We have previously described a C-terminally truncated form of CPSF6 (CPSF6-358) that has potent antiviral activity against different HIV-1, HIV-2, and SIV isolates, but no activity against EIAV, FIV, or MLV. CPSF6-358 interferes with the nuclear entry of HIV-1. We selected for HIV-1 replication in cells expressing CPSF6-358 and identified a mutation of N74D of CA as sufficient to confer viral resistance. We examined the nuclear pore dependencies for wild-type (WT) and N74D HIV-1 and found the isolates to require some of the same pore proteins and differ in requirements for others. In particular, N74D HIV-1 was less sensitive to depletion of TNPO3 (a karyopherin), NUP153, and NUP358/RanBP2 than WT HIV-1. These experiments established a genetic interaction between HIV-1 CA and the nuclear pore.We are now attempting to define the route that HIV-1 takes to the nucleus: the cell factors it interacts with and how it interacts with these factors. Our data suggest a direct interaction of wild-type CPSF6 with CA. We have also discovered TNPO3 to regulate CPSF6 nuclear localization. We are examining whether CPSF6 normally interacts with HIV-1 CA in the cytoplasm and whether this interaction is important for nuclear entry of the virus. Although experiments with transformed cells have not revealed a role for endogenous CPSF6 in HIV-1 infection, experiments with primary macrophages suggest a positive role in HIV-1 infection.