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Detection of Inferred CCR5- and CXCR4-Using HIV-1 Variants and Evolutionary Intermediates Using Ultra-Deep Pyrosequencing

Figure 3

Minimum spanning tree (MST) of V3 sequences from subject DS1.

V3 sequences generated by deep sequencing were used to construct MSTs. Identical nucleotide sequences are grouped in one node, and the circle size is proportional to the abundance of that particular V3 sequence. The length of the connecting branches corresponds to the number of nucleotide differences between the two connected nodes. Time points are color-coded, using bright colors for PBMC samples and corresponding soft colors for serum samples. (A) MST stripped of sequences from all time points except time point −12 months. For this time point, PBMCs were not available, and the MST thus consists of V3 sequences generated from serum only (shown in light red). (B) V3 sequences from PBMCs (shown in bright orange) and serum (shown in light orange) obtained at time point −9 months were added to the MST presented in panel A. (C) MST containing V3 sequences generated from −6 months serum (shown in light yellow) in addition to the sequences present in the MST in panel B. (D) Sequences from PBMCs (bright green) and serum (light green) at timepoint −3 months have been added to the MST in panel C. (E) Complete MST containing PBMC and serum V3 sequences from all time points. V3 amino acid sequences are shown relative to the majority sequence in PBMCs at the first time point (shown in blue box). Sequences of the major nsi/r5 variant, the major si/x4 variant, and all intermediates are additionally shown in the bottom left. In all panels, nodes containing V3 sequences with an si phenotype as inferred by PSSMNSI/SI and an x4 phenotype as inferred by geno2pheno[coreceptor] are indicated in red, all other sequences were predicted to be nsi/r5. The phenotype of V3 sequences of which the corresponding Env clone was analyzed in the Trofile assay is given in bright blue (for R5 sequences) or in bright red (for Dual-X sequences). Also shown are the results of phenotyping of PBMC samples by the MT-2 assay (NSI or SI) and of serum samples by ESTA (R5 or D/M), and the percentages si/x4 sequences obtained from PBMC and serum samples by deep sequencing (panel E, right side). N.t., not tested; mo, months.

Figure 3