An Increasing Proportion of Monotypic HIV-1 DNA Sequences during Antiretroviral Treatment Suggests Proliferation of HIV-Infected Cells

Abstract

Understanding how HIV-1 persists during effective antiretroviral therapy (ART) should inform strategies to cure HIV-1 infection. We hypothesize that proliferation of HIV-1-infected cells contributes to persistence of HIV-1 infection during suppressive ART. This predicts that identical or monotypic HIV-1 DNA sequences will increase over time during ART. We analyzed 1,656 env and pol sequences generated following single-genome amplification from the blood and sputum of six individuals during long-term suppressive ART. The median proportion of monotypic sequences increased from 25.0% prior to ART to 43.2% after a median of 9.8 years of suppressive ART. The proportion of monotypic sequences was estimated to increase 3.3% per year (95% confidence interval, 2.3 to 4.4%; P < 0.001). Drug resistance mutations were not more common in the monotypic sequences, arguing against viral replication during times with lower antiretroviral concentrations. Bioinformatic analysis found equivalent genetic distances of monotypic and nonmonotypic sequences from the predicted founder virus sequence, suggesting that the relative increase in monotypic variants over time is not due to selective survival of cells with viruses from the time of acute infection or from just prior to ART initiation. Furthermore, while the total HIV-1 DNA load decreased during ART, the calculated concentration of monotypic sequences was stable in children, despite growth over nearly a decade of observation, consistent with proliferation of infected CD4؉ T cells and slower decay of monotypic sequences. Our findings suggest that proliferation of cells with proviruses is a likely mechanism of HIV-1 DNA persistence, which should be considered when designing strategies to eradicate HIV-1 infection.

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

“…S1). As previously reported for archived proviral DNA amplified from primary CD4 + T cells, we found unique sequences, as well as large groups of identical sequences, marking expanded cell clones (14,15,24,25). For example, in B106, 67 of 113 env sequences obtained from the two time points were unique and the remaining 46 were members of clones.…”

“…In addition, there is little evidence for viral evolution even after prolonged periods of ART (36). In contrast, the idea that the reservoir is maintained, at least in part, by the proliferation of latently infected cells is supported by the observation of increasing proportions of identical proviral sequences in circulating CD4 + T cells (15).…”

“…Finally, the genetic characteristics of the reservoir have been studied primarily by sequencing integrated proviral DNA or cell-associated RNAs, many of which are defective and thus not representative of the replication-competent reservoir (12)(13)(14)(15)(16). Phylogenetic analyses of the replication-competent reservoir are usually limited because bulk outgrowth cultures produce species with little diversity, even when analyzed by ultra-deep sequencing (17)(18)(19)(20).…”

Potential Implication of Residual Viremia in Patients on Effective Antiretroviral Therapy

“…In essence, these data revealed the clonal expansion of some CD4 T cells with integrated vDNA in vivo during long-term suppressive therapy, similar to the observations made previously. [68][69][70] However, these studies provided a potential mechanistic explanation about how a population of CD4 T cells with integrated vDNA could undergo clonal expansion in vivo.…”

Epigenetic Metabolite Acetate Inhibits Class I/II Histone Deacetylases, Promotes Histone Acetylation, and Increases HIV-1 Integration in CD4+T Cells

“…Cellular proliferation and activation are known to have an impact on the susceptibility of CD4 ϩ T cells to HIV-1 infection. For example, cellular proliferation plays an important role in the HIV-1 life cycle by promoting virus dissemination, which helps to maintain viral reservoirs (50,51), whereas cell activation allows the translocation of host transcription factors to the nucleus, where they trigger genes implicated in immune response and virus production (52,53). Thus, cell proliferation was evaluated by the use of a dilution assay that is based on the fluorescent cell staining dye carboxyfluorescein succinimidyl ester (CFSE).…”