Two HIV-1 variants resistant to small molecule CCR5 inhibitors differ in how they use CCR5 for entry.
ABSTRACT: HIV-1 variants resistant to small molecule CCR5 inhibitors recognize the inhibitor-CCR5 complex, while also interacting with free CCR5. The most common genetic route to resistance involves sequence changes in the gp120 V3 region, a pathway followed when the primary isolate CC1/85 was cultured with the AD101 inhibitor in vitro, creating the CC101.19 resistant variant. However, the D1/86.16 escape mutant contains no V3 changes but has three substitutions in the gp41 fusion peptide. By using CCR5 point-mutants and gp120-targeting agents, we have investigated how infectious clonal viruses derived from the parental and both resistant isolates interact with CCR5. We conclude that the V3 sequence changes in CC101.19 cl.7 create a virus with an increased dependency on interactions with the CCR5 N-terminus. Elements of the CCR5 binding site associated with the V3 region and the CD4-induced (CD4i) epitope cluster in the gp120 bridging sheet are more exposed on the native Env complex of CC101.19 cl.7, which is sensitive to neutralization via these epitopes. However, D1/86.16 cl.23 does not have an increased dependency on the CCR5 N-terminus, and its CCR5 binding site has not become more exposed. How this virus interacts with the inhibitor-CCR5 complex remains to be understood.
Project description:We have described previously the generation of an escape variant of human immunodeficiency virus type 1 (HIV-1), under the selection pressure of AD101, a small molecule inhibitor that binds the CCR5 coreceptor (A. Trkola, S. E. Kuhmann, J. M. Strizki, E. Maxwell, T. Ketas, T. Morgan, P. Pugach, S. X. L. Wojcik, J. Tagat, A. Palani, S. Shapiro, J. W. Clader, S. McCombie, G. R. Reyes, B. M. Baroudy, and J. P. Moore, Proc. Natl. Acad. Sci. USA 99:395-400, 2002). The escape mutant, CC101.19, continued to use CCR5 for entry, but it was at least 20,000-fold more resistant to AD101 than the parental virus, CC1/85. We have now cloned the env genes from the the parental and escape mutant isolates and made chimeric infectious molecular clones that fully recapitulate the phenotypes of the corresponding isolates. Sequence analysis of the evolution of the escape mutants suggested that the most relevant changes were likely to be in the V3 loop of the gp120 glycoprotein. We therefore made a series of mutant viruses and found that full AD101 resistance was conferred by four amino acid changes in V3. Each change individually caused partial resistance when they were introduced into the V3 loop of a CC1/85 clone, but their impact was dependent on the gp120 context in which they were made. We assume that these amino acid changes alter how the HIV-1 Env complex interacts with CCR5. Perhaps unexpectedly, given the complete dependence of the escape mutant on CCR5 for entry, monomeric gp120 proteins expressed from clones of the fully resistant isolate failed to bind to CCR5 on the surface of L1.2-CCR5 cells under conditions where gp120 proteins from the parental virus and a partially AD101-resistant virus bound strongly. Hence, the full impact of the V3 substitutions may only be apparent at the level of the native Env complex.
Project description:Fitness is a parameter used to quantify how well an organism adapts to its environment; in the present study, fitness is a measure of how well strains of human immunodeficiency virus type 1 (HIV-1) replicate in tissue culture. When HIV-1 develops resistance in vitro or in vivo to antiretroviral drugs such as reverse transcriptase or protease inhibitors, its fitness is often impaired. Here, we have investigated whether the development of resistance in vitro to a small molecule CCR5 inhibitor, AD101, has an associated fitness cost. To do this, we developed a growth-competition assay involving dual infections with molecularly cloned viruses that are essentially isogenic outside the env genes under study. Real-time TaqMan quantitative PCR (QPCR) was used to quantify each competing virus individually via probes specific to different, phenotypically silent target sequences engineered within their vif genes. Head-to-head competition assays of env clones derived from the AD101 escape mutant isolate, the inhibitor-sensitive parental virus, and a passage control virus showed that AD101 resistance was not associated with a fitness loss. This observation is consistent with the retention of the resistant phenotype when the escape mutant was cultured for a total of 20 passages in the absence of the selecting compound. Amino acid substitutions in the V3 region of gp120 that confer complete AD101 resistance cause a fitness loss when introduced into an AD101-sensitive, parental clone; however, in the resistant isolate, changes elsewhere in env that occurred prior to the substitutions within V3 appear to compensate for the adverse effect of the V3 changes on replicative capacity. These in vitro studies may have implications for the development and management of resistance to other CCR5 inhibitors that are being evaluated clinically for the treatment of HIV-1 infection.
