HIV-1 infects host cells by fusion at the plasma membrane, leading to cytoplasmic entry of the viral capsid encasing the genome and replication machinery. The capsid eventually needs to disassemble, but time and location of uncoating are not fully characterized and may vary depending on the host cell. To study the fate of the capsid by fluorescence and superresolution (STED) microscopy, we established an experimental system that allows discrimination of subviral structures in the cytosol from intact virions at the plasma membrane or in endosomes without genetic modification of the virus. Quantitative microscopy of infected SupT1-R5 cells revealed that the CA signal on cytosolic HIV-1 complexes corresponded to ∼50% of that found in virions at the cell surface, in agreement with dissociation of nonassembled CA molecules from entering capsids after membrane fusion. The relative amount of CA in postfusion complexes remained stable until they reached the nuclear pore complex, while subviral structures in the nucleus of infected cells lacked detectable CA. An HIV-1 variant defective in binding of the host protein cleavage and polyadenylation specificity factor 6 (CPSF6) exhibited accumulation of CA-positive subviral complexes close to the nuclear envelope without loss of infectivity; STED microscopy revealed direct association of these complexes with nuclear pores. These results support previous observations indicating capsid uncoating at the nuclear pore in infected T-cell lines. They suggest that largely intact HIV-1 capsids dock at the nuclear pore in infected SupT1-R5 cells, with CPSF6 being a facilitator of nucleoplasmic entry in this cell type, as has been observed for infected macrophages.IMPORTANCE The HIV-1 capsid performs essential functions during early viral replication and is an interesting target for novel antivirals. Thus, understanding molecular and structural details of capsid function will be important for elucidating early HIV-1 (and retroviral in general) replication in relevant target cells and may also aid antiviral development. Here, we show that HIV-1 capsids stay largely intact during transport to the nucleus of infected T cells but appear to uncoat upon entry into the nucleoplasm. These results support the hypothesis that capsids protect the HIV-1 genome from cytoplasmic defense mechanisms and target the genome toward the nucleus. A protective role of the capsid could be a paradigm that also applies to other viruses. Our findings raise the question of how reverse transcription of the HIV-1 genome is accomplished in the context of the capsid structure and whether the process is completed before the capsid is uncoated at the nuclear pore.

中文翻译：

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SupT1-R5细胞中早期HIV-1复制复合物的CA含量和CPSF6依赖性分析。

HIV-1通过融合在质膜上感染宿主细胞，从而导致病毒衣壳的胞质进入，从而包裹了基因组和复制机制。衣壳最终需要拆卸，但是脱衣的时间和位置尚未完全确定，并且可能会因宿主细胞而异。为了通过荧光和超分辨率（STED）显微镜研究衣壳的命运，我们建立了一个实验系统，该系统可以从质膜或内体的完整病毒粒子中分辨出细胞质中的亚病毒结构，而无需对该病毒进行遗传修饰。感染的SupT1-R5细胞的定量显微镜检查显示，胞质HIV-1复合物上的CA信号相当于细胞表面病毒体中CA信号的约50％，与膜融合后未组装的CA分子解离进入衣壳的方式一致。融合后复合物中CA的相对量保持稳定，直到到达核孔复合体为止，而被感染细胞核中的亚病毒结构缺乏可检测的CA。在宿主蛋白裂解和聚腺苷酸化特异性因子6（CPSF6）结合方面存在缺陷的HIV-1变体在靠近核被膜的区域显示出CA阳性亚病毒复合物的积累，而不会丧失感染力。STED显微镜显示这些复合物与核孔直接相关。这些结果支持了先前的观察结果，表明在感染的T细胞系中核衣壳处于脱壳状态。他们认为，完整无缺的HIV-1衣壳位于受感染的SupT1-R5细胞的核孔中，CPSF6可以促进这种细胞类型的核质进入，正如感染巨噬细胞所观察到的那样。重要信息HIV-1衣壳在早期病毒复制过程中起着至关重要的作用，是新型抗病毒药物的有趣靶标。因此，了解衣壳功能的分子和结构细节对于阐明相关靶细胞中的早期HIV-1（通常为逆转录病毒）复制非常重要，也可能有助于抗病毒的发展。在这里，我们表明，HIV-1衣壳在转运到被感染的T细胞核期间基本上保持完整，但在进入核质后似乎会脱壳。这些结果支持了这样的假说：衣壳保护HIV-1基因组免受细胞质防御机制的侵害，并将基因组靶向细胞核。衣壳的保护作用可能是一种范例，也适用于其他病毒。我们的发现提出了一个问题，即在衣壳结构的背景下如何完成HIV-1基因组的逆转录，以及该过程是否在衣壳未在核孔中被包被之前完成。