Characterization of a Human Immunodeficiency ... - Journal of Virology

JOURNAL OF VIROLOGY, Apr. 2009, p. 3798–3809 0022-538X/09/$08.00⫹0 doi:10.1128/JVI.01751-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 83, No. 8

Characterization of a Human Immunodeficiency Virus Type 1 V3 Deletion Mutation That Confers Resistance to CCR5 Inhibitors and the Ability To Use Aplaviroc-Bound Receptor䌤 Katrina M. Nolan, Gregory Q. Del Prete, Andrea P. O. Jordan, Beth Haggarty, Josephine Romano, George J. Leslie, and James A. Hoxie* Department of Medicine, Hematology-Oncology Division, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received 19 August 2008/Accepted 25 January 2009

The human immunodeficiency virus type 1 (HIV-1) V3 loop is essential for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. Using the dual-tropic virus HIV-1R3A, we previously made an extensive panel of V3 deletions and identified subdomains within V3 that could differentially mediate R5 and X4 tropism. A deletion of residues 9 to 12 on the N-terminal side of the V3 stem ablated X4 tropism while leaving R5 tropism intact. This mutation also resulted in complete resistance to several small-molecule CCR5 inhibitors. Here, we extend these studies to further characterize a variant of this mutant, ⌬9-12a, adapted for growth in CCR5ⴙ SupT1 cells. Studies using coreceptor chimeras, monoclonal antibodies directed against the CCR5 amino terminus (NT) and extracellular loops, and CCR5 point mutants revealed that, relative to parental R3A, R5-tropic ⌬9-12a was more dependent on the CCR5 NT, a region that contacts the gp120 bridging sheet and V3 base. Neutralization sensitivity assays showed that, compared to parental R3A, ⌬9-12a was more sensitive to monoclonal antibodies b12, 4E10, and 2G12. Finally, cross-antagonism assays showed that ⌬9-12a could use aplaviroc-bound CCR5 for entry. These studies indicate that increased dependence on the CCR5 NT represents a mechanism by which HIV envelopes acquire resistance to CCR5 antagonists and may have more general implications for mechanisms of drug resistance that arise in vivo. In addition, envelopes such as ⌬9-12a may be useful for developing new entry inhibitors that target the interaction of gp120 and the CCR5 NT. infected people, it is important to study mechanisms by which resistance to these compounds can be acquired. Of particular concern is the possibility that selective pressure from these inhibitors could result in the emergence of X4-tropic viruses since, while R5-tropic viruses are characteristically transmitted (15, 54, 71, 75, 83, 91), the evolution of X4-tropic viruses correlates with a more rapid CD4⫹ T-cell decline and a faster progression to AIDS (11, 43, 44, 64), although it is unclear whether the emergence of X4-tropic viruses represents a cause or a consequence of CD4⫹ T-cell decline. Resistance to CCR5 antagonists has been generated in vitro by passaging primary isolates in the presence of increasing concentrations of drug (3, 47, 56, 60, 63, 79, 87). With the single exception of a vicrivirocresistant variant of the primary isolate CC1/85 (56), mutations associated with resistance map to the V3 loop, a region that is critical for the entry process, the principal determinant of R5 or X4 tropism (34), and a primary target for neutralizing antibodies (30, 31, 39, 45, 69). Despite changes in V3, the in vitro-derived resistant variants remained R5 tropic. In general, accelerated evolution of X4-tropic viruses has not been observed in clinical trials with CCR5 inhibitors; however, in vivo escape from maraviroc in a small number of patients has been associated with the emergence of preexisting X4-tropic variants (86). The main path to resistance to CCR5 antagonists appears to be continued use of CCR5 in the presence of the inhibitor, either via scavenging for low levels of inhibitor-free CCR5 or via use of inhibitor-bound CCR5 (63, 87). While these findings imply that resistance to small-molecule CCR5 inhibitors is associated with alterations in CCR5 utilization, the underlying mechanisms remain unclear.

Human immunodeficiency virus type 1 (HIV-1) entry into host cells is a multistep process that requires sequential interactions between envelope glycoprotein (Env) trimers on the virion with receptors on the target cell surface (7, 8, 18, 48, 85), namely, CD4 and either the CCR5 (2, 10, 17, 20, 22) or the CXCR4 (29) chemokine coreceptor. The interactions between the gp120 subunit of Env and coreceptor likely include the bridging sheet (a four-stranded antiparallel ␤ sheet on the gp120 core) and base of the V3 loop with the coreceptor amino terminus (NT) and more distal regions of V3 with the coreceptor extracellular loops (ECLs) (12, 14, 35–37, 41, 59, 65, 66, 78). Recently, a new class of HIV-1 inhibitors has been developed that bind to CCR5 and prevent entry of R5-tropic viruses (46, 81, 88). Rather than directly inhibiting binding of Env, these small-molecule CCR5 inhibitors act by an allosteric mechanism, altering the conformation of CCR5 such that it is not recognized by CD4-bound gp120 (4, 76, 80, 84). Examples of these inhibitors include maraviroc, which was recently approved by the FDA for use in treatment-experienced patients (21, 28), vicriviroc, which has advanced to phase III human clinical trials (32, 77), and aplaviroc, whose development was halted in phase II/III clinical trials due to hepatotoxicity (55, 58). Since CCR5 inhibitors are now being used to treat HIV-1-

* Corresponding author. Mailing address: University of Pennsylvania, 356 Biomedical Research Building II/III, 421 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 898-0261. Fax: (215) 573-7356. E-mail: hoxie @mail.med.upenn.edu. 䌤 Published ahead of print on 4 February 2009. 3798

