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Journal of Virology, December 2009, p. 12151-12163, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.01351-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Structure-Function Analysis of Human Immunodeficiency Virus Type 1 gp120 Amino Acid Mutations Associated with Resistance to the CCR5 Coreceptor Antagonist Vicriviroc{triangledown} ,{dagger}

Robert A. Ogert,1 Lei Ba,1 Yan Hou,1 Catherine Buontempo,1 Ping Qiu,2 Jose Duca,3 Nicholas Murgolo,2 Peter Buontempo,1 Robert Ralston,1 and John A. Howe1*

Departments of Biological Sciences-Virology,1 Molecular Design and Informatics,2 Drug Design, Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-4945, Kenilworth, New Jersey 070333

Received 1 July 2009/ Accepted 14 September 2009


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ABSTRACT
 
Vicriviroc (VCV) is a small-molecule CCR5 coreceptor antagonist currently in clinical trials for treatment of R5-tropic human immunodeficiency virus type 1 (HIV-1) infection. With this drug in development, identification of resistance mechanisms to VCV is needed to allow optimal outcomes in clinical practice. In this study we further characterized VCV resistance in a lab-adapted, VCV-resistant RU570 virus (RU570-VCVres). We show that K305R, R315Q, and K319T amino acid changes in the V3 loop, along with P437S in C4, completely reproduced the resistance phenotype in a chimeric ADA envelope containing the C2-V5 region from RU570 passage control gp120. The K305R amino acid change primarily impacted the degree of resistance, whereas K319T contributed to both resistance and virus infectivity. The P437S mutation in C4 had more influence on the relative degree of virus infectivity, while the R315Q mutation contributed to the virus concentration-dependent phenotypic resistance pattern observed for RU570-VCVres. RU570-VCVres pseudovirus entry with VCV-bound CCR5 was dramatically reduced by Y10A, D11A, Y14A, and Y15A mutations in the N terminus of CCR5, whereas these mutations had less impact on entry in the absence of VCV. Notably, an additional Q315E/I317F substitution in the crown region of the V3 loop enhanced resistance to VCV, resulting in a stronger dependence on the N terminus for viral entry. By fitting the envelope mutations to a molecular model of a recently described docked N-terminal CCR5 peptide consisting of residues 2 to 15 in complex with HIV-1 gp120 CD4, potential new interactions in gp120 with the N terminus of CCR5 were uncovered. The cumulative results of this study suggest that as the RU570 VCV-resistant virus adapted to use the drug-bound receptor, it also developed an increased reliance on the N terminus of CCR5.


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INTRODUCTION
 
CCR5 antagonists inhibit human immunodeficiency virus type 1 (HIV-1) entry by binding within a pocket formed by the transmembrane domains of CCR5. The binding of these agents locks the receptor in a conformation the virus is unable to recognize (14, 25, 32, 37, 51, 54). The CCR5 coreceptor antagonists most advanced in development are maraviroc (MVC) and vicriviroc (VCV). MVC, marketed as Selzentry, is approved for use in treatment-experienced adult patients with R5-tropic HIV-1 infection that is resistant to multiple antiretroviral agents (18), and VCV is currently being evaluated in phase II and phase III clinical trials (19, 50). With the ongoing clinical development of HIV-1 coreceptor antagonists, further studies are needed regarding the biology of HIV-1 resistance to these agents and the ability to assess resistance based on changes within the envelope glycoprotein.

The CCR5 coreceptor antagonists are unique in that they bind to the CCR5 coreceptor on the surface of the host cell, whereas most HIV-1 medicines interfere with virus propagation by inhibiting one of the essential virus-encoded enzymes. Signature mutations associated with resistance to HIV-1 reverse transcriptase, protease, and integrase inhibitors, as well as compensatory mutations allowing the virus to overcome a loss in fitness, have been identified (6, 21, 44). However, similar information on resistance mutations has not been identified with respect to the CCR5 coreceptor antagonists.

It has been established that CCR5 coreceptor antagonists block HIV-1 entry after the virus has bound to CD4. The initial interaction between CD4 and the envelope glycoprotein gp120-gp41 homotrimers induces a conformational change in gp120 (48, 49) that enables binding to CCR5 (53, 58). The interaction of the V3 loop and bridging sheet region of gp120 with CCR5 (22, 23, 46, 47, 59, 60) is believed to induce a series of further rearrangements in gp120 that expose the gp41 ectodomain and trigger fusion of virus and cell membranes (4, 5). Thus, both the complexity of the entry process and the sequence heterogeneity of the envelope glycoprotein complicate the identification of resistance mechanisms for CCR5 coreceptor antagonists.

Mutations associated with resistance to CCR5 coreceptor antagonists for in vitro derived HIV-1 R5-resistant variants have, in most cases, mapped to the V3 loop region of gp120 (2, 26, 35, 40, 56); however, one resistant variant with no mutations in the V3 loop (33) was recently shown to have mutations in the N-terminal fusion peptide of gp41 that conferred resistance (1). In clinical trials of MVC (41) and VCV (55), subjects that experienced virologic failure and demonstrated phenotypic resistance to the CCR5 coreceptor antagonists based on the PhenoSense Entry assay for coreceptor tropism (57) all developed resistance mutations that mapped to amino acid substitutions in the V3 loop region during therapy.

In this study, we have further examined resistance mutations in the V3 and bridging sheet regions of a laboratory-adapted RU570 VCV-resistant (RU570-VCVres) variant (40) using site-directed mutagenesis to reintroduce the amino acid changes into an ADA chimeric envelope containing the cognate C2-V5 region from a passage control envelope. We then sought to analyze the effect of these mutations, either alone or in various combinations, on pseudovirus infectivity, susceptibility to VCV, and interaction with CCR5.


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MATERIALS AND METHODS
 
Reagents. VCV was synthesized at Schering-Plough Research Institute, Kenilworth, NJ. Lectin from Galanthus nivalis, insolublized on 4% cross-linked beaded agarose, was obtained from Sigma-Aldrich, St. Louis, MO. pCD4 plasmid was obtained from Origene Technologies Inc., Rockville, MD.

