Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
Institute for Biomedical Technologies, CNR, Segrate, Milan, Italy.
J Virol. 2018 Sep 12;92(19). doi: 10.1128/JVI.00648-18. Print 2018 Oct 1.
Human immunodeficiency virus type 1 (HIV-1) displays the unique ability to infect nondividing cells. The capsid of HIV-1 is the viral determinant for viral nuclear import. To understand the cellular factors involved in the ability of HIV-1 to infect nondividing cells, we sought to find capsid mutations that allow the virus to infect dividing but not nondividing cells. Because the interaction of capsid with the nucleoporin protein 153 (Nup153) is important for nuclear import of HIV-1, we solved new crystal structures of hexameric HIV-1 capsid in complex with a Nup153-derived peptide containing a phenylalanine-glycine repeat (FG repeat), which we used to guide structure-based mutagenesis of the capsid-binding interface. HIV-1 viruses with mutations in these capsid residues were tested for their ability to infect dividing and nondividing cells. HIV-1 viruses with capsid N57 substitutions infected dividing but not nondividing cells. Interestingly, HIV-1 viruses with N57 mutations underwent reverse transcription but not nuclear translocation. The mutant capsids also lost the ability to interact with Nup153 and CPSF6. The use of small molecules PF74 and BI-2 prevented the interaction of FG-containing nucleoporins (Nups), such as Nup153, with the HIV-1 core. Analysis of integration sites in HIV-1 viruses with N57 mutations revealed diminished integration into transcriptionally active genes in a manner resembling that of HIV-1 in CPSF6 knockout cells or that of HIV-1-N74D. The integration pattern of the N57 mutant HIV-1 can be explained by loss of capsid interaction with CPSF6, whereas capsid interaction with Nup153 is required for HIV-1 to infect nondividing cells. Additionally, the observed viral integration profiles suggested that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin. One of the key advantages that distinguish lentiviruses, such as HIV-1, from all other retroviruses is its ability to infect nondividing cells. Interaction of the HIV-1 capsid with Nup153 and CPSF6 is important for nuclear entry and integration; however, the contribution of each of these proteins to nuclear import and integration is not clear. Using genetics, we demonstrated that these proteins contribute to different processes: Nup153 is essential for the HIV-1 nuclear import in nondividing cells, and CPSF6 is important for HIV-1 integration. In addition, nuclear factors such as CPSF6 and the state of the chromatin are known to be important for integration site selection; nevertheless, the preferential determinant influencing integration site selection is not known. This work demonstrates that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin.
人类免疫缺陷病毒 1 型（HIV-1）表现出感染非分裂细胞的独特能力。HIV-1 的衣壳是病毒核内输入的病毒决定因素。为了了解 HIV-1 感染非分裂细胞的能力涉及的细胞因子，我们试图寻找允许病毒感染分裂但不非分裂细胞的衣壳突变。由于衣壳与核孔蛋白 153（Nup153）的相互作用对于 HIV-1 的核内输入很重要，我们解决了六聚体 HIV-1 衣壳与包含苯丙氨酸-甘氨酸重复（FG 重复）的 Nup153 衍生肽复合物的新晶体结构，我们使用该结构来指导衣壳结合界面的基于结构的诱变。测试了具有这些衣壳残基突变的 HIV-1 病毒感染分裂和非分裂细胞的能力。具有衣壳 N57 取代的 HIV-1 病毒可感染分裂但不非分裂细胞。有趣的是，具有 N57 突变的 HIV-1 病毒进行了逆转录但未进行核转位。突变的衣壳也失去了与 Nup153 和 CPSF6 相互作用的能力。小分子 PF74 和 BI-2 的使用阻止了包含 FG 的核孔蛋白（Nups）与 HIV-1 核心的相互作用，如 Nup153。对具有 N57 突变的 HIV-1 病毒整合位点的分析表明，整合到转录活跃基因中的程度类似于 CPSF6 敲除细胞中的 HIV-1 或 HIV-1-N74D。N57 突变 HIV-1 的整合模式可以通过衣壳与 CPSF6 的相互作用丧失来解释，而衣壳与 Nup153 的相互作用对于 HIV-1 感染非分裂细胞是必需的。此外，观察到的病毒整合图谱表明，整合位点选择是一个多参数过程，取决于核因子和细胞染色质的状态。区分 HIV-1 等慢病毒与所有其他逆转录病毒的一个关键优势是其感染非分裂细胞的能力。HIV-1 衣壳与 Nup153 和 CPSF6 的相互作用对于核进入和整合很重要；然而，这些蛋白质中的每一个对核输入和整合的贡献尚不清楚。通过遗传学，我们证明这些蛋白质参与不同的过程：Nup153 对于非分裂细胞中的 HIV-1 核输入是必需的，而 CPSF6 对于 HIV-1 整合很重要。此外，核因子（如 CPSF6）和染色质状态已知对整合位点选择很重要；然而，影响整合位点选择的优先决定因素尚不清楚。这项工作表明，整合位点选择是一个多参数过程，取决于核因子和细胞染色质的状态。
