Abstract
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.
Highlights
In the HIV-1 replication cycle, a DNA intermediate integrates into the host cell’s genome
Such approaches for HIV-1 eradication have not yet shown therapeutic promise, possibly because many assumptions about virus persistence are based on studies involving a limited number of infected cell types, the averaged behavior of cells in diverse populations, or snapshot views
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
Summary
In the HIV-1 replication cycle, a DNA intermediate integrates into the host cell’s genome. The resulting latently infected cells persist throughout antiretroviral treatment, and their sporadic reactivation can lead to virus rebound after antiretroviral cessation This source of persistent virus is called the latent reservoir and is believed to consist largely of transcriptionally silent proviruses integrated into resting memory T cells [1] [2] [3]. Infectious virus can be produced by T lymphocytes from such patients when the cells are activated or treated with certain chromatin remodeling drugs ex vivo These observations inspired “shock and kill” HIV cure strategies, which involve pharmacologically inducing provirus expression to promote the recognition and clearance of latently infected cells [4] [5]. While intervention that reactivates silenced proviruses can activate HIV-1 gene expression in cell culture models of latency, such treatments have far failed to fulfill their promise in the clinic, suggesting much remains to be learned about the establishment and maintenance of the latent reservoir [6] [7] [8]
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