In Vitro Selection of Clinically Relevant Bevirimat Resistance Mutations Revealed by “Deep” Sequencing of Serially Passaged, Quasispecies-Containing Recombinant HIV-1

Abstract

Initial in vitro studies of bevirimat resistance failed to observe mutations in the clinically significant QVT motif in SP1 of HIV-1 gag . This study presents a novel screening method involving mixed, clinically derived gag-protease recombinant HIV-1 samples to more accurately mimic the selection of resistance seen in vivo . Bevirimat resistance was investigated via population-based sequencing performed with a large, initially antiretroviral-naïve cohort before ( n = 805) and after ( n = 355) standard HIV therapy (without bevirimat). The prevalence of any polymorphism in the motif comprising Q, V, and T was ∼6%, 29%, and 12%, respectively, and did not change appreciably over the course of therapy. From these samples, three groups of 10 samples whose bulk sequences were wild type at the QVT motif were used to generate gag-protease recombinant viruses that captured the existing diversity. Groups were mixed and passaged with various bevirimat concentrations for 9 weeks. gag variations were assessed by amplicon-based “deep” sequencing using a GS FLX sequencer (Roche). Unscreened mutations were present in all groups, and a V370A minority not originally detected by bulk sequencing was present in one group. V370A, occurring together with another preexisting, unscreened resistance mutation, was selected in all groups in the presence of a bevirimat concentration above 0.1 μM. For the two groups with V370A levels below consistent detectability by deep sequencing, the initial selection of V370A required 3 to 4 weeks of exposure to a narrow range of bevirimat concentrations, whereas for the group with the V370A minority, selection occurred immediately. This approach provides quasispecies diversity that facilitates the selection of mutations observed in clinical trials and, coupled with deep sequencing, could represent an efficient in vitro screening method for detecting resistance mutations.