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We previously described a V3 mutant of the clade B dualtropic primary isolate R3A that, as a result of a four-residue deletion on the N-terminal side of the V3 stem (⌬9-12), became exclusively R5 tropic and completely resistant to a panel of CCR5 inhibitors, including vicriviroc and aplaviroc (59). In contrast to previous reports of in vitro-derived drug-resistant variants, this mutant was never passaged in the presence of a CCR5 inhibitor. Since the proposed model of allosteric inhibition has suggested that CCR5 antagonists bind to and alter the conformation of the ECLs, we hypothesized that the resistance of ⌬9-12 resulted from a disruption of the V3-ECL interaction, leaving this Env more reliant on the CCR5 NT. In the present study, we describe a variant of this mutant, termed ⌬9-12a, that was adapted for growth in CCR5⫹ SupT1 cells in the absence of any small-molecule CCR5 inhibitor. We report that ⌬9-12a remained R5 tropic; was indeed more dependent on the CCR5 NT, specifically, on the sulfated tyrosines at positions 3, 10, 14, and 15; was more sensitive to monoclonal antibodies b12, 4E10, and 2G12; and was completely resistant to all of the CCR5 inhibitors evaluated, including aplaviroc. In addition, by using combinations of aplaviroc and CCR5 monoclonal antibodies that could distinguish between drug-bound and free CCR5, we show that ⌬9-12a was capable of using aplaviroc-bound receptor for entry. These findings indicate that increased dependence on the CCR5 NT, most likely in association with decreased dependence on the V3-ECL interaction targeted by aplaviroc and other small-molecule CCR5 inhibitors, can lead to drug resistance. This shift in coreceptor engagement exhibited by ⌬9-12a may represent a more general functional mechanism by which resistance to CCR5 antagonists is acquired in vivo. MATERIALS AND METHODS Plasmids. The CCR5/CCR2b and CCR5/CCR1 chimera plasmids have been described previously (19, 68). For construction of the ⌬9-12 and ⌬9-12a point mutants (⌬9-12 N406D, ⌬9-12 E424K, ⌬9-12 A431T, ⌬9-12 A509V, ⌬9-12a D406N, ⌬9-12a K424E, ⌬9-12a T431A, and ⌬9-12a V509A), the pHSPG plasmid encoding Env was subjected to PCR-based site-directed mutagenesis (Stratagene) according to the manufacturer’s instructions. CCR5 point mutants (Y3A, Y10A, Y14A, Y15A, K171A, E172A, Y176A, Y184A, and Y187A) in pcDNA3.1 containing human CCR5 (19) were similarly created by PCR-based site-directed mutagenesis (Stratagene). All mutations in Env and CCR5 were confirmed by DNA sequencing. Cells. The Japanese quail fibrosarcoma cell line QT6, the human embryonic kidney cell line 293T, the canine thymocyte cell line Cf2Th (kindly provided by Dana Gabuzda, Harvard University), and the human astrocytoma cell line NP-2 stably expressing CD4 and CCR5 (NP2.CD4.CCR5) were maintained in Dulbecco’s modified Eagle medium (DMEM) (high glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 2 mM penicillinstreptomycin. The human T-cell lines SupT1, SupT1 stably transfected with CCR5 (SupCCR5), SupT1 stably transfected with CCR5 and DC-SIGNR (SupT1.CCR5.DCSIGNR or SupR5R), CEM, CEMss stably transfected with CCR5 (CEMss.CCR5) (kindly provided by Michael Malim, King’s College London), MT-2, Hut-78, and Jurkat.tat stably transfected with CCR5 (Jurkat.tat.CCR5; kindly provided by Quentin Sattentau, University of Oxford) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 2 mM glutamine, and 2 mM penicillin-streptomycin. Adaptation of the ⌬9-12 Env. A replication-competent virus containing the ⌬9-12 Env was generated by electroporating (250 V, 950 ␮F) SupR5R cells (5 ⫻ 106 cells in a 4-mm cuvette) with 20 ␮g of pNL4-R3A ⌬9-12. Viral growth was monitored by immunofluorescence microscopy with 25.4, an anti-p24 murine monoclonal antibody (kindly provided by Jan McClure, University of Washington). After 3 weeks of coculture with uninfected cells, cells were 100% positive for p24. Supernatant was harvested and used to infect naive SupR5R cells. Cell-free supernatant was passaged five times onto uninfected SupR5R cells and

