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HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.

Objectives: Resistance of HIV-1 to CCR5 antagonists can occur without coreceptor switching by mutations in envelope glycoproteins that enable virus entry using the inhibitor-bound form of CCR5. We investigated whether mutations in the V3 region of HIV-1 from subjects naive to maraviroc could be associated with primary resistance to this drug., Methods: The frequency of CCR5-tropic HIV-1 subtype B isolates harbouring putative V3 maraviroc resistance mutations was assessed among the HIV tropism database of Toulouse University Hospital, France. Phenotypic assessment of maraviroc susceptibility was performed for 14 isolates representative of the main mutation patterns and 14 controls. V3 mutations were reversed or introduced by site-directed mutagenesis., Results: Ninety-three of 951 (9.8%) isolates harboured V3 mutations assumed to be associated with maraviroc resistance. Maraviroc completely blocked virus entry for all but 1 of the 14 isolates harbouring V3 mutations [IC50 8.6 nM; 95% CI (6.6-47.4)], as in the 14 control isolates [IC50 13.4 nM; 95% CI (7.7-50.3)] (P = 0.24). Primary resistance to maraviroc, with a plateau in entry inhibition, was found in one isolate (harbouring a 20F/21I genotype). Site-directed mutagenesis showed that V3 mutations are necessary but not sufficient to induce maraviroc resistance., Conclusions: The impact of V3 mutations depended on the env context in which they occurred. Simple assessment of the V3 genotype thus cannot accurately predict maraviroc resistance. Rather, phenotypic assessment of virus particles expressing the envelope glycoprotein as a whole is required. This approach revealed that primary resistance of CCR5-tropic HIV-1 subtype B isolates to maraviroc seems uncommon.

Background: HIV-1 subtype D infections have been associated with rapid disease progression and phenotypic assays have shown that CXCR4-using viruses are very prevalent. Recent studies indicate that the genotypic algorithms used routinely to assess HIV-1 tropism may lack accuracy for non-B subtypes. Little is known about the genotypic determinants of HIV-1 subtype D tropism., Results: We determined the HIV-1 coreceptor usage for 32 patients infected with subtype D by both a recombinant virus phenotypic entry assay and V3-loop sequencing to determine the correlation between them. The sensitivity of the Geno2pheno10 genotypic algorithm was 75% and that of the combined 11/25 and net charge rule was 100% for predicting subtype D CXCR4 usage, but their specificities were poor (54% and 68%). We have identified subtype D determinants in the V3 region associated with CXCR4 use and built a new simple genotypic rule for optimizing the genotypic prediction of HIV-1 subtype D tropism. We validated this algorithm using an independent GenBank data set of 67 subtype D V3 sequences of viruses of known phenotype. The subtype D genotypic algorithm was 68% sensitive and 95% specific for predicting X4 viruses in this data set, approaching the performance of genotypic prediction of HIV-1 subtype B tropism., Conclusion: The genotypic determinants of HIV-1 subtype D coreceptor usage are slightly different from those for subtype B viruses. Genotypic predictions based on a subtype D-specific algorithm appear to be preferable for characterizing coreceptor usage in epidemiological and pathophysiological studies.

We assessed the performance of genotypic algorithms for predicting the tropism of human immunodeficiency virus type 1 coreceptor usage in 52 patients infected with the CRF02-AG subtype. The combined criteria of the 11/25 and net charge rules accurately detected CXCR4-using CRF02-AG viruses, whereas the Geno2pheno tool lacked sensitivity and the position-specific scoring matrix (PSSM) tool WebPSSM lacked specificity.

Objective: Replacing phenotypic assays with simple genotypic predictions of HIV-1 coreceptor usage would make the clinical use of CCR5 antagonists easier., Design: Paired genotypic and phenotypic determination of HIV-1 coreceptor usage was performed to assess several genotypic approaches for detecting CXCR4-using and CCR5-using viruses in a clinical setting., Methods: HIV-1 coreceptor usage was prospectively assessed using plasma samples from 103 patients who were candidates for treatment with a CCR5 antagonist. Direct sequencing of the V3 region and a sensitive recombinant virus phenotypic entry assay were performed in parallel for each patient from the same bulk env PCR product., Results: The 103 patients had a median CD4+ T lymphocyte count of 268 x 10(6)cells/l and nadirs of 98 x 10(6)cells/l. Paired genotypic and phenotypic data were obtained for 98 of the 103 patients. For detecting CXCR4-using viruses, the genotypic rule based on amino-acid residues at positions 11/25 and the overall net charge of V3 was 77% sensitive and 96% specific. The Geno2pheno bioinformatic tool was 88% sensitive and 87% specific. The WebPSSM tool prediction with the SI/NSI matrix was 77% sensitive and 94% specific. The global concordance between genotypic and phenotypic data was 91% with the rule combining the amino-acid residues at positions 11/25 and V3 net charge., Conclusion: Genotypic predictions performed well in paired genotypic and phenotypic assessment of HIV-1 coreceptor usage. Multicenter studies analyzing the correlations between the genotypic determination of HIV-1 tropism and clinical response to CCR5 antagonists are needed to validate this approach in clinical practice.

Genotypic population-based methods could be faster and less expensive than phenotypic recombinant assays for determining human immunodeficiency virus type 1 (HIV-1) coreceptor usage in patient samples, but their clinical use requires good genotype-phenotype correlation and concordance with clonal analyses. We have assessed these requirements by clonal analysis of the V1 to V3 env PCR products of 26 patients infected with subtype B HIV-1. We used the resulting set of molecular clones, all sequenced and characterized using a single-cycle recombinant virus phenotypic entry assay, to reevaluate genotype-phenotype correlations. Combining the previously described 11/25 and net charge rules for the V3 genotype improved the prediction of HIV-1 coreceptor usage. We also evaluated the concordance of population-based and clonal analyses for predicting the coreceptor usage of HIV-1 quasispecies. Our population-based recombinant phenotypic assay and direct sequencing of V3 were similarly sensitive for detecting the presence of minor species in the virus population, and both correlated well with clonal analysis. The improved genotype-phenotype correlation obtained by combining two simple genotypic rules and the good concordance with clonal analyses suggest that direct sequencing of V3 is a valuable alternative to population-based recombinant phenotypic assays.