The cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules towards the nucleus. While it was initially believed that the reverse transcribed genome is released from the capsid in the cytosol, recent observations indicate that a high amount of capsid protein (CA) remains associated with subviral complexes during import through the nuclear pore complex (NPC). Observation of post-entry events via microscopic detection of HIV-1 CA is challenging, since epitope shielding limits immunodetection and the genetic fragility of CA hampers direct labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes revealed an amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells consistent with a complete capsid and showed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes.

中文翻译：

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通过遗传密码扩增对感染性 HIV-1 的直接衣壳标记允许检测大部分完整的核衣壳，并建议 HIV-1 复合物通过常见途径进入核

锥形成熟的 HIV-1 衣壳是早期病毒复制的主要协调者。进入细胞溶质后，它将病毒复制复合物沿着微管运送到细胞核。虽然最初认为逆转录的基因组是从胞质溶胶中的衣壳中释放出来的，但最近的观察表明，在通过核孔复合物 (NPC) 输入过程中，大量的衣壳蛋白 (CA) 仍然与亚病毒复合物相关。通过观察进入后的事件HIV-1 CA 的显微镜检测具有挑战性，因为表位屏蔽限制了免疫检测，并且 CA 的遗传脆弱性阻碍了直接标记方法。在这里，我们提出了一种基于遗传密码扩展和点击化学的微创策略，允许对 HIV-1 CA 进行定点荧光标记，同时保留病毒形态和传染性。因此，我们可以使用先进的显微镜直接观察病毒粒子和亚病毒复合物，包括纳米显微镜和相关成像。亚病毒复合物信号强度的量化揭示了与完整衣壳一致的 HeLa 衍生细胞和原代 T 细胞中的核复合物相关的 CA 量，并表明用小分子抑制剂 PF74 治疗不会导致衣壳从核复合物中解离。通过电子断层扫描在细胞核中检测到的锥形物体通过相关显微镜清楚地识别为衣壳衍生结构。高分辨率成像揭示了核衣壳的剂量依赖性聚集，这表明进入的粒子可能遵循共同的进入路线。