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. 2013 Jun;57(6):2640–2650. doi: 10.1128/AAC.02511-12

Multifaceted Mechanisms of HIV Inhibition and Resistance to CCR5 Inhibitors PSC-RANTES and Maraviroc

Michael A Lobritz a,b,*, Annette N Ratcliff a,b,*, Andre J Marozsan b,c,*, Dawn M Dudley a,*, John C Tilton d, Eric J Arts b,✉
PMCID: PMC3716150  PMID: 23529732

Abstract

Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated. In single-cycle assays, these HIV-1 clones displayed equal sensitivity to PSC-RANTES inhibition, suggesting effective receptor downregulation. Prolonged PSC-RANTES exposure resulted in desensitization of the receptor to internalization such that increasing virus concentration (substrate) could saturate the receptors and overcome PSC-RANTES inhibition. In contrast, resistance to MVC was observed with the MVC-resistant HIV-1 (R3 versus S2) in both multiple- and single-cycle assays and with altered virus concentrations, which is indicative of allosteric inhibition. MVC could also mediate inhibition and possibly resistance through competitive mechanisms.

INTRODUCTION

HIV-1 entry involves sequential interaction of the viral envelope glycoprotein (gp120/gp41) with human CD4 and a chemokine receptor, either CCR5 or CXCR4. Pharmacologic efforts to interrupt the coreceptor-dependent entry process have yielded a wide variety of molecules which inhibit through divergent mechanisms. Studies aimed at uncovering mechanism(s) of action have shown that small-molecule CCR5 antagonists (i.e., maraviroc [MVC], vicriviroc, and aplaviroc) bind to an allosteric site within the transmembrane helices of CCR5 (1–3). Inhibitor binding prevents interactions between HIV-1 envelope and CCR5 primarily through a noncompetitive mechanism (4, 5), although one review article also suggests the possibility of competitive inhibition between MVC and HIV-1 for the CCR5 receptor (6). However, little is known about the mechanism(s) of HIV-1 inhibition by chemokines (or their derivatives) or monoclonal CCR5 antibodies. PSC-RANTES [N-nonanoyl, des-Ser1[l-tioproline2, l-cyclohexylglycine3]-RANTES(2-68)] is a chemokine analogue with potent antiviral activity in vitro (7, 8) and in the SHIV-macaque vaginal challenge model (9). In contrast to CCR5 antagonists, chemokine analogues trigger rapid internalization of CCR5 through a clathrin-dependent endocytic process (10). Downregulation of the receptor from the cell surface by these CCL5 (RANTES) derivatives is prolonged relative to the native chemokine (11). Previous studies have concluded that CCR5 internalization by chemokine analogues is the dominant mechanism for inhibition of HIV-1 entry (7, 8). However, we and others have previously identified PSC-RANTES-resistant virus that showed a difference in sensitivity to PSC-RANTES depending upon whether the virus was tested in an assay allowing a single cycle of viral replication or multiple cycles of replication. This is in stark contrast to MVC-resistant viruses that exhibit the same sensitivity to drug regardless of the number of viral replication cycles in an assay. These observations prompted the present study on the mechanisms of inhibition and resistance to the CCR5 antagonist, MVC, and the CCR5 agonist, PSC-RANTES.

The concentration of entry inhibitor (e.g., RANTES derivatives, enfuvirtide, maraviroc, vicriviroc, and AMD3100) required to inhibit 50% of viral replication in culture (IC50) can vary 10- to 1,000-fold when comparing primary HIV-1 isolates that have never been exposed to these drugs (12–16). In contrast, primary HIV-1 isolates from treatment-naive patients display minimal variations in susceptibility to protease or reverse transcriptase inhibitors (17). Variation in the “intrinsic” susceptibility to entry inhibitors is related to the extreme variability and plasticity of the envelope glycoproteins compared to more conserved viral enzymes (16). Among primary viral isolates, we have observed >30-fold variation in sensitivity to AOP-RANTES, a predecessor of PSC-RANTES (16). Mapping of single nucleotide polymorphisms related to this differential sensitivity revealed that specific amino acids at positions 318 and 319 in the V3 loop stem of gp120 could modulate PSC-RANTES susceptibility up to 50-fold (17). The proposition that CCL5 analogues inhibit HIV-1 replication solely through receptor downregulation (7) is in conflict with the observation of differential sensitivity to these inhibitors (16, 17). Complete receptor downregulation is typically observed at the same PSC-RANTES concentration that inhibits wild-type R5 HIV-1. However, PSC-RANTES-resistant HIV-1, that maintains absolute CCR5 usage for entry, can still replicate in the presence of PSC-RANTES concentrations responsible for complete receptor downregulation.

Variable inhibition of HIV-1 replication by PSC-RANTES would suggest an alternative, overriding mechanism such as competitive binding for CCR5. In this study, we addressed the role of competitive binding in the inhibition of HIV-1 entry by maraviroc and PSC-RANTES in multiple- versus single-replication-cycle assays using viruses with differential sensitivities to these drugs. Although allosteric binding and inhibition was observed for MVC, two distinct inhibitory pathways for PSC-RANTES were segregated by comparing PSC-RANTES inhibition in cells exposed to drug for short versus long periods of time. The inhibitory activity of PSC-RANTES in the absence of receptor downregulation was further characterized using the mutant M7-CCR5 receptor (18). We examined viral replication in the presence of PSC-RANTES, under increasing viral concentrations as a substrate, to test a competitive inhibition mechanism of drug sensitivity. MVC binding to CCR5 appears to mediate inhibition of R5 HIV-1, while resistance to MVC was dependent on the use of an MVC-bound CCR5 for host cell entry. However, increasing virus concentration reduced MVC inhibition, suggesting a competition between virus and MVC for CCR5 binding. The potential for competitive MVC and PSC-RANTES inhibition is consistent with the resistance mechanisms observed with different MVC-resistant and PSC-RANTES-resistant viruses (19, 20). Overall, we provide here a detailed characterization of inhibition and resistance to the U.S. Food and Drug Administration-approved CCR5 antagonist, MVC, and the CCR5 agonist, PSC-RANTES. PSC-RANTES is one of the most potent antiretroviral drugs available but, as a modified peptide, its therapeutic use has been abandoned in favor of possible microbicide use.

MATERIALS AND METHODS

Cells, viruses, and inhibitors.

