Project Summary/Abstract Current anti-HIV-1 therapies prevent progression to AIDS but do not cure infection. HIV-1 persists in long-lived memory CD4+ T cells as a transcriptionally silent provirus, where it is undetectable by the immune system, and therefore resistant to extirpation. Recently, we reported that primate immunodeficiency virus Vpx and Vpr proteins activate HIV-1 provirus transcription by degrading the three proteins of the human silencing hub (HUSH) complex. Disruption of the HUSH complex in bulk CD4+ T cells increased transcription from HIV-1 proviruses and kinetics of HIV-1 spreading infections, indicating that the HUSH complex plays a dominant role in HIV-1 provirus silencing. Nonetheless, examination of individual clones showed heterogeneity in response to HUSH disruption, and inconsistent correlation with known silencing factors such as SETDB1. Aim 1 will be to identify requirements for HUSH complex silencing of the HIV-1 provirus. Sets of CD4+ T cell clones bearing HIV-1 proviruses that exhibit a range of HUSH responsiveness will be subjected to loss-of-function screens to identify host silencing factors that distinguish clones with different HUSH phenotypes. Such factors will be characterized independently for effects on provirus transcription and provirus chromatin features. From these experiments we expect to better understand how HUSH is recruited to, and maintains transcriptional silencing of, HIV-1 proviruses. Aim 2 will be to examine the role of the HUSH complex in CD4+ T cell transcription and development. Disruption of the HUSH complex activates LINE-1 expression in certain cell lines raising questions about possible consequences of HUSH complex disruption. Global transcription and chromatin profiling will be performed on primary human CD4+ T cells in which HUSH complex components are disrupted. Increased expression of particular retrotransposons is expected, but also immune-related genes of relevance to HIV-1, and markers that may be used to monitor HUSH complex activity in cells. Examination of transcription factor motifs within lost ATAC-Seq peaks will aid identification of DNA-binding proteins that recruit the HUSH complex. The HUSH complex will also be disrupted in cord blood human CD34+ hematopoietic stem cells used to reconstitute an immune system in mice. These experiments will tell us whether the HUSH complex is essential for human hematopoietic development generally or for CD4+ T cells specifically. Aim 3 will assess the contribution of the HUSH complex to HIV-1 latency in vivo. The effect of HUSH complex inactivation on HIV-1 provirus reactivation will be examined with CD4+ T cells harvested from HIV-1+ individuals on anti-HIV-1 suppressive therapy an...