Background:  Evolutionary studies of the HIV-1 pol gene in viruses isolated from the resting CD4⁺ T-cell reservoir of patients on HAART have largely shown retention of archival variants, including wild type HIV-1. These findings provide evidence for a stable, long-lived reservoir though replenishment of the reservoir with virus harbored in drug sanctuary sites cannot be excluded. In this analysis, we extended our studies to include the env gene. We hypothesized that viral variants maintained by ongoing replenishment should experience immune selective pressure that may be manifest by alterations in env such as N-linked glycosylation (N-gly) sites for neutralizing antibodies.

Methods:  We conducted a longitudinal study of the evolutionary features of the HIV-1 pol and env (V3-V5 region) genes of near full-length (9kb) HIV-1 cDNA. The cDNA clones were amplified from early-passage biologic clones cultured from the resting CD4⁺ T-cell reservoir of 4 children treated with up to 5 years of suppressive HAART. Three of the 4 children had prolonged suppression of virus replication, and the fourth developed treatment failure and drug resistance after 1 year on HAART. Viral sequences were analyzed for drug resistance mutations in pol, shifts in predicted N-gly sites in env, and phylogenetic properties. 

Results:  Durable suppression of virus replication on HAART was associated with evolutionary arrest in both the env and pol genes, including a notable absence of new N-gly sites in env, and lack of development of drug resistance in pol. However, in the setting of treatment failure, the evolution of env and pol was concordant and readily detectable. That is, both regions showed parallel divergence, with development of new drug resistance mutations and shift in N-gly patterns in env. 

Conclusions:  In both durable suppressive HAART and treatment failure, the evolutionary patterns in env and pol are concordant as shown by analysis of replication-competent HIV-1 in the resting CD4⁺ T cell. During effective HAART, the arrest in virus evolution is associated with lack of drug resistance mutations and stability in N-gly sites. In contrast, during treatment failure, new N-gly sites were observed to coincide with the development of new drug resistance mutations. These findings suggest that suppressive HAART can result in stasis in the latent reservoir of even the most variable regions of HIV, arguing against dependence on continuous replenishment through replication.

Keywords: HIV-1; envelope; evolution