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, particularly those carrying replication-competent proviruses. 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). CARD-SGS can be used to determine (a) the fraction of total HIV-infected cells that express HIV RNA, (b) the levels of HIV RNA expression in single cells, (c) the levels of HIV RNA expression in expanded clones, and (d) the genetic relationship of these RNAs to proviral DNA. This assay uses improved isolation of intracellular RNA from total PBMCs modified from Hong et al. (J. Clin. Microbiol. 54:902-911, 2016) and endpoint dilution of HIV-expressing cells. We used CARD-SGS to investigate changes in the HIV expression profiles over the course of treatment with ART, to identify cellular sources of persistent and rebound viremia, and to investigate the expression levels in expanded proviruses that constitute an important part of the reservoir for HIV. _____Populations of identical intracellular RNA sequences suggest either that multiple cells carrying identical proviruses were expressing viral RNA or that single cells (or their descendants) were undergoing higher levels of viral expression in comparison to other infected cells. The finding that the RNA variants were all defective implies that their expression is either from a proliferating cell population or from a single cell, but not from local spread through viral replication. Our previous studies included determining the fraction of cells within specific expanded clones that express HIV RNA and identifying the cell subsets that support proviral expression (i.e. central, transitional, or effector memory T cells) (Musick et al., Front. Microbiol. 10:2204, 2019). To address this question, we determined the fraction of HIV-1 proviruses within infected cell clones that express unspliced HIV-1 cell-associated RNA during ART. 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 are currently sorting PBMCs collected from patients with viremia fully suppressed on ART into resting (DR-) and activated (DR+) subsets. We are isolating single infected cells from the subsets to estimate the fraction of cells within them that have HIV RNA and we are measuring the levels of HIV RNA in each of the single cells. Our preliminary data suggest 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). _____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) (Boltz et al., Viruses 13:799. 2021). 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.