experienced patients as well as the transmission of resistant virus has driven the need for
the development of more effective treatments.
The complex, multi-step process of entry provides ample opportunities to
intervene in virus-cell interactions and prevent entry of the viral core into new target
cells. In addition to preventing the spread of infection within an individual, entry
inhibitors also represent a unique opportunity to minimize the transmission of virus between individuals when applied as a microbicide. An arsenal of inhibitors have been
developed to target the specific stages of entry and are discussed in the following sections
(Table 2).
1.8.1 Attachment Inhibitors
As the major receptor required for HIV entry, CD4 represents an attractive target
for preventing virus infection. Multiple approaches have been attempted to inhibit viral
attachment including targeting the CD4 receptor as well as the CD4 binding site on
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gp120. Although no CD4-targeting inhibitors are currently approved, some have
progressed to clinical trials with promising safety and efficacy results.
Early efforts to block gp120-CD4 interaction focused on a soluble form of the
CD4 receptor (sCD4), consisting of either all four (D1-D4) or the first two (D1D2) extracellular domains. Dependent on concentration and temperature, the binding of sCD4 to gp120 in in vitro experiments can have multiple effects on the envelope complex. In addition to competing with membrane anchored CD4, sCD4 can inhibit gp120 by inducing inactivation events which prevent normal entry processes. These inactivation events include 1) the decay of the CD4-induced conformation to an inactive conformation and 2) sCD4-induced shedding of gp120 from the envelope trimer
[144,145]. Although sCD4 showed promising efficacy against diverse HIV strains in vitro, therapy with sCD4 in vivo did not achieve reductions in viral load in HIV-infected patients [42]. It was later determined that higher dosages of sCD4 were required to inhibit virus entry of primary isolates than those required to inhibit laboratory adapted strains [158]. Despite the failure to suppress viral load in vivo, the successful targeting of the CD4 binding site in vitro launched efforts to develop derivatives of CD4 and mimetic compounds with improved treatment profiles.
One such CD4 derivative is a recombinant fusion protein between the D1D2
regions of CD4 and human IgG2 antibody developed by Progenics Pharmaceuticals, Inc.
called PRO 542 [150]. The heterotetrameric structure of PRO 542 enables it to bind up to
four gp120 subunits simultaneously, blocking gp120 binding to cellular CD4 and locking
gp120 into a nonfunctional conformation. Demonstrating activity against a diverse range
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of HIV strains in cell culture, PRO 542 proceeded to phase I/II clinical trials in 2003.
Further progress has not been reported since the completion of phase II trials in 2005.
Based on the specific interactions of CD4 with gp120, mimetic compounds have
been synthesized to target the CD4 binding site on gp120. These include the NBD-556
and NBD-557 compounds which were designed to project into the F43 cavity of gp120.
The NBD compounds were found to inhibit viral replication in both CCR5 and CXCR4
expressing cell lines, indicating inhibition of viral entry is not dependent upon viral
tropism [247]. These compounds were not able to inhibit a mutant, CD4-independent
viral strain suggesting their mechanism of action involved blocking the CD4-gp120
interaction. Structural modeling of NBD-556 with gp120 crystal structures indicated that
the chloro-phenyl ring of the compound projects even more deeply into the F43 cavity on
gp120 than the phenyl ring of F43 of cellular CD4 [132].
Another CD4 mimic, BMS-378806 and similar compounds have been developed
by Bristol-Myers Squibb. They too are believed to inhibit gp120 attachment to CD4 by
interacting with the F43 cavity. Although BMS-378806 was halted in phase I trials it is being pursued as a potential vaginal microbicide. A related compound, BMS- 663068, is currently undergoing phase IIa investigations with reports indicating good efficacy and safety profiles [175].
An alternative strategy to block viral attachment is to target the CD4 cellular receptor itself through antibody binding. A humanized monoclonal antibody, Ibalizumab
(previously TNX-355), targets the D2 extracellular domain of CD4 but does not prevent
CD4-gp120 binding. Instead it is believed to block conformational changes in gp120 induced by CD4 binding [146]. Clinical testing in HIV-infected patients of Ibalizumab in
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combination with optimized therapy has proved encouraging with sustained viral load suppression over long-term therapy [106]. However, the need for intravenous infusion may limit its use in clinical settings.
The cyclotriazadisulfonamide (CADA) compounds are a novel class of inhibitors which prevent nascent CD4 translocation from the ER membrane and thus result in CD4 down-modulation from the cell surface [223]. Medicinal chemistry efforts have resulted in a panel of CADA analogues with lead compounds demonstrating potent and broad inhibition of diverse HIV-1 strains. HIV-1 escape from the CADA compounds has been associated with changes in the C4 region of gp120 [222]. Although not directly located within the CD4 binding site, the S463P mutation observed by Vermeire et al, may stabilize a liganded conformation of gp120, increasing the ability to scavenge low CD4 surface receptor density. However, this CADA resistant virus was also found to be more sensitive to neutralization by patient serum. Indeed, increased sensitivity to neutralization has also been described for CD4-independent HIV-1 suggesting mutations which stabilize CD4-liganded conformations of gp120 do so at the risk of increasing neutralization potential for the virus [89]. In addition to increased sensitivity to neutralizing antibodies, the CADA resistant virus demonstrated increased replicative fitness, a trait also associated with resistance to the CD4 binding site monoclonal antibody b12 [26].
A major concern in targeting the CD4 binding site through CD4 derivatives or mimetics is the induction of the CD4-bound “activated” conformation of gp120, permitting coreceptor binding and potentially enhancing infection. Indeed, some of the first studies utilizing sCD4 noted the ability of viruses to enter CD4-negative cells in the
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presence of low nanomolar concentrations [35,209]. However, it has also been observed
that prolonged exposure to sCD4 mimics induces a transient activated state of gp120
which rapidly decays to a stable, nonfunctional conformation [82].
Selections of resistant variants in tissue culture have indicated a primary
resistance mechanism to CD4 mimetic compounds involving changes in the CD4 binding
site on gp120. Mutants resistant to NBD-556 were found to possess two mutations in gp120, S375N in the C3 region and A433T in the C4 region [244]. A previous study
indicated that mutations in gp120 residues surrounding or comprising the F43 cavity
negatively impacted the inhibitory effect of NBD-556 [132]. Similarly, mutations that
rendered a subtype B viral strain resistant to BMS-378806 (M426L and M475I) were
situated in the F43 binding cavity [122]. Thus, it appears despite the conserved nature of
this cavity, modification of residues to override drug inhibition is well tolerated.
1.8.2 Fusion Inhibitors
Since the early 1990s it has been known that synthetic peptides derived from the
amino acid sequences of the helical repeat regions of gp41 can inhibit HIV replication in
vitro [235,236]. The transitional exposure of the HR regions during pre-hairpin
formation presents an opportunity for competitive binding of these synthetic peptides to
prevent six-helix bundle formation.
The first entry inhibitor approved for use in patients was a peptide mimetic of a
portion of the HR2 region of gp41. This inhibitor, named enfuvirtide (T20) and marketed
as Fuzeon by Hoffmann-La Roche Ltd., interferes with conformational changes in gp41
necessary for mediation of viral and host cell membrane fusion. By mimicking the HR2
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region, T20 is able to bind the N-terminal heptad repeat (HR1) and prevent formation of the stable 6-helix bundle structure intermediate which aids in the stabilization of fusion
pore formation. Interference by T20 prevents delivery of the viral genome into the cell
and thereby prevents infection.
Resistance to T20 has been well characterized both in vitro and in vivo, with mutations in the HR1 region of gp41 most commonly associated with resistance. The
HR1 region is the target binding site for T20. Mutations conferring T20 resistance have mapped to positions 36-45 of gp41, with a GIV motif from 36-39 playing a predominate
role in resistance [47,179]. It has been shown that the amino acid at position 36 can
modulate the fusion kinetics of the envelope [100]. Mutations that reduce T20 binding
also appear to reduce six-helix bundle formation and overall fusion rates. Resistance to
T20 was associated with an increase in sensitivity to neutralization by antibodies
targeting gp41 [177].
In addition to T20, new fusion inhibitors are currently under development
including VIR-576, a peptide that targets the gp41 fusion peptide. VIR-576 demonstrated efficacy in a small group of treatment-naïve patients by reducing viral loads an average of
95% over ten days of monotherapy [66]. However, it is unlikely that this inhibitor or other peptide fusion inhibitors will be developed for clinical use due to high production costs and intravenous administration costs. Currently, small molecule fusion inhibitors
targeting gp41 are under investigation.
1.8.3 Inhibitors of gp120-CXCR4 Interaction
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Targeting the CXCR4 chemokine coreceptor presents unique challenges given
that genetic deletion of the cxcr4 gene or its ligand, CXCL12 (previously SDF-1)
severely impairs development and causes embryonic lethality in mice [152,212]. In
adults, CXCL12 is a strong chemotactic signal and through CXCR4 signaling regulates
hematopoietic stem cell development and migration.
Attempts to target CXCR4 for the prevention of HIV usage as a coreceptor have
resulted in multiple small molecule inhibitors; however, none are currently approved for
clinical use due to poor bioavailability and toxicity issues. The bicyclam AMD3100
efficiently inhibits X4 tropic virus entry with IC50s in the nanomolar range and is used as
a research tool to block CXCR4 virus entry [195]. Multiple derivatives of AMD3100 in
addition to other CXCR4 targeting inhibitors are in development, however none are
approaching clinical acceptance for HIV treatment.
1.8.4 Inhibitors of gp120-CCR5 Interaction
The first indication that CCR5 was a viable target for HIV therapeutic
intervention was the discovery of a 32 base-pair deletion in the ccr5 gene resulting in dysfunctional surface receptor expression in individuals homozygous for the ccr5Δ32
allele [124]. Homozygosity bestows relative protection against HIV infection by R5
tropic virus strains, although infection by X4 or dual tropic virus is still permissible [191].
Individuals heterozygous for ccr5Δ32 exhibit lower receptor expression and those
infected with HIV tend to progress less rapidly to disease. Despite the lack of surface
CCR5 expression, no apparent immunologic disadvantageous effect has been associated
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with ccr5Δ32. These observations led the way for the development of inhibitors that
could target and block CCR5 and HIV envelope interactions.
1.8.4.1 Chemokine Analog Inhibitors
Among the first attempts to block gp120-CCR5 binding was the development of
chemokine analogs by modification of the N-terminus of the natural CCR5 ligand
RANTES. Chemokine ligand binding to receptor induces signaling cascades resulting in lymphocyte activation and chemotaxis. The aim of modifying the RANTES chemokine was to limit induction of signal transduction cascades that would result in activation while retaining high affinity binding to the receptor. The N-terminus was chosen for modification because the N-terminus of chemokine ligands contributes to the induction of the signaling cascade while chemokine receptor specificity is attributed to the core of the ligand [22]. Addition of an aminooxypentane moiety to the N-terminus of RANTES resulted in an analog with increased potency against HIV replication compared to the native ligand [204]. Although this AOP-RANTES derivative did not induce chemotaxis upon binding it did induce calcium flux [182]. This could potentially result in activation of HIV replication and limit the efficacy of these chemokine analogs. Further modification of RANTES N-terminus resulted in an analog with even higher potency than AOP-RANTES [85]. The PSC-RANTES analog has been shown to block vaginal
HIV transmission in the SHIV-macaque model and is being developed as a potential microbicide [116].
Chemokine ligand binding not only induces a signal cascade but also results in receptor internalization through a clatherin-dependent endocytic pathway [203]. Similar
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to the natural ligand, the RANTES derivatives are likewise able to induce internalization of CCR5 although intracellular receptor sequestration was prolonged for PSC-RANTES.
It was believed that receptor internalization was the primary mechanism of action for
PSC-RANTES and is likely the mode of inhibition in single cycle drug sensitivity assays.
However, recent studies have shown differential sensitivity to this inhibitor in multiple cycle assays lending support to a competitive inhibition model [126]. These issues will be addressed in greater detail in Chapter 2.
Resistance to PSC-RANTES was described in the context of a SHIV virus used in vaginal transmission challenge of macaque monkeys [53]. Mutations associated with resistance were identified in the V3 loop (K315R) as well as the ectodomain of gp41
(N640D). The PSC-RANTES resistant virus was also less sensitive to inhibition by the allosteric inhibitor TAK-779 than was the control virus. The V3 mutation at 315 is in a location of gp120 that binds to the same site on CCR5 as does PSC-RANTES and could therefore alter gp120 interactions with CCR5 at this site. The gp41 mutation at 640 is located within HR2 region and may influence fusion kinetics of this virus.
1.8.4.2 Small Molecule Antagonists
Several small molecule antagonists of CCR5 are in various stages of development. These compounds have been shown to effectively inhibit HIV replication in vivo resulting in the first CCR5 antagonist approved for use in patients, maraviroc
(MVC), receiving FDA approval in 2007. This class of inhibitors functions through an allosteric mechanism where receptor binding induces altered conformations of the ECLs and prevents HIV envelope recognition and coreceptor engagement. Maraviroc and other
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CCR5 antagonists including vicriviroc (VVC), aplaviroc (APL), and TAK-779 share a common binding site within the transmembrane cavity of CCR5 (Fig. 9). This binding site does not overlap the binding site of either CCR5 agonists or the HIV envelope.
Alanine scanning mutagenesis of the transmembrane domains of CCR5 revealed key residues in domains 1, 2, 3 and 7 comprise the small molecule binding cavity [51]. The
conformational changes in the ECLs induced by binding of these individual inhibitors are
predicted to differ [104]. This may explain why some viral strains resistant to one
inhibitor have been shown to retain sensitivity to another inhibitor in this class.
However, binding induces receptor conformations that are not recognized by either CCR5
ligands or the HIV envelope glycoproteins.
Identified in a chemokine binding assay screen of a Pfizer compound library,
maraviroc is an imidazopyridine that antagonized β-chemokine binding and signaling with IC50s in the nanomolar range (Fig. 12) [49]. Maraviroc inhibited RANTES, MIP-1α,
and MIP-1β induced signaling of intracellular calcium redistribution, however did not
trigger calcium signaling or receptor internalization upon binding. Reductions in basal γ-
S-GTP binding suggested some inverse agonist activity for maraviroc potentially related
to formation of inactive states of CCR5. In the same study, maraviroc was shown to have
potent antiviral activity against a diverse panel of primary R5 HIV-1 isolates with a mean
IC90 of 2nM.
The MOTIVATE 1 (conducted in North America) and MOTIVATE 2 (conducted
in Europe, Australia, and the US) phase III clinical trials sought to study the safety and
efficacy of maraviroc in treatment experienced patients. MOTIVATE stands for
Maraviroc versus Optimized Therapy In Viremic Antiretroviral Treatment Experienced
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patients. Results from these trials published in 2008 indicated patients receiving maraviroc with an optimized background therapy (OBT) showed significant reductions in
HIV-1 RNA levels and increases in CD4 cell counts versus patients receiving OBT alone
[61,81]. Preliminary data resulted in FDA approval in 2007 for clinical use in salvage therapy regimens. Since then the indication has expanded to allow use in first line drug
regimens in combination with nucleoside analogs. It is currently marketed as Selzentry
by Pfizer, Inc.
In addition to maraviroc, vicriviroc and aplaviroc have both been shown to inhibit
HIV replication in humans. However, vicriviroc development was discontinued after
preliminary phase III clinical data while hepatic toxicity issues halted development of
aplaviroc [155].
1.8.4.3 Resistance to CCR5 Antagonists
Since entry inhibitors bind to host receptors and not directly to viral proteins,
unique and complex resistance profiles are likely to emerge. Potential pathways of
resistance include 1) alternative coreceptor usage (utilization of CXCR4 instead of CCR5
for entry), 2) enhanced entry kinetics, 3) increased receptor affinity and 4) utilization of
inhibitor-bound receptor for entry.
A primary concern in targeting CCR5 was that resistance would emerge as a
change in coreceptor usage for entry. As previously mentioned, multiple receptors have
been identified as potential coreceptors, however CCR5 and CXCR4 are the only known
coreceptors utilized in vivo. With rare exceptions, R5 HIV virus establishes new infections and predominates in asymptomatic stages of disease. However, in
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approximately half of patients, X4 variants emerge during the course of disease. The underlying mechanisms and implications of this coreceptor switching remain poorly understood at this time. Whether the emergence of X4 variants are a consequence or cause of disease progression remains unclear. In vitro selections of entry inhibitor resistant viruses have largely utilized PBMC cultures which express both CCR5 and
CXCR4. However, when dual/X4 tropism was not pre-existing in the viral swarm, mutations conferring altered coreceptor usage were not the favored resistance pathway.
Resistant viruses retained R5 tropism and were not able to infect CCR5 negative cell lines. Clinically, early reports from the MOTIVATE trials describing failure to maraviroc were attributed to coreceptor switching; however, advancements in the sensitivity of tropism testing revealed the pre-existence of X4 viruses in patients prior to the start of treatment [61]. Of the 133 patients who failed maraviroc treatment, tropism testing revealed that 76 patients had dual/mixed tropic virus. Interestingly, the level of dual/X4 virus diminished rapidly in these patients once maraviroc treatment ceased [232].
A study specifically evaluating the effects of maraviroc in patients with non-R5 tropic virus found no benefit to maraviroc treatment versus OBT [187]. Given the uncertainty regarding consequences of X4 virus emergence for disease progression, these studies recommended maraviroc treatment be limited to patients with only R5 HIV. As a result, tropism testing is required by the FDA for all patients under consideration for maraviroc therapy.
If altered coreceptor usage is not the preferred resistance pathway to CCR5 agonists or antagonsits, than alternative mechanisms must represent more attractive avenues for viral escape. Two models for entry inhibitor resistance have been proposed:
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competitive and noncompetitive. In the competitive resistance model (Fig. 13A), gp120
binds to CCR5 with a given affinity. Inhibitors, such as PSC-RANTES, that bind similar
regions of the coreceptor demonstrate higher binding affinity for those regions than
gp120 and prevent envelope engagement. Resistance to these inhibitors manifests
through acquired mutations in gp120 which increase coreceptor affinity and promote
inhibitor displacement. Competitive resistance is exhibited in drug sensitivity assays as
increases in inhibitor concentration required to achieve half-maximal inhibition (IC50).
This shift in IC50 value is typical of resistance to most other antiretroviral classes such as
reverse transcriptase and protease inhibitors. However, resistance to inhibitors that bind
allosteric regions of CCR5, such as small molecule antagonists, are predicted to follow
noncompetitive resistance pathways (Fig. 13B). The binding of allosteric inhibitors such
as maraviroc alter coreceptor conformation, preventing recognition and binding of gp120.
Mutations in gp120 that confer resistance to this type of inhibitor may permit gp120
recognition and binding of inhibitor-bound forms of the coreceptor. An HIV envelope
capable of utilizing inhibitor-bound receptor could maintain a level of entry despite increasing concentrations of drug, exhibiting a plateau effect where the maximum inhibition achieved remains steady at high drug concentrations but never reaches 100%.
The highest level of inhibition achieved is termed maximal percent inhibition (MPI). The
MPI level is modulated based on the efficiency with which the viral envelope is able to use the inhibitor-bound versus inhibitor-free forms of the coreceptor.
The dynamic relationship between CD4 and coreceptor binding affinity as well as the overall kinetic rate of the entry process could play a major role in the sensitivity of primary isolates to entry inhibitors. Studying the specific contributions of these
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processes separately has proven difficult; however general observations and inferences have been made. The intrinsic sensitivity of primary isolates to entry inhibitors can vary as much as 1000-fold in IC50 values [216], a level not observed for other antiretrovirals such as PIs or RTIs. Factors such as the extreme diversity in the env coding region, flexibility of the envelope glycoproteins, differential affinity for CD4 and CCR5, and variable rates of six-helix bundle formation may account for some of this variation.
Indeed, differential sensitivity to the fusion inhibitor T-20 as well as the CCR5 antagonist
TAK-779 have been attributed to kinetic fusion rate nuances [176], while V3 loop polymorphisms have been shown not only to contribute to coreceptor affinity and viral fitness differences but also differences in sensitivity to PSC-RANTES [126].
In addition to influencing entry inhibitor sensitivity, receptor affinity and entry kinetics are thought to contribute significantly to viral replicative fitness. Fitness is defined as the replication capacity of a virus in a given environment. Viral fitness in vivo is influenced by many parameters including host immune response, virus mutation/turnover rates, and the presence of antiretrovirals. Reduced fitness is often associated with resistance to RTIs and PIs when measured in ex vivo competitions in
PBMCs [173]. However, viral entry plays a major role in determining overall replication efficiency and therefore significantly impacts overall fitness. Mutations in the envelope that influence receptor affinity and entry rates, such as might be acquired in the development of resistance to entry inhibitors, may also significantly influence viral fitness. For example, a virus able to outcompete inhibitors such as PSC-RANTES for receptor occupancy would demonstrate reduced sensitivity to this inhibitor but may also demonstrate increased rates of entry and increased viral fitness. Indeed, studies of
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primary HIV-1 isolates have described a direct relationship between replicative fitness
and sensitivity to entry inhibitors. Envelopes of viruses taken from individuals with elite
suppression displayed reduced entry efficiency, slower entry kinetics and increased
sensitivity to entry inhibitors [114]. Likewise, a strong correlation between replicative
fitness and sensitivity to T-20, PSC-RANTES, and the CCR5 antibody 2D7 was
described in viruses containing V3 loop polymorphisms [126]. These data would suggest
that competitive binding and overall rates of the entry process not only account for
differences in sensitivity to entry inhibitors, but also influence overall replicative fitness.