HIV-1 plasmids and HIV-1 primary isolates. The HIV-1 clade G RU570 primary isolate was obtained from the National Institutes of Health AIDS Research and Reference Reagent program and was passaged in PM-1 cells in the presence of escalating concentrations of VCV to generate VCV-resistant cultures, as described previously (40). The pNL4-3E-Luc+ and pSV7d-ADA gp160 plasmids were obtained from John Moore at the Weill Cornell Medical College of Cornell University, New York, NY. Homologous recombination of HIV-1 RU570 gp120 fragments into pSV7d-ADAgp160 was performed as previously described (40).

Cell lines. The neoplastic T-cell line PM-1 was obtained from the National Institutes of Health AIDS Research and Reference Reagent program. U87 astroglioma cells expressing CD4 and CCR5 were obtained from Dan Littman at New York University and were maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 500 µg/ml G418, and 1 µg/ml puromycin. 293T cells (CRL-11268) were purchased from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 300 µg/ml G418.

HIV-1 RU570 VCV resistance generated in PM-1 cells. Generation of RU570 HIV-1 VCVres was previously described (40). RU570 stocks for phenotypic analysis were prepared by infecting PM-1 cells in the presence of 10 µM VCV. VCV was removed from the cultures at 48 h postinfection, which was followed by incubation in culture medium for 72 h. Virus stocks were stored as 1-ml aliquots at –70°C.

HIV-1 single-round virus infection assay. The HIV-1 single-round virus infection assay is based on a previously described method (34) and was modified as follows. U87 cells were seeded into 96-well collagen-coated plates 24 h prior to assay. CCR5 antagonists were prepared as 100x stocks in 100% dimethyl sulfoxide and diluted to 1x assay concentration in medium (final concentration, 1% dimethyl sulfoxide). Cells were treated with compound 2 h prior to infection. Pretreatment medium was aspirated and replaced with the virus pool supplemented with compound. Virus was allowed to adsorb in the presence of compound for 2 h at 37°C. Cultures were washed with phosphate-buffered saline (PBS), and incubation was continued in culture medium supplemented with compound and 2 µM amprenavir for 48 h. Cells were harvested from plates by trypsinization and, after neutralization with growth medium, transferred to V-bottom plates. Cells were centrifuged at 700 x g for 3 min, washed with PBS, and fixed with 4% paraformaldehyde (Cytofix; BD Biosciences) for 15 min. Following fixation, cells were washed twice with PBS supplemented with 0.2% bovine serum albumin (Stain Buffer; BD Biosciences).

For intracellular staining of p24 antigen, cells were centrifuged and suspended in permeabilization buffer (Perm/Wash buffer; BD Biosciences) for 15 min. Phycoerythrin-conjugated mouse anti-p24 monoclonal antibody ([MAb] KC57-RD1; Beckman Coulter) was diluted to a final concentration of 1:160 in permeabilization buffer. Following incubation at 4° for 1 h, cells were washed three times with 5 min between washes. Stained cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences), and data analysis was performed with CellQuest Pro software (BD Biosciences). The ratio of p24-positive (p24+) cells was determined by using a bivariate plot of FL-2 versus FL-1 fluorescence, with the gate being set on mock-infected cells. A total of 10,000 events were acquired per data point, giving a limit of quantification (99% confidence interval) of 10 p24+ cells. Percent inhibition for each virus concentration was defined as the ratio of p24+ cells in CCR5 antagonist-treated culture normalized to the ratio of p24+ cells in nontreated control times 100%. Inhibition greater than 100% indicates that the fraction of p24+ cells in the CCR5 antagonist-treated cultures was greater than fraction of p24+ cells of the nontreated control. The 50% effective concentration and maximal percent inhibition (MPI) for the passaged viruses were determined using GraphPad Prism software, version 4 (GraphPad Software, Inc., San Diego, CA).

SDM of gp120 amino acids. Site-directed mutagenesis (SDM) of individual, gp120 amino acids in the pADA-C2-V5pc clone (where the C2-V5 region of gp120 in the ADA envelope was replaced with the RU570 passage control [pc] sequence) was performed using a QuikChange SDM kit (Stratagene, La Jolla, CA). Amino acid changes corresponding to HXB2 gp120 amino acid coordinates were as follows: K305R, R315Q, K319T, and P437S. All sequence changes were verified by DNA sequence analysis. SDM of gp120 amino acids (Q315E and I317F) in the pADA-C2-V5res (where the C2-V5 region of gp120 in the ADA envelope was replaced with the RU570-VCVres sequence) was performed as described above.

SDM of CCR5. The pcDNA3.1 CCR5 expression plasmid was obtained from Dan Littman, New York University. The CCR5 construct with an N-terminal deletion of residues 2 to 17 ({Delta}2-17) was generated using overlapping PCR with the forward primer 5'-CTAAGCTTACCATGGATGAGCCCTGCCAAAAAATCAATG-3' and reverse primer 5'-CCACCACCCAAGTGATCACACTTG-3'. The resulting PCR product was cloned into the parental expression vector pcDNA3.1-CCR5 using HindIII and AleI restriction sites. Individual mutations in the N terminus of CCR5 (Y3A, Y10A, D11A, N13A, Y14A, and Y15A) and in the extracellular loop 2 (ECL2) of CCR5 (R168A, K171A, E172A, L174A, and C178A) were generated in pcDNA3.1-CCR5 using a QuikChange SDM kit (Stratagene, La Jolla, CA). All mutations were confirmed by DNA sequence analysis.

