Viral Replication and Interactions with Host Factors

Division Of Basic Sciences - Nci

Investigators

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Abstract

To identify viral determinants that are critical for nuclear import, we have screened many HIV-1 capsid protein (CA) mutants and have identified some CA mutants that exhibit a longer NE residence time before translocating into the nucleus. Interestingly, the delay in nuclear import kinetics also correlated with increased proviral integrations in chromatin near the NE, suggesting that some mutants uncoat while docked at the NE and their DNA integrates into nearby chromatin. To identify host factors that facilitate HIV-1 core nuclear import, we have carried out a high-throughput imaging-based siRNA screen and have identified several NPC-associated and non-NPC-associated host factors that facilitate the process. The central channel of the NPC is filled with disordered protein sequences enriched in phenylalanine-glycine (FG)-repeat motifs, which form a permeability barrier that restricts the diffusion of proteins >40 kD; cargos larger than 40 kD must bind to nuclear transport receptors (NTRs) to be escorted through the NPC. We have focused on NUP98, one of the strongest hits in the siRNA screen, and a major contributor of FG repeats to the NPC channel permeability barrier, to identify specific FG motifs of NUP98 that play a critical role in viral core nuclear import. We have found that while most of the FG motifs of NUP98 are dispensable, some of the FG motifs are essential for efficient HIV-1 core nuclear import. Our long-term goal is to understand which structural features of some FG motifs facilitate interactions with the HIV-1 core and their nuclear import. Additionally, in collaboration with Dr. Yong Xiong (Yale University, New Haven, CT), we determined that a single FG motif and a patch of basic residues near the C-terminus of NUP153 bind to HIV-1 cores and facilitates their nuclear entry. We will determine whether the C-terminal basic residues of other NUPs also facilitate HIV-1 core nuclear import. It is known that CPSF6 facilitates the import of viral cores from the NPC to the nucleus, but the mechanism of CPSF6-mediated nuclear import remains unclear. We are carrying out mutational analysis of CPSF6 to identify the CPSF6 determinants that are critical for binding to viral cores at the NPC and viral core translocation into the nucleus. We will test the hypothesis that HIV-1 cores remain intact until shortly before integration, in part, to evade host nuclear DNA sensors and suppress an interferon-mediated innate immune response.