PROJECT SUMMARY Cleavage and polyadenylation specificity factor 6 (CPSF6) is an HIV host factor recruited to incoming viral cores during the early stage of the lifecycle. The interaction between HIV capsid (CA) and CPSF6 is known to dictate the cellular determinants of nuclear translocation and influence integration site preference, but its impact on overall viral infectivity is unclear. While overexpression of cytoplasmic CPSF6 has been shown to restrict viral replication, knock-down of CPSF6 or disruption of the CA-CPSF6 interaction has been reported to have a broad range of infectivity phenotypes. Recently, our lab found that knock-out of CPSF6 in primary CD4+ T cells dramatically increases HIV replication with minimal impact on cell viability. This increase in replication correlates with decreased induction of interferon-stimulated genes, contrary to prior reports that suggest CPSF6 acts to shield the virus from immune recognition. In this proposal, we are testing the overall hypothesis that CPSF6 plays a critical role in regulating the innate immune response to HIV infection and that the virus recruits CPSF6 in part to circumvent this response. This hypothesis will be tested in three aims that broadly seek to understand the mechanism by which loss of CPSF6 dampens the immune response to infection, the impact of HIV infection on CPSF6 function, and the potential role of CPSF6 regulatory pathways in controlling infection. In Aim 1, we will test the hypothesis that loss of CPSF6 acts to dampen the immune response directly by induction of alternative polyadenylation (APA) or indirectly by allowing enhanced recruitment of Cyclophilin A (CYPA) to protect the core from restriction by the antiviral factor TRIM5. CPSF6 normally acts as a member of the CFIm cleavage factor complex to direct polyadenylation to distal sites of the 3' untranslated region (UTR). Inhibition of CFIm activity triggers APA, which has been previously implicated in the regulation of the innate immune response, and could explain the observed phenotype. Alternatively, there is evidence to suggest that CPSF6 and CYPA compete for core binding and loss of CYPA binding has been previously linked to enhanced restriction and innate sensing. In Aim 2, we will test the hypothesis that CPSF6 recruitment by incoming viral cores can alter overall CFIm function and induce APA. Regardless if this is linked to dampening of the immune response above, it is well established that other viruses hijack the APA pathway to enhance their replication, though this hasn't been explored during HIV infection. Finally, in Aim 3, we will test the hypothesis that perturbation of the CPSF6 regulatory network can control viral infectivity and the immune response to infection. CPSF6 activity is regulated by post-translational modification and nuclear-cytoplasmic shuttling. Truncation mutants of CPSF6 that force cytoplasmic localization have been shown to restrict HIV infection, and we will test if we can mimic ...