Telesnitsky, A. | bioRxiv Science

bioRxiv2021

Vpr shapes the proviral landscape and polyclonal HIV-1 reactivation patterns in cultured cells

Cell culture models suggest that the HIV-1 viral protein R (Vpr) is dispensable for latency establishment. However, whether Vpr affects the persistent proviral landscape and responsiveness to latency reversing agents (LRAs) is unclear. Here, integration site landscape, clonal dynamics, and latency reversal effects of Vpr were studied by comparing barcoded vpr+ and vpr- populations arising after infection of Jurkat cells in vitro. The results showed that individual integrant clones differed in fractions of LTR-active daughter cells: some clones gave rise to few to no LTR-active cells while for others almost all daughter cells were LTR-active. Integrant clones with at least 60% LTR-active cells (high LTR-active clones) contained proviruses positioned closer to preexisting enhancers (H3K27ac) and promoters (H3K4me3) than clones with <30% LTR-active cells (low LTR-active clones). Comparing vpr+ and vpr- populations revealed that the vpr+ population was depleted of high LTR-active clones. Complementing vpr-defective proviruses by transduction with vpr 16 days after infection led to rapid loss of high LTR-active clones, indicating that the effect of Vpr on proviral populations occurs post-integration. Comparing vpr+ and vpr- integration sites revealed that predominant vpr+ proviruses were farther from enhancers and promoters. Correspondingly, distances to these marks among previously reported intact HIV proviruses in ART-suppressed patients were more similar to those in the vpr+ pool than to vpr- integrants. To compare latency reactivation agent (LRA) responsiveness, the LRAs prostratin and JQ1 were applied separately or in combination. vpr+ and vpr- population-wide trends were similar, but combination treatment reduced virion release in a subset of vpr- clones relative to when JQ1 was applied separately, an effect not observed in vpr+ pools. Together, these observations highlight the importance of Vpr to proviral population dynamics, integration site landscapes, and responsiveness to latency reversing agents. One Sentence Summary Expression properties and responsiveness to latency reactivation agents of individual HIV-1 proviral clones within polyclonal populations are masked by dominant clones and influenced by proviral proximity to certain epigenetic marks and by Vpr, a viral factor not previously known to affect latency and reactivation.

microbiology↗

bioRxiv2019

Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis

HIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication. Initial findings in immortalized cells were validated using CD4+ cells from donor blood. Tracking clonal behavior by proviral \"zip codes\" indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in the nef open reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP-cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in genes sense orientation were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP-population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics.\n\nSummaryVery few HIV-1 infected cells persist in patients for more than a couple days, but those that do pose life-long health risks. Strategies designed to eliminate these cells have been based on assumptions about what viral properties allow infected cell survival. However, such approaches for HIV-1 eradication have not yet shown therapeutic promise, possibly because much of the research underlying assumptions about virus persistence has been focused on a limited number of infected cell types, the averaged behavior of cells in diverse populations, or snapshot views. Here, we developed a high-throughput approach to study hundreds of distinct HIV-1 infected cells and their progeny over time in an unbiased way. This revealed that each virus established its own pattern of gene expression that, upon infected cell division, was stably transmitted to all progeny cells. Expression patterns consisted of alternating waves of activity and inactivity, with the extent of activity differing among infected cell families over a 1000-fold range. The dynamics and variability among infected cells and within complex populations that the work here revealed has not previously been evident, and may help establish more accurate correlates of persistent HIV-1 infection.

microbiology↗