Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

Significance

A reservoir of latently infected cells poses the greatest challenge to HIV-1 eradication. Efforts to develop strategies to eliminate the reservoir have been hampered, in part, by the lack of a precise understanding of the cellular and molecular nature of this reservoir. We describe a new method to analyze the replication-competent latent reservoir quantitatively and qualitatively. We find that over 50% of the replication-competent viruses in the reservoir form part of groups with identical env sequences. However, a negative correlation exists between integrated proviral clones and replication-competent viruses, such that the larger the proviral clone, the lower is its probability of representing a replication-competent virus.

Abstract

HIV-1–infected individuals harbor a latent reservoir of infected CD4 ⁺ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.