Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

Ex vivo activation of CD4+ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells.

The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, et al. recently showed that CD4+ T-cells containing intact proviruses can clonally expand in vivo and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4+ T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an ex vivo cell culture system involving stimulation of CD4+ T-cells from donors on suppressive antiretroviral therapy (ART) with PMA/ionomycin (day 1-7), followed by rest (day 7-21), and then repeat stimulation (day 21-28), always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS) of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA) and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4+ T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.

Changes in HIV reservoirs during long-term antiretroviral therapy.

PURPOSE OF REVIEW: To review current knowledge about the impact of long-term combination antiretroviral therapy (cART) on HIV reservoirs. RECENT FINDINGS: The number of HIV-infected cells that persist during long-term antiretroviral therapy is associated with the stage of HIV infection at the time of treatment initiation. Initiation of cART reduces the number of infected cells over the first 4 years of therapy, but thereafter there is no further decline despite long-term effective cART. The remarkable stability of infected cell numbers is likely due to a balance among homeostatic or antigen-driven proliferation of infected memory T-cells subsets, clonal expansion of a subset of infected cells as a consequence of specific retroviral integration sites, and death of other infected cells. At present, there is no effective means of accelerating the decay of infected cells in individuals initiated on cART during chronic HIV infection. SUMMARY: Given the stability and difficulty in eliminating HIV-infected cells, early initiation of cART in treatment-naïve HIV-infected patients is currently the most effective way to limit the size and diversity of HIV reservoirs.

Impact of Antiretroviral Therapy on HIV-1 Persistence: The Case for Early Initiation.

Combination antiretroviral therapy suppresses HIV-1 replication, but is not curative because of the persistence of both residual viremia and long-lived cells carrying latent, intact proviruses. Recent data, however, have highlighted the substantial impact of early initiation of combination antiretroviral therapy on the number of HIV-1-infected cells that persist after treatment and on the possibility of post-treatment control of viral replication. The well-publicized case of the "Mississippi baby" has fueled great interest in outcomes following immediate initiation of combination antiretroviral therapy in both infants and adults. Here, we review current knowledge of the impact of combination antiretroviral therapy on HIV-1 reservoirs, with specific focus on the timing of treatment initiation. We propose that early initiation of combination antiretroviral therapy is likely to promote the efficacy of strategies to achieve long-term, therapy-free remission of HIV-1 and that this possibility should be considered in decisions about when to initiate the therapy.