A single-residue change in the HIV-1 V3 loop associated with maraviroc resistance impairs CCR5 binding affinity while increasing replicative capacity

Abstract

BackgroundMaraviroc (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.ResultsHere, 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.ConclusionThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0177-1) contains supplementary material, which is available to authorized users.

Maraviroc and reverse transcriptase inhibitors combinations as potential preexposure prophylaxis candidates

“…Transmitted founder clade C isolates, CH042, CH198, CH067, CH162 and CH164 were kindly provided by C. Ochsenbauer and J. Kappes at University of Alabama (Birmingham, Alabama, USA) [27]. The sequences encoding the MVC-sensitive (MVC-Sens), MVC-resistant (MVC-Res) and MVC-Sens with the V3 loop of MVC-Res [MVC-Sens (V3R)] Envs have been previously reported [28]. MVC-Sens and MVC-Res sequences come from the dominant circulating viruses isolated from a patient of the MOTIVATE study before and after MVC therapy, respectively [29].…”

“…The V3 loop of MVC-Res Env contains two changes (P308S and G310_P311InsAla) that confer high MVC resistance [28,30,31]. Chimeric viruses carrying the full-length envelope from MVC-Sens, MVC-Res and MVC-Sens (V3R) were generated as previously described [28]. Briefly, the three gp160 described above were digested with KspI and NotI from parental constructions and cloned into the pNL-KspI/Env/NotI vector derived from the HIV-1 proviral clone pNL4-3 to produce replication-competent viruses.…”

“…MVC-Sens and MVC-Res sequences come from the dominant circulating viruses isolated from a patient of the MOTIVATE study before and after MVC therapy, respectively [29]. The V3 loop of MVC-Res Env contains two changes (P308S and G310_P311InsAla) that confer high MVC resistance [28,30,31]. Chimeric viruses carrying the full-length envelope from MVC-Sens, MVC-Res and MVC-Sens (V3R) were generated as previously described [28].…”

“…Briefly, the three gp160 described above were digested with KspI and NotI from parental constructions and cloned into the pNL-KspI/Env/NotI vector derived from the HIV-1 proviral clone pNL4-3 to produce replication-competent viruses. The KspI and NotI restriction sites were introduced at the nucleotide positions 6214 and 8796 respectively in pNL4-3 as previously described [28]. DNA sequences of the cloned full-length Envs were confirmed by sequencing.…”

Trends in antiviral strategies

“…The mutants with amino acid substitutions in loop 9.2 Practiced and proposed strategies to confront the moving target challenge with antiviral inhibitors V3 of Env protein were selected to use CXCR4 rather than CCR5 as a coreceptor (see also Section 4.4 in Chapter 4). V3 loop mutants associated with maraviroc resistance may exhibit increase replicative capacity (Garcia-Perez et al, 2015).…”