Generation and characterization of HIV-1 pseudoviruses. HIV-1 pseudoviruses were produced in 293T cells by calcium phosphate transfection of pNL4-3E-Luc+ and HIV-1 envelope expression vectors using a ProFection Mammalian Transfection System (Promega Corp., Madison, WI). HIV-1 pseudovirus was harvested in culture supernatants at 48 h posttransfection. Supernatants were clarified of cell debris by centrifuging at 1,500 x g for 10 min. Single-cycle infection assays were generally performed on the same day as harvesting of HIV-1 pseudovirus. HIV-1 p24 concentrations in pseudovirus stocks were measured using a commercial enzyme-linked immunoassay (Alliance HIV-1 p24 Ag Kit; Perkin Elmer, Waltham, MA). Pseudovirus stocks were normalized by p24 prior to testing. In addition, some assays were also performed by normalizing relative light units (RLU) to the amount of p24 input per well, and the linearity of results to virus input was verified for each assay. To assess susceptibility of pseudoviruses to CCR5 coreceptor antagonists, U87-CD4-CCR5 cells were seeded into 96-well luminometer plates (Perkin Elmer, Inc., Waltham, MA) using 5,000 cells/well. Plates were incubated at 37°C. The next day serial 10-fold dilutions of inhibitor in cell culture medium (10 µM -> 0.01 nM) were added to wells 1 h prior to the addition of HIV-1 pseudovirus plus inhibitor. Antibody neutralization of HIV-1 pseudovirus infection with CTC5, an anti-N terminus CCR5 MAb (catalog number MAB1802; R&D Systems Inc., Minneapolis, MN), was also performed in the same manner using serial twofold dilutions of antibody (25 µg/ml) in the presence and absence of 10 µM VCV. Plates were incubated for 72 h, and luciferase activity was analyzed by adding 50 µl of BrightGlo luciferase assay buffer (Promega Corp., Madison, WI). Plates were read on a Dynex luminometer (300 ms/well). The number of RLU was normalized to virus dose, measured as ng of p24, and percent inhibition was calculated as follows: 100 – [average normalized RLU for HIV-1 pseudovirus plus drug/average normalized RLU for HIV-1 pseudovirus from control wells without drug] x 100. Dose-response data were analyzed using a nonlinear regression, four-parameter logistic curve fit program with GraphPad Prism software, version 4.0 (GraphPad Software Inc., San Diego, CA).

Expression of CCR5 mutants in 293T Cells. 293T cells were transfected with 10 µg of pcDNA3.1-CD4 and 20 µg of pcDNA3.1-CCR5 per 10-cm plate using a ProFection Mammalian Transfection System (Promega Corp., Madison, WI). The CCR5 and CD4 cell surface expression in 293T cells was analyzed by fluorescence-activated cell sorting using murine anti-human CD195 (clone 2D7) CCR5 antibody and anti-human CD4 antibody (catalog number 555346; BD Pharmingen, San Diego, CA). 293T cells at 24 h posttransfection were seeded into 96-well luminometer plates (Perkin Elmer) at 5,000 cells/well, and plates were incubated overnight at 37°C. HIV-1 pseudovirus infection of 293T cells was performed as described above.

Western blot analysis of gp120 incorporated into HIV-1 pseudovirus. HIV-1 pseudovirus stocks were generated as described above. p24 levels in pseudovirus stocks were measured by enzyme-linked immunosorbent assay. Equivalent levels of p24 (3 µg) from each stock were batch absorbed using lectin-agarose from G. nivalis overnight at 4°C. Lectin-agarose beads were washed with 0.25% Tween in PBS. HIV-1 envelope incorporated into eluted pseudovirus was analyzed by Western blotting with rabbit anti-gp120 polyclonal antibody (ImmunoDiagnostics, Inc., Woburn, MA).


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RESULTS
 
VCV resistance mutations located in the CCR5 coreceptor binding region of RU570 gp120 influence pseudovirus infectivity. We previously demonstrated that the C2-V5 region of gp120 (amino acids 270 to 468) from the laboratory-adapted RU570-VCVres virus was sufficient to confer VCV resistance when recombined into the susceptible ADA envelope (40). While this insertion does create a chimeric coreceptor binding domain, this region contains the V3 loop and C4 domains, which together compromise the primary CCR5 coreceptor binding determinants used during virus entry (7, 20, 22, 23, 31, 36, 46, 47). Envelope clones amplified from the RU570 passage control and RU570-VCVres virus cell cultures revealed three predominant point mutations in the V3 loop (K305R, R315Q, and K319T) and one predominant change in C4 (P437S). These mutations were present in all envelope clones from the RU570-VCVres virus compared to the passage control virus. RU570 is a clade G virus, and analysis of 896 clade G envelope sequences present in the Los Alamos HIV-1 database revealed that amino acid residues at each of the positions described here were conserved. The percentage of envelopes with the indicated amino acids at positions 305 (K, 77%; R, 19%), 315 (Q, 86%; R, 10%), 317 (F, 88%; I, 6%), 319 (A, 84%; T, 13%; K, 2%), and 437 (P, 92%; S, 4%) was determined.

Our previous studies focused on analyzing the effect of back mutations at these four positions within the V3 loop and C4 domain on resistance to VCV. To complement these studies, we built forward mutations at each position in the ADA C2-V5pc chimeric envelope as diagramed in Fig. 1A and examined their effects on both gain of resistance and pseudovirus infectivity. The ADA C2-V5pc envelope in pseudovirus assays had minimal activity (Fig. 1B), and in our previous study the T319K back mutation in the ADA C2-V5res envelope dramatically reduced pseudovirus activity (40). Therefore, we first analyzed the effects of the K319T forward mutation in the ADA C2-V5pc envelope. To measure the relative infectivity of pseudoviruses, pseudovirus stocks were normalized to equivalent levels based on p24 measurements, and assays were performed using 25 ng of p24 input per well of each pseudovirus stock.


Figure 1
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  		FIG. 1. The RU570 chimeric envelope (ADA C2-V5pc) was generated by replacing the C2-V5 region of gp120 in the ADA envelope with the RU570 passage control sequence. (A) Schematic diagram depicting the RU570 C2-V5pc region encompassing amino acids 270 to 468 of gp120 (hatched bars) within the background of ADA gp160 (black bars). The four primary amino acid changes in the coreceptor binding region of RU570 gp120 associated with VCV resistance are delineated (in V3, K305R, R315Q, K319T; in C4, P437S). (B) Effect of forward mutations generated in the ADA C2-V5pc chimeric envelope on the relative infectivity of HIV-1 pseudoviruses. Pseudovirus stocks were normalized by p24, and 25 ng of p24 of each pseudovirus was used to infect U87-CD4-CCR5 cells. Data are representative of at least three independent experiments and represent the average ± standard deviations of four replicates. The relative change in expression is in relation to ADA C2-V5res pseudovirus. (C) Western blot detection of gp120 present in HIV-1 pseudoviruses generated with the ADA C2-V5pc envelope and ADA C2-V5pc envelopes containing forward mutations. Lane 1, ADA; lane 2, ADA C2-V5pc; lane 3, ADA C2-V5pc P437S; lane 4, ADA C2-V5pc K319T; lane 5, ADA C2-V5pc K319T/P437S; lane 6, ADA C2-V5pc K305R/K319T/P437S; lane 7, ADA C2-V5pc K305R/R315Q/K319T/P437S; and lane 8, ADA C2-V5res.