Project description:Resistance to small-molecule CCR5 inhibitors arises when HIV-1 variants acquire the ability to use inhibitor-bound CCR5 while still recognizing free CCR5. Two isolates, CC101.19 and D1/85.16, became resistant via four substitutions in the gp120 V3 region and three in the gp41 fusion peptide (FP), respectively. The binding characteristics of a panel of monoclonal antibodies (MAbs) imply that several antigenic forms of CCR5 are expressed at different levels on the surfaces of U87-CD4-CCR5 cells and primary CD4(+) T cells, in a cell-type-dependent manner. CCR5 binding and HIV-1 infection inhibition experiments suggest that the two CCR5 inhibitor-resistant viruses altered their interactions with CCR5 in different ways. As a result, both mutants became generally more sensitive to inhibition by CCR5 MAbs, and the FP mutant is specifically sensitive to a MAb that stains discrete cell surface clusters of CCR5 that may correspond to lipid rafts. We conclude that some MAbs detect different antigenic forms of CCR5 and that inhibitor-sensitive and -resistant viruses can use these CCR5 forms differently for entry in the presence or absence of CCR5 inhibitors.
Project description:Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.
Project description:Maraviroc (MVC) is an allosteric CCR5 inhibitor used against HIV-1 infection. While MVC-resistant viruses have been identified in patients, it still remains incompletely known how they adjust their CD4 and CCR5 binding properties to resist MVC inhibition while preserving their replicative capacity. It is thought that they maintain high efficiency of receptor binding. To date however, information about the binding affinities to receptors for inhibitor-resistant HIV-1 remains limited.Here, we show by means of viral envelope (gp120) binding experiments and virus-cell fusion kinetics that a MVC-resistant virus (MVC-Res) that had emerged as a dominant viral quasispecies in a patient displays reduced affinities for CD4 and CCR5 either free or bound to MVC, as compared to its MVC-sensitive counterpart isolated before MVC therapy. An alanine insertion within the GPG motif (G310_P311insA) of the MVC-resistant gp120 V3 loop is responsible for the decreased CCR5 binding affinity, while impaired binding to CD4 is due to sequence changes outside V3. Molecular dynamics simulations of gp120 binding to CCR5 further emphasize that the Ala insertion alters the structure of the V3 tip and weakens interaction with CCR5 ECL2. Paradoxically, infection experiments on cells expressing high levels of CCR5 also showed that Ala allows MVC-Res to use CCR5 efficiently, thereby improving viral fusion and replication efficiencies. Actually, although we found that the V3 loop of MVC-Res is required for high levels of MVC resistance, other regions outside V3 are sufficient to confer a moderate level of resistance. These sequence changes outside V3, however, come with a replication cost, which is compensated for by the Ala insertion in V3.These results indicate that changes in the V3 loop of MVC-resistant viruses can augment the efficiency of CCR5-dependent steps of viral entry other than gp120 binding, thereby compensating for their decreased affinity for entry receptors and improving their fusion and replication efficiencies. This study thus sheds light on unsuspected mechanisms whereby MVC-resistant HIV-1 could emerge and grow in treated patients.
Project description:The binding of protein HIV-1 gp120 to coreceptors CCR5 or CXCR4 is a key step of the HIV-1 entry to the host cell, and is predominantly mediated through the V3 loop fragment of HIV-1 gp120. In the present work, we delineate the molecular recognition of chemokine receptor CCR5 by a dual tropic HIV-1 gp120 V3 loop, using a comprehensive set of computational tools predominantly based on molecular dynamics simulations and free energy calculations. We report, what is to our knowledge, the first complete HIV-1 gp120 V3 loop : CCR5 complex structure, which includes the whole V3 loop and the N-terminus of CCR5, and exhibits exceptional agreement with previous experimental findings. The computationally derived structure sheds light into the functional role of HIV-1 gp120 V3 loop and CCR5 residues associated with the HIV-1 coreceptor activity, and provides insights into the HIV-1 coreceptor selectivity and the blocking mechanism of HIV-1 gp120 by maraviroc. By comparing the binding of the specific dual tropic HIV-1 gp120 V3 loop with CCR5 and CXCR4, we observe that the HIV-1 gp120 V3 loop residues 13-21, which include the tip, share nearly identical structural and energetic properties in complex with both coreceptors. This result paves the way for the design of dual CCR5/CXCR4 targeted peptides as novel potential anti-AIDS therapeutics.