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an additional eight times onto uninfected SupCCR5 cells. To derive env clones from the final passage of infected cells, genomic DNA was prepared with the QIAamp DNA Mini kit (Qiagen). Sequences were then PCR amplified from genomic DNA, TOPO cloned into pCR2.1 (Invitrogen), and confirmed by DNA sequencing. The clone chosen for further characterization (⌬9-12a) was then digested with EcoRI and XhoI and ligated to both the similarly digested pHSPGR3A backbone (57) and the similarly digested pNL4-3 proviral backbone. Ligations were transformed into Stbl2 competent cells (Invitrogen), and recombinant clone identity was confirmed by restriction digestion and DNA sequencing. Cell-cell fusion assay. To quantify cell-cell fusion events, we used the gene reporter fusion assay previously described in detail (25, 67). Briefly, to generate effector cells, QT6 cells in six-well plates were infected with recombinant vaccinia virus strain vTF1.1 expressing T7 polymerase (1) at a multiplicity of infection of 10 for 1 h at 37°C and then transfected for 4 h by the standard calcium phosphate method with 2 ␮g of the desired env-expressing plasmid. Cells were then incubated overnight at 32°C with rifampin at 100 ␮g/ml. To generate target cells, QT6 cells in 48-well plates were transfected for 4 h by the standard calcium phosphate method with the desired receptors and the T7.luciferase reporter plasmid in a total of 2 ␮g. Cells were incubated overnight at 37°C. At approximately 18 h posttransfection, effector cells were mixed with target cells in the presence of 100 ␮g/ml rifampin and 100 nM cytosine arabinoside and cell-cell fusion was assessed 7 to 8 h later by lysing cells with 0.5% Triton X-100–phosphate-buffered saline. After addition of luciferase substrate (Promega), luciferase activity was quantified with a Thermo Labsystems Luminoskan Ascent luminometer. For inhibition experiments, various concentrations of aplaviroc (kindly provided by Celia LaBranche, GlaxoSmithKline) were added to the target cells at the time of mixing with the effector cells and fusion inhibition was measured as the percent reduction of luciferase activity. Luciferase reporter viruses. Luciferase reporter viruses were prepared by transfecting 293T cells for 5 h by the standard calcium phosphate method with the desired env-expressing plasmid and a plasmid encoding the NL4-3 luciferase virus backbone (pNL-Luc-E⫺R⫺) (9, 11). Supernatants were harvested 48 to 72 h posttransfection and stored at ⫺80°C. Virus concentrations were determined by an enzyme-linked immunosorbent assay for the viral p24 antigen (Perkin-Elmer). Equivalent amounts of virus were used to spin infect cells for 1 h at 1,300 rpm. Following spin infections, cells were incubated at 37°C and lysed at 72 h postinfection with 0.5% Triton X-100–phosphate-buffered saline. After addition of luciferase substrate (Promega), luciferase activity was quantified with a Thermo Labsystems Luminoskan Ascent luminometer. For inhibition experiments, various concentrations of CCR5 monoclonal antibodies (3A9 and 2D7 were obtained from BD Pharmingen; 45531 was obtained from R&D Systems) or the CCR5 inhibitor aplaviroc were added to NP2.CD4.CCR5 cells 1 h prior to the addition of pseudotyped virus. For cross-antagonism experiments, NP2.CD4.CCR5 cells were preincubated with 10 ␮M aplaviroc for 1 h, preincubated with serial dilutions of monoclonal antibody 2D7 or 45531 for 1 h, and then infected with pseudotyped virus. For neutralization assays, virus was incubated with serial dilutions of patient serum or antibody 1 h prior to infecting NP2.CD4.CCR5 cells. Sera from HIV-1-infected patients (110170, 110371, 110798, and B7B5) were obtained from the Clinical Core of the University of Pennsylvania Center for AIDS Research; all patients had CD4 counts of ⬎400/␮l. Immunoglobulin G (IgG) b12, 17b, and 2G12 monoclonal antibodies were obtained from the NIH AIDS Research and Reference Reagent Program; 4E10 was obtained from the International AIDS Vaccine Initiative Neutralizing Antibody Consortium Repository. For all experiments, pseudotype inhibition was measured as the percent reduction in luciferase activity. Viral growth curves. Plasmids encoding recombinant NL4-3 viruses containing R3A Envs were transfected into 293T cells for 5 h by the standard calcium phosphate method. Virus was collected at 48 h posttransfection and stored at ⫺80°C. The YU2 viral stock was obtained from the Virus and Molecular Core of the University of Pennsylvania Center for AIDS Research. Virus concentrations were determined by an enzyme-linked immunosorbent assay for the viral p24 antigen (Perkin-Elmer), and equivalent amounts of virus were added to T-cell lines. After 18 h at 37°C, excess virus was removed by washing the cells twice in RPMI 1640 medium supplemented with 2.5% FBS. Viral replication was monitored over 2 weeks by measuring viral reverse transcriptase (RT) activity in culture supernatants. For inhibition experiments, 10 ␮M aplaviroc was added to the cells 3 h prior to the addition of virus and maintained at the desired concentration throughout the time course of infection. Flow cytometry. Confluent NP2.CD4.CCR5 cells were lifted with 2.5 mM EDTA and washed twice with DMEM supplemented with 0.5% FBS. Cells were resuspended in DMEM–0.5% FBS and divided into eight aliquots (⬃4 ⫻ 106 cells/aliquot). The aliquots were incubated either with dimethyl sulfoxide at a final concentration of 0.1% or with various concentrations of aplaviroc for 30 min

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FIG. 1. Effects of adaptation of the ⌬9-12 mutant on coreceptor tropism. (A) Fusion activity in a cell-cell fusion assay is shown for parental R3A, unadapted ⌬9-12, and the adapted ⌬9-12a Env clone. Percent fusion was calculated by using luciferase activity normalized to R3A fusion on CD4⫹ CXCR4⫹ or CD4⫹ CCR5⫹ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (B) The infectivities of the R3A, ⌬9-12, and ⌬9-12a Envs, obtained with a single-cycle luciferase reporter virus on Cf2Th cells transiently transfected with CD4 and the indicated coreceptors, are shown. Percent infection was calculated by using luciferase activity normalized to R3A infection on CD4⫹ CXCR4⫹ or CD4⫹ CCR5⫹ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (C) Growth curves of viruses containing the R3A and ⌬9-12a Envs on SupT1 and SupCCR5 cells are shown. RT activity in culture supernatants was measured at the indicated time points. The results of one of three independent experiments are shown. (D) A schematic representation of the R3A, ⌬9-12, and ⌬9-12a Envs is shown. ⌬9-12 and ⌬9-12a contain a four-residue deletion in V3, indicated by ⌬. ⌬9-12a contains four additional mutations: N406D in V4, E424K and A431T in the C4 domain of the ␤21 strand of the bridging sheet, and A509V at the amino terminus of gp41 (see the text for the corresponding positions in the HXB reference Env). GFP, green fluorescent protein.

at room temperature. Each aliquot was then divided into four tubes, and cells were pelleted and stained with antibodies on ice for 30 min (anti-CD4 monoclonal antibody #19 was described previously [26], conjugated anti-CCR5 monoclonal antibody 2D7-FITC [fluorescein isothiocyanate] was obtained from BD Pharmingen, and anti-CCR5 monoclonal antibody 45531 was obtained from R&D Systems). For #19 and 45531, staining was followed by secondary detection with an affinity-purified, FITC-conjugated goat anti-mouse antibody (1:40 dilution; Invitrogen). Fluorescence-activated cell sorter analysis was performed on a Becton Dickinson FACScan flow cytometer with the CellQuest software (Becton Dickinson). Nucleotide sequence accession numbers. The ⌬9-12a sequence from this article has been deposited in GenBank under accession number FJ040213. The parental R3A sequence has GenBank accession number AY608577.

RESULTS A variant of ⌬9-12 adapted for in vitro growth remains R5 tropic. We previously reported that deletion of residues 9 to 12 within the N-terminal stem of the dual-tropic R3A V3 loop, removing the sequence Arg-Lys-Arg-Val, selectively ablates X4 tropism (59). Although the ⌬9-12 Env induced fusion with CCR5⫹ cells at levels 83% ⫾ 10% of those of parental R3A

(Fig. 1A), this Env functioned poorly when pseudotyped onto an HIV reporter virus (Fig. 1B). To overcome this obstacle, we employed a previously described adaptation strategy (49, 53). Proviral DNA was electroporated into SupR5R cells, which stably express CCR5 and DCSIGN-R. Upon the establishment of a spreading infection, virus was serially passaged initially in SupR5R cells and then eight times in SupCCR5 cells to select for increasing fitness, as demonstrated by rapid growth kinetics and syncytium induction (data not shown). An env clone from the final passage, termed ⌬9-12a, was obtained for further studies described below. ⌬9-12a gained four mutations upon passage: N406D (position 411 in the HXB reference Env), resulting in loss of a potential N-linked glycosylation site in the V4 loop; E424K and A431T (positions 429 and 436, respectively, in HXB), located within the ␤21 strand of the bridging sheet; and A509V (position 512 in HXB), at the amino terminus of the gp41 fusion peptide. Importantly, ⌬9-12a retained the four-residue deletion in V3 (Fig. 1D). We sought to determine whether ⌬9-12a recapitulated the