Pseudoviruses generated with the ADA C2-V5pc K319T envelope demonstrated dramatically improved pseudovirus infectivity to a level only threefold lower than that of ADA C2-V5res pseudoviruses (Fig. 1B). The lysine residue at position 319 was present only in envelopes amplified from the PM-1 passage control virus, whereas both the original RU570 primary isolate and RU570-VCVres viruses contained a threonine at position 319. In contrast to the K319T mutation, pseudoviruses generated with the single P437S forward mutation in C4 continued to demonstrate impaired infectivity (>1,000-fold), as shown in Fig. 1B. However, the P437S mutation in combination with K319T or K305R/K319T mutations further increased pseudovirus infectivity to a level twofold higher than ADA C2-V5res pseudoviruses (Fig. 1B). The infectivity of pseudoviruses generated with ADA C2-V5pc envelopes containing all four forward mutations (K305R/R315Q/K319T/P437S) was similar to ADA C2-V5res pseudoviruses (Fig. 1B).

Reduced infectivity is not associated with decreased envelope levels. To eliminate the possibility that the reduced infectivity observed with the ADA C2-V5pc envelope was a result of decreased virion-associated envelope levels, HIV-1 pseudoviruses were purified from cell culture supernatants using lectin-agarose from G. nivalis. Equivalent pseudovirus particles based on p24 were probed for HIV-1 envelope incorporation following affinity purification. Figure 1C depicts the Western blotting results for detection of HIV-1 gp120 in pseudoparticles using rabbit anti-gp120 immunoglobulin G. The difference in gp120 migration between ADA and the chimeric ADA envelopes is due to an additional 18 amino acids present in ADA gp120. The amount of envelope incorporated into pseudoviruses was not significantly reduced for either ADA C2-V5pc (Fig. 1C, lane 2) or ADA C2-V5pc P437S (Fig. 1C, lane 3) envelopes even though they were minimally infectious. Appreciable levels of virion-associated envelope were observed compared to ADA C2-V5res pseudovirus (Fig. 1C, lane 8) or pseudoviruses generated with the ADA C2-V5pc envelope containing combinations of two or more forward mutations (Fig. 1C, lanes 5 to 7). Therefore, the reduced infectivity observed with these envelopes is not associated with major reductions in virion envelope incorporation.

Individual mutations differently affect resistance and/or virus infectivity. In order to determine the effect on VCV resistance of different combinations of the V3 loop mutations, we analyzed pseudoviruses generated with the ADA C2-V5pc K319T envelope in which the additional forward mutations were added in a step-wise fashion. Because of the nature of the allosteric effect these inhibitors have on the conformation of CCR5, which blocks HIV-1 infection, resistance is primarily established by the ability of HIV-1 to gain entry using the inhibitor-bound form of the receptor (1, 35, 43, 56). Therefore, complete inhibition of resistant viruses in drug susceptibility assays is not achieved. Instead, a reduction in the MPI, which plateaus at higher concentrations of inhibitor (1, 35, 40, 43, 56), is used as a representative measure of resistance to coreceptor antagonists. Interestingly, pseudoviruses generated with the ADA C2-V5pc K319T envelope displayed an average MPI of 85% (Fig. 2B). Since this mutation was found only in the passage control virus, it likely represents an adaptation to in vitro passage in PM-1 cells. Notably, the T319K substitution also occurred in an RU570 passage control virus in peripheral blood lymphocytes (56). Therefore, detection of low-level resistance with this chimeric RU570 envelope (ADA C2-V5pc) containing T319 likely reflects the reduced susceptibility phenotype previously observed with the RU570 primary isolate (11, 52).


Figure 2
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  		FIG. 2. VCV dose-response curves for drug susceptibility assays performed in U87-CD4-CCR5 cells with HIV-1 pseudoviruses (amounts of p24 are as indicated by the symbols on the figure) generated with the following envelopes: ADA (A), ADA C2-V5pc K319T (B), ADA C2-V5pc K319T/P437S (C), ADA C2-V5pc K305R/K319T/P437S (D), ADA C2-V5pc K305R/R315Q/K319T/P437S (E), and ADA C2-V5res (G). In these assays, pseudovirus stocks were normalized by p24 levels, and equivalent amounts of pseudovirus per well were added to U87-CD4-CCR5 cells. Data were analyzed using nonlinear regression, four-parameter logistic curve fit analysis with GraphPad Prism software, version 4.0, and are representative of at least three independent assays. Data represent the average ± standard deviation of four replicates. The insets for panels A to E and G depict the linear dose response observed with increasing amounts of HIV-1 pseudovirus input using the p24-normalized stocks. (F) Dose-response curves for VCV susceptibility assays performed in U87-CD4-CCR5 cells using pseudovirus stocks normalized by p24 and cells infected with 25 ng of p24 of each pseudovirus stock per well. Data represent the average ± standard deviation of four replicates. (H) VCV dose-response curves generated using the laboratory-adapted RU570-VCVres and RU570 passage control virus from the original PM-1 cultures. Replicating virus was analyzed in a single-cycle virus infection assay as described in the Materials and Methods section using RU570-VCVres virus and RU570 passage control virus (inocula are as indicated on the figure and are given as RNA copies). The 50% effective concentration and MPI values for the passaged viruses were determined using nonlinear regression, four-parameter logistic curve fit analysis with GraphPad Prism software. Data are representative of the average ± standard deviation of three replicates and three independent experiments.

Pseudoviruses generated with the ADA C2-V5pc envelope containing the combined K319T/P437S forward mutations also displayed a similar level of resistance, with an average MPI of 82% (Fig. 2C). However, when K305R was added to K319T/P437S (Fig. 2D) or R315Q/K319T/P437S (Fig. 2E) mutations, an increase in resistance was observed, characterized by a further reduction in the average MPI to 71% for inocula ranging from 1 to 100 ng of p24. In addition, when 25 ng of p24 input/well of ADA C2-V5pc K319T, ADA C2-V5res R305K, and ADA C2-V5res R305K/S437P pseudovirus from p24-normalized stocks was assayed, resistance to VCV showed overlapping dose-response curves with similar MPI plateau levels (88% to 91%), further emphasizing the importance of Arg at position 305 for resistance to VCV (Fig. 2F).