While competitive mechanisms, evident as shifts in IC50 concentrations, have been observed for viruses resistant to PSC-RANTES [53] and T-20 [176], viruses resistant to small molecule antagonists have largely exhibited noncompetitive resistance profiles. An inability to achieve 100% inhibition at high drug concentration has been observed in multiple instances of CCR5 antagonist resistance. Despite these reductions in MPI, no apparent shifts in IC50 value have been noted.
There is limited data available from in vitro studies of CCR5 antagonist resistant viruses. Selection of entry inhibitor resistant viruses requires longer passage in tissue culture than previous studies selecting for PI or RTI resistance. This is in part due to the necessity of utilizing R5 HIV-1 primary isolates for tissue culture passage as opposed to
X4-tropic laboratory adapted strains which are commonly used to generate resistant variants to PIs and RTIs. R5-tropic viruses typically replicate slower than X4-tropic strains potentially due to coreceptor affinity differences and variable receptor density in cell lines [173]. These issues have significantly limited the selection of small molecule resistant viruses.
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Currently, only two reports have generated maraviroc resistant variants by
sequential passage of virus in the presence of inhibitor. The first report of R5 HIV maraviroc resistance came in 2006 when Westby et al. described resistant variants derived from two different HIV-1 primary isolates; RU570 (clade G) and CC1/85 (clade
B) [233]. Each resistant virus contained mutations in the V3 loop region (Fig. 14). The
CC1/85-res virus possessed A316T and I323V mutations while the RU570-res virus had a three amino acid (QAI) deletion from residues 315-317 in the V3 loop. While both viruses had mutations in gp120 regions outside the V3 loop, site-directed mutagenesis studies implicated the V3 mutations in the resistance phenotype. The observed reduction in MPI for the resistant viruses was the first indication that utilization of inhibitor-bound receptor to mediate entry may be a viable resistance pathway to small molecule antagonists. The only other report of maraviroc resistance isolated in vitro utilized
HIVV3Lib, a V3 loop library virus containing combinations of polymorphisms in this
region to isolate resistant variants [245]. Similarly to the Westby et al study, resistance
manifested as a reduction in MPI with V3 loop mutations
(I304V/F312W/T314A/E317D/I318V) associated with the resistance phenotype.
Since clinical approval of maraviroc, rare instances of R5 tropic viral resistance in patients have been reported. The first reports of treatment failures not related to X4 virus came from the MOTIVATE trials. Clonal analyses of envs from viruses of four patients failing maraviroc therapy who remained R5 tropic revealed reductions in MPI.
Genotypic analyses revealed unique V3 loop mutations for each virus [148]. However, it is important to note here that the cause of approximately one-third of R5 virus maraviroc treatment failures remain undetermined at this time.
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Studies have indicated resistance to other CCR5 antagonists (i.e. aplaviroc, vicriviroc, AD101, TAK-779) developed through similar mechanisms. Although cross- resistance has not been fully described for this class of inhibitors, mutations that alter sensitivity to one inhibitor may impact envelope binding affinity and entry efficiency and therefore influence sensitivity to another inhibitor.
Vicriviroc resistant primary isolates vary widely in adaptive amino acid changes observed in the envelope glycoproteins. Resistance in a subtype C infected individual participating in a phase IIb clinical trial of vicriviroc was attributed to the S306P mutation in the V3 loop [219]. Different V3 loop mutations (K305R/R315Q/ K319T) in combination with other changes in gp120 (A281T/ T413N/ P437S/ I467T) were identified in resistant virus derived from the clade G primary isolate RU570 (Fig. 14) [157]. In contrast to these studies, other reports described vicriviroc resistant viruses that lacked changes in the V3 loop. A resistant isolate derived in vitro from virus CC1/85 developed multiple changes in gp120 (V169M/K171R/G354P/N355Δ) but lacked changes in the V3 loop region [138]. Unexpectedly, mutations in the gp41 region
(G518V/M520V/F521I/L604I/ D626N/N638D/N646S/E664A) were identified in this resistant virus, although the specific mutations conferring resistance were not fully identified [6]. This same primary virus was cultured with the pre-clinical precursor of vicriviroc, AD101, and the resultant resistant clone possessed four amino acid mutations in the V3 loop region (K305R/H308P/A316V/G321E) necessary and sufficient for
AD101 resistance [105].
Several of the studies describing generation of resistant viruses have utilized either primary isolate CC1/85 or the clade G virus RU570 to generate resistance to
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maraviroc, vicriviroc, or AD101. Astonishingly, use of these two viruses has generated multiple resistant variants with dissimilar genetic pathways to resistance; however, despite the breadth of genotypic mutations described, a mechanism involving utilization of inhibitor-bound receptor has emerged in multiple instances.
Resistant variants have also been characterized for the CCR5 antagonists TAK-
779 and TAK-652. Passage of virus for over one year with dose escalations of TAK-652 resulted in a mutant exhibiting greater than 200,000-fold resistance to this inhibitor in
PBMC cultures [11]. Twelve mutations were identified in the resistant virus that were absent in the parental virus population including mutations in the V3 loop
(T306K/Q309E) as well as mutations in other regions of gp120
(K221N/T401I/T403I/M422I/A424N) and gp41(M506V/S637A/L690I/V766A/I769S).
The specific contributions of these mutations to resistance were not analyzed, however the reduction in MPI observed would again suggest a noncompetitive mechanism of resistance. Similarly, resistance to TAK-779, a compound structurally related to TAK-
652, developed over the course of several weeks of incubation with a HIVJR-FL V3 library virus containing a combination of V3 loop mutations [246]. Five V3 loop mutations
(I304V/H305N/I306M/F312L/E317D) were associated with 15-fold resistance to TAK-
779 (Fig. 14).
Resistance to aplaviroc has been described from clinical trials with resistant viruses demonstrating reductions in MPI as well [101,214]. Mutations associated with resistance to aplaviroc were scattered across the gp120 and gp41 regions [101], however clonal analyses revealed viruses capable of utilizing aplaviroc bound conformations of receptor even prior to initiation of treatment [214].
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While the particular mechanisms by which resistant viruses that maintain the
ability to use CCR5 for entry remain poorly defined, two potential mechanisms can be
suggested from this data and the location of resistance mutations in the V3 loop (Fig. 14).
First, resistant viruses could have an increased dependence on CCR5 Nt interactions and
greater tolerance for inhibitor-induced alterations to the ECLs (Fig. 11). Several studies
have indicated increased dependence on Nt interactions for resistant viruses
[18,156,157,166]. A second mechanism would involve mutations permitting the V3 loop
to interact with conformations of ECL2 that result from drug binding. Tilton et al.
suggested a model in which a combination of these mechanisms can result in resistant
viruses requiring ECL2 interactions but these requirements limit cross resistance to other
small molecule antagonists due to the differences in ECL2 conformations induced by
each inhibitor [215]. In this study, a maraviroc resistant virus isolated from a patient,
harboring V3 mutations (P/T308H, T320H, and I322aV), remained sensitive to inhibition
by aplaviroc and vicriviroc but was cross resistant to TAK-779. This resistant virus was
not only sensitive to alanine substitutions in CCR5 mutant receptors located in the Nt, as occurs with most resistant viruses, but was also sensitive to mutations in the ECLs.
These data suggest viruses that adapt to depend more heavily on interactions with the Nt, which is relatively unaffected by inhibitor binding, may demonstrate significant cross resistance to other antagonists while viruses that adapt to interact with ECL conformations induced by inhibitor binding may not recognize ECL structures induced by different inhibitors.
In summary, mutations associated with resistance to CCR5 antagonists in vitro have predominantly mapped to the V3 region of gp120 with the rare exception of viruses
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with resistant mutations in other regions of gp160. However, resistance in patients where
X4 virus outgrowth did not play a role has largely been attributed to V3 mutations arising during therapy, resulting in usage of inhibitor-bound conformations of CCR5 for viral entry.
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Figure 1. HIV-1 Genome. The HIV-1 genome consists of three ORFs and encodes fifteen proteins. The genome is approximately 9.7 kilobases and contains the necessary structural and enzymatic proteins for viral replication. The genome exists in a mature virion as single-stranded, positive sense RNA which is reverse transcribed to double- stranded proviral DNA in host cells.
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Figure 2. HIV-1 Virion. A mature virion contains two copies of ssRNA genome inside a conical shaped core structure. Also contained within this core are reverse transcriptase, protease, integrase, and nucleocapsid (not shown). The core is surrounded by matrix proteins supporting a lipid bilayer membrane. Trimeric envelope spikes composed of gp120 and gp41 monomers are embedded within the membrane.
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Figure 3. HIV Replication Cycle. 1. The replication cycle begins with envelope binding to CD4 and coreceptor, either CCR5 or CXCR4. Membrane fusion results in release of the core containing genomic RNA into the cell cytoplasm. 2. The process of reverse transcribing the ssRNA genome into dsDNA takes place. 3. Once reverse transcribed, the DNA, associated with viral and cellular proteins as part of a pre- integration complex, is actively transported to the nucleus where it is integrated into cellular DNA. 4. Cellular transcription/splicing machinery produce mRNA transcripts from which viral proteins are produced. 5. At the cell membrane, gag and gag-pol polyproteins aggregate and associate with full length viral RNA while envelope glycoproteins associate and stud the membrane surface to promote budding and assembly of new virions. Following budding, the immature virion undergoes maturation with cleavage of polyproteins and formation of the core structure resulting in an infectious virus particle.
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Figure 4. Model of HIV Entry. (1) The first step in the HIV entry process involves attachment of the envelope glycoprotein gp120 to a CD4 molecule on a target cell. Engagement with CD4 induces structural rearrangements in gp120, exposing the V3 loop (yellow) and coreceptor binding site. (2) Following CD4 attachment, the V3 loop and bridging sheet interact with the N-terminal tail and extracellular loops of a chemokine coreceptor, either CCR5 or CXCR4. Coreceptor binding induces further conformational rearrangements leading to exposure of the gp41 fusion peptide. (3) The fusion peptide inserts into the cellular membrane leading to destabilization of the bilipid membrane. (4) To simplify, CD4, coreceptor, and gp120 have been excluded. The N terminal and C terminal heptad repeat regions of gp41 fold onto each other creating a stable six-helix bundle structure. This folding brings the viral and cellular membranes into close proximity, permitting lipid mixing and creation of a fusion pore through which the HIV core enters the cell cytoplasm.
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Figure 5. Model of Reverse Transcription. 1. The tRNALys3 binds to the primer binding site on the ssRNA genome. 2. Minus-strand DNA synthesis proceeds from the tRNA in a 5’ to 3’ direction. 3. The RNaseH domain of RT degrades the genomic RNA bound to the newly synthesized DNA. 4. The minus-strand strong stop DNA is transferred to the 3’ end of the RNA genome where the homologous repeat region, R, binds to the RNA. 5. DNA synthesis proceeds from the R region. 6. RNaseH activity degrades the genomic RNA except the polypurine tract, PPT, located in the env region. 7. Plus-strand DNA synthesis proceeds 5’ to 3’ from the PPT. 8. RNaseH digests the PPT and tRNA from the DNA. 9. The second strand transfer event results in binding at the complementary PBS site. 10. The minus-strand DNA folds over the plus-strand DNA so that the U5, R, and U3 regions bind. Second strand synthesis concludes with the resultant dsDNA genome containing long terminal repeat (LTR) regions at both the 5’ and 3’ ends.
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Figure 6. Nuclear Import and Integration. (A) The pre-integration complex (PIC) consists of the reverse transcribed dsDNA genome (black), reverse transcriptase (RT, blue), integrase (IN, red), matrix (MA, green), nucleocapsid (NC, orange), vpr (yellow), and cellular nuclear import factors such as importin α (brown). The PIC complex translocates from the cellular cytoplasm to the nucleus through a nuclear pore complex. (B) Integration initiates with integrase cleaving the last two nucleotides from the 3’ ends of each DNA strand. During strand transfer, integrase joins the 3’ recessed ends of HIV DNA with cellular DNA strands through a staggered cleavage event. Cellular DNA repair machinery fills in the gap of unpaired cellular nucleotides. The 5’ unpaired nucleotides of the HIV DNA are cleaved and the cellular and viral DNA ligated together. The fully integrated viral DNA is referred to as the provirus.
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Figure 7. HIV mRNA Transcription and Splicing. (A) The transcription complex includes the HIV tat protein as well as cellular RNA polymerase II complex. Proccessive elongation is promoted by cyclin T1 recruitment of CDK9 and hyperphosphorylation of RNA polymerase II. (B) Multiple transcription products are produced from the HIV genome. Incompletely spliced mRNAs are transported out of the nucleus in a rev dependent manner while fully spliced products are transported through cellular mechanisms.
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Table 1. List of FDA Approved HIV Antiretrovirals.
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Figure 8. Structure of gp120. The structure of gp120YU-2 solved in complex with CD4, the N-terminus of CCR5 and monoclonal antibody 412d is shown in cartoon (PDB: 2QAD, [91]). CD4 and 412d are not shown. (A) The inner domain (red), outer domain (yellow), bridging sheet (blue), and third variable loop (green) are indicated. (B) The gp120 core is shown in blue with disulfide cysteine bonds shown in red and N-linked glycosylation sites indicated by yellow spheres. (C) The approximate CD4 binding site (yellow) spans the inner and outer domains as well as the bridging sheet. The putative CCR5 binding sites (green) are located on the bridging sheet and third variable loop. (D) The CD4 and coreceptor binding sites comprise a target area for neutralizing antibody binding (brown). The heavily glycosylated outer domain makes up an immunologically silent face (blue) while the inner domain is relatively unexposed in the envelope trimer and therefore elicits a non-neutralizing response (red).
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Figure 9. Structure of CD4 and Role in Immune Activation and HIV Entry. (A) The D1/D2 fragment of surface CD4 is shown with residues F43 and R59 indicated (PDB: 1CDJ; [238]). (B) CD4 contributes to MHC class II complex recognition by T cell receptor complex in antigen presentation and immune activation. In HIV entry, gp120 binds the same region of the D1 fragment of CD4 as MHC II molecules.
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Figure 10. Model of CCR5 Structure and Antagonist Binding Site. Cartoon representation of predicted CCR5 structure. Transmembrane helices are numbered and marked. The putative small molecule binding site is indicated (brown). Adapted with permission from [225].
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Figure 11. Model of Interactions Between CCR5 and HIV gp120. HIV’s gp120 envelope glycoprotein interacts with the CCR5 receptor at two main sites: the N-terminal tail (Nt) and the 2nd extracellular loop (ECL2). The diversity of the gp120 coding sequence as well as the plasticity of the glycoprotein structure contribute to differential binding affinities for these sites for diverse envelopes. Hypothetically, HIV envelopes may fall within a spectrum regarding the importance of these interactions in inducing the conformational changes within gp120 to mediate entry. In the model, viruses that depend more heavily on interactions with sulfonated tyrosine residues found on the Nt tail of CCR5 would fall on the left side of the spectrum while those relying heavily on interactions between the V3 loop tip and ECL2 would fall on the right side of the spectrum. Those viruses relying equally on Nt and ECL2 would fall in the middle.
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Table 2. HIV-1 Entry Inhibitors.
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Figure 12. Chemical Structure of Small Molecule CCR5 Antagonists. The chemical structure of five CCR5 antagonists are shown. Reprinted with permission from American Society for Pharmacology and Experimental Therapeutics [104], copyright (2008).
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Figure 13. Mechanisms of Resistance to Entry Inhibitors. (A) Competitive resistance model in which shifts in IC50 concentration are indicative of resistance. Resistance to inhibitors that bind the same region of CCR5 as ligands and HIV gp120 (ex. PSC-RANTES) are predicted to follow competitive resistance pathways. (B) Noncompetitive resistance model in which maximal inhibition is not achievable despite increasing inhibitor concentrations. The maximal level of inhibition achieved is called maximal percent inhibition (MPI). Inhibitors that bind to allosteric regions (ex. maraviroc) and induce altered receptor conformations are predicted to follow noncompetitive resistance pathways.
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Figure 14. V3 Loop Mutations Associated with CCR5 Antagonist Resistance. (A) The V3 loop sequence of laboratory adapted strain HXB2 is shown. Sequence numbering is relative to this virus. Mutation sites associated with resistance to the CCR5 antagonists TAK-652 (yellow), maraviroc (blue), and vicriviroc (red) are indicated. Mutation sites associated with resistance to multiple inhibitors including maraviroc, vicriviroc, aplaviroc, TAK-779 and TAK-652 are indicated in green. (B) The HIV-1YU-2 V3 loop structure is depicted with resistance mutation sites identified as in panel A (PDB:2QAD; [91]).
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Summary of Thesis Work
HIV-1 antiretrovirals that target viral entry via CCR5 are a burgeoning new class
of inhibitors which have experienced extensive investigation over the last decade. The
studies herein sought to determine the mechanisms of inhibition of these CCR5
agonists/antagonists. In addition, a major aim of this investigation was to identify
mechanisms of HIV-1 resistance to these inhibitors.
Inhibitors targeting CCR5 fall within two categories: agonists and antagonists.
The primary mode of inhibition of agonists such as PSC-RANTES was believed to involve downregulation and prolonged sequestration of the receptor from the cell surface thus depriving the viral envelope of the ability to use CCR5 for entry. In contrast, antagonists such as maraviroc are considered allosteric inhibitors, binding the receptor and blocking entry by inducing structural changes which impair viral envelope recognition.
Differential sensitivity of chimeric viruses containing V3 loop mutations to PSC-
RANTES suggested an alternative mode of inhibition for this compound [126]. Here we demonstrate that although receptor downregulation does contribute to PSC-RANTES inhibition, the primary mode of inhibition involves competitive binding of CCR5.
Sensitivity of viruses to PSC-RANTES inhibition can be modulated by mutations that impact CCR5 binding affinity and viral resistance to PSC-RANTES may develop via competitive resistance pathways.
Resistance to small molecule CCR5 antagonists has been demonstrated to occur via genetic pathways that permit recognition and binding of structurally altered receptor conformations. However, despite significant efforts to identify common pathways to
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resistance, the few instances of maraviroc resistance identified have each differed in
adaptive mutations leading to the resistance phenotype. We sought to evaluate resistance
to maraviroc through the isolation of an escape mutant generated in vitro. Over six months, a primary isolate developed resistance to maraviroc through reduced maximal inhibition as well as increased half-maximal inhibition concentrations. Characterizations of gp120 chimeric virus derived from the resistant variant implicated a single mutation in the CD4 binding site of gp120 as responsible for resistance. Unlike previously described resistant viruses, resistance was not associated with use of inhibitor-bound CCR5 but was rather attributed to enhanced CD4 binding affinity through a competitive mechanism.
Our data suggests a novel mechanism for CCR5 antagonist resistance related to enhanced
CD4 binding affinity.
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CHAPTER 2
MECHANISTIC STUDIES OF HIV-1 ENTRY INHIBITORS REVEAL DIFFERENTIAL SENSITIVITY OF HIV-1 TO PSC-RANTES INHIBITION INVOLVES COMPETITIVE CCR5 BINDING
Authors: Michael A. Lobritz1,2*, Annette N. Ratcliff1,2*, Andre J. Marozsan2,3, John C.
Tilton4, Eric J. Arts2
1Department of Molecular Biology and Microbiology
2Division of Infectious Diseases, Department of Medicine
3Department of Pharmacology
4Center for Proteomics and Bioinformatics
Case Western Reserve University, Cleveland, OH, USA 44106
* Authors contributed equally to the work presented herein
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2.1 Preface
Prior to my involvement in this study, a previous graduate student (Michael
Lobritz, M.D., Ph.D) performed a series of experiments using chimeric viruses harboring
V3 loop polymorphisms to evaluate the mechanism of PSC-RANTES inhibition. He
observed a 10-fold difference between chimeric viruses to inhibition in multiple
replication cycle assays as well as in the absence of CCR5 receptor internalization
leading to a hypothesis that PSC-RANTES inhibits through a competitive binding mechanism. We aimed to expand these findings by examining inhibition of a PSC-
RANTES resistant virus previously isolated from a SHIV-macaque challenge by another graduate student (Dawn Dudley, Ph.D.). We also wanted to compare PSC-RANTES
inhibition with that of the CCR5 antagonist maraviroc to better understand differences
between competitive and allosteric inhibition.
Chimeric viruses were in part produced by Michael Lobritz who also generated
mutant M7-CCR5 cell line and performed PSC-RANTES inhibition, prolonged
incubation, and time of drug addition assays [125]. Andre Marozsan generated chimeric
V3 viral genomes. Viruses R3 and S2 were kindly provided by John C. Tilton.
Generation of gp120 and ecto-MSD chimeric viruses as well as PSC-RANTES and
maraviroc inhibition assays were performed by the author.
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2.2 Abstract
Small molecule CCR5 antagonists, such as maraviroc, block HIV-1 replication
through allosteric, noncompetitive inhibition of viral envelope binding to coreceptor.
However, the mechanism(s) by which anti-CCR5 antibodies, chemokines, or chemokine
derivatives inhibit the use of CCR5 by HIV-1 have not been resolved. The chemokine analogue PSC-RANTES has the potential to inhibit HIV-1 envelope by mediating CCR5 receptor downregulation from the cell surface or by occupying CCR5 on the cell surface and occluding envelope binding. We explored the inhibition mechanisms of maraviroc and PSC-RANTES by employing viruses with differential sensitivities to these inhibitors.
Two CCR5-tropic HIV-1 clones that differ by two amino acids in the V3 crown demonstrated equivalent sensitivity to PSC-RANTES inhibition in single-cycle replication assays, indicating effective receptor downregulation. However, in multiple- cycle assays or with a CCR5 mutant that is not downregulated, CCR5-PSC-RANTES
complexes became recalcitrant or simply unable (respectively) to internalize over time.
