Background:  HIV-1 co-receptor usage plays a critical role in viral tropism and pathogenesis. We found previously that ART preferentially suppresses CXCR4-specific (X4) strains of HIV-1. CCR5 (R5) strains predominate following ART’s initiation and may evolve under selective pressure from therapy. We examined the evolution of drug resistance mutations and co-receptor utilization after ART to test the hypothesis that recombination played a role in the evolution of the viral strains.

Methods:  We performed longitudinal sequence analyses of HIV-1 RNA variants isolated from 5 women following initiation of ART. Sequences >6.6 kb were obtained from single HIV-1 genomes by limiting dilution real-time polymerase chain reaction (RT-PCR) and direct sequencing. To obtain single molecules for amplification, virion-derived RNA was reverse transcribed and the cDNA diluted to an endpoint that yielded a PCR amplicon <40% of the time. To identify recombinants, we analyzed the sequences by using a Bayesian Dual Multiple Changepoint Framework.

Results:  Variants displayed considerable heterogeneity, with both wild type and resistant pol sequences linked to env genes encoding either R5 or X4 usage. We identified 4 intra-patient recombinants from 3 patients; these molecules were each derived from 2, 3, or 4 parental sequences. To control for convergent evolution, sequences were analyzed in parallel after stripping V3 regions, and the phylogenetic patterns remained the same. Recombinant breakpoints were localized primarily to pol. The co-receptor usage of the parental sequences often differed, with the recombinant strains displaying R5 or X4 usage.

Conclusions:  Drug-resistant HIV-1 strains that evolved during ART utilized either R5 or X4. Recombination among multiple HIV-1 molecules played a significant role in shaping the evolution of env and pol sequences in patients taking ART.