Our previous study demonstrated that resistance to VCV (as MPI) for ADA C2-V5res pseudoviruses correlated with the amount of pseudovirus input. As virus input increased, MPI decreased, reaching a plateau around 50% at the highest dose of pseudovirus (Fig. 2G). In the context of the ADA C2-V5pc envelope, it is interesting that both K319T (Fig. 2B) and the K319T/P437S mutations (Fig. 2C) appear to have minimal effects on the virus dose-dependent resistance phenotype, even at the highest concentrations of p24 input (compare Fig. 2B and C with E and G). However, when the R315Q mutation is present, a virus concentration-dependent decrease in MPI is apparent (compare Fig. 2E and G). When high-titer pseudovirus stocks were used, ADA C2-V5res (Fig. 2G) and ADA C2-V5pc K305R/R315Q/K319T/P437S (Fig. 2E) pseudoviruses always produced MPI values that plateaued near 50% at ≥100 ng of p24/well, as shown. As an additional control, we also analyzed ADA pseudovirus susceptibility to VCV over the same range of pseudovirus input. ADA pseudoviruses are threefold more infectious (Fig. 1B) than ADA C2-V5res pseudoviruses, and no change in MPI was observed even at higher input levels of ≥100 ng of p24 (Fig. 2A). To verify that the RLU results for each of the different pseudoviruses were completely within the linear range of the assay, luciferase activity was measured over a >100-fold range in virus input. All pseudoviruses demonstrated r2 values of >0.95 (see inset graphs in Fig. 2A to G).

Since this virus dose-dependent phenotype appeared to be unique, we analyzed resistance with the RU570-VCVres virus from the laboratory-adapted culture in a single-cycle virus infection assay. Strikingly, the same phenotype was also observed with replicating virus. U87-CD4-CCR5 cells were infected with RU570-VCVres virus using a 10-fold range in virus input (Fig. 2H). With a high virus inoculum (6 x 109 RNA copies), a plateau in the MPI at 50% occurred. However, when virus infection was carried out using an inoculum of >10-fold less virus (4 x 108 RNA copies), the MPI was 85%. Using the same virus input with the passage control virus, assay results showed MPIs at 91% and 95%, respectively (Fig. 2H).

Amino acid changes in the N terminus of CCR5 prevent entry of RU570 VCV-resistant pseudovirus with the drug-bound receptor. To determine whether the RU570-VCVres pseudoviruses interact differentially with either the N terminus or ECL2 region of CCR5, we analyzed virus entry using cells transfected with a set of CCR5 mutants containing single CCR5 alanine substitutions in these regions. In the N terminus of CCR5, single point mutations Y3A, Y10A, D11A, N13A, Y14A, and Y15A and an N-terminal ({Delta}2-17) CCR5 deletion mutant were analyzed in pseudovirus assays in both the absence and presence of VCV. In the ECL2 region of CCR5, single point mutations R168A, K171A, E172A, L174A, and C178A were also analyzed. The infectivity of pseudoviruses generated with ADA, ADA C2-V5pc K319T, ADA C2-V5res, and ADA C2-V5pc K305R/R315Q/K319T/P437S envelopes were analyzed in 293T cells transiently expressing CD4 and the CCR5 mutants in the absence and presence of 1.0 µM VCV. The expression levels on the surface of 293T cells for each CCR5 mutant as well as coexpressed CD4 was analyzed by fluorescence-activated cell sorting using anti-CCR5 and anti-CD4 MAbs. The expression levels observed for CD4 and each CCR5 mutant were similar to wild-type (wt) CCR5/CD4 expression (data not shown).

ADA pseudoviruses were susceptible to mutations at tyrosine residues Y10A, Y14A, and Y15A in the N terminus of CCR5 and to the {Delta}2-17 N-terminal mutation (Fig. 3A, left panel). In comparison to wt CCR5, ADA pseudovirus infectivity was reduced by 50% with Y10A, Y14A, and Y15A mutants and by 75% with the {Delta}2-17 N-terminal CCR5 mutant. The Y3A and D11A mutations did not significantly affect the ability of ADA pseudoviruses to gain entry, whereas the N13A mutation reduced infectivity by only 25%. The ECL2 mutations R168A and K171A reduced ADA infection by approximately 50%, whereas the C178A mutation, which previously was shown to link ECL1 to ECL2 (3), significantly reduced ADA pseudovirus entry >75% compared to wt CCR5. As expected, ADA pseudoviruses were still susceptible to inhibition by VCV with each of the N-terminal and ECL2 CCR5 mutants (Fig. 3A, right panel).


Figure 3
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  		FIG. 3. The effect of alanine substitutions in the N terminus of CCR5 (Y3A, Y10A, D11A, N13A, Y14A, and Y15A and a {Delta}2-17 N-terminal mutant) and alanine substitutions in ECL2 (R168A, K171A, E172A, L174A, and C178A) on HIV-1 pseudovirus entry. HIV-1 pseudovirus infection is expressed as a percentage of wt CCR5 infection in the absence (left panel) and presence of 1.0 µM VCV (right panel). (A) ADA HIV-1 pseudovirus. (B) ADA C2-V5pc K319T HIV-1 pseudovirus. (C) ADA C2-V5res HIV-1 pseudovirus. (D) ADA C2-V5pc K305R/R315Q/K319T/P437S HIV-1 pseudovirus. Data represent the average ± standard deviation of at least five independent assays.

The ADA C2-V5pc K319T pseudoviruses exhibited sensitivity levels to the N-terminal mutations in CCR5 for virus entry in the absence of VCV (Fig. 3B, left panel) similar to those of the ADA pseudoviruses. Virus entry for ADA C2-V5pc K319T pseudoviruses in cells expressing CCR5 (Y10A), CCR5 (Y14A), and the {Delta}2-17 N-terminal mutation was reduced more than 50% compared to wt CCR5 infection. In the presence of 1.0 µM VCV, ADA C2-V5pc K319T pseudovirus entry with cells expressing the Y3A and N13A CCR5 mutations was similar to levels seen for wt CCR5 with 1.0 µM VCV (Fig. 3B, right panel). In contrast, pseudovirus entry in the presence of VCV was dramatically reduced for CCR5 (Y10A), CCR5 (D11A), CCR5 (Y14A), and the {Delta}2-17 N-terminal CCR5 mutant (Fig. 3B, right panel). The only mutation in ECL2 that affected ADA C2-V5pc K319T pseudovirus entry was C178A. Virus entry was reduced by approximately 50% in both the absence and presence of VCV.