Project description:We have investigated the mechanism of resistance of a HIV type 1 (HIV-1) R5 primary isolate, D1/85.16, to the small molecule CCR5 inhibitor, vicriviroc (VVC). Unlike other viruses resistant to this class of compound, D1/85.16 lacks sequence changes in the V3 region of the gp120 surface glycoprotein. Inspection of env sequences from D1/85.16 compared with those derived from the parental, inhibitor-sensitive virus, CC1/85, revealed a cluster of 3 conservative changes in the fusion peptide (FP) of the gp41 transmembrane glycoprotein that tracked with the resistance phenotype. Studies with engineered Env-chimeric and point-substituted viruses confirmed that these 3 FP residues were substantially responsible for VVC resistance without altering coreceptor usage, as assessed in both peripheral blood mononuclear cells and the TZM-bl cell line. VVC resistance is manifested differently in the 2 cell types, and there are assay-dependent complexities to the dose-response curves for the engineered resistant viruses. To explain them, we created a model for resistance and generated theoretical VVC inhibition curves that closely mimic the experimental data for the resistant viruses. The basis for the model is the existence of distinct forms of CCR5, with varying affinities for small molecule CCR5 inhibitors that are presumed to be present in different proportions on different cell types, and are used selectively by resistant HIV-1 variants when ligated with an inhibitor. Together, the experimental results and theoretical model may help understand how HIV-1 uses CCR5 to enter target cells under various conditions.
Project description:To study HIV-1 escape from a coreceptor antagonist, the R5 primary isolate CC1/85 was passaged in peripheral blood mononuclear cells with increasing concentrations of the CCR5-specific small molecule inhibitor, AD101. By 19 passages, an escape mutant emerged with a >20,000-fold resistance to AD101. This virus was cross-resistant to a related inhibitor, SCH-C, and partially resistant to RANTES but still sensitive to CCR5-specific mAbs. The resistant phenotype was stable; the mutant virus retained AD101 resistance during nine additional passages of culture in the absence of inhibitor. Replication of the escape mutant in peripheral blood mononuclear cells completely depended on CCR5 expression and did not occur in cells from CCR5-Delta32 homozygous individuals. The escape mutant was unable to use CXCR4 or any other tested coreceptor to enter transfected cells. Acquisition of CXCR4 use is not the dominant in vitro escape pathway for a small molecule CCR5 entry inhibitor. Instead, HIV-1 acquires the ability to use CCR5 despite the inhibitor, first by requiring lower levels of CCR5 for entry and then probably by using the drug-bound form of the receptor.
Project description:While CCR5 is the principal coreceptor used by macrophage (M)-tropic HIV-1, not all primary CCR5-using (R5) viruses enter macrophages efficiently. Here, we used functionally-diverse R5 envelope (Env) clones to characterize virus-cell interactions important for efficient CCR5-mediated macrophage entry. The magnitude of macrophage entry by Env-pseudotyped reporter viruses correlated with increased immunoreactivity of CD4-induced gp120 epitopes, increased ability to scavenge low levels of cell-surface CCR5, reduced sensitivity to the CCR5 inhibitor maraviroc, and increased dependence on specific residues in the CCR5 ECL2 region. These results are consistent with an altered and more efficient mechanism of CCR5 engagement. Structural studies revealed potential alterations within the gp120 V3 loop, the gp41 interaction sites at the gp120 C- and N-termini, and within the gp120 CD4 binding site which may directly or indirectly lead to more efficient CCR5-usage. Thus, enhanced gp120-CCR5 interactions may contribute to M-tropism of R5 HIV-1 strains through different structural mechanisms.
Project description:HIV CCR5 antagonists select for env gene mutations that enable virus entry via drug-bound coreceptor. To investigate the mechanisms responsible for viral adaptation to drug-bound coreceptor-mediated entry, we studied viral isolates from three participants who developed CCR5 antagonist resistance during treatment with vicriviroc (VCV), an investigational small-molecule CCR5 antagonist. VCV-sensitive and -resistant viruses were isolated from one HIV subtype C- and two subtype B-infected participants; VCV-resistant isolates had mutations in the V3 loop of gp120 and were cross-resistant to TAK-779, an investigational antagonist, and maraviroc (MVC). All three resistant isolates contained a 306P mutation but had variable mutations elsewhere in the V3 stem. We used a virus-cell β-lactamase (BlaM) fusion assay to determine the entry kinetics of recombinant viruses that incorporated full-length VCV-sensitive and -resistant envelopes. VCV-resistant isolates exhibited delayed entry rates in the absence of drug, relative to pretherapy VCV-sensitive isolates. The addition of drug corrected these delays. These findings were generalizable across target cell types with a range of CD4 and CCR5 surface densities and were observed when either population-derived or clonal envelopes were used to construct recombinant viruses. V3 loop mutations alone were sufficient to restore virus entry in the presence of drug, and the accumulation of V3 mutations during VCV therapy led to progressively higher rates of viral entry. We propose that the restoration of pre-CCR5 antagonist therapy HIV entry kinetics drives the selection of V3 loop mutations and may represent a common mechanism that underlies the emergence of CCR5 antagonist resistance.