The human embryonic kidney cell line 293T was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, provided by Andrew Rice (21) and maintained in complete media. U87MG human glioblastoma cells stably expressing CD4 and CCR5 were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, provided by HongKui Deng and Dan Littman (22) and maintained in complete media supplemented with 300 μg G418/ml and 1 μg of puromycin/ml (both from Invitrogen, Carlsbad, CA). Complete media consists of Dulbecco modified Eagle medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 100 μg of penicillin-streptomycin/ml (Gibco). U87.CD4.M7-CCR5 cells were generated by stable lentiviral transduction of U87.CD4 cells with pBABE.M7-CCR5 and puromycin selection. Cells were maintained in complete medium supplemented with 300 μg of G418/ml and 1 μg of puromycin/ml.

Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood extracted from HIV-negative donors by Ficoll-Hypaque centrifugation. PBMC cultures were stimulated in RPMI 1640 medium (Gibco) with 10% FBS, 100 μg penicillin-streptomycin/ml, and 1 μg of phytohemagglutinin/ml for 48 to 72 h. The cells were maintained thereafter in RPMI 1640 medium with 10% FBS, 100 μg of penicillin-streptomycin/ml, and 1 ng of interleukin-2/ml. CD4+ T cells were isolated from activated total PBMCs using a Miltenyi CD4+ T cell isolation kit. The purity of the CD4+ cell population was determined to be >95% by flow cytometry.

The generation and production of NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) chimeric viruses have been previously described (17). These viruses contain specific polymorphisms at 318/319 in the V3 loop (YA or RT) which were found to modulate sensitivity to PSC-RANTES by 10-fold. Replication-competent virus was generated by transfection of 293T cells by using the Effectene transfection system (Qiagen, Germantown, MD) with 2 μg of each molecular clone. The cells were washed after 24 h, and virus in the supernatant was collected after 72 h. Virus was propagated on U87.CD4.CCR5 cells, and supernatants were monitored for reverse transcriptase activity. Virus samples were collected and frozen, and infectious titers determined by limiting-dilution 50% tissue culture infective dose measurements on U87.CD4.CCR5 and activated CD4+ T cells. PCR amplification and sequencing of the C2-V3 region from proviral DNA confirmed that reversion of the V3 mutations did not occur during viral propagation. Pseudovirus particles were generated either by cotransfection of 2 μg of pREC_env [V3A1-92RW009(YA) and V3A1-92RW009(RT)] envelope expression vectors with 2 μg of the pseudotyping vector pNL.Luc.AM or cotransfection of pREC_env vectors with pNL4-3.E− in 293T cells. Pseudovirus particles produced with pNL.Luc.AM contain genomic copy of firefly luciferase gene, while pseudovirus particles produced with pNL4-3.E− do not contain a reporter gene and infection must be scored by a reporter in trans. Pseudovirus particles were quantified by limiting dilution reverse transcriptase assay.

Isolation of the PSC-RANTES resistant (M584) and sensitive (P3-4) viruses during SHIV macaque challenge has been previously described (19). The isolation and characterization of the MVC-resistant (R3) and MVC-sensitive (S2) viruses has previously described (23). The envelope region from the ectodomain of gp120 to the membrane spanning domain of gp41 (ecto-MSD) of viruses M584 and P3-4 were PCR amplified and cloned by yeast homologous recombination in Saccharomyces cerevisiae MYA-906 cells (ATCC) into pREC_NFL_Δecto-MSD/URA3 vector. The gp120 region of viruses R3 and S2 were PCR amplified and cloned by homologous recombination into pREC_NFL_Δgp120/URA3. Infectious chimeric virus was produced by cotransfection of 3 μg of each vector with 3 μg of pCMV_cplt in 293T cells using a Fugene transfection system (Promega, Madison, WI). Pseudovirus particles were generated by cotransfection of these pREC_NFL vectors with the pseudotyping vector pNL.Luc.AM in 293T cells. Virus particles were quantified by using a limiting-dilution reverse transcriptase assay.

PSC-RANTES was kindly provided by Oliver Hartley. Stock solutions of PSC-RANTES and MVC were diluted in phosphate-buffered saline (PBS) and filter sterilized. Stock solutions of enfuvirtide were diluted in ethanol. Stock solutions of TAK-779, lamivudine (3TC), nevirapine (NVP), and saquinavir were diluted in dimethyl sulfoxide.

Plasmids.

pBABE-M7-CCR5 was kindly provided by Nathaniel Landau (18). The M7-CCR5 receptor harbors seven mutations in the cytoplasmic tail of CCR5 (S336/S337/Y339/T340/S342/T343/S349), rendering it deficient in receptor internalization (18). This vector was used to generate U87.CD4.M7-CCR5 cells. The pseudotyping vector pNL.Luc.AM was kindly provided by John Moore (5). This vector contains the firefly luciferase gene in place of env in a nearly full-length (NFL) NL4-3 genome. Pseudoviruses produced with this vector encode the luciferase enzyme and are restricted to a single round of replication. The pseudotyping vector pNL4-3.E− was constructed by digestion of pNL4-3 with NheI, blunting, and religation. HIV-1 envelope pseudotyped particles produced with this vector lack the envelope gene and are restricted to a single round of replication. Plasmid pDM128-LTR-fluc2 (a gift from David McDonald) contains the HIV-1 LTR and the firefly luciferase gene. Luciferase expression from this plasmid is HIV-1 Tat and Rev dependent (24).

Generation of cloning vectors pREC_NFL_Δgp120/URA3 and pREC_NFL_Δecto-MSD/URA3 and complementary plasmid pCMV_cplt are described in reference 25). Briefly, pREC_NFL/URA3 vectors consist of the NFL proviral genome of the NL4-3 HIV-1 laboratory-adapted strain with the specified regions (i.e., Δgp120) replaced by the orotidine-5′-phosphate decarboxylase gene (URA3) for the selection of homologous recombination in yeast cells. Complementing plasmid, pCMV_cplt, contains R, U5, and a gag fragment under a cytomegalovirus promoter. In cotransfections, packaging of RNA copies of both plasmids can produce infectious chimeric virus in subsequent propagations in cell lines. Vectors for expressing the NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) envelopes have been previously described (17).

Reverse transcriptase activity assay.

The reverse transcriptase activity is a measure of virus production in the supernatant and is used to quantify the level of infection (16). The supernatant (10 μl) was collected and incubated for 2 h at 37°C with buffer (25 μl) containing nucleotides dATP, dGTP, dCTP, and α-32P-labeled dTTP. A volume of 10 μl was spotted on DEAE filtermats, dried, and washed five times for 5 min each with 1× saline-sodium citrate buffer and twice with 85% ethanol. Filtermats were dried, and the radioactivity measured either by a Packard beta-counter to quantify the counts per minute (cpm) or the filtermats were exposed to phosphorimaging screen for 2 h at room temperature and densitometry quantified by use of an Imager FX.

