Characterization of full-lengh CPSF6 and its interaction with HIV capsid

PROPOSAL SUMMARY / ABSTRACT: HIV is a retrovirus that achieves infection through integration of its viral DNA into the host genome of non- dividing cells. To execute this function, HIV must ensure the nuclear import of its reverse-transcribed genetic material, which is protected inside of a proteinaceous fullerene cone known as the capsid. As the dimensions of the capsid are larger than the conventionally accepted width of the nuclear-pore-complex (NPC), it was long thought that disassembly of the capsid in the host cytoplasm was essential for HIV’s genomic material to gain access to the nucleus. Recent work has demonstrated that the central channel of the NPC is highly dynamic and able to accommodate an intact HIV capsid; subsequently several groups have directly observed assembled capsids within the nuclear compartment. Work from the last decade suggests that the cellular protein CPSF6 (Cleavage and Polyadenylation Specificity Factor 6) governs the nuclear import of viral genomic material through interactions with the HIV capsid. The work outlined in this proposal, broken into three independent aims, seeks to provide biochemical and structural understanding of this interaction. In AIM1, I will use real-time binding techniques to provide insight into the biophysical principles of the CPSF6-capsid interactions. I hypothesize that CPSF6 has a previously unappreciated binding site at the tri-hexamer interface of the HIV capsid. In AIM2, I will employ our library of capsid assemblies to determine the structure of CPSF6 bound to the capsid protein and confirm these structural features with functional assays. Finally, in AIM3, I will employ a DNA origami mimic of the NPC in an in vitro reconstitution dissect CPSF6’s contribution to nuclear import of the HIV capsid. I hypothesize that CPSF6 can compete with Nup153 and thereby release the capsid from the nuclear basket.