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CHARACTERIZATION OF APLAVIROC-RESISTANT VIRUS TABLE 1. Host range of A9-12aa

T-cell line

Coreceptor

R3A

⌬9-12a

SupT1 CEM Hut-78 MT-2b SupCCR5c SupR5Rc CEMss.CCR5c Jurkat.tat.CCR5c

CXCR4 CXCR4 CXCR4 CXCR4 CCR5 CCR5 CCR5 CCR5

⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

⫺ ⫺ ⫺ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

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pseudovirions were poorly infectious on cells expressing 2555, but each infected cells expressing 5222, with more efficient entry seen for ⌬9-12a than for R3A (31% ⫾ 8% versus 15% ⫾ 5% of parental R3A on CCR5⫹ cells). For the CCR5/CCR1 chimeras, R3A, but not ⌬9-12a, infected cells expressing 1555, while ⌬9-12a, but not R3A, exhibited low but detectable levels of infection on 5111, again suggesting that ⌬9-12a is more dependent on the CCR5 NT.

a RT activities relative to the background are shown as follows: ⫺, no increase; ⫹, 1-log increase; ⫹⫹, 2-log increase; ⫹⫹⫹, 3-log increase. b Cells express low levels of CCR5 by 2D7-FITC. c 1,000 nM AMD3100 added to block endogenous CXCR4.

strictly R5-tropic phenotype of unadapted ⌬9-12. In cell-cell fusion assays, parental R3A mediated fusion with both CXCR4⫹ and CCR5⫹ cells while ⌬9-12a, like unadapted ⌬912, only induced fusion with CCR5⫹ cells (Fig. 1A). CCR5dependent fusion for this adapted Env was markedly enhanced (442% ⫾ 57% of that of parental R3A). Notably, in contrast to ⌬9-12, which was poorly functional on luciferase reporter viruses, ⌬9-12a pseudovirions were able to infect Cf2Th cells that expressed CCR5 (61% ⫾ 6% of that of R3A) but were unable to infect CXCR4-expressing cells (Fig. 1B). Next, the ⌬9-12a Env was introduced into an NL4-3 backbone and infectivity was evaluated on SupT1 cells, which express only CXCR4, and on SupCCR5 cells, which express both CXCR4 and CCR5 (Fig. 1C). Whereas R3A infected both SupT1 and SupCCR5 cells, ⌬9-12a infected SupCCR5 cells but was unable to infect SupT1 cells lacking CCR5. Finally, ⌬9-12a virus was tested for the ability to replicate in several CCR5⫹ and CXCR4⫹ T-cell lines (Table 1). As expected, this virus infected all of the CCR5⫹ cell lines tested (SupCCR5, SupR5R, CEMss.CCR5, and Jurkat.tat.CCR5) but did not infect CXCR4⫹ CCR5⫺ cell lines (SupT1, CEM, Hut-78, and MT-2). These data indicate that the adapted ⌬9-12a Env, like unadapted ⌬9-12, remained exclusively R5 tropic. ⌬9-12a exhibits increased dependence on the CCR5 NT for entry. We previously reported that ⌬9-12 is completely resistant to a panel of small-molecule CCR5 inhibitors in cell-cell fusion assays and proposed a mechanism by which the mutation perturbs the V3-ECL interaction, resulting in an increased reliance on the CCR5 NT (59). To address this possibility, the R3A and ⌬9-12a Envs were first evaluated in cell-cell fusion assays on a panel of chimeric CCR5/CCR2b and CCR5/CCR1 coreceptors (Fig. 2A). While R3A and ⌬9-12a mediated fusion poorly with cells expressing 2555 (i.e., CCR2b NT with CCR5 ECLs), both Envs mediated fusion with cells expressing 5222 (i.e., CCR5 NT with CCR2b ECLs), with ⌬9-12a exhibiting fusion levels 10-fold greater than those of parental R3A and 161% ⫾ 37% of those of R3A on CCR5⫹ cells. R3A mediated low-level fusion with both 5111 (i.e., CCR5 NT with CCR1 ECLs) and 1555 (i.e., CCR1 NT with CCR5 ECLs). However, in sharp contrast, ⌬9-12a mediated fusion with cells expressing 5111 (9.5% ⫾ 1.8% of parental R3A on CCR5⫹ cells) but was unable to induce fusion with cells expressing the 1555 construct. To validate these results in infection assays, pseudovirions were used to infect QT6 cells transiently expressing CD4 and these chimeric coreceptors (Fig. 2B). R3A and ⌬9-12a

FIG. 2. Dependence of ⌬9-12a on the CCR5 amino terminus. (A) The fusion activities of the R3A and ⌬9-12a Envs with cells expressing CD4 and the indicated coreceptors are shown. Percent fusion was calculated by using luciferase activity normalized to R3A fusion on CD4⫹ CCR5⫹ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (B) Infectivities of the R3A and ⌬9-12a Envs in a pseudotype infection assay on QT6 cells expressing CD4 and the indicated coreceptors are shown. Percent infection was calculated by using luciferase activity normalized to R3A infection on CD4⫹ CCR5⫹ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (C) NP2.CD4.CCR5 cells were preincubated with increasing concentrations of 3A9 and infected with R3A and ⌬9-12a pseudovirions. Percent infection was calculated by using luciferase activity normalized to infection in the absence of antibody for each virus. The values are means ⫾ the standard errors of the means. The data shown are the averages of three experiments. GFP, green fluorescent protein.

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FIG. 3. Effects of adaptive mutations in ⌬9-12a on coreceptor tropism. Fusion activity on cells expressing CD4, CD4 plus 5222, or CD4 plus 2555 is shown for R3A, the panel of add-back mutations (N406D, E424K, A431T, and A509) in which ⌬9-12a mutations were introduced individually into ⌬9-12, and the panel of takeaway mutations (D406N, K424E, T431A, and V509A) in which ⌬9-12a mutations were individually removed. Percent fusion was calculated by using luciferase activity normalized to ⌬9-12a fusion on CD4⫹ 5222⫹ cells. The values are means plus the standard errors of the means. The data shown are the averages of four experiments.

