Background:  Suppressive ART lowers viremia 10,000-fold or more, but its impact on HIV-1 genetic diversity and divergence has not been well characterized. We therefore analyzed HIV-1 genetic variation and population shift in patients before and during suppressive ART.

Methods:  HIV-1-infected patients (N = 10) were sampled frequently for as long as 5 years after initiating ART. A total of 1300 HIV-1 gag-pro-pol (p6-RT) sequences were obtained by single-genome sequencing (SGS). Diversity was measured by average pair-wise difference (APD), genetic variation over time was assessed by phylogenetic analyses and a test for panmixia, and polymorphic changes were characterized using Highlighter.

Results:  Before ART, HIV-1 sequences had intra-patient APD values of 0.2 to 2.5% per site. ART reduced plasma viral RNA levels to undetectable (<75 copies/mL) in all patients within 5 months of initiation. In 8 of 10 patients, phylogenetic analyses and measurements of intra-patient APD revealed no change in viral diversity or population structure between pre- and post-ART samples despite up to 4-log₁₀ decreases in HIV-1 RNA levels. In 2 patients, divergence of HIV-1 was evident after ART resulting from the replacement of pre-therapy virus populations containing mostly unique viral variants with populations of dominant species with frequent G to A mutations, stop codons, and shifts in CTL escape mutation profiles. In one of these 2 patients, a variant found 1 day after initiating ART became a predominant plasma clone after 4 years on suppressive therapy, similar to that described by Bailey et al. (2006). The predominant plasma clone was significantly different (P <10^-9) from the pre-therapy population by tests for panmixia and divergence (2.9%), resulting primarily from accumulation of G to A mutations.

Conclusions:  HIV-1 diversity did not change significantly despite 10,000-fold reduction in viremia with suppressive ART. Viral divergence during ART in 2 patients resulted from the replacement of viral populations containing many unique variants with populations containing dominant species having frequent G to A mutations. These results suggest that both short- and long-lived cells are infected with diverse virus populations before therapy, that viral replication is completely blocked by ART, and that diverse replication-competent viral variants may decay with long-term suppressive therapy in favor of progeny from defective proviruses in surviving cells.