Impact of Mutations Outside the V3 Region on Coreceptor Tropism Phenotypically Assessed in Patients Infected with HIV-1 Subtype B

Abstract

HIV coreceptor tropism (CTR) testing is a prerequisite for prescribing a coreceptor antagonist. CTR is increasingly deduced by analyzing the V3 loop sequence of gp120. We investigated the impact of mutations outside V3 on CTR as determined by the enhanced-sensitivity Trofile assay (ESTA). Paired ESTA and gp120 sequencing (population sequencing; from codon 32 of the conserved C1 to the variable V5 domains) were obtained from 60 antiretroviral treatment (ART)-naïve patients (15 with AIDS) infected with subtype B HIV-1. For gp120 sequence analysis, nucleotide mixtures were considered when the second highest electropherogram peak was >25%; sequences were translated into all possible permutations and classified as X4, dual/mixed (DM), and R5 based on coincident ESTA results. ESTA identified R5 and DM viruses in 72 and 28% of patients, respectively; no pure X4 was labeled. Forty percent of AIDS patients had R5 strains. Thirty-two positions, mostly outside V3, were significantly (P < 0.05) different between R5 and DM sequences. According to multivariate analysis, amino acid changes at 9 and 7 positions within the C1 to C4 and V1 to V5 regions, respectively, maintained a statistical significance, as did the net charge of V3 and C4. When analyzing only R5 sequences, 6 positions in the variable regions were found which, along with the V4 net charge, were significantly different for sequences from early-and end-stage disease patients. This study identifies specific amino acid changes outside V3 which contribute to CTR. Extending the analysis to include pure X4 and increasing the sample size would be desirable to define gp120 variables/changes which should be included in predictive algorithms.Based on coreceptor usage, HIV-1 isolates currently are classified as R5 tropic (using only the CCR5 receptor to enter cells), X4 tropic (using only CXCR4), and dual/mixed (DM) (able to use both coreceptor types). Maraviroc (MVC), the only currently licensed CCR5 antagonist, is ineffective in patients harboring non-R5 strains (28), therefore its prescription requires that the presence of R5-tropic virus is ascertained.Until recently, the only recommended method for tropism determination was by phenotypic assay (Trofile; Monogram Biosciences), which has been extensively used to provide tropism information in MVC clinical trials (6, 11). Trofile is a recombinant virus assay in which a pseudovirus is generated from the full-length env gene amplified from the patient's virus population and subsequently used to infect U87 cell lines expressing either the CXCR4 or CCR5 receptor on their surfaces. A new version of the test (enhanced-sensitivity Trofile assay [ESTA]) with a 0.3% sensitivity for X4 variants (24) was made available in 2008; however, the Trofile assay is not suitable for routine patient care. In fact, the assay is expensive and labor-intensive and is performed in only a single reference laboratory in the United States. Genotypic methods represent a more feasible alternative due to their greater accessibility, lower cost, and fast...

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“…Different amino acid positions were found to be significantly different in R5-tropic and X4-using viruses. Although most positions were found in the V2 region, none was similar as described in the previous study [38]. Despite these findings no significant improvement could be reached by incorporating these data into geno2pheno[coreceptor] [32,37], and the benefit of these approaches compared to the cost of sequencing additional regions seems low.…”