PROJECT SUMMARY This longstanding NIH grant over its lifetime has made seminal discoveries on the mechanisms of intranuclear HIV-1 trafficking and nuclear localization. In prior funding cycles, we determined that the nuclear pore complex protein nucleoporin 153 interacted with the viral protein capsid to affect the translocation of incoming viral replication complexes into the cell nucleus and sites of viral DNA integration in the human genome. We also first identified the cellular alternate polyadenylation protein cleavage and polyadenylation specificity factor 6 (CPSF6) as a direct binding partner of the viral capsid and revealed an important role for this interaction in integration of the viral reverse transcript into active chromatin. Over the most recent funding cycle, our work has clarified that the capsid-CPSF6 interaction is critical for viral replication complexes to travel into the nuclear structure, where they colocalize with nuclear organelle structures that are known as nuclear speckles. Using novel bioinformatic tools, our work moreover revealed the significant preference for HIV-1 to integrate into regions of our genome that physically associate with nuclear speckles. In this way, our most recent research has clarified the granularity of nuclear architectural structure that most favors and most attracts HIV-1 to its chromosomal sites of integration. Moving forward, we will determine several unknown aspects of this fundamental virus-host interaction, including the mechanistic basis of CPSF6 action in HIV-1 intranuclear targeting as well as the functional consequences of mistargeting, wherein HIV-1 is known to uncharacteristically integrate into heterochromatic lamina-proximal sequences out towards the periphery of the nuclear structure. We furthermore will investigate the participations of other host factors that we have earmarked as behaving biochemically similar to CPSF6 for their roles in HIV-1 trafficking in the nucleus to preferred sites of integration. We will also investigate the functional consequences of these virus-host interactions in monocytic cell types, wherein HIV-1 replication complexes can be sensed by cellular innate machinery to counteract the infection process. The translational relevance of this basic scientific research is highlighted through the mechanism of action of capsid protein inhibitors such as lenacapavir, which are in late stage clinical trials and are known to inhibit the interaction of the HIV-1 capsid with Nup153 and CPSF6 proteins in vitro, and to induce HIV-1 integration retargeting in cells. Our proposed work in total will address fundamental aspects of virus-host interactions that occur during HIV-1 infection to shield the virus from sensing in innate immune cells, navigate integration to preferred genomic DNA sites, as well as the consequences of misguided integration on virus function and reactivation from latency.