Bimodal Expression Patterns, and Not Viral Burst Sizes, Predict the Effects of Vpr on HIV-1 Proviral Populations in Jurkat Cells.

Integration site landscapes, clonal dynamics, and latency reversal with or without vpr were compared in HIV-1-infected Jurkat cell populations, and the properties of individual clones were defined. Clones differed in fractions of long terminal repeat (LTR)-active daughter cells, with some clones containing few to no LTR-active cells, while almost all cells were LTR active for others. Clones varied over 4 orders of magnitude in virus release per active cell. Proviruses in largely LTR-active clones were closer to preexisting enhancers and promoters than low-LTR-active clones. Unsurprisingly, major vpr+ clones contained fewer LTR-active cells than vpr- clones, and predominant vpr+ proviruses were farther from enhancers and promoters than those in vpr- pools. Distances to these marks among intact proviruses previously reported for antiretroviral therapy (ART)-suppressed patients revealed that patient integration sites were more similar to those in the vpr+ pool than to vpr- integrants. Complementing vpr-defective proviruses with vpr led to the rapid loss of highly LTR-active clones, indicating that the effect of Vpr on proviral populations occurred after integration. However, major clones in the complemented pool and its vpr- parent population did not differ in burst sizes. When the latency reactivation agents prostratin and JQ1 were applied separately or in combination, vpr+ and vpr- population-wide trends were similar, with dual-treatment enhancement being due in part to reactivated clones that did not respond to either drug applied separately. However, the expression signatures of individual clones differed between populations. These observations highlight how Vpr, exerting selective pressure on proviral epigenetic variation, can shape integration site landscapes, proviral expression patterns, and reactivation properties. IMPORTANCE A bedrock assumption in HIV-1 population modeling is that all active cells release the same amount of virus. However, the findings here revealed that when HIV-infected cells expand into clones, each clone differs in virus production. Reasoning that this variation in expression patterns constituted a population of clones from which differing subsets would prevail under differing environmental conditions, the cytotoxic HIV-1 protein Vpr was introduced, and population dynamics and expression properties were compared in the presence and absence of Vpr. The results showed that whereas most clones produced fairly continuous levels of virus in the absence of Vpr, its presence selected for a distinct subset of clones with properties reminiscent of persistent populations in patients, suggesting the possibility that the interclonal variation in expression patterns observed in culture may contribute to proviral persistence in vivo.

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, with limited findings using CD4+ cells from donor blood consistent with observations in immortalized cells. 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 the sense orientation of genes 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.