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

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.

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

“…1B ). Consistent with previously reported differences in bimodal expression patterns among proviral clones ( 53 ), the results showed that each clonal pool was comprised of both GFP + and GFP − cells, with LTR-active proportions being discernible by GFP + percentages (%GFP + ) that differed among the clones: mostly GFP + cells for some clones (high-%GFP + clones) and distinctly different GFP + percentages for others ( Fig. 1B ).…”

“…2C are represented in light gray; regions that appear to display darker shading indicate the presence of multiple clones at the same coordinates. We and others have previously reported significant differences among HIV-1-infected cell clones in the amount of virus released per active cell ( 23 , 53 ), and the results here indicated that burst sizes for the integrant clones in the current study ranged over 4 orders of magnitude.…”

“…To study the effects of Vpr on the responsiveness of individual proviruses in the absence of virus spread, Jurkat cells were infected with distinguishable vpr -positive ( vpr + ) or vpr -negative ( vpr − ) versions of the NL4-3 derivative shown in Fig. 1A ( 53 ). The use of the EF1α promoter to drive the constitutive expression of the puromycin resistance gene allowed the selection of infected cells independent of LTR expression.…”

“…It has long been recognized that at least some HIV-1 integrants establish clones in which a fraction of the total daughter cells possess active LTRs while proviruses are silenced in other sibling cells ( 54 ). Previous work using zip-coded vpr − derivatives of the vector used here has shown that for each integrant, the clone gives rise to a mixture of cellular progeny that includes some GFP + cells and some GFP − cells and that over time sibling cells switch between LTR-active (GFP + ) and -inactive (GFP − ) expression phenotypes while maintaining LTR-active and -inactive cells in stable equilibrium proportions ( 53 ). To confirm that individual integrant clones in the current study also contained clone-specific mixtures of GFP + and GFP − cells, single cells were isolated by limiting dilution from the vpr − pool and expanded, and cells from each clone were then subjected to flow cytometry ( Fig.…”

Maraviroc as a potential HIV-1 latency-reversing agent in cell line models and ex vivo CD4 T cells

“…Interestingly, control CD4 T cell cultures in the absence of stimulation had negligible CFR levels at T0 through T14, although infectious particles were revealed when supernatants were passed onto the CD4/MOLT-4 CCR5 virus amplification co-culture system used in TZA. This highlights the need to control for spontaneous HIV-1 reactivation in any experiment on latency reversal, both in vitro [45] and in vivo [46].…”