Delving into a Unique Transcription Factor Family to Regulate HIV Transcription

ABSTRACT The persistence of latently infected memory CD4+ T cells releasing a low-level trickling of viral RNAs and proteins in people living with HIV (PLWH) on antiretroviral therapy (ART) provides a strong rationale for the development of cure strategies. HIV functional cure approaches are being explored, at the transcriptional level, to achieve sustained ART-free viral remission that would result from epigenetic changes over time. These approaches are supported by evidence in PLWH who spontaneously control their viral load below the limit of detection, in the absence of ART (elite controllers, ECs). These ECs were shown to have a large proportion of their proviruses in condensed chromatin (heterochromatin) loci, with virus refractory to reactivation. The chromatin environment surrounding the HIV promoter is believed to be regulated by host transcription factors, host chromatin remodelers and the viral Tat protein. Our hypothesis is that the pioneer factor (PF) family may also remodel the HIV locus chromatin, via their unique epigenetic modulator properties, resulting in the regulation of HIV transcription. PFs play critical roles in chromatin remodeling, mostly during development and disease phenotypes. Unique properties of PFs include 1) trigger assembly of regulatory factors on target DNA in heterochromatin; 2) bookmark genes for rapid reactivation/repression and 3) induce persistent epigenetic modulation of the cellular chromatin. A few PFs were reported to modulate HIV latency and persistence in primary CD4+ T cells but were poorly studied mechanistically. Preliminary data using shRNAs targeting 13 PFs in the HIV latently infected CD4+ T cells, revealed 3 novel hits that hindered HIV transcription. This data further highlights the significance of uncovering the role played by the PFs on HIV transcription in CD4+ T cells, with the long-term goal to harness them for HIV cure approaches and/or diagnostic purposes. Here, we propose to: Aim 1. Screen a shRNAmir library targeting 31 PFs (and controls) by flow cytometry, taking advantage of the HIV reporter pMorpheus-V5 vector in Jurkat T cells. This vector allows the distinction of productively infected, latently infected, and uninfected cells. The shRNAmir relative abundance in these populations will reflect their activity on HIV transcription. The follow-up of the candidates will be based namely on reproducibility and novelty. Aim 2. Validate the hits identified in preliminary data and Aim 1. Their activity will be confirmed in cell line models of latency with different HIV transcriptional environments, and in primary blood CD4+ T cells from uninfected and virally suppressed ART treated female and male donors since estrogen modulates certain PFs’ activity. Their impact on CD4+ T cell proliferation, activation and cytotoxicity will also be measured. Aim 3. Characterize the 3 hits identified in preliminary data. Their recruitment to the host/HIV promoters (ChIP-Seq analysis), their modulation of the HIV chromatin (MNase nucleosomal mapping, enChIP-MS) and transcriptional regulation (RNA Seq analysis) in cell models of HIV latency will be studied for their comprehensive transcriptional regulatory profiling. Cell based- and in vitro- assays will define their dependence to HIV Tat.