RU570 chimeric envelopes ADA C2-V5res (Fig. 3C, left panel) and ADA C2-V5pc K305R/R315Q/K319T/P437S (Fig. 3D, left panel) showed reductions in pseudovirus infection to less than 50% of wt CCR5 for Y10A, Y14A, and Y15A CCR5 mutants in the absence of VCV. The reduced infectivity was similar to the extent of decrease observed for both ADA and ADA C2-V5pc K319T pseudoviruses. The level of infectivity for D11A and N13A CCR5 mutants was approximately 75% of wt CCR5, whereas the Y3A mutation had a minimal effect on RU570-VCVres pseudovirus entry. While ADA and ADA C2-V5pc K319T pseudovirus infection with cells expressing the CCR5 {Delta}2-17 N-terminal mutation was reduced to approximately 25% of wt CCR5, RU570-VCVres pseudoviruses were unable to infect cells expressing the CCR5 {Delta}2-17 N-terminal mutant. In addition, the only ECL2 mutation that affected RU570-VCVres pseudovirus entry was C178A. Virus entry was reduced by >50% with the drug-free receptor (Fig. 3C and D, left panels).

The CCR5 mutations had a much greater effect on the ability of RU570-VCVres pseudoviruses to enter cells in the presence of VCV than the drug-free receptor. ADA C2-V5res (Fig. 3C, right panel) and ADA C2-V5pc K305R/R315Q/K319T/P437S pseudoviruses (Fig. 3D, right panel) were significantly more sensitive to CCR5 amino acid mutations at positions Y10, D11, Y14, and Y15 with the drug-bound receptor. The RU570-VCVres pseudoviruses were unable to infect cells expressing CCR5 (Y10A), CCR5 (Y14A), and CCR5 (Y15A) and demonstrated minimal infectivity (<5% of wt CCR5) for the CCR5 (D11A) receptor in the presence of 1.0 µM VCV. Similar to the results for the drug-free receptor, C178A was the only ECL2 mutation that reduced virus entry by 50% in the presence of VCV. These findings suggest that interactions between RU570-VCVres gp120 and the N terminus of CCR5 are more critical for virus entry using the drug-bound receptor than for native CCR5. RU570-VCVres pseudoviruses were also unable to infect cells expressing the CCR5 {Delta}2-17 N-terminal mutant in the presence of VCV, further confirming that this resistant virus is completely dependent on the intact N terminus of CCR5. In contrast to these results, the RU570-VCVres pseudoviruses were able to infect cells expressing either the Y3A or N13A CCR5 mutations in the drug-bound state.

Further confirmation of the reliance on the N terminus of CCR5 for RU570-VCVres pseudovirus entry using the drug-bound receptor was demonstrated with a MAb specific for the N terminus of CCR5 used to neutralize virus infection (29). RU570-VCVres pseudovirus entry was reduced by approximately 80% at the highest concentration of the anti-N terminus CCR5 MAb CTC5 in the presence of VCV compared to only 40% in the absence of VCV with ADA C2-V5res (Fig. 4A) and ADA C2-V5pc K305R/R315Q/K319T/P437S (Fig. 4B) pseudoviruses.


Figure 4
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  		FIG. 4. RU570-VCVres pseudoviruses are more sensitive to neutralization with a MAb specific to the N terminus of CCR5 in the presence of VCV. The sensitivity of ADA C2-V5res (A) and ADA C2-V5pc K305R/R315Q/K319T/P437S (B) HIV-1 pseudoviruses to neutralization by MAb CTC5 in the absence (–) or presence (+) of VCV was determined in U87-CD4-CCR5 cells. Data are representative of two independent assays and represent the percent inhibition of control infection without MAb (mean ± standard deviation; n = 4). Ab, antibody.

Amino acid substitutions in the V3 loop crown enhance VCV resistance in RU570. Previously, we identified an amino acid substitution at position 317 (I317F) in <50% of the RU570 envelope clones that were amplified from the RU570-VCVres virus culture (40). Based on this observation and the presence of a double Q315E/L317F amino acid substitution in a patient isolate resistant to VCV (28), we generated both single and combined Q315E and I317F substitutions in the ADA C2-V5res chimeric envelopes. Pseudoviruses generated with these envelopes were analyzed for resistance to VCV in drug susceptibility assays in U87-CD4-CCR5 cells (Fig. 5A and B).


Figure 5
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  		FIG. 5. VCV dose-response curves for drug susceptibility assays performed in U87-CD4-CCR5 cells with 5 ng of p24 HIV-1 pseudovirus (A) and 25 ng of HIV-1 pseudovirus (B). HIV-1 pseudoviruses were generated with the envelopes indicated on the figure. In these assays, pseudovirus stocks were normalized by p24 levels, and equivalent amounts of pseudovirus per well were added to U87-CD4-CCR5 cells. Data were analyzed using nonlinear regression, four-parameter logistic curve fit analysis with GraphPad Prism software, version 4.0, and are representative of at least three independent assays.

As previously described, pseudovirus stocks were normalized by p24, and 5 ng and 25 ng of p24 input from pseudovirus stocks were analyzed. Interestingly, when both amino acid substitutions were added to the ADA C2-V5res chimeric envelopes, we observed an increase in resistance to VCV, as demonstrated by the increased reduction in the MPI. The MPI plateau decreased from 71% to 47% with 5 ng of p24 input (Fig. 5A) and from 57% to 33% with 25 ng of pseudovirus input (Fig. 5B) for ADA C2-V5res and ADA C2-V5res Q315E/I317F pseudoviruses, respectively. The single amino acid change Q315E had no significant effects on ADA C2-V5res phenotypic resistance, whereas the single I317F mutation did influence resistance but to a lesser degree than the combined mutations, as shown by the decrease in the MPI from 71% to 64% for 5 ng (Fig. 5A) and 57% to 44% for 25 ng (Fig. 5B) of pseudovirus input.