Multiple-cycle drug susceptibility assays.

Sensitivity to HIV-1 inhibitors was assessed in either U87.CD4.CCR5 cells or in activated, purified CD4+ T lymphocytes. Cells were plated on 96-well plates (2 × 104 U87.CD4.CCR5 cells/well or 2 × 105 CD4+ T cells/well). Drugs were added to wells in serial 10-fold dilutions (PSC-RANTES, 100 to 10−5 nM; MVC, 100 to 10−5 μM; enfuvirtide [ENF], 10 to 10−6 μM; TAK-779, 10 to 10−6 μM; 3TC, 10 to 10−6 μM; NVP, 10 to 10−6 μM), followed by incubation for 1 h at 37°C. Virus was added to the cells at a multiplicity of infection (MOI) of 0.001 infectious units/cell unless otherwise specified, and the cells and virus were incubated for 24 h at 37°C. Input virus was removed, and the cells were washed with PBS. Fresh medium containing the appropriate concentration of drug was added back to the cells. Virus production into the supernatant was monitored by use of a radioactive reverse transcriptase assay and plotted versus the drug concentration to determine the 50% inhibitory concentration (IC50) or maximal percent inhibition (MPI) for each virus and drug. All drug susceptibility assays were performed in a minimal of three independent experiments, generating three drug susceptibility curves for each virus, each drug, and each condition.

Single-cycle drug susceptibility assays.

Single-cycle assays were performed using replication-defective particles containing a genomic copy of the firefly luciferase gene (pNL.Luc.AM) pseudotyped with different HIV-1 envelopes and used to infect U87.CD4.CCR5 or U87.CD4.M7-CCR5 cells. Pseudovirus particles were determined by use of limiting-dilution reverse transcriptase activity assays (26), and the infectivity of the stock pseudoviruses was assessed by limiting-dilution infection of U87.CD4.CCR5 cells. Drug sensitivity assays were performed using a volume of pseudovirus in the linear range of infection.

Flow cytometry.

Fluorescence-activated cell sorting (FACS) analysis of CCR5 expression was performed on U87.CD4.CCR5 and U87.CD4.M7-CCR5 cells. Cells were pelleted at 2,000 rpm for 5 min, washed with PBS, and washed in FACS staining buffer (PBS, 5% FBS, 1% bovine serum albumin, 0.1% sodium azide). The cells were again pelleted (2,000 rpm for 5 min) and resuspended in FACS staining buffer in addition to antibodies: CCR5 was detected with phycoerythrin (PE)-conjugated anti-CCR5 (clone 2D7 or clone 3A9; Becton Dickinson) or with isotype controls (PE-conjugated IgG2aκ). All antibodies were incubated at 12.8 μg/ml at room temperature for 30 min. The cells were diluted in an additional 150 μl of FACS staining buffer, pelleted again (2,000 rpm for 5 min), and resuspended in 400 μl of FACS staining buffer for analysis. For internalization assays, cells were incubated with antibody as described and subsequently treated with PSC-RANTES (10 nM to 1 pM) for 2 h prior to the detection of CCR5. The cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson).

RESULTS

Dichotomous sensitivity to PSC-RANTES in multiple- and single-cycle assays.

PSC-RANTES and other chemokine analogues have been shown to inhibit HIV-1 by downregulating CCR5 from the cell surface (8). This mechanism suggests that, when comparing a variety of HIV-1 isolates, all viruses should be inhibited to a similar degree by the same concentration of PSC-RANTES. In contrast to this hypothesis, we have observed a >30-fold difference in sensitivity to inhibition by AOP-RANTES against a panel (n = 14) of diverse primary HIV-1 isolates (16) and a 10-fold variation in sensitivity to PSC-RANTES inhibition (Fig. 1A). Polymorphisms in the V3 region, specifically at positions 318 and 319 in V3 (HXB2 numbering) can alter the sensitivity of the virus by as much as 50-fold to RANTES derivatives (17). We generated two viruses—NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT)—that are isogenic except for two amino acid polymorphisms at positions 318 and 319 in the V3 crown (17). NL4-3-V3A1-92RW009(YA) was less sensitive to inhibition by PSC-RANTES than was NL4-3-V3A1-92RW009(RT) by a factor of 10 in multiple-cycle replication assays using U87.CD4.wt-CCR5 cells (Fig. 1A) (P < 0.01). Likewise, HIV-1 env chimeric viruses derived from PSC-RANTES-resistant (M584) versus sensitive (P3-4) SHIV variants demonstrated a significant 5.3-fold decreased sensitivity to PSC-RANTES inhibition (Fig. 1B) (P < 0.01) (19). Resistance to PSC-RANTES was 7-fold in the same cells expressing macaque CCR5, suggesting a receptor adaptation contributing to drug resistance (19). We also compared the sensitivity of these viruses to inhibition by PSC-RANTES using single-cycle virus replication. In contrast to the multiple-cycle assays, similar IC50s for PSC-RANTES were observed for NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) (Fig. 1A and Table 1) and P3-4 and M584 (Fig. 1B and Table 1). We postulate that the differences between multiple- and single-cycle assays may be a consequence of cell surface CCR5 downregulation by PSC-RANTES. It is important to note that we previously analyzed the sensitivity to the YA, RT, M584, and P3-4 viruses to inhibition by different RT and entry inhibitors as controls (19). Both the wild-type and the PSC-RANTES-resistant viruses showed similar sensitivities to drugs not targeting the host cell entry step in replication. Finally, all entry inhibitors and each of these viruses (in the present study) have been tested using human PBMCs in the past and to confirm resistance, as observed in the U87 cells (16, 19, 20, 23, 27). The present study is designed to carefully define drug mechanism. Variations in the virus (outside of env) via the use of primary HIV-1 isolates and in target cells of different donors (with varying expression of receptors) would likely hamper comparisons of drug mechanism.

Fig 1.