Differential PSC-RANTES inhibition of the V3 clones was now mediated by competitive
binding for CCR5. These results were recapitulated using a PSC-RANTES resistant virus isolated from SHIV macaque vaginal challenge. Increasing virus concentration could saturate the receptors on cell surfaces and overcome PSC-RANTES inhibition. In contrast, viruses resistant to maraviroc demonstrated differential sensitivity under all assay conditions, indicating allosteric inhibition and noncompetitive resistance mechanisms. We find that potent inhibitory activity of PSC-RANTES relates to
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competitive CCR5 binding with receptor downregulation providing minimal contribution to overall HIV-1 inhibition under physiological conditions.
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2.3 Introduction
HIV-1 entry involves the sequential interaction of the viral envelope glycoprotein
(gp160) with human CD4 and a chemokine receptor, either CCR5 or CXCR4.
Pharmacologic efforts to interrupt the coreceptor-dependent entry process have yielded a
wide variety of molecules which inhibit through divergent mechanisms. Studies aimed at
uncovering mechanism(s) of action have shown that small molecule CCR5 antagonists
(i.e. maraviroc, vicriviroc, aplaviroc) bind to an allosteric site within the transmembrane
helices of CCR5 [51,198,218]. Inhibitor binding prevents interactions between HIV-1
envelope and CCR5 through a noncompetitive mechanism [170,233]. However, little is
known about the mechanism(s) of HIV-1 inhibition by chemokines (or their derivatives)
or monoclonal CCR5 antibodies. PSC-RANTES [N-nonanoyl, des-Ser1[L-tioproline2,
L-cyclohexylglycine3]-RANTES(2-68)] is a fourth generation chemokine analogue
(preceded by Met-RANTES, NNY-RANTES, and AOP-RANTES) with potent antiviral activity in vitro [85,162] and in the SHIV-macaque vaginal challenge model [116]. In
contrast to CCR5 antagonists, chemokine analogues trigger rapid internalization of CCR5
through a clathrin-dependent endocytic process [203]. Intracellular sequestration of the
receptor by these RANTES derivatives is prolonged and return of CCR5 to the cell
surface is delayed relative to the native chemokine [131]. Previous studies have
concluded that CCR5 internalization by chemokine analogues is the dominant mechanism
for inhibition of HIV-1 entry [85,162]. However, these mechanistic studies have not
thoroughly evaluated the effects of possible competitive binding between the chemokine
derivative and the virus particle for receptor occupancy.
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The concentration of entry inhibitor (i.e. T20, maraviroc, vicriviroc, AMD3100)
required to inhibit 50% of viral replication in culture (IC50) can vary 10-1000 fold when comparing primary HIV-1 isolates that have never been exposed to these drugs
[47,111,188,207]. In contrast, treatment naïve primary HIV-1 isolates display minimal variations in susceptibility to protease inhibitors or reverse transcriptase inhibitors [126].
Variation in the “intrinsic” susceptibility to entry inhibitors appears to be related to the
extreme variability and plasticity of the envelope glycoproteins as compared to the more
conserved viral enzymes [216]. Regarding RANTES derivatives, a panel of 14 divergent
primary isolates demonstrated a 31-fold variation in sensitivity to AOP-RANTES [216].
Mapping of sites related to differential sensitivity revealed that polymorphisms at amino
acid positions 318 and 319 in the V3 loop of gp120 were sufficient to modulate PSC-
RANTES susceptibility by as much as 50-fold in three separate envelope genetic
backgrounds [126]. The proposed mechanism of inhibition by RANTES analogues (i.e.
receptor sequestration) [85] is in conflict with the observed differential sensitivity to these
inhibitors [126,216]. If receptor downregulation is the principal mechanism of inhibition,
inhibition would be determined by the absence versus the presence of receptor, a
condition equally impacting all CCR5-tropic HIV-1 isolates.
Variable HIV-1 inhibition by PSC-RANTES would suggest an alternative,
overriding mechanism such as competitive binding for CCR5. In this study, we
compared inhibition of HIV-1 entry by maraviroc and PSC-RANTES in multiple versus
single replication cycle assays using viruses with differential sensitivities to these drugs.
While allosteric binding/inhibition was observed for maraviroc, two inhibitory pathways
for PSC-RANTES were segregated and compared. PSC-RANTES inhibition was
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examined using short- versus long-term exposure of cell cultures to the drug as well as in
the absence of receptor downregulation using a previously characterized mutant CCR5
(M7-CCR5) [24]. Competitive inhibition was tested by attempting to titrate out the
inhibitory effects of PSC-RANTES by increasing virus concentration. Overall, we have
found that receptor sequestration by PSC-RANTES results in the incomplete but immediate inhibition of HIV-1 entry (<12-24 h) but that inhibition is mediated by competitive CCR5 binding in more physiological conditions of prolonged drug exposure
(>12-24 h). Furthermore, receptor sequestration is not necessary for potent inhibition of
HIV-1 replication by PSC-RANTES, and resistance to PSC-RANTES is consistent with a competitive model of inhibition.
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2.4 Materials and Methods
2.4.1 Cells, Viruses and Inhibitors. The human embryonic kidney cell line 293T
was obtained through the AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH, provided by Dr. Andrew Rice [80] and maintained in complete
media. U87MG human glioblastoma cells stably expressing CD4 and CCR5 were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS,
NIAID, NIH, provided by Dr. HongKui Deng and Dr. Dan Littman [20] and maintained in complete media supplemented with 300µg/mL G418 and 1µg/mL puromycin.
Complete media consists of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100µg/mL penicillin/streptomycin. U87.CD4.M7-CCR5 cells were generated by stable lentiviral transduction of U87.CD4 cells with pBABE.M7-CCR5 and puromycin selection. Cells were maintained in complete media supplemented with 300µg/mL G418 and 1µg/mL puromycin.
Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood extracted from a HIV negative donor by Ficoll-hypaque centrifugation. PBMC cultures were stimulated in RPMI 1640 medium with 10% FBS, 100µg/mL penicillin/streptomycin, and 1µg/mL phytohemagglutinin (PHA) for 48-72 hours. Cells were maintained thereafter in RPMI 1640 medium with 10% FBS, 100µg/mL penicillin/streptomycin, and 1ng/mL interleukin-2 (IL-2). CD4+ T cells were isolated from activated total PBMCs using the Miltenyi CD4+ T cell isolation kit. The purity of the CD4+ cell population was determined to be > 95% by flow cytometry.
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Generation and production of the NL4-3/A1-92RW009 V3 chimeric viruses have
been previously described [126]. These viruses contain specific polymorphisms at
318/319 in the V3 loop (YA or RT) which were found to modulate sensitivity to PSC-
RANTES by 10-fold. Replication competent virus was generated by transfection of 293T cells using the Effectene transfection system with 2µg of each molecular clone. Cells were washed after 24 hours and virus in supernatant was collected after 72 hours. Virus was propagated on U87.CD4.CCR5 cells and supernatants were monitored for reverse transcriptase activity. Viruses were collected, frozen, and infectious titers determined by limiting dilution TCID50 measurements on U87.CD4.CCR5 and activated CD4+ T cells.
PCR amplification of the C2-V3 region from proviral DNA confirmed that reversion of
the V3 mutations did not occur during viral propagation. Psuedovirus particles were
generated by cotransfection of these vectors with the psuedotyping vector pNL.Luc.AM
in 293T cells. Psuedovirus particles were quantified by limiting dilution reverse
transcriptase assay.
Isolation of the PSC-RANTES resistant (M584) and sensitive (P3-4) viruses
during SHIV macaque challenge has been previously described in [53]. Isolation and
characterization of the MVC resistant (R3) and sensitive (S2) viruses has been previously
described in [215]. The envelope region from the ectodomain of gp120 to the membrane
spanning domain of gp41 (ecto-MSD) of viruses M584 and P3-4 were PCR amplified
and cloned by yeast homologous recombination in Saccharomyces cerevisiae MYA-906
cells (ATCC) into pREC_NFL_Δecto-MDS/URA3 vector. The gp120 region of viruses
R3 and S2 were PCR amplified and cloned by homologous recombination into pREC_NFL_Δgp120/URA3. Infectious chimeric virus was produced by cotransfection
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of 3µg each of these vectors with 3µg of pCMV_cplt in 293T cells using Fugene transfection system. Psuedovirus particles were generated by cotransfection of these vectors with the psuedotyping vector pNL.Luc.AM in 293T cells. Virus particles were quantified by limiting dilution reverse transcriptase assay.
PSC-RANTES was kindly 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 MVC and PSC-RANTES were diluted in PBS and filter sterilized. Stock solutions of T-20 were diluted in ethanol. Stock solutions of
TAK-779, 3TC, nevirapine and saquinavir were diluted in DMSO and filter sterilized.
2.4.2 Plasmids. pBABE-M7-CCR5 was a kind gift of Dr. Nathaniel Landau[24].
The M7-CCR5 receptor harbors seven mutations in the cytoplasmic tail of CCR5
(S336/S337/Y339/T340/S342/T343/S349) rendering it deficient in receptor internalization [24]. This vector was used to generate U87.CD4.M7-CCR5 cells. The psuedotyping vector pNL.Luc.AM was a kind gift of Dr. Andre Marozsan and Dr. John
Moore [170]. This vector contains the firefly luciferase gene in place of env in a near full length NL4-3 genome. Psuedoviruses produced with this vector encode the luciferase enzyme and are restricted to a single round of replication.
Generation of cloning vectors pREC_NFL_Δgp120/URA3 and pREC_NFL_Δecto-MSD/URA3 and complementary plasmid pCMV_cplt are described in [52]. Briefly, pREC_NFL /URA3 vectors consist of the near full length (NFL) proviral genome of the NL4-3 HIV-1 laboratory adapted strain with the specified regions (i.e.
Δgp120) replaced by the orotidine-5′-phosphate decarboxylase gene (URA3) for
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selection of homologous recombination in yeast cells. Complementing plasmid, pCMV_cplt, contains R, U5, and a gag fragment under a CMV promoter. In cotransfections, packaging of RNA copies of both plasmids can produce infectious chimeric virus in subsequent propagations in cell lines.
2.4.3 Reverse Transcriptase Activity Assay. Reverse transcriptase activity is a measure of virus production in supernatant and is used to quantify the level of infection.
Supernatant (10µL) was collected and incubated for 2 hr at 37ºC with buffer (25µL) containing nucleotides dATP, dGTP, dCTP and [α32P]-labeled dTTP. A volume of 10µL was spotted on DEAE filtermats, dried, and washed five times for five minutes each with
1X saline-sodium citrate buffer and twice with 85% ethanol. Filtermats were dried and radioactivity measured either by a Packard beta-counter to quantify counts per minute
(cpm) or filtermats were exposed to phosphoimaging screen for 2 hr at room temperature and densitometry quantified by Imager FX.
2.4.4 Multiple-Cycle Drug Susceptibility Assays. Sensitivity to HIV-1 inhibitors was assessed in either U87.CD4.CCR5 cells or in activated, purified CD4+ T lymphocytes. Cells were plated on 96-well plates (2 x 104 U87.CD4.CCR5 cells/well or
2 x 105 CD4+ T cells/well). Drugs were added to wells in serial 10-fold dilutions (PSC-
RANTES: 100 – 10-5 nM; MVC: 100 – 10-5 μM; T-20: 10 – 10-6 μM; TAK-779: 10 – 10-6
μM; 3TC: 10 – 10-6 μM; Nevirapine 10 – 10-6 μM) and incubated for 1 h at 37oC. Virus was added to the cells at a multiplicity of infection of 0.001 IU/cell unless otherwise specified, and the cells and virus were incubated for 24 h at 37oC. Input virus was
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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 and plotted versus drug
concentration to determine the 50% inhibitory concentration (IC50) or maximal percent inhibition (MPI) for each virus and drug.
2.4.5 Single-Cycle Drug Susceptibility Assays. Single-cycle assays were performed using replication-defective particles containing a genomic copy of the firefly
luciferase gene (NLLuc.AM) pseudotyped with different HIV-1 envelopes and used to infect U87.CD4.CCR5 or U87.CD4.M7-CCR5 cells. Psuedovirus particles were determined by limiting dilution reverse transcriptase activity [137], 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.
2.4.6 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,
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
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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 hr prior to detection of CCR5. Cells were analyzed on a FACScalibur flow cytometer (Beckton Dickinson).
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2.5 Results
2.5.1 Dichotomous Sensitivity to PSC-RANTES in Multiple- and Single-cycle
Assays. PSC-RANTES, as well as other chemokine analogues, has been shown to inhibit
HIV-1 by downregulating CCR5 from the cell surface [162]. 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 [216]. 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 [126]. 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 [126]. 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 (Fig. 15A) (p<0.01). Likewise, HIV-1 chimeric viruses generated using envelopes
derived from sensitive (P3-4) or PSC-RANTES resistant (M584) SHIV variants demonstrated 4-fold variation in PSC-RANTES sensitivity under these same conditions
(Fig. 15B) (p<0.01). We next assessed sensitivity of these viruses to inhibition by PSC-
RANTES using single-cycle drug susceptibility assays. In contrast to the multiple cycle assays, similar IC50 values for PSC-RANTES were observed for NL4-3-V3A1-
92RW009(YA) and NL4-3-V3A1-92RW009(RT) (Fig. 15A) and P3-4 and M584 (Fig. 15B). We postulate that the difference we observe between multiple- and single-cycle assays may
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be a consequence of cell surface CCR5 downregulation. For comparison, multiple- and single-cycle assays were performed using viruses (S2 and R3) with differential sensitivity to maraviroc, an entry inhibitor that does not affect steady-state levels of CCR5 on the surface. Reductions in the maximal percent inhibition of the maraviroc resistant virus,
R3, were compared with maximal inhibition observed for the sensitive virus, S2 (Fig.
15C). A similar level of maximal inhibition was observed for virus R3 under multiple- and single-cycle conditions (61 and 56%, respectively), indicating inhibition by maraviroc was the same under both assay conditions.
2.5.2 Inhibition of HIV-1 Replication with a Downregulation-Defective
Mutant of CCR5. Endocytosis of CCR5 occurs through phosphorylation of its C- terminal domain by G protein coupled receptor kinases (GRKs) and recognition of the phophorylated receptor by β-arrestin [24]. M7-CCR5 cells express a mutant CCR5 where the serine phosphorylation site on the C-terminal domain has been replaced with alanine.
This surface expressed M7-CCR5 cannot be downregulated upon binding to PSC-
RANTES [24]. To assess the inhibitory activity of PSC-RANTES in the absence of receptor downregulation, we generated stable U87.CD4.M7-CCR5 cells. Flow cytometry analyses revealed these cells expressed similar levels of CCR5 as U87.CD4.wt-CCR5 cells (Fig. 16) and supported HIV-1 replication, albeit to slightly reduced levels compared to the wild type U87.CD4.wt-CCR5 cells (data not shown). Using two specific monoclonal antibodies to CCR5, clone 2D7 and clone 3A9, we measured the effect of
PSC-RANTES downregulation of wild type and M7-CCR5. Clone 2D7 recognizes an epitope in the second extracellular loop of CCR5, a region reported to overlap the PSC-
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RANTES binding site [170]. Alternatively, the epitope for clone 3A9 is found on the N- terminus of CCR5 and binding of this antibody is not inhibited by PSC-RANTES binding
[170]. Staining with clone 3A9 in the presence of PSC-RANTES indicated efficient downregulation of wild type CCR5 but not the M7-CCR5 receptor (Fig. 16B). In contrast, binding of clone 2D7 was inhibited by PSC-RANTES in both the wild type and
M7-CCR5 cells, suggesting PSC-RANTES can efficiently bind M7-CCR5 (Fig. 16A).
We assessed PSC-RANTES inhibition in U87.CD4.M7-CCR5 cells in both multiple- and single-cycle assays and observed a 10-fold difference between NL4-3-V3A1-92RW009(YA)
and NL4-3-V3A1-92RW009(RT) in both conditions (Fig. 15A). This contrasts with our
previous observation in U87.CD4.wt-CCR5 cells in single cycle where no difference in
sensitivity to PSC-RANTES was detected for these viruses. The IC50 values obtained in
U87.CD4.M7-CCR5 cells after multiple-cycles of replication did not differ significantly
from those PSC-RANTES IC50 values derived from U87.CD4.wt-CCR5 cells in multiple- cycle assays. This observation suggests that PSC-RANTES can potently inhibit HIV-1
replication in the absence of receptor downregulation. We performed an additional
single-cycle assay in U87.CD4.M7-CCR5 cells, this time using viruses P3-4 and M584.
As occurred with viruses NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT), we
observed a difference in sensitivity (5-fold) to PSC-RANTES in single-cycle using the
M7-CCR5 mutant whereas no difference had been detected using wild type CCR5 (Fig.
15B). By comparison, inhibition by maraviroc in U87.CD4.M7-CCR5 cells using viruses
S2 and R3 recapitulated results observed using wild type CCR5 in both multiple- and
single-cycle conditions, suggesting the absence of downregulation did not significantly
impact inhibition by this drug (Fig. 15C).
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To confirm that the difference in sensitivity we observed between multiple- and
single-cycle assays was specific to inhibition by PSC-RANTES, using NL4-3-V3A1-
92RW009(YA) and NL4-3-V3A1-92RW009(RT) we performed drug sensitivity assays under
both conditions with the fusion inhibitor T20, the CCR5 antagonist TAK-779 and reverse
transcriptase inhibitors 3TC and nevirapine (NVP) (Fig. 17). Differential sensitivity to
T20 and TAK-779 were observed in both multiple- and single-cycle assays whereas both viruses were equally sensitive to PSC-RANTES in single cycle. The viruses, having identical RT coding regions, did not differ from each other in sensitivity to 3TC or nevirapine in either multiple- or single-cycle assays. Thus it appeared the differences observed in multiple- and single-cycle assays for PSC-RANTES were specific to the mode of inhibition of this drug.
2.5.3 Prolonged Incubation with PSC-RANTES Recapitulates Multiple-cycle
Assays. Dissimilar inhibition by PSC-RANTES in the multiple- and single-cycle assays suggests an alternative mechanism at play with prolonged incubations in tissue culture.
As described in Figure 18, a 2hr incubation of U87.CD4.wt-CCR5 cells with 10 nM PSC-
RANTES removes over 95% of CCR5 from the cell surface. However, there was only
20% reduction of CCR5 from the same cells when exposed to 10 nM PSC-RANTES for five days (Fig. 18A). Previous studies have shown that CCR5 returns to the cell surface after 4-6 days of PSC-RANTES exposure [162]. Thus, we hypothesized that the differential inhibition to PSC-RANTES observed in multiple-cycle assays was a result of competitive binding for CCR5 between the inhibitor and virus particle. Verification of this hypothesis requires the eventual abrogation of CCR5 downregulation by PSC-
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RANTES with prolonged incubation. To this end, we performed single-cycle assays after a five day incubation of target U87.CD4.CCR5 cells with PSC-RANTES (Fig. 18D).
Luciferase was measured 48 hr post-infection. A 10-fold variation in sensitivity to PSC-
RANTES was observed between NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT)
(Fig. 18D), similar to the multiple-cycle results (Fig. 18B), but not to single-cycle
experiments performed after only 1 hour of PSC-RANTES exposure (Fig. 18C). Using
parallel cultures of U87.CD4.CCR5.Luc cells, a second prolonged incubation experiment
was performed to assess the antiviral decay of PSC-RANTES (Fig. 18E). In one culture,
cells were incubated with 10-fold dilutions of PSC-RANTES for five days while a
parallel culture was incubated in media alone. After five days, the media containing
PSC-RANTES was transferred to cells that had been incubated with media alone. The
cells preincubated with PSC-RANTES were provided fresh media containing PSC-
RANTES at the appropriate concentrations. All cultures were incubated for 1 hour and
then infected with virus for single-cycle assays. We observed differential sensitivity to
PSC-RANTES between NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) in the
cultures exposed to PSC-RANTES for 5 days, washed and then provided with fresh PSC-
RANTES (Fig. 18E). However, no difference in PSC-RANTES sensitivity was observed between the viruses in the cultures that received five day old PSC-RANTES for 1 hour prior to infection. The potency of the five day old PSC-RANTES was reduced by 10-fold as compared to fresh PSC-RANTES, however it should be noted that the remaining PSC-
RANTES could still downregulate the CCR5 receptor (data not shown) and thereby
inhibit NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) during a single cycle of
replication.
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2.5.4 PSC-RANTES Inhibits HIV-1 Entry by Competitive CCR5 Binding.
HIV-1 inhibition is not mediated by receptor downregulation when treating cells
expressing M7-CCR5 with PSC-RANTES and therefore may be exclusively related to
blockade of gp120 binding to CCR5 by PSC-RANTES occupancy through a competitive
mechanism. In our assay system, the inhibitory potential of PSC-RANTES in an exclusively competitive mechanism is dependent upon the concentration of virus which is
CD4-bound and primed for coreceptor interaction. This virus concentration can be manipulated by adjusting the initial multiplicity of infection (Fig. 19). We assessed sensitivity to PSC-RANTES and maraviroc in U87.CD4.M7-CCR5 cells at a multiplicity
of infection of 0.1, 0.02, 0.004, and 0.0008 (IU/cell) using a single virus [NL4-3-V3A1-
92RW009(YA)] and at each day post infection (Fig. 19A, B). With each 5-fold increase in
multiplicity of infection, the level of PSC-RANTES inhibition was reduced and as a
consequence the IC50 value increased (Fig. 19A). Likewise, inhibition by maraviroc was reduced as the virus inoculum increased (Fig. 19B). Supernatant reverse transcriptase activity accumulated over the course of infections with peak virus production observed at day 6 (data not shown). The IC50 values increased not only with increasing virus
inoculums, but within each multiplicity of infection the IC50 value increased each day as
virus grew out in the culture. Thus, in the absence of receptor downregulation, the
increasing IC50 values observed for both PSC-RANTES and maraviroc reflect the increasing concentration of virus competing with inhibitor for CCR5 (Fig. 19C,D).
