PROJECT SUMMARY / ABSTRACT There are more than 38 million people living with HIV-1 infection worldwide, and more than 1.1 million in the United States. HIV-1 acute infection and latency reversal are both dependent on viral expression of full-length unspliced viral RNAs (US-vRNAs) that serve dual roles in the cytoplasm as either (1) viral mRNAs encoding Gag and Gag-Pol proteins that drive virus particle assembly or (2) viral RNA genome substrates bound by Gag/Gag- Pol for packaging into virions. This project combines live cell and superresolution imaging, biochemical assays, and other research tools to address key foundational issues in HIV-1 US-vRNA nuclear export and virion assembly relevant to informing the development of new antiviral strategies to target these crucial stages. Aim 1 will define where and when in the nucleus US-vRNAs interface with viral and cellular components of the nuclear export machinery. Our preliminary data have demonstrated surprisingly little overlap between the subcellular trafficking behaviors of retroviral US-vRNAs and the Rev and Rex proteins that mediate their nuclear export through the cellular Exportin-1 (XPO1, also known as CRM1) nuclear export pathway. We will test the hypothesis that key interactions between HIV-1 Rev and US-vRNAs are highly transient, using single molecule detection coupled to an innovative “cell expansion” superresolution light microscopy technique. Our goal is to precisely define the subnuclear sites of Rev-US-vRNA and Rev-XPO1 interaction and study the effects of prescribed perturbations to Rev function on these essential processes. Based on our progress from prior studies of a species-specific block to Rev function in mouse cells, Aim 2 will determine why nuclear Rev-US-vRNA complexes need to recruit multiple XPO1 proteins to achieve efficient nuclear export. We will test the hypothesis that Rev drives XPO1 dimerization in human cells but not mouse cells and attempt to achieve cell-intrinsic resistance to HIV-1 in human CD4+ T cells by inactivation of the putative species-specific XPO1 multimerization domain using precision gene editing. For Aim 3, we will use live cell imaging to test the hypothesis that Rev-dependent nuclear export licenses viral RNAs for enhanced stability and genome packaging in the cytoplasm. These experiments will take advantage of newly described mutant forms of HIV-1 that allow us to, for the first time, study US-vRNAs programmed for either translation or packaging independently. Collectively, these studies will provide new mechanistic insights into the viral and cellular machines that drive HIV-1 genome trafficking, advance the development of new cell-based assays and imaging technologies for studying HIV-host interactions, and test translationally relevant strategies for the targeted inactivation of HIV-1 genomes in vivo.