Fig 1

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PSC-RANTES and maraviroc sensitivity in multiple- and single-cycle assays. The fold difference in sensitivity to PSC-RANTES using wild-type (wt-CCR5) and M7-CCR5 variants in multiple cycles and a single cycle of NL4-3-V3A1-92RW009(YA) (dark gray, IC50 set to 1) and NL4-3-V3A1-92RW009(RT) (white) (A) and M584 (light gray, IC50 set to 1) and P3-4 (white) (B) was determined. Error bars represent standard deviations from the IC50 calculations from two independent experiments performed in triplicate. ND, IC50s not determined. *, P < 0.01 (one-tailed t test).

Table 1.

IC50s for PSC-RANTES in single- and multiple-cycle assays using U87.CD4.wt-CCR5 or U87.CD4.M7-CCR5 cells

Cell type and virus Assaya Drugb IC50 (nM)c
Mean SD
wt-CCR5 cells
    YA MC PSC 0.265 0.039
    YA SC PSC 0.028 0.0062
    YA MC ENF 219 23
    YA SC ENF 5.9 2.7
    YA MC TAK 105 17
    YA SC TAK 31 5.2
    YA MC 3TC 162 26.4
    YA SC 3TC 647 93
    YA MC NVP 17 7.5
    YA SC NVP 39 7.8
    RT MC PSC 0.024 0.0012
    RT SC PSC 0.023 0.0036
    RT MC ENF 3.3 0.019
    RT SC ENF 0.4 0.015
    RT MC TAK 2.2 0.14
    RT SC TAK 0.29 0.015
    RT MC 3TC 120 14.1
    RT SC 3TC 510 102
    RT MC NVP 20 7.8
    RT SC NVP 41 13
    P3-4 MC PSC 0.086 0.036
    P3-4 SC PSC 2.1 0.57
    P3-4 MC ENF 0.39 0.038
    P3-4 SC ENF ND ND
    P3-4 MC TAK 37 15
    P3-4 SC TAK ND ND
    M584 MC PSC 0.47 0.25
    M584 SC PSC 1.9 0.2
    M584 MC ENF 0.11 0.043
    M584 SC ENF ND ND
    M584 MC TAK 63 5.1
    M584 SC TAK ND ND
    S2 MC MVC 10 2.4
    S2 SC MVC 21 3.5
    R3 MC MVC 7400 1200
    R3 SC MVC 93 15
M7-CCR5 cells
    YA MC PSC 0.32 0.045
    YA SC PSC 4.4 0.51
    YA MC ENF 42 6.3
    YA SC ENF 18 5.5
    YA MC TAK 120 44
    YA SC TAK 45 18
    YA MC 3TC 74 12.2
    YA SC 3TC 96 17.3
    YA MC NVP 22 4.54
    YA SC NVP 27 9.4
    RT MC PSC 0.027 0.009
    RT SC PSC 0.38 0.05
    RT MC ENF 0.81 0.14
    RT SC ENF 0.24 0.036
    RT MC TAK 3.7 0.13
    RT SC TAK 0.88 0.1
    RT MC 3TC 55 2.2
    RT SC 3TC 67 6.1
    RT MC NVP 18 1.4
    RT SC NVP 31 2.9
    P3-4 MC PSC ND ND
    P3-4 SC PSC 0.6 0.35
    M584 MC PSC ND ND
    M584 SC PSC 3.1 0.14
    S2 MC MVC 0.3 0.11
    S2 SC MVC 4 1.06
    R3 MC MVC 101 27
    R3 SC MVC NA NA
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a

MC, multiple cycle; SC, single cycle.

b

PSC, PSC-RANTES [N-nonanoyl, des-Ser1[l-tioproline2, l-cyclohexylglycine3]-RANTES(2-68)]; ENF, enfuvirtide; TAK, CCR5 antagonist TAK-779; NVP, nevirapine; 3TC, lamivudine; MVC, maraviroc.

c

ND, not determined; NA, not available due to lack of inhibition at maximal drug concentration.

Resistance to MVC is typically characterized by the ability of the resistant virus to utilize a drug-bound receptor and thus reduce the maximal inhibition of the drug below 100%. Based on our findings using flow cytometry, MVC does not affect steady-state levels of CCR5 on the surface (data not show). The sensitivity of HIV-1 to MVC was determined under multiple- and single-cycle assay conditions using MVC-resistant and MVC-sensitive virus clones (R3 and S2, respectively) (Fig. 2A and B) (23). With R3 HIV-1, the virus can replicate to a 50% level (compared to the absence of drug) with MVC at a 10 μM concentration, which is typically >IC99 concentration for wild-type virus (Fig. 2A and B). The MPI of the maraviroc-resistant virus, R3, was compared to maximal inhibition observed for the sensitive virus, S2, in the single- and multiple-cycle assays (Fig. 2C). A similar level of maximal inhibition was observed for virus R3 under multiple- and single-cycle conditions (61 and 56%, respectively), indicating that inhibition by MVC was the same under both assay conditions.

Fig 2.

Fig 2

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Sensitivity of the S2 and R3 HIV-1 strains to maraviroc in single-cycle (A) and multiple-cycle (B) drug susceptibility assays using U87.CD4.M7-CCR5 cells and U87.CD4.wt-CCR5 cells. Percent inhibition curves for PSC-RANTES (A) and MVC (B) were generated using data collected on day 6 postinfection. The data represent the means of triplicate values from three independent experiments. Error bars were removed to decrease complexity, but the standard deviations fell within 10% of the average values. (C) Maximal percent inhibition for maraviroc using wt-CCR5 and M7-CCR5 variants in multiple cycles and a single cycle of S2 (white) and R3 (black). Error bars represent the standard deviations of values from triplicates. *, P < 0.01 (one-tailed t test).

Finally, we have previously tested resistance to various nucleoside and non-nucleoside RT inhibitors in both single- and multiple-cycle conditions. The same fold difference in drug susceptibility was observed with zidovudine, 3TC, NVP, and efavirenz resistance versus wild-type virus to the respective drugs in both single- and multiple-cycle assays (16, 17, 28, 29). In summary, the inability to observe drug resistance in a single cycle versus multiple-cycle assay appears limited to PSC-RANTES.

Inhibition of HIV-1 replication with a downregulation-defective mutant of CCR5.

Endocytosis of CCR5 occurs through phosphorylation of its C-terminal domain by G protein-coupled receptor kinases (GRKs) and recognition of the phosphorylated receptor by β-arrestin (18). M7-CCR5 cells express a mutant CCR5 with an alanine replacing the serine phosphorylation site on the C-terminal domain. This surface-expressed M7-CCR5 cannot be downregulated upon binding to any CCR5 ligand or derivative such as PSC-RANTES (18). However, M7-CCR5 still acts as coreceptor for HIV-1 entry.