Under multiple-cycle conditions, the IC50 value determination is highly dependent upon
titer of the virus and on the sampling day.
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We also evaluated the effect of input virus quantity on PSC-RANTES inhibition in the context of wild type CCR5 when receptor downregulation would be possible in multiple- and single-cycle assays. In single-cycle assays using wild type CCR5, the measured IC50 value was independent of the multiplicity of infection (Fig. 20B).
However, under multiple-cycle conditions the IC50 value increased as the amount of input
virus increased as was observed using the M7-CCR5 mutant cells (Fig. 20 A,C). We also
observed that viral titer has an effect on IC50 values in M7-CCR5 expressing cells in
single-cycle similar to that observed for these cells in multiple-cycle assays (Fig. 20D).
2.5.5 Kinetics of PSC-RANTES Inhibition of HIV-1 Entry. In order to determine at which stage in the entry process competitive inhibition by PSC-RANTES occurs, we performed time of drug addition experiments on U87.CD4.M7-CCR5 cells using virus synchronized for entry (Fig. 21). Cells were spinoculated with virus at 4ºC, a temperature not permissive for cell fusion. Viral entry was initiated by addition of 37ºC media. The cell/virus mixtures were then sequentially treated for a period of 2 hours with completely inhibitory concentrations of PSC-RANTES (10nM), TAK-779 (10µM), or
T20 (10µM). Luciferase activity was quantified 48 hours post infection and the inhibitory activity of each drug assessed relative to the no drug condition. The t1/2
inhibition observed for PSC-RANTES (3 minutes) was similar to that for TAK-779 (5
minutes) (Fig. 21). Enfuvirtide (T20) demonstrated delayed kinetics with a t1/2 of 30
minutes. This delay is expected since T20 targets formation of the six-helix bundle, an event triggered by coreceptor binding (inhibited by PSC-RANTES and TAK-779). These data suggested inhibition by PSC-RANTES, even in the absence of receptor
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downregulation, targeted a process of entry prior to membrane fusion, namely coreceptor binding.
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Figure 15. PSC-RANTES and Maraviroc Sensitivity in Multiple- and Single-cycle Assays. Fold difference in sensitivity to PSC-RANTES using wt- and M7-CCR5 variants in multiple- and single-cycle of (A) NL4-3-V3A1-92RW009(YA) (dark gray, IC50 value set to 1) and NL4-3-V3A1-92RW009(RT) (white) (B) M584 (light gray, IC50 value set to 1) and P3-4 (white). Error bars represent standard deviation from the IC50 calculations from two independent experiments performed in triplicate. (C) Maximal percent inhibition to maraviroc using wt- and M7-CCR5 variants in multiple- and single-cycle of S2 (white) and R3 (black). Error bars represent standard deviation of values from triplicates. * represents p < 0.01 (one-tailed T test).
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Figure 16. Characterization of U87.CD4.M7-CCR5 Cells. (A) Cell surface CCR5 was detected by CCR5 MAb 2D7 after 2 hour treatment with 10-fold dilutions of PSC- RANTES. (B) Cell surface CCR5 was detected by CCR5 MAb 3A9 after 2 hour treatment with 10-fold dilutions of PSC-RANTES. * represents p<0.01 (one-tailed T test).
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Figure 17. Sensitivity of V3 Chimeric Viruses to Antiretrovirals in Single- and Multiple- Replication Cycle Assays. Drug sensitivity assays were performed in U87.CD4.CCR5 cells under single- or multiple-cycle conditions using V3 chimeric viruses NL4-3-V3A1-92RW009(YA) (dark gray, IC50 value set to 1) and NL4-3-V3A1- 92RW009(RT) (light grey) for entry inhibitors PSC-RANTES, T20 (ENF), TAK-779 and reverse transcriptase inhibitors 3TC and nevirapine (NVP). Data represents mean of triplicates with standard deviations. * represents p < 0.01 (one-tailed t test).
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Figure 18. Prolonged Incubation of Cells with PSC-RANTES. Inhibition of NL4-3- V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) by PSC-RANTES was measured in single-cycle assays after prolonged incubation of cells with PSC-RANTES. (A) Flow cytometry analyses of CCR5 surface expression was performed on U87.CD4.CCR5 cells after 2 hours and 5 days of exposure to 10-fold dilutions of PSC-RANTES. (B) Multiple-cycle assays allow for approximately 4 cycles of replication over a 6 day period. (C) Single-cycle assays are completed in 48 hours. (D) U87.CD4.CCR5 cells were exposed to 10-fold dilutions of PSC-RANTES for 5 days. On day 5, cells were infected and luciferase activity measured after 48 hours. (E) Parallel cultures were plated in which culture A was exposed to 10-fold dilutions of PSC-RANTES for five days as in (D) while culture B was left untreated. After 5 days, the supernatant of culture A was transferred to the cells of culture B. The cells of culture A were then replenished with fresh media containing the appropriate concentrations of PSC-RANTES. Both cultures were incubated for 1 hour and then infected with virus. Luciferase activity was measured after 48 hours. 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). Error bars indicate standard deviation of the IC50 value as measured from two independent experiments performed in triplicate. * represents p < 0.01 (one-tailed T test).
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Figure 19. Effect of MOI and Virus Expansion on PSC-RANTES and Maraviroc IC50 Values. The relative IC50 value of virus NL4-3-V3A1-92RW009(YA) to PSC-RANTES (A) and MVC (B) was measured by reverse transcriptase assay in multiple replication cycle drug susceptibility assays in U87.CD4.M7-CCR5 cells for increasing multiplicity of infection (IU/cell) and on multiple days post infection. Percent inhibition curves for PSC-RANTES (C) and MVC (D) were generated using data collected on day 6 post infection for multiplicities of infection (MOI) indicated. Data represents mean of triplicate values. Error bars were removed to decrease complexity but standard deviations fell within 10% of the average values.
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Figure 20. Effect of MOI on Inhibition by PSC-RANTES in the Absence of Receptor Downregulation. Inhibition by PSC-RANTES was assessed in multiple- (A and C) and single- (B and D) replication cycle assays using either wild type CCR5 (A and B) or M7-CCR5 (C and D) cells. Cultures were infected with varying levels (MOI) of virus NL4-3-V3A1-92RW009(YA) and percent inhibition calculated based on the no drug condition. Data represents means of triplicates. Error bars were removed to decrease complexity, however standard deviations fell within 10% of average values.
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Figure 21. Kinetics of Entry Inhibitor-Mediated Inhibition. (A) U87.CD4.M7- CCR5 cells were spincoculated with psuedovirus of NL4-3-V3A1-92RW009(YA) at 4ºC. Unbound virus was washed away with cold PBS and cells were plated in 96-well format. Inhibitory concentrations (IC99) of entry inhibitors PSC-RANTES (10nM), TAK-779 (10µM), and T20 (ENF-10µM) were added sequentially to cells after infection was synchronized by the addition of warm media. Luciferase activity was measured 48 hours post infection. (B) Percent inhibition was calculated relative to the luciferase activity measured from the addition of drug at 120 minutes post infection. Data represents mean of triplicates with standard deviations.
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2.6 Discussion
In this study, we have addressed the mechanism(s) of HIV-1 inhibition by CCR5
entry inhibitors. While CCR5 antagonists inhibit via allosteric binding to receptor, the
partial CCR5 agonist PSC-RANTES, in addition to triggering receptor internalization,
appears to compete with HIV-1 envelope for binding to CCR5. Variable intrinsic sensitivity to PSC-RANTES observed for diverse HIV-1 viruses lends support to the role of competitive mechanism of inhibition rather than an exclusively noncompetitive mechanism involving receptor downregulation. To date, most studies have focused on the relative rate of CCR5 internalization by chemokine analogs and prolonged retention in the cell [131,140,162,203]. Thus, we compared the inhibitory effects mediated by
steric occupancy of CCR5 versus that mediated by receptor downregulation by PSC-
RANTES. Our findings indicate that PSC-RANTES inhibition primarily involves a competitive process.
The mechanism(s) of PSC-RANTES inhibition of CCR5-tropic HIV-1 isolates appear quite distinct from that of other entry inhibitors. Fusion inhibitors such as T20 block formation of the gp41 six helix bundle and inhibit at a later stage of entry [30,54].
Indeed, R5 HIV-1 isolates were immediately sensitive to PSC-RANTES whereas time of drug addition experiments indicate a short time delay for HIV-1 sensitivity to T20 (t1/2 =
30min). CCR5 antagonists (e.g. Maraviroc, Vicriviroc, TAK-779) bind to an allosteric
position in the CCR5 transmembrane helices [51,198,218]. This binding is thought to
alter CCR5 conformation and reduce HIV-1 binding. As a consequence, acquired
resistance to drugs such as maraviroc and vicriviroc often involves a mutated virus that
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can utilize both the unbound and drug-bound form of the receptor [138,156,215,233]. In contrast, R5 HIV-1 and PSC-RANTES appear to bind overlapping sites on CCR5.
Binding of PSC-RANTES (or the native RANTES) to CCR5 is thought to involve initial interaction with the N-terminus and then engagement with second extracellular loop, which promotes signaling through coupled G protein [59,190,239]. Thus, decreased susceptibility to PSC-RANTES inhibition may involve a shift in the CCR5-gp120 binding sites and/or increased HIV-1 affinity for CCR5. Primary HIV-1 isolates display the same differential sensitivity to PSC-RANTES as to other entry inhibitors including
TAK-779 and even T20 [126]. Decreasing sensitivity to these entry inhibitors was also a strong correlate of decreasing replicative fitness and entry efficiency [126]. These findings suggest a co-evolution of several HIV-1 regions in gp120/gp41 that are involved in host cell entry process. In addition or alternatively, engagement of CCR5 may be the rate limiting step in this entry process controlled by a defined region of envelope (i.e. V3 loop).
We have previously identified two natural polymorphisms in the V3 loop of the envelope glycoprotein that conferred variable sensitivity to PSC-RANTES and differential replicative fitness [126,139]. Two of these virus clones (with single substitutions in the V3 loop) displayed a 10-fold difference in PSC-RANTES susceptibility in multiple replication cycle assays but did not exhibit this difference in single-cycle assays (Fig. 15A). Repetition of these assays using viral clones derived from sensitive and PSC-RANTES resistant virus yielded similar results (Fig. 15B). In contrast, sensitivity to inhibition by maraviroc was similar regardless of assay duration
(Fig. 15C). These findings suggest mechanistic differences not only between PSC-
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RANTES and maraviroc inhibition but also in PSC-RANTES inhibition in single versus
multiple replication cycles. A major limitation to single-cycle assays is the duration of virus and drug exposure. In multiple cycle assays, a multiplicity of infection of 0.001
(IU) results in the infection of all cells in four rounds of replication (Fig. 18). Neither virus production nor the effect of CCR5 recycling to the cell surface is taken into account by single-cycle assays. Through 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 (Fig. 18A-D).
Previous studies would predict that an extended exposure to PSC-RANTES would result in CCR5 downregulation, retention of the receptor in the cell, and an establishment of lower steady–state CCR5 level on the cell surface [162]. However, following initial downregulation, CCR5 appears desensitized to re-internalization with PSC-RANTES
(Fig. 18E). If downregulation was the only or principal mechanism of inhibition, PSC-
RANTES would not be effective at blocking HIV-1 entry in a multiple cycle assay due to this desensitization. This hypothesis was tested in experiments where cells were incubated for five days with PSC-RANTES prior to the addition of virus for a single cycle assay. Most of the CCR5 receptor had returned to the cell surface in this assay (Fig.
18A) and yet, the virus was still sensitive to PSC-RANTES inhibition. Furthermore, this assay resulted in a 10-fold variation of PSC-RANTES IC50 values between NL4-3-V3A1-
92RW009(YA) and NL4-3-V3A1-92RW009(RT), which was the same difference observed in
multiple cycle assays. Together these findings support a role for competitive inhibition
of HIV-1 by PSC-RANTES.
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Using the M7-CCR5 variant, we were able to evaluate inhibition in the absence of
receptor downregulation. While the absence of receptor downregulation did not
significantly impact inhibition by maraviroc (Fig. 15C), 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 V3
mutant viruses in both multiple- and single-cycle assays (Fig. 15A). Importantly, we
observed a 5-fold difference in IC50 values between our sensitive (P3-4) and resistant
(M584) viruses in single-cycle assays using the M7-CCR5 mutant, whereas no difference
in IC50 values had been detected in single-cycle with wild type receptor (Fig. 15B).
Furthermore, increasing the virus inoculum decreased the potency of PSC-RANTES in
this system. Increasing IC50 values occurred not only when the amount of input virus
increased, but also as infected cells in the culture produced more virus into the
supernatant over time (Fig. 19A-D). These findings not only suggest that the major
difference between single- and multiple-cycle assays with PSC-RANTES is the dominance of receptor sequestration with the former and inhibition by competitive binding with the latter, but that resistance to PSC-RANTES is likely related to competitive binding with inhibitor for CCR5.
Establishing that PSC-RANTES inhibits primarily through a competitive mechanism still does explain why differences in PSC-RANTES susceptibility exist between viruses. Regarding NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT), we
have previously shown that these variants differ in replicative fitness and entry efficiency
[126]. Therefore, it is possible that the V3 loop containing Y318A319 has higher affinity
for binding to CCR5 than does the R318T319 variant. Higher binding affinity may result in
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either an increased ability to compete with PSC-RANTES for CCR5 occupancy or an increased ability to scavenge low levels of surface CCR5 that would be diminished in the presence of PSC-RANTES. Both the increased viral fitness and the acquired resistance mutations (R315 in the V3 loop and D640 in gp41) of virus M584 would support a model in which sensitivity to PSC-RANTES relates to V3 loop affinities, coreceptor association rates, and overall entry efficiency [53]. Adaptive mutations that permit escape from PSC-
RANTES inhibition may arise through mutations that modulate receptor affinity and promote competition with inhibitor for CCR5 as well as enhance the overall rate of the entry process and influence viral replicative fitness. Thus, differences in sensitivity to
PSC-RANTES relate to interactions between the envelope glycoprotein and the coreceptor and the capacity of the virus to outcompete inhibitor for CCR5 binding.
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CHAPTER 3
HIV-1 RESISTANCE TO MARAVIROC CONFERRED BY A CD4 BINDING SITE MUTATION IN THE ENVELOPE GLYCOPROTEIN GP120
Authors: Annette N. Ratcliff1,2, Wuxian Shi3, and Eric J. Arts1,2
1Department of Molecular Biology and Microbiology
2Division of Infectious Diseases, Department of Medicine
3Center for Proteomics and Bioinformatics
Case Western Reserve University, Cleveland, OH, USA 44106
Copyright © 2012 American Society for Microbiology, Journal of Virology, Jan. 2013, doi:10.1128/JVI.01863-12
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3.1 Preface
Having investigated the mechanism of inhibition of CCR5 agonists/antagonists
and observed competitive inhibition for PSC-RANTES, we sought to explore pathways of HIV-1 resistance to CCR5 entry inhibitors. Noncompetitive resistance mechanisms through usage of inhibitor bound CCR5 had been described for CCR5 antagonists such as maraviroc. However, different genetic pathways to resistance had been observed. We wished to explore alternative resistance pathways and mechanisms to maraviroc by isolating and characterizing resistant variants.
The work presented herein was performed by the author with the exception of structural modeling analyses which were performed by Wuxian Shi.
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3.2 Abstract
Maraviroc (MVC) is a CCR5 antagonist that inhibits HIV-1 entry by binding to the coreceptor and inducing structural alterations in the extracellular loops. In this study, we isolated MVC resistant variants from an HIV-1 primary isolate that arose after 21 weeks of tissue culture passage in the presence of inhibitor. Gp120 sequences from passage control and MVC-resistant cultures were cloned into NL4-3 via yeast-based recombination followed by sequencing and drug susceptibility testing. Using 140 clones, three mutations were linked to MVC resistance but none appeared in the V3 loop as was the case with previous HIV-1 strains resistant to CCR5 antagonists. Rather, resistance was dependent upon a single mutation in the C4 region of gp120. Chimeric clones bearing this N425K mutation replicated at high MVC concentrations and displayed significant shifts in IC50 values, characteristic of resistance to all other antiretroviral drugs but not typical of MVC resistance. Previous reports on MVC resistance describe an ability to use a drug-bound form of the receptor leading to reduction in maximal drug inhibition. In contrast, our structural models on K425-gp120 suggest this resistant mutation impacts CD4 interactions and highlights a novel pathway for MVC resistance.
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3.3 Introduction
Human immunodeficiency virus type-1 (HIV-1) entry into host cells is a complex process characterized by three distinct stages: viral attachment to CD4, coreceptor
(CCR5 or CXCR4) binding, and membrane fusion. The viral envelope spike is comprised of trimer of heterodimer subunits of extracellular glycoprotein (gp120) and transmembrane gp41 held together by noncovalent interactions and disulfide bridging
[123]. During entry, binding of gp120 to CD4 induces conformational changes in the envelope exposing a coreceptor binding site. Models of these interactions suggest multiple regions in gp120 are involved with coreceptor binding. The tip of the third variable loop (V3 loop) of gp120 interacts with the second extracellular loop of the coreceptor (ECL2) while the N-terminus of the coreceptor interacts with the V3 loop stem, the bridging sheet (between the V1/V2 stem), and the fourth conserved region (C4) of gp120 [41,91]. Coreceptor engagement drives further conformational changes which results in insertion of gp41 fusion peptide [69], formation of the gp41 six alpha helix
bundle [230], viral and host cell membrane fusion, and release of the viral RNA
containing core into the cell cytoplasm.
As the newest class of antiviral compounds targeting HIV-1 infection, small
molecule entry inhibitors represent a novel generation of drugs targeting a host cell
protein rather than an enzymatic process unique to the virus. Although the development
of entry inhibitors includes compounds targeting gp41 (T20) as well as gp120
(chemokine derivatives, MAbs, CD4-IgG2), the observation that naturally occurring polymorphisms in CCR5 can render homozygous individuals resistant to R5-tropic HIV-
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1 infection [124,165,191] inspired the development of small molecule inhibitors of
CCR5. CCR5 is the main coreceptor for HIV strains transmitted between individuals and that predominate in early infection. Thus, occluding gp120 engagement of CCR5 was an attractive target for drug development [16]. Maraviroc (MVC) became the first and so far only FDA approved small molecule HIV inhibitor/CCR5 antagonist for use in HIV- infected patients. Other CCR5 antagonists, agonists, and binding antibodies reached various stages of preclinical and clinical development but were eventually abandoned due to off target complications [155], poor pharmacodynamics/kinetics [58], and difficulties in screening appropriate patients for treatment due to FDA requirements to counter screen for CXCR4-using HIV-1 [1,169].
Maraviroc is an imidazopyridine that binds a hydrophobic transmembrane cavity of CCR5, altering the conformation of the extracellular loops of the receptor and disrupting chemokine binding as well as interactions with the gp120 envelope glycoprotein [51,104]. Vicriviroc (VCV), AD101, TAK-779 and aplaviroc (APL) are additional small molecule CCR5 inhibitors that bind a similar transmembrane region as maraviroc and likewise induce altered receptor conformations [104]. HIV-1 resistance to such inhibitors is likely to entail unique escape mechanisms given a host receptor, not a viral enzyme, is the drug target. Potential resistance pathways to these inhibitors include coreceptor switching to CXCR4-using viruses, increased affinity and binding to CD4 and/or CCR5, use of inhibitor-bound conformations of CCR5, and increased kinetics of membrane fusion. Although outgrowth of CXCR4-using virus remains a concern for the therapeutic administration of CCR5 antagonists and is why patients are screened for X4- tropic virus prior to starting a maraviroc regimen, de novo mutations altering coreceptor
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tropism do not appear to be the preferential pathway for resistance [163,217]. Rather, resistant viruses emerging from in vivo and in vitro mutational pathways have been characterized as utilizing an inhibitor-bound conformation of CCR5 for entry
[170,215,233].
Resistance to MVC and a variety of other small molecule CCR5 inhibitors has been generated in vitro by passage of inhibitor sensitive viral isolates in sequential dose escalations of drug [138,170,217,233]. Resistance is typically characterized as a reduction in the maximal percent inhibition (MPI) indicating usage of an inhibitor-bound conformation of CCR5 for entry. Although resistance is associated with a variety of amino acid changes observed in both gp120 and gp41, changes in the V3 loop have been identified as major contributors to the resistance phenotype to nearly all CCR5 agonists and antagonists [127]. To date, no signature pattern of mutations has been identified as predictive of CCR5 antagonist resistance. Of greater significance, very few specific mutations have been observed more than once in MVC resistant strains suggesting that each diverse HIV-1 env gene may provide a different genetic pathway for resistance to coreceptor inhibitors.
In the present study, we report the isolation of MVC resistant variants from a subtype A HIV-1 primary isolate which developed resistance over 21 weeks of tissue culture passage in the presence of dose escalations of inhibitor. Resistance in this virus appeared to map to a mutation in the C4 region of gp120. Drug sensitivity assays of gp120 chimeric virus clones derived from the resistant virus did not display reductions in maximal percent inhibition (MPI) but rather showed increases in IC50 values up to 40- fold compared to the parental virus. These pronounced shifts in IC50 value as well as the
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location of the primary resistance mutation in structural modeling predictions suggest a novel MVC resistance mechanism related to altered CD4 binding.
