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Micro RNA Targets in HIV Latency: Insights into Novel Layers of Latency Control.

ABSTRACT: Despite the considerable progress that has been made in identifying cellular factors and pathways that contribute to establishment and maintenance of the latent HIV reservoir, it remains the major obstacle to eradicating this virus. Most recently, noncoding genes have been implicated in regulation of HIV expression. In this study, small RNA sequencing was used to profile expression of microRNAs (miRNAs) in a primary CD4⁺ T cell in vitro model of HIV latency. Previously, we have shown that protein-coding genes dysregulated in this model were enriched for the p53 signaling pathway, which was confirmed experimentally. We further found a link between p53 signaling and dysregulated long noncoding RNAs. In this study, we hypothesized that miRNAs may provide an additional level of regulation of the p53 signaling pathway during HIV latency. Twenty-six miRNAs were identified to be dysregulated in our latency model. A subset of these miRNAs was validated by real-time quantitative polymerase chain reaction. Predicted messenger RNA (mRNA) targets and cellular pathways enriched for mRNA targets were identified using several analytical methods. Our analyses showed that many protein-coding genes and pathways targeted by dysregulated miRNAs have relevance to regulation of HIV expression or establishment of HIV latency. The p53 signaling pathway was found among pathways that were targeted by dysregulated miRNAs at a greater level than expected by chance. This study provides a mechanistic insight into regulation of the p53 pathway through miRNAs that may contribute to the establishment of latency.

SUBMITTER: Heinson AI

PROVIDER: S-EPMC7876363 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCurrent antiretroviral therapy is effective in controlling HIV-1 infection. However, cessation of therapy is associated with rapid return of viremia from the viral reservoir. Eradicating the HIV-1 reservoir has proven difficult with the limited success of latency reactivation strategies and reflects the complexity of HIV-1 latency. Consequently, there is a growing need for alternate strategies. Here we explore a "block and lock" approach for enforcing latency to render the provirus unable to restart transcription despite exposure to reactivation stimuli. Reactivation of transcription from latent HIV-1 proviruses can be epigenetically blocked using promoter-targeted shRNAs to prevent productive infection. We aimed to determine if independent and combined expression of shRNAs, PromA and 143, induce a repressive epigenetic profile that is sufficiently stable to protect latently infected cells from HIV-1 reactivation when treated with a range of latency reversing agents (LRAs).ResultsJ-Lat 9.2 cells, a model of HIV-1 latency, expressing shRNAs PromA, 143, PromA/143 or controls were treated with LRAs to evaluate protection from HIV-1 reactivation as determined by levels of GFP expression. Cells expressing shRNA PromA, 143, or both, showed robust resistance to viral reactivation by: TNF, SAHA, SAHA/TNF, Bryostatin/TNF, DZNep, and Chaetocin. Given the physiological importance of TNF, HIV-1 reactivation was induced by TNF (5 ng/mL) and ChIP assays were performed to detect changes in expression of epigenetic markers within chromatin in both sorted GFP- and GFP+ cell populations, harboring latent or reactivated proviruses, respectively. Ordinary two-way ANOVA analysis used to identify interactions between shRNAs and chromatin marks associated with repressive or active chromatin in the integrated provirus revealed significant changes in the levels of H3K27me3, AGO1 and HDAC1 in the LTR, which correlated with the extent of reduced proviral reactivation. The cell line co-expressing shPromA and sh143 consistently showed the least reactivation and greatest enrichment of chromatin compaction indicators.ConclusionThe active maintenance of epigenetic silencing by shRNAs acting on the HIV-1 LTR impedes HIV-1 reactivation from latency. Our "block and lock" approach constitutes a novel way of enforcing HIV-1 "super latency" through a closed chromatin architecture that renders the virus resistant to a range of latency reversing agents.

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Micro RNA Targets in HIV Latency: Insights into Novel Layers of Latency Control.

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