To assess the inhibitory activity of PSC-RANTES in the absence of receptor downregulation, we generated stable U87.CD4.M7-CCR5 cells. Flow cytometry analyses revealed these cells expressed similar levels of CCR5 as U87.CD4.wt-CCR5 cells and supported HIV-1 replication, albeit to slightly reduced levels compared to the wild-type U87.CD4.wt-CCR5 cells (data not shown). Using two specific monoclonal antibodies to CCR5, clone 2D7 and clone 3A9, we measured the effect of PSC-RANTES downregulation of wild-type and M7-CCR5 cells (Fig. 3). Clone 2D7 recognizes an epitope in the second extracellular loop of CCR5, a region reported to overlap the PSC-RANTES binding site (5). Alternatively, the epitope for clone 3A9 is found on the N terminus of CCR5, and binding of this antibody is not inhibited by PSC-RANTES (5). Staining with clone 3A9 in the presence of PSC-RANTES indicated efficient downregulation of wild-type CCR5 but not the M7-CCR5 receptor (Fig. 3B). In contrast, binding of clone 2D7 was inhibited by PSC-RANTES in both the wild-type and the M7-CCR5 cells, suggesting that PSC-RANTES can efficiently bind M7-CCR5 (Fig. 3A). We assessed PSC-RANTES inhibition in U87.CD4.M7-CCR5 cells in both multiple- and single-cycle assays and observed a 10-fold difference between NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) under both conditions (Fig. 1A). This difference in sensitivity contrasts with our previous observation in U87.CD4.wt-CCR5 cells in a single cycle where no difference in sensitivity to PSC-RANTES was detected for these viruses.

Fig 3.

Fig 3

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Characterization of U87.CD4.M7-CCR5 cells. (A) Cell surface CCR5 was detected by CCR5 monoclonal antibody (MAb) 2D7 after 2 h of treatment with 10-fold dilutions of PSC-RANTES. (B) The cell surface CCR5 was detected by CCR5 MAb 3A9 after 2 h of treatment with 10-fold dilutions of PSC-RANTES. The data represent the means of three independent experiments with the standard deviations. *, P < 0.01 (one-tailed t test).

The IC50s obtained in U87.CD4.M7-CCR5 cells after multiple cycles of replication did not differ significantly from those PSC-RANTES IC50s derived from U87.CD4.wt-CCR5 cells in multiple-cycle assays (Table 1). This observation suggests that PSC-RANTES can potently inhibit HIV-1 replication in the absence of receptor downregulation. We performed an additional single-cycle assay in U87.CD4.M7-CCR5 cells, this time using viruses P3-4 (sensitive) and M584 (resistant). As with the YA and RT viruses, we observed a difference in sensitivity (5.1-fold) to PSC-RANTES in a single cycle using the M7-CCR5 mutant, whereas no difference had been detected using wild-type CCR5 (Fig. 1B). By comparison, inhibition by maraviroc in U87.CD4.M7-CCR5 cells using viruses S2 (sensitive) and R3 (resistant) recapitulated results observed using U87 cells expressing wild-type CCR5 in both multiple- and single-cycle conditions, suggesting the mutations in CCR5 that eliminated receptor internalization did not significantly impact inhibition by MVC (Fig. 2C).

To further confirm that differences in viral sensitivity were specific to PSC-RANTES inhibition in multiple- and single-cycle assays, we performed drug sensitivity assays under both conditions with the fusion inhibitor ENF, the CCR5 antagonist TAK-779 and the reverse transcriptase inhibitors 3TC and NVP (Fig. 4). As previously described, NL4-3-V3A1-92RW009(YA) shows decreased sensitivity to ENF and TAK-779, as well as PSC-RANTES in multiple-cycle assays. Decreased sensitivity to ENF and TAK-779 was also observed with the YA compared to the RT virus in single-cycle assays. In contrast, these two viruses showed similar sensitivity to PSC-RANTES in single-cycle conditions. The RT and YA viruses displayed the same sensitivity to 3TC and NVP in either multiple- or single-cycle assays.

Fig 4.

Fig 4

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Sensitivity of V3 chimeric viruses to antiretrovirals in single- and multiple-replication-cycle assays. Drug sensitivity assays were performed in U87.CD4.wt-CCR5 cells under single- or multiple-cycle conditions using V3 chimeric viruses NL4-3-V3A1-92RW009(YA) (dark gray, IC50 set to 1) and NL4-3-V3A1-92RW009(RT) (light gray) for the entry inhibitors PSC-RANTES, enfuvirtide (ENF), and TAK-779 and the reverse transcriptase inhibitors 3TC and nevirapine (NVP). The data represent the means of three independent experiments with the standard deviations. *, P < 0.01 (one-tailed t test).

Finally, it should be noted that all of the latter single-cycle assays involved using a Env-pseudotyped Luc-in cis virus. Virus for these assays was produced from 293T cells cotransfected where the Env was expressed from pREC_env vectors (see Materials and Methods) cotransfected with pNL.Luc.AM which expresses the HIV-1 proteome except for Env, which is substituted with firefly luciferase (flLUC). Upon de novo infection, the proviral DNA integrates into the genome and expresses flLUC. To ensure that the system was not affecting the differential effects of PSC-RANTES, we repeated the drug susceptibility studies under single-cycle conditions with the Env pseudotyped virus (lacking in cis Luc) and with replication-competent chimeric virus containing the YAA1-92RW009 or RTA1-92RW009 Env V3 loop (i.e., same configuration of Env as in the pseudotyped virus). To detect single-cycle infection, the target U87.CD4.wt-CCR5 cells were transiently transfected with the pDM128-LTR-fluc2 construct, which expresses flLUC in trans under the control of both HIV-1 Tat and Rev derived from the infecting virus. A protease inhibitor (1 μM saquinavir) was added to prevent reinfection of the U87 cell after a single cycle with the chimeric virus. Again, similar sensitivity to PSC-RANTES was observed using these different single-cycle systems with different virus configuration and expression of flLUC in trans (see Fig. S1 in the supplemental material).

Prolonged incubation with PSC-RANTES recapitulates multiple-cycle assays.