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3.4 Materials and Methods
3.4.1 Cells, Viruses and Inhibitors. U87.CD4.CCR5 and 293T cells were
maintained as described in 2.4.1. Peripheral blood mononuclear cells (PBMCs) were
obtained from whole blood extracted from a HIV negative donor by Ficoll-Paque centrifugation. PBMC cultures were stimulated with 1µg/mL phytohemagglutinin (PHA) for 48 hrs in RPMI 1640 medium containing 10% FBS and 100µg/mL penicillin/streptomycin. Cells were maintained thereafter in RPMI 1640 medium with
10% FBS, 100µg/mL penicillin/streptomycin, and 1ng/mL interleukin-2 (IL-2).
HIV-1 primary isolates were propagated in U87.CD4.CCR5 cells and titers were determined by the Reed-Muench endpoint titration method [137].
Stock solutions of maraviroc (MVC) and TAK-779 were diluted in PBS and filter
sterilized while enfuvirtide (T20) was diluted in ethanol.
3.4.2 Resistant Virus Isolation. Three viruses (A74, B6, and C8) were propagated on U87.CD4.CCR5 cells in a 6-well plate format (1.5 X 105 cells/well) in
either the presence or absence of MVC. Inhibitor concentration was increased when viral
replication was detected using a radio-labeled reverse transcriptase assay as previously described [13]. Equivalent volumes of supernatant (50-100µL) were passaged onto fresh
U87.CD4.CCR5 cells either with or without inhibitor each week. Weekly supernatants
were collected and frozen.
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3.4.3 Multiple-Cycle Drug Susceptibility Assays. U87.CD4.CCR5 cells were plated in 96-well format (1x104 cells/well) for 24 hr. Cells were then treated for 2 hr with
5-fold dilutions of MVC (100µM to 2x10-6 µM) and infected at a multiplicity of infection
(MOI) of either 0.01 or 0.001 infectious units (IU)/cell as indicated. All assays were performed in triplicate. After 24 hr, cells were washed in 1X PBS to remove free virus and media containing the appropriate MVC drug dilutions was added. Viral replication was measured daily by radio-labeled reverse transcriptase assay using a Packard beta counter to quantify counts per minute (cpm). Drug sensitivity curves were generated using non-linear regression curve fitting features of GraphPad Prism 5 software.
3.4.4 Construction of gp120 Chimeric Viruses. Gp120 chimeric viruses were cloned into an NL4-3 backbone from passage control and virus passaged in the presence of MVC escalations using the yeast homologous recombination-gap repair technique [52].
From this point forward, the viruses and clones are referred to as PC (passage control) or
MVC (maraviroc selected) . 21 (number of passages) . 1 (clone number) or PC.21.1 or
MVC.21.1. The gp120 regions of the PC.1 (starting virus), PC.21 and MVC.21 viruses were PCR amplified using primers F_gp120 5’-GACAGGTTAATTGATAGACTA-3’ and B_gp120 5’-CTTCCTGCTGCTCCCAAGAAC-3’. Briefly, these gp120 PCR products were then co-transformed with a SacII-linearized pREC_NFL_Δgp120/URA3 into Saccharomyces cerevisiae MYA-906 yeast cells (ATCC). Following homologous recombination, plasmids were extracted from the yeast cells and transformed into electrocompetent Escherichia coli Stbl4 cells (Invitrogen, Carlsbad, CA). Individual bacterial colonies were grown and plasmids were extracted using Qiagen miniprep kits.
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Chimeric pREC_NFL_gp120 plasmids were co-transfected into 293T cells (3x104
cells/well) along with the complementing plasmid pCMV_cplt using Fugene 6 reagent
(Roche) as described [52]. Heterodiploid virus particles containing one copy of the NFL
and cplt HIV-1 RNAs represent approximately half of the virus derived from 293T cell
transfections and can be propagated on U87.CD4.CCR5 cells to produce replication
competent virus with a complete virus genome. Virus stocks of each clone were titered
as described previously and in [137].
3.4.5 Oligonucleotide Ligation Assay. The gp120 regions of the bulk passage control (PC.21) and resistant virus (MVC.21) were RT-PCR amplified using envend 5’-
CTTTTTGACCACTTGCCACCCAT-3’ for reverse transcription and F_gp120/B_gp120
for subsequent PCR amplification. Oligonucleotides were designed for ligase
discrimination reactions to bind upstream or downstream of the mutation sites observed
in MVC.21 for envelope codons 117, 396, and 425. For each mutation site, we employed
two upstream interrogator oligonucleotides to discriminate between and quantify the
sequences RCGA and QCAA at codon 117, sequences LTTA, GGGA, VGTA at codon 396 and sequences NAAT and KAAA at codon 425. A complete description of this ligase
discrimination assay has been described previously [213]. The oligonucleotides designed
specifically for this study are listed in Figure 28.
3.4.6 Site-directed Mutagenesis PCR Method. The K425 mutation was
introduced into the MVC.21.122 gp120 clone by a nested PCR amplification method.
Two PCR fragments were generated from the pREC_NFL_gp120/MVC.21.122 plasmid
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using primers F_gp120 with B_K425 5’-
CTACTCTTTGCCACATCTTTATAATTTGCTTTATTCTGCATGGAAGA-3’ and F_K425 5’-
TCTTCCATGCAGAATAAAGCAAATTATAAAGATGTGGCAAAGAGTAG-3’ with B_gp120. Using
products from these PCR amplifications as templates, a nested PCR was performed with
primers F_gp120 and B_gp120 to generate the full gp120 region which was subsequently
cloned into pREC_NFL_Δgp120/URA3 by yeast homologous recombination as
described above. Individual colonies were sequenced and a single clone containing the
K425 mutation was identified and named pREC_NFL_gp120/MVC.21.122.425K.
Replication competent virus was generated from this plasmid by cotransfection in 293T
cells and propagation in U87.CD4.CCR5 cells as described above.
3.4.7 In vitro Fitness Assays. The replicative fitness of PC.21 and MVC.21
gp120 chimeric viruses were assessed in head to head competition experiments in
U87.CD4.CCR5 cells as described in [13]. Cells were plated in 48-well format (2x105
cells/well) and infected in triplicate with viruses at an MOI of 0.0001 IU/cell. Infected
cells were collected five days post infection and genomic DNA was isolated using
Qiagen kit. The gp120 region was amplified from cellular DNA using F_gp120/B_gp120
as described earlier. Amplicons were probed for sequence identity at gp120 residue 117
and 425 OLA as described [113,213]. Frequency of wild type or mutant residue identity versus total signal was used to determine viral fitness. Fitness difference (WD) was
calculated as the fitness of the resistant virus divided by the fitness of the sensitive virus.
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3.4.8 Structural Modeling. The structural model of N425K mutant was generated from the crystal structure of gp120 in complex with CD4 and a tyrosine- sulfated antibody 412d (PDB ID: 2QAD;[91]). The mutation of N425 to K425 was made in the model building program COOT [56] and the local structure around the mutation was regularized using the same program. By a slight torsion of the K425 side chain, two close contacts can be made from the Nε of K425 with residues from CD4 including a cation-π interaction with F43 of CD4.
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3.5 Results
3.5.1 HIV-1 Primary Isolates Demonstrate Variable Sensitivity to Maraviroc.
The concentration of entry inhibitor required to inhibit viral replication by 50% (IC50) can
vary up to 1000 fold when comparing HIV-1 primary isolates that have never been exposed to these drugs [111]. In the present study, we assessed “intrinsic” susceptibilities of HIV-1 subtype A, B, C and D primary isolates to maraviroc. Multiple cycle drug susceptibility assays were performed in U87.CD4.CCR5 cells in the presence of 5-fold
decreasing concentrations of MVC starting at 100µM. Sensitivity varied up to 100-fold
for the twenty primary isolates tested (Fig. 22A). As discussed in Chapter 2 and [126],
mutations in the V3 loop of gp120 can modulate sensitivity to entry inhibitors. Multiple
cycle sensitivity assays performed using NL4-3 V3 chimeric viruses demonstrated that differences in the V3 region alone can confer up to 50-fold variation to maraviroc (Fig.
22B). However, env sequence context has been shown to influence the impact of V3
mutations on sensitivity to entry inhibitors [126]. In Figure 22A, the primary isolates
A74 and B6 demonstrate differential sensitivity with IC50s of 98nM and 1.6nM, respectively. However, chimeric viruses (NL4-3V3-A74 and NL4V3-B6) bearing the V3 regions of these primary isolates in an isogenic env background demonstrated similar sensitivity to maraviroc (Fig. 22B). This data would suggest that multiple regions of envelope can influence viral sensitivity to maraviroc.
3.5.2 Maraviroc Resistant Mutant Generated in Cell Culture. Since viral titer and sampling day can influence IC50 values when measured in multiple-cycle assays,
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the optimal multiplicity of infection (0.001 IU/cell) and sampling day (5 days post
infection) were determined in U87.CD4.CCR5 cells (data not shown). These conditions
were used throughout this study unless otherwise indicated. Given the differential
sensitivity to MVC, three primary isolates (A74, B6, and C8) were chosen for selection of maraviroc resistance in culture. When sensitivity assays were repeated for viruses
A74, B6 and C8 using these criteria, the subtype A primary isolate (A74) was less sensitive to MVC inhibition (IC50 = 10 nM) compared with the subtype B (B6) and C
(C8) viruses which had similar sensitivity (mean IC50 = 2 nM) (Fig. 23A). These three
primary isolates were passaged weekly in the presence of increasing MVC concentrations
in U87 human glioma cells expressing CD4 and CCR5 to generate a maraviroc escape
mutant (Fig. 23B). As a control, viruses were also passaged in the absence of inhibitor
to differentiate changes that occurred as a result of drug pressure versus selection during
long term culture. Reverse transcriptase (RT) activity was monitored in the cell-free supernatant for each weekly passage and inhibitor concentration increased when RT activity reached 2-fold over background. Virus C8 cultures were abandoned after failure to produce robust RT activity in weeks 15 and 16. Although virus B6 cultures continued to produce RT activity through week 21, viruses collected from this passage remained sensitive to MVC with IC50 values for control and inhibitor treated cultures 8 nM and 14
nM, respectively (Fig. 23C). It is important to note that only one other study has selected
for MVC resistance in vitro using primary HIV-1 isolates and in this case, only 2 of 6 viruses challenged with drug developed resistance [233]. Of all CCR5 agonists and antagonists, there are only seven published reports of in vitro resistance selection to this
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drug class as mediated by altered CCR5 usage rather than outgrowth of a pre-existing
X4-using variant [11,138,157,170,217,233,245].
Drug susceptibility analyses of virus A74 at week 21 (MVC.21) in
U87.CD4.CCR5 cells indicated this virus was able to escape inhibition by MVC at
concentrations >1,000 fold higher than the IC50 value for the input virus (Fig. 24A). At
the highest drug concentration tested (100µM), MVC.21 was inhibited by 81%,
indicating incomplete suppression of virus entry despite saturating levels of inhibitor. In
addition to this reduction in the maximal percent inhibition (MPI), a shift in IC50 value to
216 nM was also observed for MVC.21 virus representing a 20-fold level in resistance
compared to the inoculating virus. In contrast, the passage control virus (PC.21)
demonstrated a slight hypersensitivity to MVC with an IC50 of 1 nM. Tropism testing in
U87.CD4.CXCR4 cells indicated no dual or X4-tropic viruses arose during the 21 passages (data not shown).
The U87 human glioma cell line is stably transfected to express high levels of
CD4 and CCR5 which does not accurately reflect levels on natural HIV-1 targets, e.g.
CD4+ T cells and macrophages. Therefore, multiple cycle drug sensitivity assays were performed in peripheral blood mononuclear cells (PBMC) isolated from an HIV negative donor with PC.21 and MVC.21. The results confirmed those observed in the
U87.CD4.CCR5 cells with a reduction in MPI of MVC.21 to 73% and an IC50 value of
300 nM compared to 5 nM for PC.21 (Table 3).
3.5.3 MVC.21 Cross Resistant to Another CCR5 Antagonist. MVC.21 showed evidence of cross resistance to another CCR5 antagonist, TAK-779, as indicated
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by replication in U87.CD4.CCR5 cells treated with an IC99 inhibitory concentration
(10µM) that completely blocked PC.21 virus replication (Fig. 24B). Since TAK-779 (as
well as VCV and APL) has been shown to bind a similar CCR5 transmembrane region as
maraviroc and alter coreceptor conformation [104], it is likely a similar mechanism of
resistance is employed to overcome inhibition by TAK-779. In contrast, 10µM of
enfuvirtide (IC99 concentration) mediated similar levels of MVC.21 and PC.21 inhibition in U87.CD4.CCR5 cells. As a fusion inhibitor, enfuvirtide or T20 inhibits entry by targeting formation of the six helix gp41 bundle and subsequent membrane fusion [32].
Lack of enfuvirtide resistance suggests MVC resistance in MVC.21 virus is mediated by
a mechanism that precedes membrane fusion and likely relates to receptor interactions.
3.5.4 Multiple Mutations Throughout gp120 in Resistant Virus. Population
sequencing of the envelope region of the PC.21 and MVC.21 viruses identified several
amino acid substitutions related to MVC selection in the gp120 region but none in gp41.
However, it was difficult to determine a distinct linkage of mutations due to the high
genetic diversity in the PC.21 and MVC.21 population. To identify specific mutations
and linkage in gp120, the gp120 region of the input (PC.1), passage control (PC.21) and
MVC treated virus from passages 7, 14 and 21 were PCR amplified from viral cDNA and
cloned into the pREC_NFL_Δgp120/URA3 construct by yeast homologous
recombination/gap repair (Fig. 25A) [53].
Approximately 10 clones from each passage control (PC.1, PC week 7 or PC.7,
PC.14, PC.21) and 25 clones from each MVC treated passage (MVC week 7 or MVC.7,
MVC.14, MVC.21) were randomly chosen and sequenced to identify mutations in >140
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virus clones from all conditions and to determine the linked amino acids selected under
MVC pressure. Sequence alignments of gp120 clones identified nine amino acid
substitutions across gp120 that were more prevalent in the MVC.21 clones than in the
PC.1 and PC.21 clones (data not shown). To simplify sequence datasets, Figure 25B
summarizes the mutational pattern of clones from PC.1, PC.21 and MVC.21. We utilized
a color coding system where a black box at a specific position indicates the
predominate/average amino acid in the inoculum virus (PC.1). When the
predominate/average sequence in the MVC-treated virus (MVC.21) was a different
amino acid, a red box is represented for this site. For example, the average sequence at
position 33 in gp120 was a glutamate (E) in PC.1 and PC.21 (black box) but was a
glycine (G) in MVC.21 virus (red box). A total of twenty-three unique mutational
patterns were identified based on this average sequence analyses. We did observe some
“mutant” amino acids (red box; Fig. 25B) in PC.1 and PC.21 clones and likewise, some
wild type amino acids in the MVC.21 clones. Overall, the linkage pattern of these
mutations in the MVC.21 population was complex with only six of twenty-three clones
(Fig. 25B) having all nine mutations. Only one of nine clones in the input or PC.1 virus had wild type amino acids at all nine positions.
The frequencies of the wild type versus mutant residues at these individual mutation sites were determined using the total number of sequenced clones. Enhanced frequencies in the MVC.21 versus PC.21 virus should provide some indication of which mutations may contribute to the resistance phenotype (Fig. 26). Three mutations
(R117Q, L/G396V, and N425K) were found at high frequency in the MVC.21 clones but at low frequency in either the input or PC.21 clones (Fig. 26A, C, H, respectively)
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suggesting their role in MVC resistance. In contrast to previous studies, the V3 mutation
Q315R was present at the same frequency in the PC.21 clones as the MVC.21 clones
(Fig. 26G) suggesting this mutation emerged with normal adaptation to U87.CD4.CCR5 tissue culture and not selected under drug pressure. The remaining mutations E33bG,
KG138-39ΔΔ, Q290K, and D461E appeared at high frequency in the MVC.21 clones but were also found at frequencies >25% in the input population (Fig. 26E, F, B, D,
respectively). In regards to linkage, the R117Q, L396V and N425K were found together
in twenty-two of twenty-three MVC.21 clones but never linked in the PC.1 or PC.21
virus. Strikingly, the N425K mutation was found in all but one of the twenty-three
MVC.21 clones (except MVC.21.122) but was absent from all seventeen of the PC.1 or
PC.21 clones. An additional twenty-two clones from MVC.7 and thirteen clones from
MVC.14 were sequenced in the gp120-C4 region. The N425K mutation was not found in
any of the MVC.7 clones but was present in 8 of 13 MVC.14 clones, suggesting this
mutation arose between passages 7 and 14 (Fig. 27).
To confirm the frequency of the putative MVC-resistant mutations in the viral populations, an oligonucleotide ligation assay (OLA) [53,112] was performed using interrogator oligos specific to the mutations R117Q, L/G396V and N425K on gp120 PCR products from cDNA of PC.21 and MVC.21 virus (Fig. 28A-E). In this study, we could only predict the probability of linkage. Using this quantitative OLA, we found that the
Q117 and V396 sequences were detected at 0.94 and 0.97 frequency in MCV.21 but at
0.55 and 0.32 frequency in the PC.21 virus population, respectively (Fig. 28C, D). Based on the very low frequency of K425 in the PC.1 and PC.21 populations (~7%) (near the limit of detection at 1%) (Fig. 28E), it appears that the N425K mutation either arose as a
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de novo mutation in the MVC.21 population or was selected for from a very small
fraction of the input virus.
3.5.5 Sensitivity of Selected A74 HIV-1 Clones to MVC Inhibition. Based on
our cloning strategy, we have a diverse array of gp120 clones with different mutational
patterns that represent nearly all combinations of single and multiple substitutions
associated with MVC resistance (namely, R117Q, Q290K, L/G396V, D461E, E33bG,
N425K and the deletion of K138/G139). This limits the need for site-directed
mutagenesis on virus to assess the contribution of each mutation (alone or in
combination) to relative MVC resistance. Seven gp120 clones were selected to produce
virus from 293T transfections (Fig. 29A), which were then tested for MVC sensitivity in
U87.CD4.CCR5 cells (Fig. 29B-D). Although MVC.21.119 and MVC.21.132 share the
same mutation profile for the nine MVC-associated amino acid sites in gp120, they do
vary slightly in residues at other gp120 sites and are therefore not identical clones (data
not shown).
Clones from the input virus (PC.1.1) and week 21 passage control (PC.21.109 and
PC.21.111) were sensitive to MVC inhibition with similar IC50 values (Fig. 29B, D).
However, clonal viruses derived from MVC.21 varied greatly in IC50 values.
MVC.21.132 had a moderate 10-fold shift in IC50 value to 8.8nM while MVC.21.119 and
MVC.21.128 had pronounced 40-fold shifts in IC50 to 34 and 37nM, respectively (Fig.
29B, D). Importantly, MVC.21.123 retained the wild type V3 mutation Q315 yet still demonstrated a moderate 11-fold IC50 shift and was able to replicate at high
concentrations (Fig. 29C), suggesting Q315R mutation is not associated with resistance
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in MVC.21. All four of these clones (119, 123, 128, and 132) had the triad of R117Q,
L396V and N425K linked mutations. Clone MVC.21.122 had an IC50 value of 0.9nM
similar to that observed for the PC.21 clones despite having the R117Q and L396V
mutations.
3.5.6 Relative Fitness of MVC-Resistant and Sensitive HIV-1 Clones. The
R117Q, Q290K, L/G396V, and D461E mutations have a higher frequency (5-30 %) in the HIV-1 subtype A population in the human epidemic whereas the lysine at 425 was found in less than 2% of HIV-1 Subtype A envelope sequences (Table 4) (Los Alamos
HIV Sequence Database: http://www.hiv.lanl.gov/content/hiv-db/mainpage.html). The prevalence of N425K was even lower in other HIV-1 subtypes (B, C, and D) with an occurrence of < 0.2% (Table 4). These findings suggest that R117Q, Q290K, L/G396V,
D461E, and E33bG have a lower genetic barrier for mutation and less impact on replicative fitness than the N425K mutation. However, we have previously shown that env mutations conferring resistance to entry inhibitors may not always confer a replicative fitness cost. Their low frequency in the HIV-1 population (i.e. low population fitness) may be attributable to other factors such as cellular tropism and sensitivity to neutralizing antibodies or cell-mediated cytotoxic T cell killing. We performed pairwise competitions between three MVC sensitive clones (PC.1.1, PC.21.109, and MVC.21.122) and three MVC resistant clones (MVC.21.119, MVC.21.128, and MVC.21.132) in
U87.CD4.CCR5 cells (Fig. 30). It is important to note that even though MVC.21.122 was isolated from the MVC selection, this clone remained MVC sensitive (Fig. 29B-D).
Interestingly, all of the MVC resistant clones were actually more fit than MVC sensitive
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clones PC.1.1 and PC.21.109. The clone (MVC.21.132) with the lowest level of MVC
resistance (10-fold) was also the least fit of the MVC resistant clones losing the
competition against PC.21.109. In contrast, the MVC sensitive clone, MVC.21.122
(lacking N425K) could compete against the MVC resistant clones suggesting that the
mutations (R117Q, Q290K, L/G396V, D461E, and E33bG) may actually confer fitness
increases and compensate for the loss of fitness conferred by N425K.
3.5.7 K425 is Primary Resistance Mutation. MVC.21.122 was the only clone from the MVC resistant population that lacked the N425K mutation in the C4 region of gp120 and the only clone not to exhibit an MVC resistance phenotype (Fig. 29C). This observation indicated that the K425 mutation was likely essential for resistance in
MVC.21. Site directed mutagenesis was performed to introduce the K425 mutation into the MVC.21.122 clone and to confirm the role of this mutation on MVC resistance.
