Genotypic prediction of HIV-1 CRF01-AE tropism

Abstract

HIV-1 subtype CRF01-AE predominates in south Asia and has spread throughout the world. The virus tropism must be determined before using CCR5 antagonists. Genotypic methods could be used, but the prediction algorithms may be inaccurate for non-B subtypes like CRF01-AE and the correlation with the phenotypic approach has not been assessed. We analyzed 61 CRF01-AE V3 clonal sequences of known phenotype from the GenBank database. The sensitivity of the Geno2pheno10 genotypic algorithm was 91%, but its specificity was poor (54%). In contrast, the combined 11/25 and net charge rule was highly specific (98%) but rather insensitive (64%). We thus identified subtype CRF01-AE determinants in the V3 region that are associated with CXCR4 use and developed a new simple rule for optimizing the genotypic prediction of CRF01-AE tropism. The concordance between the predicted CRF01-AE genotype and the phenotype was 95% for the clonal data set. We then validated this algorithm by analyzing the data from 44 patients infected with subtype CRF01-AE, whose tropism was determined using a recombinant phenotypic entry assay and V3-loop bulk sequencing. The CRF01-AE genotypic tool was 70% sensitive and 96% specific for predicting CXCR4 use, and the concordance between genotype and phenotype was 84%, approaching the concordance obtained for predicting the tropism of HIV-1 subtype B. Genotypic predictions that use a subtype CRF01-AE-specific algorithm appear to be preferable for characterizing coreceptor usage both in pathophysiological studies and for ensuring the appropriate use of CCR5 antagonists.

Fig 1

V3 amino acid sequence alignments and matched phenotypes of the 11 CRF01-AE CXCR4-using clones from the GenBank data set. V3 amino acid sequence alignments and corresponding phenotypes were selected from the GenBank database. These sequences are shown with the following abbreviation with reference to the consensus sequence: dash, gap inserted to maintain alignment. Residues at positions 11 and 25 and mutated N-linked glycosylation sites are boxed to highlight the substitutions noted. The V3 net charge (calculated by subtracting the number of negatively charged amino acids [D and E] from the number of positively charged ones [K and R]), the number of amino acids in V3, and the genotype predicted by the combined 11/25 and net charge rules built for subtype B viruses and by Geno2pheno10 are shown, together with the phenotype. Discordant genotypic predictions with reference to the phenotype are boxed.

Fig 2

Receiver operating characteristic curves of the genotypic algorithms versus the phenotype. (a) ROC curve for the data obtained on the GenBank clones. (b) ROC curve for the data obtained on the set of clinical samples. The black line represents the performance of the original geno2pheno algorithm, and the gray line represents the performance of the geno2pheno-C_NGS-Sanger algorithm, both referring to the phenotype. The point represents the performance of the combined rule for CRF01-AE.

Fig 3

V3 amino acid sequence alignments and matched phenotypes of the 44 subtype CRF01-AE viruses. V3 amino acid sequence alignments were obtained by bulk sequencing env PCR products from the 44 subtype CRF01-AE-infected patients. These sequences are shown with the following abbreviations with reference to the CRF01-AE consensus sequence: dot, identity with amino acid baseline sequence; dash, gap inserted to maintain alignment; slash, amino acid position related to dual-virus population. Replacements are indicated by the appropriate code letters. Residues at positions 11 and 25 and mutated N-linked glycosylation sites are boxed to highlight the substitutions noted. The V3 net charge (calculated by subtracting the number of negatively charged amino acids [D and E] from the number of positively charged ones [K and R]), the number of amino acids in V3, and the genotype predicted by the combined 11/25 and net charge rules built for subtype B viruses and by Geno2pheno10 are shown, together with the observed phenotype. Discordances between the genotypic predictions and the phenotype are boxed.