Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. Human immunodeficiency virus type 1 (HIV-1) and other lentiviruses mediate the infection of non-dividing cells through the ability of the capsid protein¹ to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex^2,3,4. Although recent studies have observed the presence of the capsid protein in the nucleus during infection^5,6,7,8, reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore complex blockade to monitor the kinetics of HIV-1 nuclear import and define the biochemical staging of these steps of infection. Surprisingly, we observe that nuclear import occurs with relatively rapid kinetics (<5 h) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription is completed in the nucleus. We also observe that HIV-1 remains susceptible to the capsid-destabilizing compound PF74 following nuclear import, revealing that uncoating is completed in the nucleus. Additionally, we observe that certain capsid mutants are insensitive to a Nup62-mediated nuclear pore complex blockade in cells that potently block infection by wild-type capsid, demonstrating that HIV-1 can use distinct nuclear import pathways during infection. These studies collectively define the spatio-temporal staging of critical steps of HIV-1 infection and provide an experimental system to separate and thereby define the cytoplasmic and nuclear stages of infection by other viruses.

逆转录病毒感染涉及将病毒 RNA 基因组逆转录为 DNA，然后将其整合到宿主细胞基因组中。^{人类免疫缺陷病毒 1 型 (HIV-1) 和其他慢病毒通过衣壳蛋白1}参与靶细胞的细胞核输入途径并通过核成分介导其核易位的能力介导非分裂细胞的感染。孔隙复合物^2,3,4。尽管最近的研究已经观察到感染期间细胞核中存在衣壳蛋白^5,6,7,8，病毒核心的逆转录和分解通常被认为是细胞质事件。在这里，我们使用诱导型核孔复合物阻断来监测 HIV-1 核输入的动力学并定义这些感染步骤的生化分期。令人惊讶的是，我们观察到核输入以相对快速的动力学（<5 小时）发生并且在靶细胞中完成逆转录之前，表明逆转录在细胞核中完成。我们还观察到 HIV-1 在核输入后仍然容易受到衣壳不稳定化合物 PF74 的影响，这表明脱壳是在细胞核中完成的。此外，我们观察到某些衣壳突变体对细胞中 Nup62 介导的核孔复合物阻断不敏感，这种阻断可有效阻断野生型衣壳的感染，这表明 HIV-1 在感染期间可以使用不同的核输入途径。这些研究共同定义了 HIV-1 感染关键步骤的时空分期，并提供了一个实验系统来分离并由此定义其他病毒感染的细胞质和细胞核阶段。