Nucleoporin NUP153 Phenylalanine-Glycine Motifs Engage a Common Binding Pocket within the HIV-1 Capsid Protein to Mediate Lentiviral Infectivity

Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153C). NUP153C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153C) potently restricted HIV-1, providing an intracellular readout for the NUP153C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.

慢病毒（Lentiviruses）可感染非分裂细胞，多种细胞转运蛋白在慢病毒核输入与整合过程中发挥关键功能。我们此前的研究表明，HIV-1感染过程中，病毒衣壳（capsid, CA）蛋白依赖细胞源性核孔蛋白（nucleoporin, NUP）153；本研究进一步证实，CA N端结构域与富含苯丙氨酸-甘氨酸（FG）重复序列的NUP153 C端结构域（NUP153C）之间存在直接相互作用。将NUP153C与恒河猴Trim5α限制因子的效应结构域融合（即Trim-NUP153C）可强效抑制HIV-1，该体系可作为逆转录病毒感染过程中NUP153C与CA相互作用的细胞内检测指标。在此实验条件下，灵长类慢病毒与马传染性贫血病毒（equine infectious anemia virus, EIAV）均可结合NUP153C，该结果与体外纯化蛋白间的直接结合实验结果一致。此外，这类结合表型与病毒感染过程中内源性NUP153蛋白的需求具有相关性。诱变实验一致鉴定出了对结合作用与慢病毒感染性至关重要的NUP153C与CA残基。NUP153C内不同的FG基序分别介导HIV-1与EIAV衣壳的结合。HIV-1 CA的结合位点定位于共同的α螺旋3/4疏水口袋区域，该区域同时可结合小分子PF-3450074（PF74）抑制剂以及剪切与聚腺苷酸化特异性因子6（cleavage and polyadenylation specific factor 6, CPSF6），其中天冬酰胺57（Asn57，在EIAV中为Asp58）发挥尤为关键的作用。据此，PF74与CPSF6均可竞争性结合HIV-1 CA口袋，进而与NUP153C竞争结合HIV-1 CA；当细胞表达Trim-NUP153C或敲低NUP153时，需使用浓度显著更高的PF74才能抑制HIV-1感染。CA突变病毒的细胞周期依赖性与NUP153依赖性之间的相关性进一步表明，NUP153C与CA的相互作用是HIV-1感染非分裂细胞能力的基础。我们的研究结果揭示了不同宿主因子在病毒入侵过程中结合HIV-1 CA同一区域的相似机制。综上，部分慢病毒CA蛋白可直接结合NUP153上的FG基序，进而调控病毒的核输入过程。