Dissimilar inhibition by PSC-RANTES in the multiple- and single-cycle assays suggests an alternative mechanism at play with prolonged incubations in tissue culture. As shown in Fig. 5A, a 2-h incubation of U87.CD4.wt-CCR5 cells with 10 nM PSC-RANTES removes >95% of CCR5 from the cell surface as detected with the 3A9 antibody. However, after a 5-day exposure to 10 nM PSC-RANTES, there was only 20% reduction of CCR5 from the same cells (Fig. 5A). Previous studies have shown that CCR5 returns to the cell surface after 4 to 6 days of PSC-RANTES exposure (8). Thus, we hypothesized that the differential inhibition to PSC-RANTES observed in multiple-cycle assays was a result of competitive binding for CCR5 between the inhibitor and virus particle rather than receptor downregulation. Verification of this hypothesis requires the eventual abrogation of CCR5 downregulation by PSC-RANTES with prolonged incubation. To this end, we performed single-cycle assays after a 5-day incubation of target U87.CD4.wt-CCR5 cells with PSC-RANTES (Fig. 5D). Luciferase was measured 48 h postinfection. When single-cycle assays were performed after a 5-day PSC-RANTES incubation, a 10-fold variation in sensitivity to PSC-RANTES was observed between NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) (Fig. 5D), similar to the multiple-cycle assay results (Fig. 5B). In contrast, there was considerable inhibition but no difference in PSC-RANTES sensitivity between the YA and RT virus in a single-cycle assay performed after 1 h of PSC-RANTES exposure (Fig. 5C).

Fig 5.

Fig 5

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Prolonged incubation of cells with PSC-RANTES. The inhibition of NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) by PSC-RANTES was measured in single-cycle assays after prolonged incubation of cells with PSC-RANTES. (A) Flow cytometry analyses of CCR5 surface expression was performed on U87.CD4.wt-CCR5 cells after 2 h and 5 days of exposure to 10, 1, and 0.1 nM PSC-RANTES. (B) Multiple-cycle assays allow for ∼4 cycles of replication over a 6-day period, and virus was quantified based on the RT activity in the supernatant. (C) Single-cycle assays were completed in 48 h, and virus infection was measured by luciferase activity. (D) U87.CD4.wt-CCR5 cells were exposed to 10-fold dilutions of PSC-RANTES for 5 days. On day 5, the cells were infected, and the luciferase activity was measured after 48 h. (E) Parallel cultures were plated in which culture A was exposed to 10-fold dilutions of PSC-RANTES for 5 days, as in panel D, whereas culture B was left untreated. After 5 days, the supernatant of culture A was transferred to the cells of culture B. The cells of culture A were then replenished with fresh media containing the appropriate concentrations of PSC-RANTES. Both cultures were incubated for 1 h and then infected with virus. The luciferase activity was measured after 48 h. Plots indicate the fold difference in IC50 between NL4-3-V3A1-92RW009(YA) (gray bars, set to 1) and NL4-3-V3A1-92RW009(RT) (white bars). Error bars indicate the standard deviations of the IC50, as measured from two independent experiments performed in triplicate. *, P < 0.01 (one-tailed t test).

Using parallel cultures of U87.CD4.wt-CCR5.Luc cells, a second prolonged incubation experiment was performed to control for drug decay over time (Fig. 5E). In one culture, cells were incubated with 10-fold dilutions of PSC-RANTES for 5 days while a parallel culture was incubated in media alone. After 5 days, the media from cells incubated with 10-fold serial dilutions with PSC-RANTES was transferred to the mock-treated cells. The cells preincubated with PSC-RANTES were provided media containing fresh PSC-RANTES at the appropriate concentrations. All cultures were incubated for 1 h and then infected with virus for single-cycle assays. We observed differential sensitivity to PSC-RANTES between NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) using single-cycle conditions in the cultures exposed to PSC-RANTES for 5 days, washed, and then provided with fresh PSC-RANTES (Fig. 5E). However, no difference in PSC-RANTES sensitivity was observed between the viruses in the cultures that received 5-day-old PSC-RANTES for 1 h prior to infection. The potency of the 5-day-old PSC-RANTES was reduced by 10-fold compared to fresh PSC-RANTES; however, it should be noted that the remaining PSC-RANTES could still efficiently downregulate the CCR5 receptor (data not shown) and thereby inhibit NL4-3-V3A1-92RW009(YA) and NL4-3-V3A1-92RW009(RT) during a single cycle of replication. A similar experimental protocol was performed using the MVC-resistant R3 and S2 HIV-1. In this experiment, resistance to MVC was observed at both 1 h or 5 days of incubation with MVC or when MVC was transferred to new U87.CD4.wt-CCR5.Luc cells. Again, MVC potency was reduced ∼5-fold with the 5-day incubation (data not shown).

PSC-RANTES Inhibits HIV-1 entry by competitive CCR5 binding.

Similar levels of HIV-1 inhibition in PSC-RANTES-treated M7-CCR5 and wt-CCR5 U87.CD4 cells in multiple-cycle assay suggests that majority inhibition by this drug is mediated by competitive mechanism rather than receptor downregulation. With competitive inhibition, the inhibitory potential of the drug (e.g., PSC-RANTES) is directly related to the competing substrate concentration, in this case, the virus bound to CD4 and primed for coreceptor interaction. This virus concentration can be manipulated by adjusting the initial multiplicity of infection (Fig. 6). We assessed sensitivity of the NL4-3-V3A1-92RW009(YA) virus to PSC-RANTES and maraviroc (IC50s; y axes, Fig. 6A and B, respectively) in U87.CD4.M7-CCR5 cells at increasing multiplicity of infections (x axes) and at each day postinfection (z axes). With each 5-fold increase in multiplicity of infection, the level of PSC-RANTES and MVC inhibition (IC50s) was reduced (Fig. 6A and B). Likewise, virus production in this multiple-cycle assay also increased virus “substrate” over time, which reduced IC50s. Thus, in the absence of receptor downregulation, increased IC50 MVC and PSC-RANTES values correlated with increasing concentration of input or produced virus, i.e., competing substrate for CCR5 (Fig. 6C and D). As discussed below, the IC50s or relative sensitivity of CCR5-tropic HIV-1 to MVC and PSC-RANTES inhibition is highly dependent upon titer of the virus and on the sampling day. With both drugs, this variable sensitivity based on substrate concentrations (or competitive inhibition) was unexpected based on previous literature, suggesting that RANTES derivatives inhibited via receptor downregulation (8) and that MVC inhibited via a noncompetitive, allosteric mechanism (2, 3, 30).

Fig 6.