Using the same drug inhibition assays described above, we observed a dramatic decrease in susceptibility of MVC.21.122.425K to maraviroc, i.e. a 36-fold shift in IC50 from
0.9nM to 33nM (Fig. 31A) as well as replication of MVC.21.122.K425 at 100µM MVC
(Fig. 31B).
3.5.8 Shift in IC50 Versus the MPI Effect Denotes MVC Resistance. As
described earlier, resistance to CCR5 antagonist is generally related to continued virus
replication at even the highest achievable drug concentrations in culture. For example,
the CC1/85 MVC-resistant virus containing the T316 and V323 V3 loop mutations is
capable of replicating at 25% level in the presence of 1µM MVC as compared to the
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absence of drug [233]. However, the CC1/85 MVC-resistant virus does not show a shift in IC50 value. With our MVC resistant virus, there is a mixed resistant phenotype. By
simply plotting the percent relative inhibition versus drug concentration, the shift in IC50
is quite clear and similar to the increases in IC50 value characteristic of resistance to all other antiretroviral drugs aside from CCR5 antagonists. In addition to this drug resistance phenotype, the MVC–resistant clones bearing the K425 mutation showed low but significant levels of virus replication at even the highest drug concentrations (Fig.
29C). However, this MPI effect was much less pronounced than with other MVC-
resistant viruses as previously reported [245]. Despite MPI values >95% for MVC.21
clones 119, 123, 128, and 132, virus replication was detectable even with 100 µM MVC
whereas no replication was observed with the MVC sensitive clones 1, 109, and 122 (Fig.
29C).
3.5.9 Model of K425 in gp120 Structure Suggests Role in CD4 Binding
Affinity. The N425 residue is located in the β20 sheet of the gp120 bridging sheet as
illustrated in Figure 32 using the crystal structure of HIV-1 gp120Yu-2 complexed with
CD4 and a tyrosine-sulfated 412d antibody [91]. These four anti-parallel beta sheets of
gp120 comprise the complete CD4 and partial coreceptor binding sites. This 425 position
as either an asparagine or lysine is found distal from the gp120 region thought to interact
with the N-terminus of CCR5. However, N425 is specifically located within a cavity
known to interact with phenylalanine 43, found within the D1 domain of CD4. N425-
gp120 and F43-CD4 are not predicted to form direct interactions. However, when K425
is modeled into the structure using the COOT modeling program, the side chain of K425
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is predicted to form a hydrogen bond with the oxygen of S42-CD4 as well as generate a
new cation-π interaction between the aromatic ring of F43-CD4 and the Nε side chain of
K425. Since F43-CD4 is known to be a critical residue for gp120 binding [10,109,143],
our model would suggest that enhanced interactions with CD4 may play a role in the
resistance mechanism of A74.MVC.21 virus.
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A
B
Figure 22. Sensitivity of HIV-1 Primary Isolates to Maraviroc. (A) IC50 values of a panel of HIV-1 group M viruses including subtype A (red), subtype B (blue), subtype C (grey), and subtype D (green) to maraviroc were determined in U87.CD4.CCR5 cells at a multiplicity of infection of 0.01 IU/cell. Alignments of the V3 loop region of gp120 with reference virus HXB2 are also shown. (B) Drug sensitivity curves of V3 loop chimeric NL4-3 viruses were performed in U87.CD4.CCR5 cells. Data represents triplicates with standard deviations shown.
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A
B
C
Figure 23. Prolonged Tissue Culture Passage to Select for MVC Resistance. (A) Drug sensitivity curves of viruses A74, B6, and C8 were measured in U87.CD4.CCR5 cells using 10-fold lower viral inoculums (0.001 IU/cell) than those used for Figure 22A. (B) Viruses A74, B6 and C8 were passaged in U87.CD4.CCR5 cells weekly in the presence of increasing concentrations of MVC to select for a MVC escape mutant. Cultures were monitored for reverse transcriptase activity as described in Materials and Methods. Cultures for virus C8 were abandoned at week 16 (*) when no RT activity was measured. (C) Virus B6 derived from passage control week 21 (PC.21) and MVC treated week 21 (MVC.21) cultures were used to infect U87.CD4.CCR5 cells in the presence of increasing concentrations of MVC as described in Materials and Methods. Data represents triplicates with standard deviations shown. Inhibition curves were generated in GraphPad Prism software version 5.
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Figure 24. MVC Resistance and Cross Resistance to TAK-779 after Prolonged Culture with MVC Inhibitor. (A) Virus A74 derived from passage control week 21 (PC.21) and MVC treated week 21 (MVC.21) cultures were used to infect U87.CD4.CCR5 cells in the presence of increasing concentrations of MVC as described in Materials and Methods. Inhibition curves were generated in GraphPad Prism software version 5 for panel A. (B) These viruses were also used to infect U87.CD4.CCR5 cells in the presence of maximal inhibitory concentrations (10µM) of CCR5 antagonists MVC and TAK-779 and fusion inhibitor enfuvirtide (T20). Reverse transcriptase activity was measured 7 days post infection. Percent infection was calculated relative to the no drug control for each virus. Data shown are means of triplicates with error bars representing standard deviations.
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Table 3. Summary of MVC Sensitivity in U87.CD4.CCR5 and PBMC Cells.
U87.CD4.CCR5 PBMC Virus IC50 (nM) MPI (%) IC50 (nM) MPI (%) A74 10 99 5 100 PC.21 1 100 6 98 MVC.21 216 81 300 73
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Full recombination recombination Viral RNA was isolated from indicated indicated from isolated was RNA Viral
(A)
Virus. f Gp120 Chimeric and Characterization o . Generation n is present. The asterisks (*) in (B) refer to clones harboring only mutations selected primarily in the MVC passage. in only (B) inMVC (*) primarily the passage. refer harboring asterisks to mutations clones The selected n is present. ure 25 method as described in Materials and Methods. Individual bacterial colonies were selected as indicated in the table. (B) of gp120 regions clones PC.1,from PC.21 sequenced. MVC.21mutations were identified and Nine were in MVC.21nes clo based virus indicated are Rowsand reference referon HXB2 numbering. columns torefer while individualto gp120 clones a red box indicates a wildresiduethat gp120 site whereas type at box indicates gp120 mutations.average black A specific mutatio clones indicated columns. of harboringbelow Number gp120 sites are mutations specific at Fig passage control MVCand culture treated supernatants the and gp120 regions amplified reverseafter transcriptase and nested PCR. Gp120 regions were recombined into pREC_NFL_Δgp120/URA3 vector using yeast homologous
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Figure 26. Frequency of gp120 Mutations in PC.21 and MVC.21 Derived Clones. The frequency of wild type and mutant amino acid are displayed and represent those individual mutations in the clones derived from PC.1, PC.21 and MVC.21 cultures (A- H). Frequencies were calculated based on number of clones with either wild type or mutant residues versus the total number of clones sequenced for that population as shown in Fig. 3B. The center panel of HIV-1 envelope gene maps the location of each mutation in gp120 conserved (C1-C5) or variable (V1-V5) regions. Wild type sequences are shown in black with mutations shown in red.
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Figure 27. Change of Codon 425 Mutation Frequency During Passage Control and MVC Selection. Using the clonal sequences (n=79) from the inoculum and MVC treated passage 7, 14, and 21, the frequency of N425 and K425 was determined.
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Figure 28. Oligonucleotide Ligation Assay Determines Change in gp120 Mutation Frequency during MVC Selection. (A) Interrogator oligonucleotides harboring bead capture sequences and single nucleotide polymorphisms are able to discriminately bind RT-PCR product and ligate to biotinylated reporter capture oligonucleotides. Ligated interrogator/reporter capture oligos bind specifically to capture beads to permit identification and quantification of sequence identity via streptavidin-phycoerythrin binding. (B) Codon specific interrogator oligonucleotides were used to discriminate between wild type and mutant codons. Discriminating nucleotides are indicated in bold. Sample specific reporter capture oligonucleotides are also shown. The frequencies of the R117Q (C), L/G396V (D), and N425K (E) were measured in the RT-PCR product from the bulk PC.1, PC.21, and MVC.21 virus populations as described in Materials and Methods.
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Figure 29. Sensitivity of gp120 Chimeric Virus to Maraviroc Inhibition. (A) Individual gp120 clones from PC.21 and MVC.21 were selected based on differential mutation patterns for MVC drug susceptibility testing in U87.CD4.CCR5 cells. Black indicates wild type residue while red indicates a mutation at that gp120 residue. IC50 fold change relative to input gp120 clone PC.1.1 are shown in (B). Data represents mean of triplicate wells with standard deviations. (C) Autoradiographs of [α-32P] TTP labeled reverse transcriptase activity of five highest MVC concentrations tested (100-0.16µM). Triplicate wells are shown. Reverse transcriptase activity was detected at 100µM MVC for all MVC.21 derived chimeric viruses except the MVC.21.122 virus. (D) Drug sensitivity curves for chimeric gp120 viruses were performed in U87.CD4.CCR5 cells in triplicate as described in Materials and Methods. The IC50 values are shown to the right of panel (D) whereas the fold change derived from these IC50 values are shown in (B) Inhibition curves were generated using GraphPad Prism software version 5.
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Table 4. Summary of Los Alamos HIV Database gp120 Sequence Identity at Codon 425.
Total number Amino Acid Frequency (%) of sequences H K N R Other Group M 61,096 0.3 0.1 97.1 2.0 0.5 Subtype A 1,065 7.8 2.0 77.0 12.3 0.9 Subtype B 42,585 0.2 0.01 99.1 0.3 0.3 Subtype C 11,451 0.1 0.1 98.5 0.9 0.4 Subtype D 1,450 0.6 0.2 98.3 0.2 0.6
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Figure 30. Replicative Fitness of HIV-1 gp120 Chimeric Virus Clones Derived from MVC Selected and Passage Control Experiments. The infectious titers of three MVC- resistant clones (MVC.21.119, MVC.21.128, and MVC.21.132) and three MVC-sensitive clones (MVC.21.122, PC.21.109, and PC.1.1) were measured using a standard TCID50 assay (34). A pairwise competition was performed competing the MVC resistant against the sensitive gp120 clones in U87.CD4.CCR5 cells using equal MOI of each virus (0.0001 IU/cell). Virus was harvested at days 4-9 and at peak viremia (day 7), the relative frequency of each virus in the competition was measured using OLA to distinguish and quantify the amount of one virus versus the other (as described; (50)). The fitness difference (or ratio of the relative fitness values) were calculated as described and the results presented in the figure.
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Figure 31. Effect of the N425K Mutation on MVC Resistance. (A) Drug sensitivity assays were performed for clone MVC.21.122 and clone MVC.21.122.425K in which the K425 mutation was introduced by site-directed mutagenesis as described in Materials and Methods. U87.CD4.CCR5 cells were infected in triplicate with standard deviations shown. Inhibition curves were generated using GraphPad Prism software version 5. (B) Autoradiographs of [α-32P] TTP labeled reverse transcriptase activity of five highest MVC concentrations tested (100-0.16µM). Triplicate wells are shown. Reverse transcriptase activity was detected at 100µM MVC for MVC.21.122.425K.
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Figure 32. Modeling of K425 in gp120 HIV-1YU-2 Virus Structure Suggests Role in CD4 Binding Affinity. The structure of gp120 HIV-1YU-2 complexed with CD4 and 412 Ab (not shown) was used to model the N425 to K mutation. N425 residue lines a cavity in gp120 into which F43 of CD4 projects (inset). Mutation of N425 to K is predicted to form new interactions with F43. (PDB: 2QAD; [91])
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3.6 Discussion
HIV-1 enters a host cell by engaging two receptors, CD4 and one of two seven transmembrane G-coupled protein receptors, CCR5 or CXCR4. With a few exceptions, nearly all new HIV-1 infections are established with a CCR5-using virus while the
CXCR4-using virus emerges only in late disease of approximately 50% of infected individuals. The clear predominance of R5 HIV-1 during asymptomatic disease led to the rapid pre-clinical development of several CCR5 agonists and antagonists. The CCR5 agonists, such as AOP-RANTES or PSC-RANTES displayed higher per mole potency than the small molecule CCR5 antagonists but also resulted in receptor downregulation, some aberrant signal transduction, and increased potential for inflammatory responses
[126,140,162,182,204,216]. Of the various CCR5 antagonists, only three reached advanced stages of clinical trials and only maraviroc was approved first for salvage-based treatment and then for first line treatment in combination with nucleoside analogs
[81,202]. For the most part, virologic failures to MVC-based treatment regimens have been poorly characterized and generally, complicated by the pre-existence of multidrug resistant genotypes in these heavily treated patients [84,210,232]. When used as a salvage-based treatment regimen, resistance commonly relates to the rapid selection of pre-existing X4 HIV-1 in the intrapatient HIV-1 population [232]. These CXCR4 using clones can be detected, even at low frequencies, by phenotypic assays [234].
Consequently, MVC resistance via altered CCR5 binding or increased receptor affinity is rarely observed in MVC treatment failures [215,233]. However, if patients were efficiently screened for X4 using virus prior to treatment, altered CCR5 usage may be a
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more common pathway for MVC resistance as compare to a de novo emergence of X4 using virus, i.e. mutations resulting in higher genetic/fitness barriers.
In addition to inefficient screening for X4 using viruses, there are several problems in measuring MVC resistance during treatment. First, nearly 30% of the MVC treatment failures (without resistance to the other drugs in the regimen) did not show characteristic resistance to MVC [61,84]. Second, current phenotypic assays using single cycle entry may be skewed for the detection of the MPI effect [232,233]. Third, a different evolutionary pathway in env was associated with every MVC resistant virus that retains CCR5 binding [215,233,245]. Fourth, it is commonly assumed that these MVC- resistant viruses can utilize drug-bound forms of the receptor based on incomplete inhibition even at the highest MVC concentrations [233]. However, there are other studies to suggest that increased kinetics of host cells could lead to resistance to CCR5 agonists and antagonists [6,53,126,138]. Two independent studies have shown that mutations in the gp41 domain may enhance viral-host membrane fusion, the last step in host cell entry
[4,53]. In these cases, rapid formation of the six alpha helix bundle may help to the transition from the rate limiting step of Env engagement with CCR5, with or without the inhibitor. Collectively, these observations, caveats, and exceptions with HIV-1 resistance to CCR5 antagonist suggest that divergent evolution within drug-sensitive env genes does not necessarily result in convergence to a specific resistance mechanism.
Our in vitro selection experiments for MVC resistance started with three primary
HIV-1 isolates but only a subtype A (A74) developed MVC resistance over a 6 month time period. This MVC-resistant virus retains CCR5 binding and does not switch co- receptor usage. Unlike previous studies [215,233,245], a V3 loop mutation is not
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associated with resistance and our MVC-resistant virus displays only a weak MPI effect.
Instead, resistance is described as a >20-fold shift in the concentrations required to inhibit
50% of the resistant versus wild type virus. This increase in IC50 concentrations is typical
for HIV-1 resistance to nearly all antiretroviral drugs but is rarely observed with HIV-1
resistant to CCR5 antagonists. As in previous studies, our MVC resistant A74 virus had
mutations scattered throughout the gp120 coding region that appeared to be selected due
to drug pressure. However, most of these mutations had minimal effect on the resistance
phenotype but may stabilize the N425K mutation found in the C4 domain. This
observation was clearly demonstrated with the MVC-sensitive MVC.21.122 clone which
harbored the R117Q, Q290K, L396V, and D461E mutations but lacked the N425K
mutation. When the N425K mutation was introduced into the MVC.21.122 clone, this
virus was now highly resistant to MVC (>36-fold increase in IC50 values).
As described above, most diverse HIV-1 isolates appear to follow different
evolutionary pathways to MVC resistance but all have been linked to emergence of a V3
loop mutation. These V3 loop mutations are often found at high frequency in the
untreated HIV-1 populations suggesting higher entropy, low cost on replicative fitness,
and a lower genetic barrier to resistance. We have previously shown that env mutations
conferring resistance to entry inhibitors were often associated with increased replicative
fitness, i.e. directly related to enhanced host cell entry efficiency [6,53,126]. This is in
sharp contrast to decreased replicative fitness observed with acquired resistance to nearly
all other antiretroviral drugs. However, drug resistance mutations conferring lower
fitness costs are often found at higher frequencies in the untreated HIV-1 population or
within the intrapatient population. For example, there is a low genetic and fitness barrier
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to NVP resistance via the K103N mutation [7,93] which reflects (1) the slow reversion of this mutation following cessation of NVP treatment [57,78,160] and (2) increasing circulation and transmission of the K103N virus within the human population where
NVP-based treatment regimens are most prevalent [220]. In this study, we have shown that the collection of E33bG, R117Q, Q290K, L/G396V, N425K, and D461E selected under MVC pressure resulted in virus of higher replicative fitness than the wild type/passage control virus. With the exception of the N425K mutation, these mutations are found as dominant sequences in relatively high proportions of HIV-1 among the subtype A population and within the group M HIV-1 population in general. Thus, these findings suggest a low genetic barrier for these mutations and limited fitness cost. We did not observe a wild type virus containing all of these natural polymorphisms. Thus, the emergence of all mutations may follow a complex fitness landscape which in this case, is directed by MVC selective pressure and emergence of resistance. In contrast to the relative high frequency of these polymorphisms, the N425K mutations is found in
<1% of all HIV-1 sequences in the Los Alamos Database (n=61,096) and has never been associated with resistance to any entry inhibitor. Interestingly, the unique MVC sensitive clone which emerged under MVC selective pressure, i.e. MVC.21.122 had a higher replicative fitness than the highly MVC resistant viruses MVC.21.119 and MVC.21.128.
All three of the viruses contained the R117Q, deletion of 138-139 and L/G396V mutations but the more fit, MVC.21.122 lacked the primary drug resistance mutation,
N425K. These findings suggest that N425K mutation may confer a fitness cost which is compensated by the linked R117Q and L/G396V mutations. However, it is important to stress that resistance to entry inhibitors based on Env mutations are less predictable in
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terms of replicative fitness costs. Nearly all drug resistant mutations to the RT inhibitors, protease inhibitors, and integrase inhibitors confer a fitness cost [227]. In contrast, higher replicative fitness is often related to resistance to CCR5 antagonists and agonists even when the drug is absent [6,53,126]. This increased replicative fitness of the virus resistant to the CCR5 agonist/anatagonist is commonly related to enhanced CCR5 binding affinity
(with or without bound inhibitor) and faster CCR5 binding kinetics which is the rate limiting step in the host cell entry process. As part of more detailed studies on the
N425K mutation and its interaction with CD4, we are currently exploring the direct impact of this mutation on replicative fitness, entry efficiency, CD4 affinity, and other parameters.
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CHAPTER 4
ENHANCED CD4 BINDING AFFINITY NOVEL MECHANISM OF HIV-1 RESISTANCE TO MARAVIROC
Authors: Annette N. Ratcliff1,2, Wuxian Shi3, Richard Gibson2, John C. Tilton3, and Eric J. Arts1,2
1Department of Molecular Biology and Microbiology
2Division of Infectious Diseases
3Center for Proteomics and Bioinformatics
Case Western Reserve University, Cleveland, OH 44106
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4.1 Preface
We have established that mutations in the CD4 binding site of the envelope
glycoprotein gp120 mediate resistance to maraviroc for virus A74.MVC.21. These
findings suggested an alternative mechanism of resistance related to gp120-CD4 binding
affinity and overall entry kinetics rather than the noncompetitive resistance often
described for inhibitors of this class. Here, we sought to investigate the CD4 binding
affinity of the maraviroc resistant virus and compare resistance of this virus with a
maraviroc resistant virus that utilizes MVC-bound CCR5 for entry.
Structural analyses were performed by Wuxian Shi. Richard Gibson aided in experimental design. Viruses S2 and R3 were kindly provided by John C. Tilton.
Remainder of experiments and analyses were performed by the author.
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4.2 Abstract
HIV-1 resistance to maraviroc as well as other CCR5 antagonists such as vicriviroc, TAK-779, aplaviroc and AD101 has been largely attributed to acquired mutations in the V3 loop of the envelope glycoprotein gp120 resulting in usage of drug- bound conformations of CCR5. We previously described the isolation of maraviroc resistant variant A74.MVC.21 harboring a K425 mutation in the CD4 binding site of gp120 and exhibiting a mixed resistance phenotype with reductions in maximal percent inhibition and shifts in half maximal inhibitory concentrations. In this study, we sought to explore the mechanism of resistance of gp120 chimeric clones harboring K425 through comparative analyses with clones bearing gp120 of the maraviroc resistant variant R3 previously characterized as utilizing MVC-bound CCR5 for entry. Drug susceptibility assays performed under single and multiple replication cycle conditions revealed significant differences with A74.MVC.21 clones demonstrating a resistance phenotype only under multiple cycle conditions. When exposed to sCD4, K425 bearing viruses were adept at infecting cells in the absence of cell surface CD4. Additionally, use of 293-
Affinofile cells revealed resistant clones were efficient at infecting cells bearing very low levels of CD4 but across a wide range of CCR5 on the cell surface. We also show that enhanced rate of entry and increased viral fitness are associated with resistant viruses bearing K425. Together, these data suggest a novel mechanism of resistance to maraviroc related to enhanced CD4 binding affinity and increased entry efficiency.
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4.3 Introduction
Engagement of CD4 by the gp120 glycoprotein of human immunodeficiency virus’s (HIV) envelope induces structural changes in the envelope complex exposing a coreceptor binding site formed by gp120’s bridging sheet and third variable (V3) loop.