RU570 Q315E/I317F substitution in the V3 loop crown abolishes virus entry in cells expressing the Y14A mutation. While the Q315E/I317F mutation in the RU570-VCVres chimeric envelope further increased resistance to VCV, it also completely eliminated the ability of pseudovirus to infect cells expressing the Y14A mutation under both drug-free and drug-bound receptor conditions (Fig. 6). This contrasts with ADA C2-V5res and ADA C2-V5pc K305R/R315Q/K319T/P437S pseudoviruses which, in the absence of VCV, still retained infectivity at levels reduced to less than 50% of wt CCR5 infectivity with the Y14A mutation. Also, both D11A and N13A mutations reduced RU570 VCVres Q315E/I317F pseudovirus entry to approximately 50% of wt CCR5 activity with the drug-free receptor. Previously described results suggest that the V3 crown most likely interacts with the ECL2 domain of CCR5 (7, 12, 17, 22, 23). The increased resistance to VCV that was observed with the Q315E/I317F mutation suggests that changes in the V3 crown likely improved recognition of the VCV-bound configuration of CCR5. Thus, we hypothesize that the Q315E/I317F mutation in the crown of the V3 loop influences the interactions with the drug-bound configuration of ECL2. By contrast, a reduced recognition of the ECL2 configuration in the native receptor with the Q315E/I317F mutation is plausible. This could potentially have created a stronger dependence on the N terminus, in particular, the O-sulfated tyrosine residue at position 14, for virus entry using native CCR5.


Figure 6
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  		FIG. 6. The effect of alanine substitutions in the N terminus of CCR5 (Y3A, Y10A, D11A, N13A, Y14A, and Y15A) and of the {Delta}2-17 N-terminal CCR5 mutant transiently expressed with CD4 in 293T cells on pseudovirus entry for HIV-1 ADA C2-V5res Q315E/I317F pseudoviruses. Pseudoviruses were analyzed for infectivity in the presence (right panel) or absence (left panel) of 1.0 µM VCV, and data are expressed as a percentage of wt CCR5 infection. Data represent the average ± standard deviation of at least 3 independent assays.

Molecular model demonstrates a potential new interaction between the CCR5 binding domain in RU570-VCVres gp120 and the N terminus of CCR5. To analyze the potential structural implications of the RU570-VCVres adaptive mutations on the CCR5 coreceptor binding region of gp120, we used homology models (built with InsightII/Discover [Accelrys, San Diego, CA] and Maestro/Prime [Schrödinger, Portland, OR]) of the RU570-VCVpc gp120 interaction with the N-terminal CCR5 peptide consisting of residues 2 to 15 (CCR52-15) and the RU570-VCVres gp120/CCR52-15 N-terminal complex based on a previously reported structure-based docked CCR52-15 N terminus/HIV-1 gp120-CD4 molecular model (22). The K305R mutation, in which the conserved Lys in the outgoing stem region of the V3 loop is replaced by Arg, primarily impacted the degree of VCV resistance in RU570. Our model demonstrates a potential for the formation of a salt bridge between Arg 305 and Asp 11 in the N terminus of CCR5 (Fig. 7B). In the passage control envelope, Lys 305 could form a single H bond with Asp 11 in the N terminus; however, Lys 319 may sterically and electrostatically hinder this interaction (Fig. 7A). The K319T mutation in the resistant V3 loop eliminates this constraint in RU570-VCVres gp120. Replacing Lys 319 with Thr, a smaller and neutral amino acid, could facilitate the interaction between Lys or Arg at position 305 with Asp 11 in the N terminus of CCR5. The P437S mutation in C4 is located in the bridging sheet region in the cavity formed at the intersection of the V3 loop and bridging sheet. Our model predicts the possibility of a hydrogen bond interaction between Ser 437 and the backbone carbonyl-group of Asn 13 in the CCR5 N terminus (Fig. 7C). The R315Q mutation influenced the virus dose-dependent effect on VCV resistance and is located in the crown region of the V3 loop, a region thought to interact with the extracellular domains of CCR5, specifically ECL2. The overall validity of this model is the subject of ongoing experimental studies.


Figure 7
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  		FIG. 7. Molecular model of the V3 loop and C4 domain of RU570-VCVres gp120 and the N-terminal CCR5 peptide based on coordinates obtained from Huang et al. (22) depicting the interaction between D11 in the N terminus of CCR5 with K305 in RU570 passage control gp120 (A) or R305 in RU570-VCVres gp120 (B). (C) The modeled interaction for Asn 13 in the N terminus of CCR5 and RU570-VCVres gp120.


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DISCUSSION
 
The observations made in this study reveal that an enhanced dependence on interactions between gp120 and the N terminus of CCR5 plays an important role in the ability of chimeric RU570-VCVres pseudoviruses to enter cells when the drug-bound receptor is used. Our data demonstrated that entry of ADA C2-V5res and ADA C2-V5pc K305R/R315Q/K319T/P437S pseudoviruses was more sensitive to neutralization by a MAb specific to the N terminus of CCR5 (29) and to individual point mutations in the N terminus of CCR5 when the receptor is drug bound. These results are similar to those obtained using HIV-1 containing an engineered deletion in V3. When V3 was deleted from HIV-2 (30) or a 4-amino-acid deletion was constructed in the dual-tropic HIV-1 R3A V3 loop, these viruses were completely resistant to CCR5 coreceptor antagonists (27, 30, 38, 39). In the case of the HIV-1 R3A V3 mutant with a deletion of residues 9 to 12 ({Delta}9-12), increased dependence on the N terminus of CCR5 was reported. The HIV-1 R3A {Delta}9-12 virus was completely dependent on Y3, Y10, Y14, and Y15 tyrosine residues in the N terminus of CCR5 (27, 38), whereas in our studies the RU570-VCVres variant was not affected by the Y3A mutation and also showed dependence on position D11 in the N terminus. While mutations at the N-terminal tyrosine positions along with D11 were critical for entry with the drug-bound receptor, these mutations had only a limited effect on RU570-VCVres pseudovirus entry in the absence of VCV. In the case of RU570, we observed a gradation of resistance, with the ADA C2-V5pc K319T virus demonstrating the least resistance and the least dependence on the N terminus. As resistance to VCV increased with the additional V3 loop mutations, a parallel increase in dependence on the intact N terminus of CCR5 was observed.

