Quantify the Fraction of Proviruses That Express HIV RNA to Better Understand Proviral Latency and Its Reversal

Division Of Basic Sciences - Nci

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Abstract

It is not fully understood what fraction of HIV-infected cells express HIV RNA at a given point in time, to what levels HIV RNA is expressed in single cells, how much variation there is among cells and clones, and to what degree expression levels and profiles change with ART and new interventions, including LRAs. Investigating the fraction of infected cells that express HIV RNA and their levels of expression in single cells prior to, during, and after interventions will provide a better understanding of HIV persistence and its rebound after stopping ART. To address these questions, we applied our HIV cell-associated RNA and DNA SGS (CARD-SGS) method (Wiegand, et al. PNAS 2017) to PBMC and lymph node samples from participants in the SCOPE cohort (protocol #NCT00187512), in a prospective study at the University of Pittsburgh (IRB# STUDY20040215), and in NIH clinical trials (08-I-0221). In our publication in PNAS 2024 (Capoferri, et al.), we demonstrated that CARD-SGS can determine the fraction of total HIV-infected cells that express HIV RNA and the levels of HIV RNA in single cells. In the absence of ART, a subset of individuals, termed HIV controllers, have levels of plasma viremia that are orders of magnitude lower than non-controllers who are at higher risk for HIV disease progression. In addition to having fewer infected cells, it was possible that lower levels of plasma viremia in controllers are due to a smaller fraction of the infected cells having HIV RNA compared with non-controllers. To directly test this possibility, we used CARD-SGS to compare the fraction of infected cells that contain HIV RNA in PBMC obtained from controllers and non-controllers. The fraction of infected cells containing HIV RNA did not differ between the two groups. Rather, the levels of viremia were strongly associated with the total number of infected cells that had HIV RNA, with controllers having 34-fold fewer infected cells per million PBMC. These results revealed that viremic control is not associated with a lower fraction of proviruses expressing HIV RNA, unlike what is reported for elite controllers, but is only related to having fewer infected cells overall, maybe reflecting greater immune clearance of infected cells. Our findings show that proviral silencing is not a key mechanism for viremic control and will help to refine strategies towards achieving HIV remission without ART. Because it has been shown in vitro that resting CD4+ T cells typically have transcriptionally silent (latent) proviruses and activated CD4+ T cells typically have transcriptionally active proviruses, especially after multiple rounds of ex vivo activation, we hypothesized that activated T cells in vivo are the source of the HIV RNA that we detect by CARD-SGS. The hypothesis was that latently infected, resting T cells are maintained by homeostatic proliferation (allowing cells to proliferate without inducing expression of the integrated provirus) but, if the cell is activated by interaction with its cognate antigen, then the integrated provirus is induced to express HIV RNA. We obtained PBMC collected by leukapheresis from 8 donors with viremia suppressed on ART who were enrolled in the ACTG A5341s cohort, the SCOPE cohort, or NIH trial 08-I-0221. To investigate whether cellular activation in vivo is associated with proviral expression, we examined HIV RNA levels in sorted, unstimulated "activated" and "resting" memory CD4+ T cells from the participants on long-term ART. To our surprise, we found that similar fractions of proviruses express HIV RNA in vivo in both HLA-DR+ and HLA-DR- cells. We also found that the levels of HIV usRNA in single cells are not different between the subsets. Similar results were observed between T-cell clones carrying intact HIV and defective proviruses. Our results showed that cellular activation and provirus transcription are unlinked in infected CD4+ cells that persist on long-term ART, indicating that ART selects for a population of proviruses that are resistant to expression of HIV RNA, even when the host cell undergoes immune activation. Natural antisense transcripts are expressed in eukaryotes, prokaryotes, and viruses and can possess regulatory functions at the transcriptional and/or post-transcriptional levels. In vitro studies have shown that HIV-1 antisense transcripts (AST) promote viral latency through epigenetic silencing of the proviral 5' LTR. However, expression of AST in vivo has not been convincingly demonstrated. In this study, we used single RNA template amplification and sequencing to demonstrate expression of AST in unstimulated PBMC collected from PWH. Our results show that expression levels of AST may be higher during ART compared to untreated individuals and that clones of infected cells persisting on ART continue to express HIV AST. This study is the first to verify HIV-1 AST expression in vivo with sequencing, documenting AST presence without cellular activation and suggesting its natural occurrence in PWH. These findings advance our understanding of HIV-1 persistence and underscore the potential of targeting viral reservoirs in efforts to manage latent infections without continuous ART (Capoferri, et al. Life Sci Alliance, 2025) In our current and future research aims, CARD-SGS, and a new method to be developed (PIT-seq) will be used to investigate differences in HIV expression profiles in donors with various levels of viremia without ART, in those with viremia suppressed on ART, and in those being exposed to therapeutic vaccines, LRAs, and other new interventions.