Fig 6

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Effect of MOI and virus expansion on PSC-RANTES and maraviroc IC50 values. The relative IC50 of virus NL4-3-V3A1-92RW009(YA) to PSC-RANTES (A) and MVC (B) was measured by reverse transcriptase assay in multiple-cycle drug susceptibility assays using U87.CD4.M7-CCR5 cells. IC50 values were assessed using different inocula of virus referred to as MOIs (IU/cell) (x axis) and on multiple days postinfection (z axis). Percent inhibition curves for PSC-RANTES (C) and MVC (D) were generated using data collected on day 6 postinfection for the MOIs indicated. The data represent the means of three independent experiments. Error bars were removed to decrease complexity, but the standard deviations fell within 10% of the average values.

We also evaluated the effect of input virus concentration on PSC-RANTES inhibition in the context of wild-type and M7 CCR5 in multiple- and single-cycle assays. In single-cycle assays using wild-type CCR5, the IC50s for PSC-RANTES were identical at all MOIs, which again indicates that inhibition within the first ∼48 h is largely dependent on receptor downregulation by PSC-RANTES (Fig. 7B). Competitive inhibition was evident with PSC-RANTES in the M7-CCR5 cells under single- and multiple-cycle conditions. In other words, increasing the amount of input virus decreased inhibition by PSC-RANTES (Fig. 7C and D).

Fig 7.

Fig 7

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Effect of MOI on inhibition by PSC-RANTES in the absence or presence of receptor downregulation. Inhibition by PSC-RANTES was assessed in multiple (A and C)- and single (B and D)-cycle assays using either wild-type CCR5 (A and B) or M7-CCR5 (C and D) cells. Cultures were infected with various levels (MOIs) of virus NL4-3-V3A1-92RW009(YA), and the percent inhibition was calculated based on the no-drug condition. The data represent the means of mean of three independent experiments. Error bars were removed to decrease the complexity; however, the standard deviations fell within 10% of the average values.

DISCUSSION

In this study, we addressed the mechanism(s) of HIV-1 inhibition by CCR5 entry inhibitors. In addition to triggering receptor internalization, the partial CCR5 agonist PSC-RANTES appears to compete with HIV-1 envelope for binding to CCR5. Diverse HIV-1 isolates display variable sensitivity to inhibition by RANTES derivatives, which provided the first evidence for CCR5 receptor competition as an inhibitory mechanism. To date, most studies on HIV-1 inhibition by CCR5 agonists have focused on the relative rate of CCR5 internalization and prolonged receptor retention in the cell (8, 10, 11, 31). In the present study, we compared the inhibitory effects mediated by steric occupancy of CCR5 versus receptor downregulation by PSC-RANTES. Our findings indicate that PSC-RANTES inhibition primarily involves a competitive process. For comparison to these agonist activities, we also examined the inhibitory mechanism of the CCR5 antagonist, maraviroc and assumed that inhibition of HIV-1 was mediated by allosteric effects upon CCR5 binding. Altered conformations of MVC-bound CCR5 can prevent HIV-1 from utilizing the coreceptor for host cell entry. In addition to of these allosteric effects, MVC can also compete with HIV-1 for CCR5 binding. Increases in virus (substrate) concentration reduced the inhibitory activity of both PSC-RANTES and MVC, as observed with classical competitive inhibition mechanisms.

CCR5 antagonists (e.g., maraviroc, vicriviroc, and TAK-779) bind to CCR5 transmembrane helices (1–3, 32) but do not necessarily occlude the HIV-1 envelope glycoprotein binding site. Resistant HIV-1 isolates are able to enter host cells via a drug-bound form of CCR5 and are capable of replicating at even the highest achievable MVC concentrations (4, 23, 33, 34). We have recently characterized an MVC-resistant virus that displays an increase in IC50 to MVC and has a minimal MPI phenotype (20), whereas the MPI phenotype has been the primary mechanism of MVC resistance with R5 HIV-1 in vitro (5). The results presented here suggest that aside from the allosteric mechanism of inhibition, MVC and R5 HIV-1 may also compete with CCR5 binding. Increasing concentrations of R5 HIV-1 (as a substrate) diminished MVC inhibition. Thus, the shift in IC50s observed with our MVC-resistant R5 variant is consistent with competitive mechanisms of MVC inhibition, in addition to allosteric inhibition. These findings also suggest that there may be shifts in MVC inhibitory mechanisms during MVC dosing in patients. During early treatment with MVC and low drug trough levels in patients with initially high viral loads, higher virus concentrations may compete with lower MVC levels for CCR5 binding. In contrast, during adequate control of viremia and prolonged MVC treatment, higher and stable MVC levels may be binding to CCR5, causing a conformational change, and thus inhibit binding by any residual HIV-1 via an allosteric mechanism.

R5 HIV-1 and PSC-RANTES appear to bind overlapping sites on CCR5. Binding of PSC-RANTES (or the native CCL5) to CCR5 is thought to involve initial interaction with the N terminus and then engagement with the second extracellular loop, which promotes signaling through a G protein-coupled receptor and CCR5 internalization (35–37). In the normal chemotaxis process, a cell bearing a chemokine receptor migrates toward a gradient of increasing chemokine concentrations but eventually stops. This directional migration appears to be mediated by cellular polarity in the signal transduction machinery associated with chemokine receptor signaling instead of polarity in the presence of CCR5 in the cellular membrane. The stop in migration appears to be associated with an exhaustion of the signal transduction machinery and, thus, the receptor becomes desensitized to ligand-induced internalization and signaling. Since PSC-RANTES and other RANTES agonists still induce Ca2+ mobilization, CCR5 signaling, and receptor internalization, we propose that the same CCR5 desensitization occurs with high concentrations of PSC-RANTES, as observed with the native RANTES. In other words, PSC-RANTES will initially bind and induce CCR5 internalization and/or signaling, but the cell will become desensitized to the downstream effects of PSC-RANTES binding to CCR5 on the cell surface.

