PROJECT SUMMARY (See instructions): Antiretroviral therapy (ART) suppresses HIV-1 viremia but does not cure HIV-1 infection. Inability to cure is attributed to HIV-1 proviruses in long-lived memory CD4+ T cells, most of which are transcriptionally silent and resistant to extirpation. Better understanding of the factors that regulate provirus transcription could offer new strategies for effective cure of HIV-1 infection. This grant proposal stems from our observation that vpx/vpr from across primate immunodeficiency virus phylogeny increase provirus transcription by degrading the Human Silencing Hub (HUSH) complex. The immediate goals are to better understand host and viral determinants of HUSH-mediated transcriptional silencing of the provirus. Aim 1 will be to use unbiased, genome-wide, HUSH loss-of-function screens to identify host factors required in trans for HUSH-mediated silencing. We will also identify cellular proteins near HUSH-silenced proviruses using a dCas9–APEX2 fusion protein (C-BERST) that is targeted to proviruses with sgRNAs. Functional relevance of hits will be confirmed and mechanistic hypotheses about how these factors contribute to provirus silencing will be tested. We will assess the contribution of our hits to HIV-1 latency by quantitating cell-associated HIV1 RNA after candidate gene disruption in stimulated CD4+ T cells from people living with HIV-1 on ART. Aim 2 will be to characterize cis-acting requirements for detection and silencing by HUSH. Promoters and transcripts from HUSH targeted proviruses and transgenes will be engineered to finely map and characterize the determinants of HUSH sensitivity. HIV-1 sequences cloned from people living with HIV-1 will be tested to determine if HUSH exerts selective pressure on the virus during establishment of HIV-1 latency in vivo. If HIV-1 infection is to be treated by perturbing HUSH, the consequences of these interventions on CD4+ T cell function and development need to be better understood. In Aim 3, the effect of HUSH on the chromatin landscape in 1° CD4+ T cells will be assessed using RNASeq, ATAC-Seq, and CUT&Tag for multiple chromatin features. HUSH-sensitive genes identified here, which include endogenous retroviruses, will be used iteratively as reporters for screens in Aim 1. HUSH components will be knocked out in human CD34+ hematopoietic stem cells (HSCs) and these cells will be used to reconstitute a model immune system in which the effect of HUSH knockout on development and chromatin landscape of CD4+ T cells will be assessed. Finally, the effect of HUSH knockout on HIV-1 replication and establishment of latency will be assessed within this experimental model of HIV-1 infection.

RELEVANCE (See instruction Anti-HIV-1 drugs prevent HIV-1-infected people from developing AIDS but the drugs do not cure infection because HIV-1 becomes a permanent genetic element in human chromosomes that is called a provirus. We discovered that host cell proteins called the HUSH complex silence the HIV-1 provirus, making it difficult for drugs, or the immune system, to detect and eliminate the virus. The experiments proposed here, which are intended to better understand how the HUSH silences the HIV-1 provirus, are hoped to provide information that will facilitate efforts to Cure HIV-1 infection.