Coreceptor (CCR5 or CXCR4) binding initiates further structural rearrangements permitting insertion of gp41 into the target cell membrane culminating in membrane fusion. CCR5 antagonists are a relatively new class of antiretrovirals that induce altered receptor conformations which prevent gp120 recognition and binding to CCR5.
Maraviroc (MVC) is thus far the only small molecule CCR5 antagonist approved for therapeutic use in patients, however other inhibitors such as vicriviroc (VVC), TAK-779, and aplaviroc (APL) likewise induce altered receptor conformations upon binding to
CCR5. As with any class of antiretrovirals, the development of drug resistant variants arising during treatment can result in virologic rebound. Understanding and predicting drug resistance is important for optimal treatment outcomes.
Resistance to CCR5 antagonists can result from the outgrowth of CXCR4-tropic strains that were pre-existing at low frequencies in the viral swarm prior to initiation of treatment [232]. Patients now undergo tropism testing and treatments with CCR5 antagonists are strictly limited to those harboring exclusively R5-tropic viruses.
Although outgrowth of X4-tropic strains remains problematic for CCR5 antagonist therapy, new mutations that result in altered coreceptor tropism have not been detected.
Rather, the limited number of viruses reported to acquire resistance either in vitro or in vivo typically developed adaptive amino acid mutations in gp120, particularly the V3
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loop, which allowed utilization of inhibitor-bound conformations of CCR5 for entry
through a noncompetitive mechanism observed in phenotypic assays as a decrease in
maximal percent inhibition (MPI) [105,138,157,170,215,217,233]. While this appears to
be the preferred mechanism for R5 virus resistance, noteworthy exceptions have been
observed including gp41 fusion peptide mutations causing VVC resistance [4] as well as
a report of MVC clinical resistance through an apparent competitive mechanism between
gp120 and MVC for CCR5 occupancy [45].
We previously reported an in vitro-derived HIV-1 subtype A MVC resistant
isolate which harbored a N425K primary resistance mutation in the C4 region of gp120
but lacked any V3 loop mutations associated with resistance. The location of residue 425
in the crystal structure of gp120YU-2 complexed with CD4 suggested a role for enhanced
CD4 binding in the mechanism of MVC resistance of this virus. In this study, we
compared resistance profiles of this subtype A virus (A74.MVC.21) with a subtype B
patient-derived MVC resistant virus (R3) previously shown to utilize drug-bound CCR5
[215]. Unlike the subtype B resistant virus, the in vitro-derived resistant virus did not exhibit a resistance profile in single cycle assays but rather demonstrated an increase in half-maximal inhibitory concentration (IC50) under multiple replication cycle conditions.
This virus was capable of binding soluble CD4 with high affinity, displayed efficient use of low level CD4 in 293 Affinofile cells, exhibited faster fusion kinetics and higher replicative fitness. Our findings suggest MVC resistance is mediated by enhanced CD4 binding affinity through a competitive resistance mechanism.
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4.4 Materials and Methods
4.4.1 Cells, Viruses and Inhibitors. U87.CD4.CCR5 and 293T cells were maintained as described in 2.4.1. 293 Affinofile cells, a kind gift of Dr. Benhur Lee, are a HEK293 derived cell line dually inducible for CD4 and CCR5 expression [95]. This cell line was generated by the sequential transduction of transactivators and the inducible promoters for expressing CD4 and CCR5. CD4 and CCR5 receptor expression can be simultaneously and independently induced with doxycycline and ponasterone A (ponA), respectively. 293 Affinofile cells were maintained in DMEM supplemented with 10% dialyzed FBS, 100µg/mL penicillin/streptomycin, and 50 µg/ml blasticidin. Prior to use, surface receptor expression of CD4 and CCR5 were determined by quantitative fluorescence-activated cytometry (qFACS) analysis using either phycoerythrin- conjugated mouse anti-human CD4 antibody (clone Q4120; Invitrogen, Carlsbad, CA) or phycoerythrin-conjugated mouse anti-human CCR5 antibody (clone 2D7; BD
Biosciences, San Jose, CA). Duplicate 6-well plates were seeded with 2x105 cells/well and CD4 and CCR5 expression were induced the following day using doxycycline and
Ponasterone A, respectively. Cells were induced using 2-fold dilutions from 0-2 ng/mL of doxycycline (CD4) and 0-2 µM of PonA (CCR5) for 24 hours. Surface receptor expression levels were quantified as previously described [95]. 293.CD4 cells, a kind gift of Dr. John C. Tilton, are a HEK293 cell line stably transduced with the MV7neo-T4 retroviral vector encoding human CD4. These cells were maintained in complete media supplemented with 300µg/mL G418.
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Gp120 chimeric replication competent viruses were generated as described in section 3.4.4. Psuedoviral particles were generated by cotransfection of 293T cells with pREC_NFL_gp120 plasmids and pNL.Luc.AM psuedotyping vector. Expression vectors encoding envelopes of viruses S2 and R3 were kindly provided by Dr. John C. Tilton
[215]. Gp120 regions of S2 and R3 were PCR amplified and cloned by yeast homologous recombination into pREC_NFL_Δgp120/URA3 as previously described. Sequencing confirmed the integrity of the cloned regions regarding MVC resistance mutations.
Maraviroc and TAK-779 were diluted in PBS and filter sterilized. C34 and recombinant human soluble CD4 (2 domain) produced and purified from Escherichia coli
were obtained through the AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH [12,71,75]. Recombinant human soluble CD4 (4 domain) produced
using recombinant baculovirus vectors and secreted from insect cells was purchased from
Protein Sciences Corporation, Meriden, CT.
4.4.2 Structural Modeling. The location of MVC resistance mutations were
identified using the crystal structure of gp120 HIV-1YU-2 with CD4 and tyrosine-sulfated
antibody 412d (PDB ID: 2QAD) [91]. Sequence alignment of gp120YU-2 with A74.PC.21
indicated relative homology in the C4 region. Using 2QAD as a template, the K425
mutation was modeled into the structure using the COOT structural modeling program
[56]. The local structure was regularized with slight torsion of the lysine side chain
resulting in two potential new interactions: a hydrogen bond with S42 and cation-π
interaction with aromatic ring of F43 of CD4. This structural model was also compared
to a high resolution structure of gp120 in complex with CD4 and a CD4i antibody 17b
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(PDB ID: 1G9M) [109]. The Nε of K425 in the structural model superimposed well with a bound water molecule in the binding pocket of F43 from the 1G9M crystal structure.
4.4.3 Drug Susceptibility Assays. For single replication cycle drug susceptibility assays, U87.CD4.CCR5 cells in 96-well format (1x104cells/well) were pretreated for 2 hours with 10-fold dilutions of MVC (1µM to 1x10-7µM) then infected in triplicate with luciferase encoding psuedovirus. After 48 hours, supernatant was removed and cells were lysed using 50µL Glo lysis buffer (Promega, Madison, WI). Cells were lysed for 20 minutes at room temperature, transferred to white polystyrene 96 well plates, and read with a PerkinElmer VICTOR plate reader using injectors to introduce 50µL Bright Glo reagent per well (Promega, Madison, WI). Luciferase activity was measured as relative light units (RLU).
For multiple replication cycle drug susceptibility assays, U87.CD4.CCR5 cells were plated as above and pretreated with 5-fold dilutions of MVC (100µM to 2x10-6 µM).
Cells were infected at an MOI of 0.001 IU/cell with NL4-3/gp120 chimeric virus in quadruplicate. After 24 hours, viral inoculums were removed and replaced with media containing the appropriate MVC dilutions. Reverse transcriptase activity in supernatant was monitored as previously described on days 4 through 7 post infection.
4.4.4 sCD4 Activation and Inhibition Assays. 293T or 293.CD4 cells (1x106 cells) were plated in 10cm2 tissue culture dishes and transfected with 6µg pBABE.CCR5 plasmid (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH [46,147]) using Fugene 6 tranfection reagent (Invitrogen). After 24 hours, cells were
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plated in 96-well plate format at a density of 1x105 cells/well. Normalized amounts of
psuedoviruses were preincubated with 20 or 200nM of 4 domain sCD4 (Protein Sciences
Corporation, Meriden, CT) at 37⁰C for 30 minutes prior to infection. Cells were infected
in triplicate and luciferase activity measured 48 hours after infection as described above.
Fold change in RLU as well as percent inhibition were measured relative to the luciferase
activity of the no sCD4 condition.
For sCD4 titration assays, 293T or 293.CD4 cells were treated in the same
manner described above. Cells were plated in 96-well plate format at 1x105 cells/well
after CCR5 transfection. Cells were treated with 4-fold dilutions of 2 domain sCD4
(25µg/mL to 0.001µg/mL) and then infected with psuedoviruses in triplicate. Luciferase
activity was measured 48 hours after infection.
4.4.5 CD4/CCR5 Receptor Affinity Assays. 293 Affinofile cells were plated in
96-well format (1.5x104 cells/well) and CD4 and CCR5 receptor expression was induced the following day. Maximal and minimal receptor expression was induced for each receptor in triplicate columns of the plate using 2ng/mL and 0.03ng/mL doxycycline for
CD4 and 2µM and 0.03 µM Ponasterone A for CCR5. In rows, the alternate receptor
expression was induced in 2-fold serial dilutions (8 dilutions) from 0-2 ng/mL
doxycycline (CD4) and 0-2µM PonA (CCR5). This induction scheme results in 32
distinct CD4 and CCR5 surface expression profiles. Cells were incubated at 37⁰C for 24
hr prior to infection. Cells were infected in triplicate with normalized psuedovirus and
luciferase activity measured 48 hours after infection as described above. Maximal
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infection was considered to be the luciferase activity measured for the highest induction
of both CD4 and CCR5 and percent infection was calculated relative to this condition.
4.4.6 Kinetic Fusion Assay. U87.CD4.CCR5 cells (5x105 cells) were
spinoculated with psuedovirus at 2500xg at 4⁰C for 90 minutes. Cells were then plated in
96-well plates at 1x105 cells/well in 50µL 4⁰C cold media. Infection was synchronized
by addition of 130uL/well media prewarmed to 37⁰C. This was considered time 0 post-
infection. At time intervals post infection, 20µL of the fusion inhibitor C34 (100µM final) was added to triplicate wells. Cells were incubated at 37⁰C for 48 hours and
luciferase activity was measured. Relative infection was calculated based on the
maximal luciferase activity observed for each virus at three hours post-infection.
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4.5 Results
4.5.1 Differential Maraviroc Resistance Profiles. Multiple studies of CCR5 antagonist resistant viruses have described an inability to completely suppress viral replication at high drug concentrations [105,215,217,219,233]. This has been attributed to mutations in the gp120 glycoprotein, particularly the V3 loop, that permit interactions with drug bound conformations of CCR5. However, we have shown that while MVC resistant virus A74.MVC.21 continues to replicate at high drug levels, the primary resistance mutation is located in a region of gp120 which comprises the CD4 binding site and is unlikely related to altered CCR5 binding. To better understand the resistance of
A74.MVC.21, we compared this virus with another MVC resistant isolate (R3) which was derived from a patient failing MVC therapy in the SCOPE cohort and was shown to utilize MVC-bound CCR5 for entry [215].
As shown in Figure 33, maraviroc resistant viruses A74.MVC.21 and R3 differ in primary resistance mutations. The primary resistance mutation for virus A74.MVC.21 was previously mapped to a K425 mutation in the C4 region of gp120 with mutations
Q117 and V396 in other regions of gp120 appearing associated with resistance in this isolate. Alternately, the primary resistance mutation identified in the R3 virus was a
H308 mutation in the V3 loop region with V3 mutations H320 and V322a as well as a V4 loop mutation G407 modulating the level of resistance [215]. When comparing the location of these resistance mutations in a structure of HIV-1 gp120YU-2 in complex with
CD4 and antibody 412d (PDB: 2QAD, [91]) we noticed that residue 425 was located proximal to the F43 residue of CD4 (Fig. 33), a residue known to be critical in gp120-
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CD4 binding interactions [8,143,186]. Modeling of the N425K transition (inset) revealed
new potential interactions between side chain of K425 and F43, specifically a cation-π
interaction with the aromatic ring of the phenylalanine. Additionally a new hydrogen bond could be formed with S42 of CD4. This structural model was also compared to a high resolution structure of gp120HXBc2 in complex with CD4 and a CD4i antibody 17b
(PDB ID: 1G9M) [108]. The Nε of K425 in the structural model superimposed well with
a bound water molecule in the binding pocket of F43 from the 1G9M crystal structure.
This model suggests a role for CD4 binding in A74.MVC.21 resistance to maraviroc. In
contrast, the H308 primary resistance mutation of virus R3 is located in the stem of the
V3 loop, a region believed to interact with the second extracellular loop of the CCR5
coreceptor. R3 has previously been shown to utilize inhibitor bound CCR5 for entry and
was sensitive to alanine substitutions in both the N terminus and extracellular loops of
mutant CCR5 receptors [215].
In addition to genotypic differences, the phenotypic resistance profile of NL4-
3/gp120 chimeric viral clones derived from A74.MVC.21 differed from those described
for other MVC resistant viruses in that resistance was observed as increases in the
concentration of MVC required to suppress viral replication by 50% (IC50). To directly
compare resistance profiles of A74.MVC.21 derived gp120 clones and clone R3 in a
similar genomic background, the gp120 regions of R3 and the pretreatment sensitive
clone S2 were PCR amplified and cloned by yeast homologous recombination into
pREC_NFL_Δgp120/URA3. Replication competent viruses as well as luciferase
expressing psuedoviruses were generated for A74.PC.21.109, A74.MVC.21.132, S2, and
R3 as described. Both single cycle and multiple replication cycle drug sensitivity assays
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were performed in U87.CD4.CCR5 cells (Fig. 34). Strikingly, resistance could not be detected for A74.MVC.21.132 in single cycle assays (Fig. 34A) but was clearly indicated by a 17-fold rightward shift in IC50 value as compared with the MVC sensitive clone
A74.PC.21.109 in multiple replication cycle conditions (Fig. 34B). In direct contrast, resistance for clone R3 was observed as a reduction in the maximal percent inhibition
(MPI) to 56% and 61% under both single and multiple cycle conditions, respectively
(Fig. 34C, D). The differential resistance profiles obtained for A74.MVC.21.132 and R3 in addition to the location of the resistance mutations in the gp120 structure suggested a mechanism of resistance for A74.MVC.21.132 unrelated to utilization of inhibitor bound
CCR5.
4.5.2 Recombinant Human Soluble CD4 Enhances Infection of
A74.MVC.21.132. Given the proximity of the K425 mutation of A74.MVC.21 to the putative binding site of CD4, we wanted to determine whether soluble CD4 (sCD4) could mediate entry of A74.MVC.21.132 into cells that did not express cell surface CD4. At non-inhibitory concentrations sCD4 can act as a surrogate for cell bound CD4, inducing the conformational changes in gp120 required for coreceptor binding and permitting infection of CD4 negative cells. This enhancement of virus infection or activation by sCD4 has been described for some SIV, HIV-2 and HIV-1 strains and seems to correlate with high CD4 binding affinity [3,35,194,197]. The concentration of sCD4 required to induce enhancement of infection can vary widely dependent on viral strain.
293T cells were transfected to express CCR5 and infected with psuedovirus that had been preincubated at 37 C with either 20nM or 200nM of sCD4 (D1-D4) (Fig. 35A).
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In the absence of sCD4, neither the sensitive or MVC resistant viruses were able to infect
cells lacking surface CD4. However, resistant virus A74.MVC.21.132 was able to infect
CD4 negative cells in the presence of 20nM and 200nM sCD4 demonstrating 3-fold and
2-fold increases in RLU, respectively (Fig. 35A). Sensitive viruses A74.PC.21.109 and
S2 were not able to infect cells even in the presence of sCD4 while resistant virus R3 displayed a 1.4-fold increase in RLU at 200nM sCD4. To determine whether sCD4 could enhance infection in cells that expressed surface CD4, 293 cells stably expressing CD4 were transfected with CCR5 and infected with psuedovirus pretreated with sCD4 in the same manner as CD4 negative cells (Fig. 35B). While all viruses were able to infect these CD4+CCR5+ cells, again the A74.MVC.21.132 virus demonstrated an enhancement of infection with 5.2-fold higher RLU activity at 20nM sCD4 and 2.7-fold higher activity at 200nM as compared to psuedovirus that was not preincubated with sCD4 prior to infection. In contrast, infection of A74.PC.21.109 was inhibited by 11 and
32% at these concentrations, respectively. Infectivity of virus S2 was relatively unaffected by these concentrations of sCD4 while the resistant virus R3 showed only a
1.6-fold change in RLU at the highest sCD4 concentration tested (Fig. 35B).
The enhancement of infection of A74.MVC.21.132 by sCD4 in both CD4
negative and CD4 positive cells suggested this virus was binding sCD4 with higher
affinity than the A74.PC.21.109 virus. However, the reduction in fold change RLU of
A74.MVC.21.132 at 200nM versus 20nM sCD4 indicated this effect was concentration
dependent and that at higher concentrations this virus may be inhibited by sCD4. To
further examine sCD4 effects on A74.MVC.21.132 and determine requirements of CD4
domains, a titration curve using the recombinant D1-D2 form of sCD4 produced in
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bacteria was performed in 293T and 293.CD4 cells transfected to express CCR5 (Fig.
36). Recapitulating the results using sCD4 (D1-D4), the 2 domain peptide permitted
infection of CD4 negative cells for virus A74.MVC.21.132. A steady enhancement of
infection was observed for A74.MVC.21.132 up to 0.4µg/mL sCD4. Beyond this
concentration, RLU activity decreased to a level observed at the lowest drug
concentration (Fig. 36A). Interestingly, in 293.CD4 cells expressing CCR5 infection also
appeared enhanced up to 0.4µg/mL at which point the level of infection declined rapidly
for A74.MVC.21.132 resulting in complete suppression of virus infection by 6.25µg/mL
(Fig. 36B). The resultant IC50 for A74.MVC.21.132 was 1.5µg/mL. Alternatively,
A74.PC.21.109 appeared less sensitive to inhibition by sCD4 with viral infection
suppressed by only 50% at the highest concentration tested (25µg/mL). Together the data in Figures 35 and 36 suggested that in a concentration dependent manner sCD4 could induce the necessary conformational changes in gp120 of the MVC resistant virus
A74.MVC.21.132 to permit entry into CD4 negative cells. Beyond a threshold concentration however, A74.MVC.21.132 became sensitive to inhibition of CD4 positive cells potentially through a competitive binding mechanism between sCD4 and cellular
CD4 for gp120 binding.
4.5.3 Efficient Infection of Cells with Low Levels of Surface CD4. We next measured the ability of A74.MVC.21.132 to infect cells that express low levels of surface
CD4 (Fig. 37). 293 Affinofile cells have dual inducible markers for variable and independent expression of both CD4 and CCR5 [95]. Based on quantitative flow cytometry we determined the concentrations of doxycycline and ponasterone A required
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to induce minimal CD4 and CCR5 expression, respectively. In Figure 37, the results of
low CD4 and low CCR5 induction across a range of alternate receptor expression levels
are depicted. The low CD4 induction condition resulted in approximately 3000 receptors
per cell. The A74.MVC.21.132 virus was particularly adept at utilizing low levels of
CD4 to mediate infection of cells across a wide range of CCR5 receptor surface
expression compared to A74.PC.21.109 which lacked the K425 mutation (Fig 37A).
Similarly, under low CCR5 surface expression levels (5000 receptors per cell),
A74.MVC.21.132 was more efficient at infecting cells at the lower CD4 expression
levels than A74.PC.21.109 (Fig. 37B).
4.5.4 Maraviroc Resistant Virus Demonstrates Increased Sensitivity to CD4
Mimetic. If K425 binds with greater affinity to F43 of CD4 then it is possible that this
resistant virus may also bind CD4 derived compounds designed to mimic the binding of
F43 to gp120 with greater affinity. Enhanced binding to a CD4 mimic would deprive the
virus of the ability to bind cell surface CD4 and inhibit entry. To test this hypothesis,
drug susceptibility assays were performed in U87.CD4.CCR5 cells in the presence of
decreasing concentrations of the attachment inhibitor BMS-806. BMS-806 is a first
generation attachment inhibitor specifically designed to mimic F43-CD4 and bind to gp120 within the F43 binding pocket. Figure 38 shows that at the highest concentrations tested (50 and 5µM), MVC.21 is inhibited to a greater extent (98%) than is the maraviroc
sensitive PC.21 virus (46%). Although inhibition by this compound is weak for the
parental virus, this data would suggest an increase in sensitivity to the attachment
inhibitor for MVC.21, likely as a result of tighter binding of the compound within the
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F43 binding pocket. Since K425 is the only amino acid different between MVC.21 and
PC.21 within this binding pocket, it is reasonable to surmise that K425 enhanced binding
affinity to BMS-806 and was thereby more easily blocked from binding surface CD4.
4.5.5 Maraviroc Resistant Virus Exhibits Faster Entry Kinetics. Mutations
affecting either CD4 or CCR5 binding have been shown to influence viral entry kinetics,
including mutations associated with CCR5 antagonist resistance [172]. To assess the
impact of the resistance mutation K425 on viral fusion kinetics we performed a time of
drug addition assay using the fusion peptide C34 in U87.CD4.CCR5 cells (Fig. 39). The
sensitive A74.PC.21.109 virus exhibited a half-maximal time of fusion (t1/2) of 120 minutes. In comparison, the rate of fusion for the A74.MVC.21.132 resistant virus was three times faster with a t1/2 of only 40 minutes. This would suggest that not only does
K425 significantly impact the interactions of this virus with CD4, but also enhances the
overall rate of the entry process.