In terms of HIV-1 inhibition, PSC-RANTES initially blocks HIV-1 entry by inducing internalization of the cell surface receptor but, with the return of CCR5 to the plasma membrane, inhibition is mediated by a competitive inhibition mechanism. This hypothesis was tested in experiments where cells were incubated for 5 days with PSC-RANTES prior to the addition of virus for a single-cycle assay. Most of the CCR5 receptor had returned to the cell surface in this assay (Fig. 5A), and yet the virus was still sensitive to PSC-RANTES inhibition. With multiple cycles of replication and virus propagation, PSC-RANTES primarily binds to a desensitized CCR5, and thus inhibition is mediated by competitive inhibition. If PSC-RANTES was used as a therapeutic agent, a trough drug concentration in plasma would be established to maintain HIV inhibition. With persistent, systemic drug administration, the target cells would likely resemble the >5-day scenario of Fig. 5, where the CCR5 receptor would be desensitized to internalization and/or signal transduction. Thus, inhibition of HIV-1 by PSC-RANTES would likely be mediated by competitive binding to CCR5 rather than the absence of receptor due to an initial CCR5 internalization. In the microbicide model, the addition of PSC-RANTES to the vaginal tract may act with effects similar to those of native RANTES during a tissue injury or infection, where the release of chemokines induces inflammation and leukocyte/monocyte infiltration. The cells that migrate to the mucosal tissue are desensitized to CCR5 internalization and ligand-mediated signal transduction. With leukocyte migration into the RANTES gradient (or possibly, the PSC-RANTES gradient), cells are known to stop at the site of inflammation due to desensitization by the high chemokine concentrations (35–37).

With only receptor downregulation as an inhibitory mechanism, all R5 HIV-1 isolates should have displayed similar sensitivities to PSC-RANTES inhibition, but this was clearly not the case in testing various RANTES derivatives with human PBMCs (17). Decreasing sensitivity to PSC-RANTES inhibition was a strong direct correlate of both entry efficiency and replicative fitness of the R5 HIV-1 isolates (17). These findings suggest a coevolution of several HIV-1 regions in gp120/gp41 that are involved in the host cell entry process. The efficiency of CCR5 engagement, mediated in part by the V3 loop of HIV-1 Env, appears to be the rate-limiting step in host cell entry. Our previous studies identified two natural polymorphisms in the V3 loop of the envelope glycoprotein that conferred variable sensitivity to PSC-RANTES and correlated with differential entry efficiency, as well as replicative fitness (17, 38). One virus clone with R318/T319 in the V3 loop compared to its isogenic match with Y318/A319 was 10-fold more sensitive to PSC-RANTES inhibition in multiple-cycle assays but did not exhibit this difference in single-cycle assays (Fig. 1A). We confirmed that PSC-RANTES resistance was only evident in multiple-cycle assays but not in single-cycle assays using another PSC-RANTES-resistant virus derived from macaques. Previous studies had identified a PSC-RANTES-resistant virus circulating in macaques that had been vaginally treated with a high dose of PSC-RANTES and then exposed vaginally to SHIVSF162-P3 (19). The macaque-derived env gene from an infected macaque was cloned into HIV and shown to contain a K315R mutation in the V3 loop and a N640D mutation in the HR2 domain of gp41. These mutations conferred both resistance to PSC-RANTES and adaptation to rhesus macaque CCR5 usage. Our initial studies involved the more physiological multiple-cycle/propagation assays with human primary T cells, followed by studies with the U87.CD4 cell line expressing human or rhesus CCR5. A subsequent study performed single-cycle assays using an Env pseudotyped construct with the same K315R and N640D mutations did not detect PSC-RANTES resistance (39). In the present study, we have confirmed those findings in that the K315R and N640D mutations in the SF162 env do not confer resistance in single-cycle assays.

A lack of PSC-RANTES resistance was observed in single-cycle assay conditions with the SF162 virus harboring K315R and N640D mutations (39). However, PSC-RANTES resistance was observed in multiple-cycle assays (19). In the present study, we show that PSC-RANTES downregulates or “strips” the CCR5 receptor from the cell surface within the first 24 to 48 h of treatment, i.e., generally the time required for single-cycle, pseudotyped-virus assays. However, when the CCR5 receptor recycles to the cell surface, it appears to be desensitized to further receptor internalization despite high-affinity interaction with PSC-RANTES. With CCR5 mutants (e.g., M7) that are recalcitrant to internalization upon binding to RANTES, the virus competes with the RANTES derivative for CCR5 binding immediately upon drug addition. Thus, PSC-RANTES resistance was evident in single-cycle assays using M7-CCR5 cells with the K315R/N640D virus and with the Y318/A319 virus. Again, all of these viruses displayed equal sensitivity to PSC-RANTES when we used cells expressing the wild-type CCR5 in single-cycle conditions (due to receptor internalization).

In summary, MVC and PSC-RANTES can inhibit R5 HIV-1 replication via competition between the virus and drug for CCR5 receptor. The primary mechanism of MVC inhibition appears to be allosteric, which is further confirmed by the use of an MVC-bound form of CCR5 by most MVC-resistant viruses. However, increasing virus concentrations in culture appear to outcompete MVC for CCR5 binding and reduce the effect of allosteric inhibition. For PSC-RANTES, inhibition via CCR5 internalization is restricted to the first 1 or 2 days of infection or, in other words, the time required for one replication cycle. In the case of PSC-RANTES treatment (e.g., microbicide use), the drug may have already desensitized the receptor prior to the arrival of virus to the susceptible cell. Nonetheless, a subnanomolar potency of this drug suggests a higher binding affinity of PSC-RANTES compared to most HIV-1 isolates for CCR5. Thus, it is not surprising that intrinsic PSC-RANTES resistance of HIV-1 correlates to higher CCR5 affinity, greater entry efficiency, and increased replicative fitness than PSC-RANTES-sensitive virus. Finally, increased replicative fitness was also observed in the MVC-resistant virus that displayed a shift in the MVC IC50s rather than just an MPI effect. In contrast, MVC-resistant HIV-1 with an MPI effect demonstrated a reduced entry efficiency, suggesting that a fitness cost was associated with the promiscuous use of both the unbound and the MVC-bound forms of the CCR5 receptor. We are currently testing the binding affinity/avidity of PSC-RANTES and HIV-1 Env to CCR5 using various biochemical approaches that involve fluorescently tagged virus or drug and the labeling of CCR5 on the cell surface.

Supplementary Material

Supplemental material
supp_57_6_2640__index.html (805B, html)

ACKNOWLEDGMENTS

This study was supported by National Institutes of Health grants AI436402, AI49170, and AI46283. All virus work was performed in the biosafety level 2 and 3 facilities of the Case Western Reserve/University Hospitals Center for AIDS Research (AI36219).

We acknowledge the AIDS Research and Reference Reagent Program for cell lines and supplies of TAK-779. We also appreciate the supplies of PSC-RANTES from Oliver Hartley, and we thank Jonathan Karn for the equipment for the luciferase measurements.

Footnotes

Published ahead of print 25 March 2013

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.02511-12.

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