In a complementary approach, we determined the relative sensitivities of R3A and ⌬9-12a pseudovirions to 3A9, a monoclonal antibody directed against the CCR5 NT. Infection by R3A and ⌬9-12a pseudovirions was determined on NP2. CD4.CCR5 cells preincubated with increasing concentrations of 3A9 (Fig. 2C). R3A was only partially inhibitable, with infection levels of 64% ⫾ 4% at the highest concentration of antibody (50 ␮g/ml). In contrast, ⌬9-12a was highly sensitive to 3A9, with infection levels of ⬍5% at this concentration and a 50% inhibitory concentration (IC50) of 4 ␮g/ml. As described later, R3A and ⌬9-12a showed similar sensitivities to CCR5 monoclonal antibodies 2D7 and 45531, which are directed against CCR5 ECL2 (see Fig. 7B and 6C). The four adaptive mutations acquired by ⌬9-12a do not affect its dependence on the CCR5 NT. Based on the resistance of the unadapted ⌬9-12 Env to small-molecule CCR5 inhibitors, we hypothesized that the V3 deletion was solely responsible for the CCR5 NT-dependent phenotype of ⌬9-12a. However, the adapted ⌬9-12a Env also contains four additional mutations, three in gp120 and one in gp41. To address the role of these adaptive changes in coreceptor utilization, we made a series of add-back mutations in the context of the ⌬9-12 Env (N406D, E424K, A431T, and A509V) and takeaway mutations in the context of the ⌬9-12a Env (D406N, K424E, T431A, and V509A). This panel of Envs was evaluated in cell-cell fusion assays on the chimeric CCR5/CCR2b coreceptors (Fig. 3). All of the mutant Envs mediated fusion only on cells expressing 5222, similar to the parental Envs, indicating that the four adaptive mutations did not affect dependence on the CCR5 NT. In addition, the N406D and A509V add-back mutations increased the fusogenicity of the unadapted ⌬9-12 Env on 5222 to a level comparable to that of the adapted ⌬9-12a Env. Conversely, the D406N, K424E, and V509A takeaway mutations decreased the fusogenicity of the adapted ⌬9-12a Env on

5222. Thus, adaptive changes acquired upon passage, particularly the loss of the V4 glycosylation site and the mutation at the N-terminal residue in gp41, contributed to improved utilization of the CCR5 NT. ⌬9-12a requires sulfated tyrosines in the CCR5 NT for entry. The CCR5 NT contains sulfated tyrosines at positions 3, 10, 14, and 15, with sulfated tyrosines 10 and 14 being particularly important for the entry of R5-tropic viruses (27). To determine the relative sensitivities of the R3A and ⌬9-12a Envs to mutations at these positions, all were individually mutated to alanine. Several residues in CCR5 ECL2, some of which have been shown to be important for some R5-tropic isolates, were also mutated to alanine (23, 50). Relative to wild-type CCR5, parental R3A exhibited decreased fusion (Fig. 4A) and infection (Fig. 4B) levels on cells expressing the Y10A, Y14A, and Y15A mutants but not on cells expressing Y3A. A more striking reduction in fusion (to levels of ⬍5% of those of R3A on CCR5⫹ cells) was observed for ⌬9-12a on Y10A, Y14A, and Y15A. In infection assays, ⌬9-12a was markedly reduced on all of the tyrosine mutants, including Y3A. R3A and ⌬9-12a behaved similarly in both cell-cell fusion and pseudotype infection assays on cells expressing the ECL2 mutants. Thus, these results indicate that tyrosines 10, 14, and 15 in the CCR5 NT, but not tyrosine 3, contribute to the fusogenicity and infectivity of R3A while, particularly in infection assays, all are absolutely required for ⌬9-12a. ⌬9-12a is more sensitive to neutralization by IgG b12, 4E10, and 2G12. The sensitivity of ⌬9-12a to several broadly neutralizing monoclonal antibodies was also evaluated. We previously reported that the R3A mutant TA1, which contains a 15residue deletion from the V3 crown and stem, exhibits enhanced neutralization sensitivity to b12 (reactive with the CD4 binding site), 17b (reactive with a CD4-inducible epitope overlapping the bridging sheet), and 4E10 (reactive with an epitope

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FIG. 4. Effects of CCR5 point mutations on ⌬9-12a. (A) Cell-cell fusion events between the R3A or ⌬9-12a Env and cells expressing the indicated CCR5 point mutants are shown. Percent fusion was calculated by using luciferase activity normalized to R3A fusion on CD4⫹ CCR5⫹ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (B) The infectivities of the R3A and ⌬9-12a Envs were determined by using a single-cycle luciferase reporter virus on Cf2Th cells expressing the indicated CCR5 point mutants. Percent infection was calculated by using luciferase activity normalized to R3A infection on CD4⫹ CCR5⫹ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. GFP, green fluorescent protein.

in the gp41 membrane-proximal region) (49). Pseudovirions containing the R3A, TA1, and ⌬9-12a Envs were preincubated with serial dilutions of antibody and used to infect NP2. CD4.CCR5 cells (Fig. 5). Compared to parental R3A, TA1 and ⌬9-12a exhibited approximately 100-fold and 10-fold increases, respectively, in neutralization sensitivity to b12 (Fig. 5A). In contrast, while TA1 was extremely sensitive to neutralization by 17b, with an IC50 of ⬍0.1 ␮g/ml, R3A and ⌬9-12a were resistant at concentrations of up to 50 ␮g/ml (Fig. 5B). Compared to R3A, TA1 and ⌬9-12a both showed an approximately 10-fold enhancement of neutralization by 4E10 (Fig. 5C). Finally, R3A and ⌬9-12a were susceptible to neutralization by 2G12, which binds to a carbohydrate-dependent epitope on gp120, with ⌬9-12a showing an approximately threefold enhancement (IC50 of 1 ␮g/ml for ⌬9-12a, compared to 2.8 ␮g/ml for R3A) (Fig. 5D). 2G12 failed to neutralize TA1, most likely because this Env contains a mutation resulting in loss of a glycosylation site at position 342 (49), which is part of the 2G12 epitope (74). While TA1 also exhibited enhanced sensitivity to neutralization by several HIV-1-infected patient sera (49), no differences were found for ⌬9-12a (data not shown). Thus, compared to parental R3A, ⌬9-12a exhibited increased neutralization sensitivity to several broadly neutralizing monoclonal antibodies directed against Env, particularly b12 and 4E10, but compared to TA1, which contains a more extensive deletion in V3, was less sensitive to b12 and unaffected by 17b. ⌬9-12a is completely resistant to aplaviroc. We previously reported that unadapted ⌬9-12 is completely resistant, in cell-