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Figure 33. Maraviroc Resistance Mutations of R3 and A74.MVC.21 Viruses. (A) The maraviroc resistant viruses R3 and A74.MVC.21 differ in their resistance mutations. Virus R3 has a primary resistance mutation H308 in the V3 loop with mutations H320, V322a, and G407 associated with the phenotype. In contrast, A74.MVC.21 contains the primary resistance mutation K425 in the C4 region of gp120 with mutations Q117 and V396 associated. (B) Modeling of K425 into the HIV-1YU-2 gp120 structure identified new interactions between K425 and F43 of CD4 (yellow lines). Alignment of the YU-2 strain and A74.MVC.21 C4 region of gp120 is shown. Amino acid differences between strains are shown in bold. Resistance mutations for R3 (blue) and A74.MVC.21 (red) are indicated as spheres in the structure.
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Figure 34. Maraviroc Resistance Profiles in Single and Multiple Cycle Assays. Sensitivity of maraviroc resistant viruses A74.MVC.21.132 (red line) and R3 (blue line) were performed in single- and multiple-replication cycle assays. Single replication cycle drug susceptibility assays (panels A and C) were performed in U87.CD4.CCR5 cells using psuedoviruses. Luciferase activity was measured 48 hours after infection and percent inhibition calculated based on the no drug condition. Multiple replication cycle drug susceptibility assays (panels B and D) were performed in U87.CD4.CCR5 cells. Reverse transcriptase activity was quantified and percent inhibition calculated based on the no drug condition. Standard deviations are shown. Curves were generated using curve fitting features of GraphPad Prism 5 software.
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Figure 35. Pre-exposure to sCD4 Enhances Infection by A74.MVC.21.132. (A) Psuedoviruses were pre-incubated with sCD4 (0, 20, or 200nM) and then used to infect 293T cells transfected to express CCR5 but lacking cellular CD4. Luciferase activity was measured and compared with the no sCD4 condition. Data represents mean of triplicates with standard deviations. (B) Psuedoviruses were treated as described and used to infect 293.CD4 cells transfected to express CCR5. Luciferase activity was measured and compared with the no sCD4 condition. Data represents mean of triplicates with standard deviations.
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Figure 36. Enhancement of Viral Infection and Inhibition by sCD4 is Concentration Dependent. (A) Psuedoviruses were pre-incubated with sCD4 in 4-fold dilutions starting at 25µg/mL and used to infect 293T cells transfected to express CCR5. Luciferase activity (RLU) was measured 48 hours post infection. (B) Psuedoviruses were pre-incubated with sCD4 in 4-fold dilutions starting at 25ug/mL and used to infect 293.CD4 cells transfected to express CCR5. Luciferase activity was measured 48 hours post infection. Percent infection was calculated based on the luciferase activity measured for the no sCD4 condition. Data represents means of triplicates with standard deviations shown.
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Figure 37. A74.MVC.21.132 Infects Cells Expressing Low Surface Density CD4. (A) 293 Affinofile cells were induced as described in Materials and Methods to express low levels of CD4 across a range of CCR5 surface expression levels. Cells were infected with psuedovirus and luciferase activity measured 48 hours post infection. Percent infection was calculated based on the luciferase activity measured for the highest induction of both receptors (not shown). (B) 293 Affinofile cells were induced to express low levels of CCR5 across a range of CD4 surface expression levels and infected with psuedovirus as described. Data represents means of triplicates with standard deviations. * represents p < 0.01 (one-tailed T test).
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Figure 38. Sensitivity to Attachment Inhibitor BMS-806. Drug susceptibility assays performed in U87.CD4.CCR5 cells with 10-fold dilutions of BMS-806 were performed with sensitive (A74.PC.21, black line) and maraviroc resistant (A74.MVC.21, red line) viruses. Reverse transcriptase activity was measured five days post infection and percent inhibition calculated based on the no drug condition. Data represents means of triplicates with standard deviations.
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Figure 39. Maraviroc Resistant Virus Demonstrates Enhanced Fusion Kinetics. Time of drug addition assays were performed in U87.CD4.CCR5 cells using luciferase encoding psuedovirus particles to score fusion. An IC99 concentration of the fusion inhibitor C34 was added to cells at the indicated time intervals post infection. Relative infection was measured based on the maximal fusion scored for each virus at 3 hours post infection. Data represents triplicates with standard deviations shown. Curves were generated using nonlinear regression curve fitting features of GraphPad Prism 5 software.
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4.6 Discussion
Studies have characterized CCR5 antagonist resistance in primary HIV-1 viruses either selected in vitro or derived from patients on entry inhibitor therapy
[4,11,138,214,215,217,219] while others have described resistant variants generated in
culture using laboratory adapted strains or mutated genomes to promote selection of
escape mutants [110,233,245,246]. Although virologic failure in patients on CCR5
antagonist therapy often relates to outgrowth of pre-existing CXCR4 tropic virus present
prior to initiation of treatment [232], in some cases viruses replicating in the presence of
CCR5 antagonists either in vitro or in vivo have evolved mechanisms to recognize and
bind inhibitor-altered conformations of coreceptor while maintaining the ability to use
drug-free conformations [5,11,105,138,156,214,215,217,233]. Despite a notable
exception involving mutations in gp41 [5], mutations in the V3 loop, have led to a
mechanism involving the acquired ability of resistant viruses to utilize inhibitor-bound
forms of CCR5 in multiple instances [138,156,170,180,214,215,217,233]. In most
instances, viruses that adapt to use inhibitor-bound forms of the coreceptor do so through
mutations in the V3 region of gp120, however mutations in other regions of gp120 and
gp41 have been associated with resistance in some viruses. In the present study, we
compared resistance of one such V3-mutant virus (R3) demonstrating the ability to use
drug-bound coreceptor with a resistant virus harboring a mutation in the CD4 binding site
of gp120. Here, we present evidence that MVC-resistant virus A74.MVC.21.132
demonstrates efficient binding of soluble CD4, a lower threshold requirement for CD4
surface density, increased sensitivity to a CD4 mimetic inhibitor, and enhanced rate of
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fusion. We propose that mutation K425 in the phenylalanine 43 binding pocket of gp120
lowers the threshold requirement for CD4 binding and leads to efficient coreceptor
binding site exposure which permits this virus to outcompete inhibitor for drug-free conformations of CCR5.
The R3 viral clone, containing V3 loop mutations as well as loss of a glycosylation site at residue 386, was previously isolated from the viral swarm of a patient failing MVC therapy during the SCOPE trial [215]. Like other resistant viruses capable of using drug-bound coreceptor, in phenotypic assays virus R3 demonstrated
incomplete suppression of replication at high MVC concentrations (Fig. 34).
Alternatively, MVC-resistant A74.MVC.21.132 exhibited a shift in half-maximal inhibitor concentration denoting a competitive mechanism of resistance. Interestingly, whereas resistance was observed under both single- and multiple- replication cycle assays for R3, a resistance phenotype was not apparent for A74.MVC.21.132 in single cycle assays where viruses were limited to a single entry event. Rather, shifts in IC50 became
more pronounced over multiple rounds of replication. The disparate drug susceptibility
curves generated in single versus multiple cycle conditions raise questions about
maraviroc-CCR5 interactions and receptor occupancy. Since receptor downregulation is
not induced by maraviroc binding an alternative mechanism must be at play.
Theoretically, in single cycle assays 100% occupancy of receptor is achievable at high
inhibitor concentrations; however, over time through an as yet unresolved mechanism
some receptors may become unoccupied and therefore available for gp120 binding. We
are currently investigating the mechanism responsible for the apparent lack of resistance
observed in single-cycle assays and how this relates to MVC-CCR5 interactions.
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Attachment of the envelope to CD4 is the critical initiating step of the entry process. Engagement of CD4 leads to structural rearrangements, exposing regions of gp120 that form a discontinuous coreceptor binding site [31,63]. For purposes of immune evasion, the coreceptor binding site and V3 loop remain concealed until after CD4 binding. However, studies have described CD4-independent HIV-2, SIV, and laboratory adapted HIV-1 strains with envelopes capable of mediating entry via CCR5 or CXCR4 in the absence of CD4 [35,55,89,103]. Increased sensitivity of these envelopes to neutralizing antibodies that target CD4 induced epitopes suggest that these envelopes exist in an open/activated state where the coreceptor binding site is exposed and available for binding without the requirement for CD4 to induce such a conformation. Similarly, viruses that efficiently bind CD4 under limiting receptor conditions have demonstrated increased exposure of coreceptor binding regions as evidenced by increased sensitivity to neutralization. Taken together these observations would suggest that viruses that acquire
CD4 independence or that demonstrate reduced threshold for CD4 receptor levels via efficient CD4 binding are able to proficiently mediate transitions to active states required for coreceptor binding and membrane fusion.
While improved CD4 binding affinity has been associated with resistance to
CCR5 antagonists in laboratory adapted viruses [110], here we note the first instance of this type of resistance mechanism occurring in a primary isolate under selective pressure.
A previous study using a virus in which the V3 loop base and crown had been deleted found this virus bound CD4 with greater affinity and was refractory to inhibition by small molecule CCR5 inhibitors [110]. Loss of the V3 base and crown likely resulted in loss of interactions with the extracellular loops of CCR5, the domains altered by CCR5
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antagonist binding. Studies of other CCR5 antagonist resistant viruses have described
altered CCR5 interactions where resistant envelopes rely on interactions with the N- terminus, a domain relatively unaffected by inhibitor binding [51,166,181]. It is therefore not surprising that adaptation of this virus to replicate without the V3 loop also resulted in a virus resistant to inhibition by these compounds. However, the MVC-resistant virus we have studied here developed enhanced CD4 binding through acquisition of mutation
K425 even in the presence of an intact V3 loop. This would suggest that mutation of the
V3 loop to accommodate CCR5 conformations induced by inhibitor binding was not the preferred mechanism as has been described for other resistant viruses. Instead, increased affinity for CD4 may enable this virus to either outcompete inhibitor for CCR5 occupancy or scavenge unbound CCR5 in a limiting environment.
There are several potential explanations for why this mode of resistance has not
yet been described in vivo. The first involves the widespread utilization of single round
entry assays to determine phenotypic resistance to CCR5 antagonists. For a variety of
reasons including ease of producing enveloped pseudovirus as compared with infectious
virus particles, standard phenotypic assays used to determine CCR5 antagonist resistance
are limited to a single round of virus entry. As described earlier, MVC resistance for
A74.MVC.21.132 was only observed under multiple cycle conditions and would be
overlooked in single-cycle assays (Fig. 34). A second explanation for why this type of
resistance has not been described in vivo is associated with neutralization potential. As mentioned, a consequence of induction of activated envelope conformations and exposure of coreceptor binding site is increased sensitivity to neutralization [89,103,244].
It is reasonable to theorize that a virus such as A74.MVC.21.132 that binds CD4 with
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great efficiency but is prevented from binding coreceptor by limiting environments of free CCR5, such as may occur in the presence of small molecule inhibitors, may result in prolonged exposure of activated conformations of gp120 and experience increased potential for immune response. Increased neutralization may limit the replication capacity of this virus and prevent it from competing with viruses within the intrapatient population that are not as easily neutralized. We hope to assess neutralization potential of this resistant envelope as part of future studies.
Addition of this type of resistance emerging under CCR5 antagonist selective pressure to the more widely reported adaptation to utilize drug-bound coreceptor suggests an intricate landscape for the development of resistance to this class of inhibitors. The high genetic diversity of env in conjunction with the complex interactions between the envelope and host receptors to mediate entry poses multiple avenues for viral escape. In addition to the predisposition to select for X4 tropic virus in the intrapatient population, the difficulty in predicting viral resistance via these avenues represents a significant dilemma in the administration of these inhibitors. Ongoing studies to understand the implications, impediments, and pathways to viral resistance for CCR5 antagonists are warranted.
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CHAPTER 5
GENERAL DISCUSSION AND FUTURE DIRECTIONS
187
GENERAL DISCUSSION AND FUTURE DIRECTIONS
HIV-1 entry is a highly cooperative process involving complex interactions between viral envelope glycoproteins and host cell receptors. These envelope-receptor interactions play a critical role in target cell tropism and influence viral replicative fitness, transmission, and ultimately disease progression. Extensive study of the mechanisms involved in the entry process has led to greater understanding of transmission, disease progression, pathogenesis and has contributed to the development of inhibitors which may improve clinical treatment of HIV-1 infection.
Critical to our understanding of the entry process are the interactions of the HIV envelope with the coreceptor CCR5. Numerous studies suggest the critical interactions between envelope and coreceptor involve at least two regions of gp120 interacting with two domains of CCR5. One interaction involves the tip of V3 loop engaging the second extracellular loop of CCR5 while interactions between portions of the bridging sheet with
O-sulfated tyrosine residues in the N-terminus of the coreceptor account for a second major site of interaction [48,59,91,184]. Although the reasons remain poorly defined, R5
HIV is transmitted between individuals while X4 HIV arises through the course of infection but only in approximately half of all patients. It was with these observations, as well as the discovery of mutant CCR5 in apparently healthy individuals, that led to the rapid development of inhibitors to target R5 HIV infection via CCR5. The studies outlined here aimed to characterize inhibition of HIV entry by inhibitors targeting CCR5 as well as explore viral resistance pathways to these inhibitors.
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5.1 Competitive Inhibition of HIV Entry Via CCR5
The amino-terminal modification of the CCR5 ligand RANTES led to molecules
which inhibited HIV entry in the nanomolar range. The most potent of these compounds,
PSC-RANTES, has been pursued as a potential topical microbicide. While it was
initially believed that the primary mode of inhibition by PSC-RANTES involved the
induction of signaling cascades leading to internalization of CCR5 from the cell surface
[162], differential sensitivity of HIV virus isogenic except for two mutations in the V3
loop of gp120 [126] as well as the rapid selection of virus resistant to PSC-RANTES in a
SHIV/macaque challenge study [53] suggested an alternative mode of inhibition
involving competitive CCR5 binding may be at play. Our investigation of PSC-
RANTES inhibition by use of mutant receptors that cannot be downregulated from the
cell surface revealed that inhibition via downregulation may participate in the first round
of infection but competitive binding likely accounts for the majority of inhibitory activity of this drug. Increasing quantity of virus resulted in shifting IC50 concentrations
indicating that as the amount of virus increased it was able to compete off PSC-RANTES
for coreceptor binding.
Competitive binding as the primary mode of PSC-RANTES inhibition has
important implications for the use of this drug as a microbicide. General mechanisms of
resistance to competitive inhibitors include increased affinity for the substrate, which in
the case of entry inhibitors would result in increased affinity of the envelope for receptor.
Indeed, resistance to PSC-RANTES which developed during a SHIV/macaque vaginal
challenge resulted in a virus harboring mutations in the V3 loop of gp120 and a mutation
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in gp41 [53]. The location of the gp120 mutation, in the tip of the V3 loop, suggests altered interaction with the extracellular loops of CCR5 while the gp41 mutation may influence the efficiency and rate of membrane fusion. The increased replicative fitness that was observed for the PSC-RANTES resistant virus [53] would lend support to this
reasoning; however receptor affinity and entry kinetic studies could confirm our
supposition. If, as we hypothesize, increased affinity for CCR5 binding is the mode of
resistance for this virus, than resistance to PSC-RANTES may have developed via a
mechanism that has been observed for both CCR5 agonists and antagonists.
The mechanistic studies presented in Chapter 4 regarding the MVC-resistant virus
MVC.21 also imply a competitive model of resistance developed to a CCR5 antagonist.
However, instead of increased affinity to CCR5 as may be the case for the PSC-RANTES
resistant virus, the resistance to maraviroc we observed for this virus was attributed to
increased affinity for CD4. In spite of this difference between enhanced binding affinity
to CCR5 or CD4, it is possible that these viruses share a common more generalized
mechanism involving competition with the inhibitor for binding to CCR5 and that the
divergence in receptor affinity exists due to the difference in CCR5 binding between
these two types of inhibitors. For example, PSC-RANTES binds to the same site of
CCR5 as does the HIV envelope, therefore, in order to overcome PSC-RANTES binding
adaptations in the envelope would need to impact binding affinity for this same site.
Alternatively, CCR5 antagonists such as maraviroc do not bind the same region of CCR5
as does the envelope [51,104,225]. Therefore, virus does not compete directly for the
same binding site of CCR5 with these inhibitors, but the virus may indirectly compete
with these compounds for general receptor occupancy. To this end, the MVC.21 resistant
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virus we describe here may have adapted to ensure maximal likelihood of coreceptor
binding, or increased coreceptor binding rate, so as to thwart maraviroc binding to CCR5
and inhibiting the ability of this virus to use MVC-CCR5 for entry. In order to accomplish this, the K425 mutation in the CD4 binding site permitted efficient binding of
CD4 which in turn triggered coreceptor binding site exposure and faster entry kinetics.
Thus, resistance to both PSC-RANTES and MVC may relate to a broader, more
generalized mechanism involving competition for CCR5.
5.2 Envelope Sequence Context Dependency of K425 Mutation
The location of the primary resistance mutation identified for A74.MVC.21 in the
phenylalanine 43 binding pocket of gp120, a highly conserved binding site among
diverse HIV-1 strains, begs the question would a lysine at residue 425 impart resistance
in the context of envelopes from other HIV-1 viruses? Our sequence data suggests that while lysine is a naturally occurring polymorphism, it is extremely rare and is only observed in 0.1% of sequences from Group M HIV-1 viruses in the Los Alamos HIV
Sequence Database (Table 4). However, when we broke down Group M viruses by subtype we found that although still rare, lysine was more prevalent (2%) in subtype A viruses than any other subtype. Since the parental virus (A74) was of subtype A lineage, is it possible that K425 only functions efficiently in a subtype A envelope background?
Since no signature mutations predictive of CCR5 antagonists resistance have been identified, and since mutations conferring resistance to these inhibitors have largely been dependent on the context of the envelope sequence in which they arose, it will be
191
important for future studies to determine whether the resistance mutation (K425) we identified here is also restricted to the subtype A sequence in which it was found. Due to the conserved nature of the binding pocket in which K425 is located, it could be that this mutation can function in diverse backgrounds as long as the other residues comprising the phenylalanine 43 pocket are conserved between strains. Of great importance will be determining whether K425 functions in the context of a subtype B genetic background as the majority of individuals on CCR5 antagonist therapy are infected with subtype B virus.
Studies are now underway to determine the functionality of envelopes from subtype B, C, and D harboring K425 and to determine sensitivity of these envelopes to maraviroc.
In addition to determining whether K425 imparts resistance to diverse primary isolates, determining the role of the linked mutations Q117 and V396 found in the resistant virus should also be addressed. The K425 mutation was not found in the absence of Q117 and V396 in any of the clonal sequences analyzed and interestingly, the single clone derived from the resistant population which lacked K425
(A74.MVC.21.122) and remained sensitive to MVC, demonstrated increased replicative fitness as compared to viral clones derived from the parental virus population. Although this clone did not contain K425, it did have the Q117 and V396 mutations. Do viruses with K425 but lacking Q117 and V396 demonstrate reduced fitness or envelope functionality? In other words, do Q117 and V396 bolster envelope functionality so as to compensate for a cost in fitness associated with acquiring K425? Using our site-directed mutagenesis and cloning strategies these considerations can be explored in more detail and may provide important insight into the viral population landscape into which K425 arose.
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5.3 Potential of K425 Bearing Envelope to Elicit Neutralizing Antibody Response
The recent identification of broadly neutralizing antibody VRC01 by Li, Y. et al
[121] suggests an opportunity for the elicitation of neutralizing antibodies that
specifically target the conserved CD4 binding site of gp120. The VRC01 antibody has
been shown to neutralize by a mechanism which mimics the interactions of CD4 with
gp120. The R71 amino acid of VRC01 forms similar interactions with gp120 residues
(i.e. D368) that bind R59 of CD4. The mimicry of R59-CD4 interactions, critical for
gp120-CD4 binding, by VRC01 may account for the broad nature of the neutralization
observed for this antibody. This data, along with the enhanced sensitivity of
A74.MVC.21 to the F43-CD4 mimetic attachment inhibitor BMS-806 (Fig. 38) would
suggest that were A74.MVC.21 able to elicit an antibody response with specificity for the
F43 binding pocket, it could potentially neutralize diverse HIV envelopes.
However, the potential of A74.MVC.21 to elicit a neutralizing response is not
limited to the F43 binding pocket. If, as we have proposed, the K425 mutation enhances
CD4 binding and as a consequence lowers the threshold for triggering conformational
changes in gp120 than it may promote exposure of the ligand-bound (CD4i)
conformation of gp120 which is primed for coreceptor engagement. Prolonged exposure
of the coreceptor binding site of gp120, as may occur in the presence of sCD4, could
expose epitopes on gp120 that are otherwise hidden. Exploitation of this consequence by
introducing the K425 mutation into diverse env backgrounds, as mentioned above, could
create idyllic immunogens with the potential to elicit unique neutralizing antibody
193
responses. Whereas the CD4 binding site may be relatively conserved, CD4i epitopes
may be more heterogeneous. By introducing K425 into diverse genetic envelopes, it may
present an attractive method for exposing CD4i epitopes with minimal manipulation of the envelope sequence.
With ongoing investigations we hope to leverage the K425 mutation to learn more about the dynamic relationship between CD4/CCR5 affinity, requirements for coreceptor occupancy by CCR5 antagonists, influences and impacts of replicative fitness on resistance, and neutralization potential of viruses that demonstrate enhanced CD4 binding.
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Figure 10. Model of CCR5 structure and antagonist binding site.[225]
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195
Figure 12. Chemical structure of small molecule CCR5 antagonists.[104]
196
Figures 16, 17, 18, 20, and 21.
197
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