HIV-1 resistance to antiretroviral drugs is dependent on primary mutations that confer resistance and secondary mutations that play a compensatory role enabling the virus to maintain replication fitness. In the case of RU570, mutations identified in gp120 also followed a similar pattern by influencing either resistance and/or pseudovirus infectivity. Pseudovirus infectivity may correlate with replication fitness, but further confirmation using replicating virus is needed to confirm this observation. The K305R mutation along with K319T in the V3 loop of the RU570-VCVres virus contributed to the resistance phenotype; however, the K319T mutation in the V3 loop also plays a major role in virus infectivity. The T319K amino acid substitution in RU570 occurred in the PM-1 passage control cultures, and interestingly this same substitution also occurred when RU570 was cultured in peripheral blood mononuclear cells (56), which provides further support that this amino acid change is a specific cell culture-derived adaptation. When the K319T substitution was made in the ADA C2-V5pc envelope, infectivity of pseudoviruses was restored, which also showed a low level of phenotypic resistance to VCV. Since RU570 was previously shown to have reduced susceptibility to coreceptor antagonists (11, 52), this amino acid change is most likely not an adaptive amino acid change associated with the evolution of laboratory-adapted VCV resistance in RU570.

The P437S mutation in the C4 region primarily appears to impact only virus infectivity. This finding agrees with previous results showing that a P437A mutation in the YU2 R5 envelope enabled a more receptor-accessible conformation, predicted to result in more efficient receptor triggering (45). Also, this same mutation enhanced binding of YU2 gp120 to CCR5 (47). The K305R amino acid substitution described here has also been observed in a cell culture-derived AD101-resistant CC1/85 virus (26) and was also identified in clinical isolates from two subjects that developed resistance to VCV in phase II clinical studies (28, 55). However, a larger data set will be required to determine if K305R is a signature mutation for VCV resistance. The R315Q substitution in the V3 crown influenced the resistance phenotype in RU570 by contributing to the virus dose-dependent effect on resistance.

The Q315E/I317F pair of mutations which contributed to VCV resistance in a subtype D HIV-1 isolate from a clinical subject (28) primarily impacted resistance when they were added to RU570-VCVres by converting the partially resistant phenotype in RU570 to a phenotype demonstrating nearly complete resistance. When this substitution was made, the dose-dependent effect observed with R315Q was still apparent. A similar resistance phenotype was observed in RU570 when the substitution at position 315 was either a Q or E, whereas the Q315E substitution combined with I317F considerably lowered the MPI when the mutations were introduced into the RU570 ADA C2-V5res envelope. The Q315E/I317F combination may make critical contacts with the extracellular domains of VCV-bound CCR5 although to date we have no experimental data to support this hypothesis. Because adaptations involved in VCV resistance have so far been found to be context dependent (35), this finding suggests that some mutations can influence resistance in an unrelated background. In addition, the degree of resistance seems to be more dependent on crown mutations that are predicted to interact with ECL regions than with mutations that affect interactions with the N terminus of CCR5.

The structure of the CCR5 N-terminal peptide complex with gp120-CD4 revealed that following engagement of gp120 with the N terminus, the flexible V3 stem was converted into a rigid β-hairpin, with the V3 crown predicted to interact with the ECL2 region of CCR5 (22, 23). The model we propose based on this structure suggests that Ser 437 in C4 could form a hydrogen bond interaction with the backbone carbonyl-group of Asn 13, and the K305R amino acid change in the V3 stem could result in a stronger contact between the stem and residue D11 in the N terminus of CCR5. In the previously published structure of the CCR5 N terminus, residue D11 forms an ionic interaction with R440 in the C4 region of gp120 from the clade B virus YU2 (22); however, the corresponding amino acid residue in RU570 is A440, the consensus clade G residue. The D11A CCR5 substitution mutation severely compromised RU570-VCVres pseudovirus entry with the drug-bound receptor but had only minor influence on virus entry in the absence of drug. This result suggests that the interaction of D11 with gp120 may compensate for the altered configuration of CCR5 when VCV is bound to the receptor. In general, CCR5 Y10, Y14, and Y15 have been shown to influence R5-tropic virus entry to various degrees, depending on the virus and cell type in which the CCR5 N-terminal tyrosine mutants are expressed (7-10, 12, 13, 15-17, 24). In the case of RU570, the Q315E/I317F mutation in the V3 crown region of RU570-VCVres gp120 increased the VCV-resistant phenotype, resulting in an increased dependence on the O-sulfated tyrosine at position 14 in the N terminus for virus entry using the native configuration of CCR5. This finding could result from a reduced interaction between the V3 crown and the ECL2 configuration of the drug-free receptor. Overall, these interactions predicted by our model provide a possible structural basis for an enhanced interaction of gp120 with the N terminus of CCR5, but these predictions require further experimentation to confirm their validity.

Another intriguing possibility suggests that adaptive amino acid changes in V3 that cause resistance to diverse entry inhibitors cluster in the gp120 trimer interface that overlies gp41 (42). These adaptive changes in V3 could reduce the allosteric hold of gp120 on gp41, thereby lowering the activation energy barrier for membrane fusion without affecting any specific binding to CCR5 (42). This idea agrees with previously described attributes of V3 that were suggested to contribute to subunit associations within the viral spike, based on the position of V3 in the crystal structure of a V3-containing gp120 core and its modeled trimer (23). Further understanding of the ordering and timing of structural rearrangements in gp120 that occur following engagement of CCR5 and the subsequent fusion events is needed to more clearly understand how these adaptive changes in the V3 loop might mechanistically impart resistance to CCR5 coreceptor inhibitors.


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FOOTNOTES
 
* Corresponding author. Mailing address: Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-E403C, MS 4945, Kenilworth, NJ 07033. Phone: (908) 740-7260. Fax: (908) 740-3032. E-mail: john.howe{at}spcorp.com Back

{triangledown} Published ahead of print on 23 September 2009. Back

{dagger} The authors have paid a fee to allow immediate free access to this article. Back


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Journal of Virology, December 2009, p. 12151-12163, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.01351-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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