cell fusion assays, to several small-molecule inhibitors of CCR5, including aplaviroc (59). To validate that the adapted ⌬9-12a Env maintains this drug-resistant phenotype, we first evaluated the aplaviroc sensitivity of the R3A and ⌬9-12a Envs in a cell-cell fusion assay (Fig. 6A). R3A was inhibited by aplaviroc with an IC50 of 6 nM and a small plateau of approximately 15%, relative to fusion in the absence of inhibitor, at high drug concentrations (100 to 10,000 nM). In striking contrast, ⌬9-12a was not only completely resistant to aplaviroc but showed enhanced fusion levels at drug concentrations of 1 to 10,000 nM. Similarly, in pseudovirion infection assays on NP2.CD4.CCR5 cells, R3A was completely inhibited by 10 nM aplaviroc (IC50, 2 nM) while ⌬9-12a exhibited a plateau of partial sensitivity (approximately 40%) at drug concentrations of 0.01 to 1,000 nM and reproducible enhancement at 10,000 nM (125% ⫾ 3% of infection in the absence of inhibitor) (Fig. 6B). Finally, the aplaviroc sensitivity of the growth of a replication-competent virus bearing the ⌬9-12a Env was assessed by infecting SupCCR5 cells in the presence or absence of 10 ␮M aplaviroc (Fig. 6C). YU2, a purely R5-tropic HIV-1, replicated well in the absence of the drug but was completely inhibited by aplaviroc at up to 13 days postinoculation. As noted previously, the ⌬9-12a virus was highly infectious on SupCCR5 cells. However, although no enhancement was noted in the presence of 10 ␮M aplaviroc, ⌬9-12a was able to replicate to high levels in the presence of the drug. Collectively, these findings in cell-cell fusion, pseudotype infection, and viral growth assays all clearly show that the ⌬9-12a Env is resistant to aplaviroc.

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FIG. 5. Sensitivity of the ⌬9-12a mutant to a panel of broadly neutralizing monoclonal antibodies. The sensitivities of R3A, TA1, and ⌬9-12a to IgG b12 (A), 17b (B), 4E10 (C), and 2G12 (D) determined in a pseudotype infection assay on NP2.CD4.CCR5 cells are shown. Percent infection was calculated by using luciferase activity normalized to infection in the absence of inhibitor for each virus. The values are means ⫾ standard errors of the means. The data shown are the averages of three experiments.

⌬9-12a can use aplaviroc-bound CCR5 for entry. To determine if ⌬9-12a could bind to aplaviroc-bound CCR5 during entry, we used a previously reported cross-antagonism assay with two different anti-CCR5 monoclonal antibodies that can distinguish between aplaviroc-bound and free CCR5 (16) (Fig. 7). NP2.CD4.CCR5 cells were treated with various concentrations of aplaviroc for 30 min and then stained with 2D7 and 45531, which recognize different epitopes within CCR5 ECL2 (Fig. 7A). As reported previously (16), fluorescence-activated cell sorter analysis revealed that 2D7 was unaffected by aplaviroc binding while 45531 was completely inhibited by aplaviroc concentrations of ⱖ10 nM. Next, NP2.CD4.CCR5 cells were infected with pseudovirions bearing the R3A or ⌬9-12a Env in the presence of increasing concentrations of 2D7 (Fig. 7B) or 45531 (Fig. 7C). Prior to the addition of virus, cells were preincubated in the presence or absence of a saturating concentration of aplaviroc (10,000 nM). R3A infection was completely inhibited by this concentration of aplaviroc and was unaffected by the addition of 2D7 or 45531 (data not shown). Both R3A and ⌬9-12a were sensitive to inhibition by 2D7, and ⌬9-12a remained sensitive in the presence of aplaviroc (Fig. 7B). In contrast, while both R3A and ⌬9-12a were inhibitable by 45531, with a 50% reduction in infectivity seen at an antibody concentration of 100 ␮g/ml, ⌬9-12a became completely

resistant to 45531 when preincubated with aplaviroc (Fig. 7C). Thus, a saturating concentration of aplaviroc, which prevented 45531 binding to CCR5, restored the infectivity of ⌬9-12a pseudovirions in the presence of this antibody, strongly suggesting that ⌬9-12a is able to use aplaviroc-bound CCR5 for entry. DISCUSSION Utilization of the CCR5 and CXCR4 coreceptors by gp120 involves interactions between (i) the gp120 bridging sheet and base of V3 with the coreceptor NT and (ii) distal regions of V3 with poorly defined conformational domains formed by the coreceptor ECLs (12, 14, 35–37, 41, 59, 65, 66, 78). We previously reported that a dual-tropic HIV-1 Env with a deletion of four residues from the N-terminal V3 stem (R3A ⌬9-12) became completely resistant to an extensive panel of small-molecule CCR5 inhibitors (59). Rather than directly inhibiting gp120 binding, these inhibitors bind to a pocket formed by CCR5 membrane-spanning domains and likely alter ECL conformations that are required to engage V3 (4, 24, 70, 76, 80, 84). We proposed that this ⌬9-12 deletion selectively reduced the contribution of the V3-ECL interaction to entry with a resulting resistance to CCR5 inhibitors and that this Env be-

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FIG. 6. Sensitivity of the ⌬9-12a mutant to aplaviroc. (A) The sensitivities of the R3A and ⌬9-12a Envs to aplaviroc in a cell-cell fusion assay on CCR5⫹ cells are shown. Percent fusion was calculated by using luciferase activity normalized to fusion in the absence of inhibitor for each Env. The values are means ⫾ the standard errors of the means. The data shown are the averages of three experiments. (B) The sensitivities of R3A and ⌬9-12a to aplaviroc in a pseudotype infection assay on NP2. CD4.CCR5 cells are shown. Percent infection was calculated by using luciferase activity normalized to infection in the absence of inhibitor for each virus. The values are means ⫾ the standard errors of the means. The data shown are the averages of three experiments. (C) Growth curves of infectious viruses containing the ⌬9-12a and YU2 Envs on SupCCR5 cells in the presence or absence of 10 ␮M aplaviroc (APL) are shown. RT activity in culture supernatants was measured at the indicated time points. The results of one of three independent experiments are shown.

