Abstract
The human immunodeficiency virus type 1 (HIV-1) can infect fully differentiated, nondividing cells by interacting with the host nuclear pore complex. The host nuclear pore protein NUP153 directly engages with the HIV-1 capsid to promote viral nuclear entry. This study used a multidisciplinary approach combining computational and experimental techniques to comprehensively map the effect of mutating the amino acids of NUP153 on HIV capsid interaction, revealing that the HIV capsid made specific contact with six amino acids of NUP153 (PxxVFTFG). The simplicity of the capsid-interacting motif (CbM.1, capsid-binding motif 1) suggested the possibility that other FxFG-containing motifs could interact with the HIV-1 capsid. Accordingly, three additional FxFG-containing motifs (CbM.2, CbM.3, and CbM.4) homologous to CbM.1 were identified on NUP153, were predicted to bind to HIV-1 capsid and target the same pocket on the capsid where CbM.1 binds to. Strikingly, the loss of CbM.3 appeared to improve the co-sedimentation of NUP153C with recombinant capsid tubes. Using cell-based viral transduction assay, in vitro binding assay, and surface binding assay, it was identified that one of the three motifs (CbM.3, P1222 - G1229) could physically bind to the HIV-1 capsid. To explore the biological relevance of these FxFG motifs of NUP153C, RNAi was first used to knock down NUP153 expression in human cells. As expected, complementation with NUP153 lacking a functional CbM.1 significantly reduced HIV-1 transduction, in addition, loss of CbM.1 can be fully rescued by CbM.3. Conversely, mutation of CbM.3 improved transduction by ~50%, and increased 2-LTR circles by 30-fold compared to the wild-type. In contrast to the supportive role of CbM.1 during HIV-1 nuclear entry, our data suggest that CbM.3 antagonizes this process. This antagonism was not a general feature of FxFG motifs as the effect was specific to CbM.3 not seen with the other two motifs. As CbM.3 binds the same pocket on the capsid as CbM.1, our data suggested that these two motifs compete for capsid binding, with the overall effect of reducing HIV-1 nuclear ingress. Our work is likely to improve our current understanding of how FxFG motifs in the nuclear pore complex contribute to HIV-1 viral nuclear import.