Effects of Antiretroviral Therapy on Transcriptional Activity of HIV Proviruses

Division Of Basic Sciences - Nci

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Abstract

BACKGROUND: We and others recently discovered that HIV infection can be maintained during antiretroviral therapy (ART) by the proliferation of cells that were infected prior to initiating ART. However, it was not known how commonly such clones express HIV RNA during ART. The findings that expanded clones can be the source of persistent viremia (Maldarelli et al., Science 345:179-183, 2014; Simonetti et al., PNAS 113:1883-1888, 2016) indicated that at least some members of clonal populations can express unspliced RNA during ART and may, therefore, result in rebound viremia if ART is interrupted. We hypothesized that the majority of infected cells that persist in individuals on ART have undergone clonal expansion and are composed of members that express HIV RNA. To test this hypothesis, we examined HIV expression levels in single cells in both treated and untreated individuals using our single-cell HIV cell-associated RNA and DNA single-genome sequencing (CARD-SGS) method (Wiegand et al., PNAS 114:E3659-E3668, 2017). We determined the fraction of HIV-1 proviruses within infected cell clones that express unspliced HIV-1 cell-associated RNA during ART (Musick et al., Front. Microbiol. 10:2204, 2019). In total, 34 different clones carrying either intact or defective proviruses were assessed. We found that about 3% of cells within clones contained unspliced HIV-1 RNA. Highest levels of HIV-1 RNA were found in the effector memory T-cell subset. The fraction of cells within clones that contained HIV-1 RNA was not different in clones with intact (median 2.3%) vs. defective (median 3.5%) proviruses (p=0.2). However, higher fractions and levels of RNA were found in cells with proviruses containing multiple drug resistance mutations, including those contributing to rebound viremia. These findings show that the vast majority of HIV-1 proviruses within expanded T-cell clones, including intact proviruses, may be transcriptionally silent at any given time, implying that infected T cells may be able to be activated to proliferate without inducing the expression of the integrated provirus or, alternatively, may be able to proliferate without cellular activation. ____ACCOMPLISHMENTS: We previously reported a replication-competent HIV-1 variant that is produced by a highly expanded cell clone and persists in the plasma of an HIV-infected donor treated with ART (Simonetti et al., PNAS 113:1883-1888, 2016). In 2020, we expanded this case study to 5 additional donors with their viremia suppressed on ART and demonstrated that clones carrying and expressing replication-competent HIV are common among donors (Halvas et al., J. Clin. Invest. 130:5847-5857, 2020). To follow up on the hypothesis that cells may differentiate and proliferate without inducing the expression of the integrated provirus, we sorted PBMCs collected from patients with viremia fully suppressed on ART into resting (DR-) and activated (DR+) subsets. We isolated single infected cells from the subsets and measured the fraction that have HIV RNA and the levels of HIV RNA in each of the single cells. Our data show that the fraction and levels of HIV RNA are not different in the resting and activated cells, indicating that infected cells that persist on ART are able to maintain proviral latency despite proliferation and activation (Groebner et al., Conference on Retroviruses and Opportunistic Infections, 2021). The results of this study are currently being assembled into a manuscript. _____Because our previous studies showed that only a small fraction of infected cells that persist on ART have proviruses that are transcriptionally active, we hypothesized that the HIV promotor in the 5' untranslated region may contain CpG islands that are DNA methylated. Such methylation may prevent the expression of the HIV provirus by inhibiting the binding of the HIV transcription factors. To test this hypothesis, we isolated single HIV proviruses that were previously shown to be transcriptionally silent in vivo, treated them with bisulfate to deaminate unmethylated CpG islands, and quantified the number of guanines that retained their animation (indicating that they were methylated). Although we found CpG islands within HIV coding regions that were methylated, we rarely identified methylated guanines within the HIV promotor of transcriptionally silent proviruses. Our findings show that methylation of the HIV promotor is not a mechanism for HIV persistence. We published these findings in Viruses in 2021 (Boltz et al., Viruses 13:799. 2021). ___Since methylation of the HIV promoter was not found to be a mechanism for HIV latency in donors on ART, we investigated the expression of an antisense gene (Ast) encoded within the HIV env gene (Sklutuis, et al. Conference on Retroviruses and Opportunistic Infections, 2022). In vitro studies showed that HIV encodes an antisense gene that recruits and retains EZH2, a component of the polychrome repressor complex-2 (PRC2) to the HIV-1 5'LTR. EZH2 catalyzes the trimethylation of lysine 27 on histone H3, a suppressive epigenetic mark that promotes nucleosome assembly and suppresses of HIV-1 transcription. This mechanism suggests that Ast acts as a lncRNA in promoting epigenetic silencing of the HIV-1 5'LTR to induce and maintain viral latency in HIV infected cells. To address these findings, our laboratory recently explored the expression levels of Ast in single infected cells isolated from donors with viremia suppressed on ART using digital reverse transcription PCR. We found that a median of 26% of infected PBMCs were detected to express HIV Ast at a given point in time. This finding confirmed the presence of detectable Ast levels in donors with their viremia suppressed on ART and suggests its potential role as a regulatory RNA. The results of our Ast study are being prepared for publication. ____ In the absence of ART, levels of plasma viremia vary in people living with HIV (PLWH). What determines levels of plasma viremia within individuals is not fully understood but is likely associated with levels of proviral expression, immune-control, and viral fitness. More than 90% of PLWH have limited natural control of HIV-1 replication without ART (non-controllers); however, a small fraction of PLWH (5%) naturally control levels of HIV-1 replication, which is reflected by lower levels of plasma viremia (controllers). However, using the CARD-SGS assay (described above), our preliminary data reveal that there is not a higher fraction of infected cells with transcriptionally active proviruses in non-controllers compared to viremic controllers (determined by measuring levels of unspliced HIV-1 RNA in single infected cells). Rather, there are fewer total infected cells in viremic controllers. These findings suggest that natural control of HIV replication is not due to a higher fraction of latently infected cells in these individuals but is due to more potent immune responses (i.e. NK cell and/or CTL responses) that efficiently recognize and kill infected cells that express HIV RNA. Our future studies will characterize the immune responses in HIV controllers and non-controllers.