came more dependent on the gp120-NT interaction. Further support for this hypothesis came from another R3A mutant, TA1, which contains a 15-amino-acid deletion of the distal half of V3 (49), was resistant to CCR5 inhibitors, and was more

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sensitive to mutations in the CCR5 NT. For reasons that remain unclear, the ⌬9-12 Env, when pseudotyped onto a luciferase reporter virus or expressed on an NL4-3 backbone, was poorly infectious. However, as we now report, ⌬9-12a, derived from an in vitro-passaged virus containing the ⌬9-12 Env, was functional in cell-cell fusion and pseudotype infection assays and was as infectious as parental R3A on CCR5⫹ T-cell lines when incorporated onto an NL4-3 proviral backbone. This adapted Env maintained the ⌬9-12 deletion and remained completely resistant to CCR5 antagonists, making it well suited to probe underlying differences in coreceptor utilization caused by this small V3 deletion. Several approaches were taken to evaluate how ⌬9-12a and R3A utilized CCR5. First, with a panel of CCR5/CCR2b and CCR5/ CCR1 chimeras, ⌬9-12a showed enhanced fusion and infection levels relative to those of parental R3A on chimeras that contained the CCR5 NT and, in contrast to R3A, was unable to function on coreceptors lacking this domain. Second, because CCR5 NT tyrosines at positions 3, 10, 14, and 15 are sulfated (13), with Y10 and Y14 being particularly important for engaging the gp120 core coreceptor binding domain (27), we used a panel of CCR5 mutants with alanine substitutions at these positions. In fusion assays, reduced but persisting fusion activity was seen for R3A on the Y10, Y14, and Y15 mutants, while ⌬9-12a fusion levels were severely reduced to ⬍5% of that of R3A on wild-type CCR5. Moreover, in infection assays, R3A was reduced on Y10A, Y14A, and Y15A but not on Y3A, while ⌬9-12a was markedly reduced on all of the tyrosine mutants. Finally, with monoclonal antibodies to the CCR5 NT or ECL2, ⌬9-12a pseudovirions were more inhibitable, relative to R3A, by an NT-specific antibody, whereas no differences were observed with ECL2-specific antibodies. Taken together, these findings confirm our hypothesis that deletion of V3 residues 9 to 12 renders this Env more dependent on the CCR5 NT and reveal that all of the sulfated tyrosines, including Y3, are critical for its function. In addition to its role in engaging the chemokine coreceptor, V3 is also a principal target for neutralizing antibodies and likely shields more conserved domains on the gp120 core from recognition by broadly neutralizing antibodies (33, 39, 61, 69). We previously reported that TA1 exhibits markedly enhanced sensitivity to neutralization by monoclonal antibodies directed to the bridging sheet and CD4 binding site, as well as to HIV-1-positive human serum samples (49). Other studies have shown that a full deletion of V3 can increase the exposure of gp120 epitopes, as determined by antibody binding assays (38, 72, 89). While the ⌬9-12 mutation removed only four residues from the V3 stem, its neutralization sensitivity was also increased for b12 and 4E10, although not for 17b. While this effect could result directly from loss of the positively charged Arg-Lys-Arg-Val sequence, it seems more likely that it reflects alterations in cooperative interactions on the gp120 trimer that are involved with protecting these sites. Global effects on neutralization sensitivity have been reported for changes in V1/V2 and in V3 on the HIV and simian immunodeficiency virus envelopes (6, 40, 42, 51). The increased sensitivity to b12 is of interest, given the importance of this epitope as a target for broadly neutralizing antibodies (73, 90) and the fact that antibodies with b12-like activity have been identified in rare HIV1-infected individuals with broadly neutralizing sera (5, 52).

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FIG. 7. Susceptibility of ⌬9-12a to anti-CCR5 monoclonal antibodies 2D7 and 45531 in the presence of a saturating concentration of aplaviroc. (A) NP2.CD4.CCR5 cells were treated with aplaviroc, stained with the indicated antibodies, and analyzed by flow cytometry. The mean channel fluorescence (MCF) is plotted against increasing concentrations of aplaviroc. (B) NP2.CD4.CCR5 cells were preincubated in the presence or absence of 10 ␮M aplaviroc, preincubated with serial dilutions of 2D7, and then infected with R3A or ⌬9-12a pseudovirions. Percent infection was calculated by using luciferase activity normalized to infection in the absence of inhibitor for each virus. The values are means ⫾ the standard errors of the means. The data shown are the averages of three experiments. (C) NP2.CD4.CCR5 cells were preincubated in the presence or absence of 10 ␮M aplaviroc, preincubated with serial dilutions of 45531, and then infected with R3A or ⌬9-12a pseudovirions. Percent infection was calculated by using luciferase activity normalized to infection in the absence of inhibitor for each virus. The values are means ⫾ the standard errors of the means. The data shown are the averages of three experiments.

Whether conformational changes in V3 induced by the ⌬9-12 mutation could serve to render this Env more capable of eliciting b12-like antibodies remains to be determined. The most intriguing findings about ⌬9-12a were its complete resistance to small-molecule CCR5 inhibitors and its ability to use aplaviroc-bound CCR5. Consistent with previously presented work (16), we showed that monoclonal antibody 45531 was completely blocked by aplaviroc at concentrations above 10 nM. Since aplaviroc inhibits the binding of 45531 to CCR5, a virus that can use aplaviroc-bound CCR5 would be less affected by the addition of 45531 when a saturating concentration of aplaviroc was also present. Indeed, ⌬9-12a was no longer inhibited by 45531 in the presence of 10,000 nM aplaviroc. Our results indicate that when aplaviroc binds to CCR5, it prevents 45531 from binding while creating an alternative, functional binding site for the aplaviroc-resistant ⌬9-12a virus. Resistance to CCR5 inhibitors has been reported for viruses passaged in vitro in the presence of these drugs (3, 47, 56, 60, 63, 79, 87) and recently obtained from patients on therapy (82). For in vitro-derived viruses, the determinants are often complex and have been reported to involve mutations in and outside of the V3 loop. Although this resistance has been associated with an increase in the IC50 (i.e., competitive inhibition), consistent with an increased affinity for CCR5, more commonly resistance occurs without a change in the IC50 (i.e., noncompetitive inhibition), characterized by dose-response curves showing a plateau in maximal inhibition (46). This plateau has been proposed to result from viral adaptation to engage drug-bound conformations of CCR5, although the underlying interactions are poorly understood. Maraviroc-resistant isolates from treatment-experienced patients have also shown properties consistent with noncompetitive resistance, suggesting that the ability of viruses to use drug-bound CCR5

molecules may be the preferred mechanism of drug resistance in vivo (62). Our findings with ⌬9-12a show that a virus engineered to contain a specific deletion in V3 became completely resistant to aplaviroc and failed to show a reduction in fusion or infection assays at any drug concentration. These findings strongly suggest that, in contrast to a “plateau effect,” where an initial component of drug sensitivity is followed by a noncompetitive, plateau phase (63, 87), ⌬9-12a can apparently recognize aplaviroc-free and aplaviroc-bound forms of CCR5 with equal affinity. Given our finding that the use of the drug-bound receptor by ⌬9-12a is associated with increased dependence on the CCR5 NT and presumably less dependence on V3-ECL interactions targeted by CCR5 antagonists, our results suggest that preferential use of the CCR5 NT may provide a more general mechanism underlying resistance to this class of drugs. Interestingly, ⌬9-12a showed reproducibly enhanced fusion and infectivity in cell-cell fusion and pseudotype infection assays, respectively, suggesting that it may actually prefer drug-bound CCR5. A similar phenotype was recently observed for a single clinical isolate from a patient who developed resistance to vicriviroc (82). This finding raises the intriguing possibility that the CCR5 NT may be better exposed for gp120 binding when CCR5 is occupied by drug. Whether ⌬9-12a could be used to derive drug-dependent Envs as a way to probe differences in drug-induced CCR5 conformations remains a subject of ongoing investigation. In summary, our findings have revealed that the mutant resulting from the removal of only four residues from the N-terminal side of the V3 stem of HIV-1R3A is strictly R5 tropic; more dependent on the CCR5 NT; more sensitive to neutralization by monoclonal antibodies b12, 4E10, and 2G12; completely resistant to small-molecule CCR5 inhibitors; and

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capable of using aplaviroc-bound CCR5. This detailed characterization is the first example of a small, engineered V3 mutation that results in complete resistance to CCR5 antagonists. The resistance correlates with a shift in CCR5 utilization with increased dependence on the CCR5 NT and decreased reliance on the CCR5 ECLs. This alteration in CCR5 usage may represent a common mechanism of resistance to CCR5 antagonists as more clinical data are obtained from patients failing therapy with small-molecule CCR5 inhibitors. Finally, given that all of the current small-molecule CCR5 inhibitors appear to target interactions of more distal portions of V3 with the CCR5 ECLs, this novel V3 mutant may provide a tool for developing new classes of antiviral drugs that target gp120 interactions with the CCR5 NT. ACKNOWLEDGMENTS This work was supported by grant AI-045378 (to J.A.H.) from the National Institutes of Health. We thank James Nolan, Jr., for his help with the figures. We thank members of the J. A. Hoxie laboratory and GlaxoSmithKline, particularly Celia C. LaBranche and Jerry L. Jeffrey, for helpful discussions. p24 assays were performed by the Virus and Molecular Core of the Penn Center for AIDS Research. We also thank GlaxoSmithKline for aplaviroc. REFERENCES 1. Alexander, W. A., B. Moss, and T. R. Fuerst. 1992. Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor. J. Virol. 66:2934–2942. 2. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1␣, MIP-1␤ receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272: 1955–1958. 3. Baba, M., H. Mikake, X. Wang, M. Okamoto, and K. Takashima. 2007. Isolation and characterization of human immunodeficiency virus type 1 resistant to the small-molecule CCR5 antagonist TAK-652. Antimicrob. Agents Chemother. 51:707–715. 4. Billick, E., C. Seibert, P. Pugagh, T. Ketas, A. Trkola, M. J. Endres, N. J. Murgolo, E. Coates, G. R. Reyes, B. M. Baroudy, T. P. Sakmar, J. P. Moore, and S. E. Kuhmann. 2004. The differential sensitivity of human and rhesus macaque CCR5 to small-molecule inhibitors of human immunodeficiency virus type 1 entry is explained by a single amino acid difference and suggests a mechanism of action for these inhibitors. J. Virol. 78:4134–4144. 5. Braibant, M., S. Brunet, D. Costagliola, C. Rouzioux, H. Agut, H. Katinger, B. Autran, and F. Barin. 2006. Antibodies to conserved epitopes of the HIV-1 envelope in sera from long-term non-progressors: prevalence and association with neutralizing activity. AIDS 20:1923–1930. 6. Cao, J., N. Sullivan, E. Desjardin, C. Parolin, J. Robinson, R. Wyatt, and J. Sodroski. 1997. Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein. J. Virol. 71:9808–9812. 7. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89:263–273. 8. Chen, B., E. M. Vogan, H. Gong, J. J. Skehel, D. C. Wiley, and S. C. Harrison. 2005. Structure of an unliganded simian immunodeficiency virus gp120 core. Nature 433:834–841. 9. Chen, B. K., K. Saksela, R. Andino, and D. Baltimore. 1994. Distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferaseencoding viruses. J. Virol. 68:654–660. 10. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J. Sodroski. 1996. The ␤-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135–1148. 11. Connor, R. I., K. E. Sheridan, D. Ceradini, S. Choe, and N. R. Landau. 1997. Change in coreceptor use correlates with disease progression in HIV-1infected individuals. J. Exp. Med. 185:621–628. 12. Cormier, E. G., and T. Dragic. 2002. The crown and stem of the V3 loop play distinct roles in human immunodeficiency virus type 1 envelope glycoprotein interactions with the CCR5 coreceptor. J. Virol. 76:8953–8957. 13. Cormier, E. G., M. Persuh, D. A. Thompson, S. W. Lin, T. P. Sakmar, W. C. Olson, and T. Dragic. 2000. Specific interaction of CCR5 amino-terminal domain peptides containing sulfotyrosines with HIV-1 envelope glycoprotein gp120. Proc. Natl. Acad. Sci. USA 97:5762–5767.

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