Viral Replication and Interactions with Host Factors

HIV-1必须穿过细胞质到达被感染细胞的核膜（NE），通过核孔进入细胞核，并将其基因组整合到宿主细胞的染色体DNA中。在这个过程中，HIV-1必须将其RNA基因组逆转录成双链DNA，保护其基因组和蛋白质免受宿主降解酶的侵害，并避免触发宿主防御，从而抑制其复制。这些进入后的事件很难用生物化学方法研究，因为每个细胞中很少有病毒复合物；此外，由于没有有效标记和跟踪病毒复合体的方法，很难从微观上研究这些事件。我们以前观察到进入细胞核的HIV-1病毒复合物停留在核外周，并不是随机分布在整个细胞核中。我们试图确定病毒复合体是否在靠近核外围的位置整合，类似于病毒复合体的位置。我们开发了原位RNA和DNA荧光原位杂交（FISH）来鉴定转录活性原病毒的核位置。我们发现，感染后24小时转录活性原病毒的分布与未整合的病毒复合体非常相似，表明病毒复合体在核周围附近的位点整合。我们还鉴定了感染后5天转录活性位点的核位置，发现转录活性原病毒是随机分布的。这些结果表明HIV-1在靠近核外周的位点整合；然而，经过几次细胞分裂后，整合位点的位置变得随机。这些结果表明，不同的基因存在于靶细胞群体的核外围，因此基本上整个基因组都可用于HIV-1。此外，结果表明基因在细胞中的位置不是固定的，在细胞之间是不同的。HIV-1病毒衣壳核、逆转录酶和RNA的复合体融合后进行逆转录。在病毒DNA整合到宿主基因组之前，病毒核心必须分解（脱衣），但病毒核心脱衣发生的地点和时间尚不清楚。由于无法直接标记病毒衣壳蛋白（CA）并量化感染后不同阶段与病毒复合物相关的CA的数量，脱包衣的研究一直受到阻碍。我们开发了一种用绿色荧光蛋白（GFP）在感染性病毒衣壳上直接标记CA的新方法，其传染性损失最小。我们对这种标记方法进行了表征，并观察到当只有5-10%的CA蛋白被GFP标记时，病毒复合物保留了大部分感染性，具有正常的核心稳定性，并与亲环蛋白a结合。这种强大的GFP-CA标记方法将有助于未来对HIV-1复制早期阶段的研究。我们使用直接gfp - ca标记方法，通过活细胞显微镜来深入了解感染细胞的病毒核心剥膜。我们观察到，与目前的细胞质或ne相关病毒核心剥离模型相反，大多数病毒CA仍然与核病毒复合物相关。核病毒复合体在整合前经历一个快速的核脱壳过程。我们还发现核病毒复合物对衣壳抑制剂PF74仍然敏感，这表明核复合物保留了CA六聚体。这些突破性的研究改变了我们对病毒复制早期阶段和病毒核心剥离的看法。我们在《美国国家科学院院刊》（Burdick et al., PNAS 117: 5486-5493, 2020）上发表的描述这项工作的论文被PNAS的编辑选为Cozzarelli奖，作为生物医学科学类别中发表的300篇论文中最重要的出版物。《美国国家科学院院刊》在https://youtu.be/6X3f-1vDeC8和https://traffic.libsyn.com/secure/pnas-science-sessions-podcast/vinayPathakPodcast.mp3上发布了对主要作者Vinay Pathak的采访视频和播客。我们之前的研究表明，病毒复合体具有较长的NE停留时间，这表明在病毒复制过程中，病毒复合体通过核孔的易位是一个困难且耗时的步骤。然而，我们对病毒复合体与宿主蛋白之间的分子相互作用知之甚少，而这种相互作用是核输入所必需的。为了深入了解病毒复制中这一重要但鲜为人知的步骤，我们已经确定了表现出NE停留时间增加和核输入延迟的CA突变体。先前的研究表明，与正常病毒复合物相比，不与宿主因子亲环蛋白A结合的病毒复合物使用不同的核输入途径。我们的结果表明，CA突变体表现出核输入延迟涉及到与病毒复合物结合的亲环蛋白a。这些研究为病毒复合体转运进入细胞核的机制提供了新的见解。

HIV-1 must travel through the cytoplasm to reach the nuclear envelope (NE) of an infected cell, transport through a nuclear pore to enter the nucleus, and integrate its genome into the chromosomal DNA of the host cell. During this process, HIV-1 must reverse transcribe its RNA genome into double-strand DNA, protect its genome and proteins from host degradative enzymes, and avoid triggering host defenses that would inhibit its replication. These post-entry events are difficult to study biochemically because there are very few viral complexes in each cell; in addition, it has been difficult to study these events microscopically because methods to efficiently label and track viral complexes have not been available. ___We previously observed that HIV-1 viral complexes that enter the nucleus remain at the nuclear periphery and are not randomly distributed throughout the nucleus. We sought to determine whether viral complexes integrate at sites near the nuclear periphery similar to the location of the viral complexes. We developed in situ RNA and DNA fluorescence in situ hybridization (FISH) to identify the nuclear locations of transcriptionally active proviruses. We found that the distribution of transcriptionally active proviruses 24 hours after infection was very similar to that of unintegrated viral complexes, indicating that the viral complexes integrate at sites near the nuclear periphery. We also characterized the nuclear locations of transcriptionally active sites 5 days after infection and found that transcriptionally active proviruses were randomly distributed. These results indicate that HIV-1 integrates at sites near the nuclear periphery; however, the locations of the integration sites become random after a few cell divisions. These results imply that different genes are present at the nuclear periphery in a population of target cells such that essentially the entire genome is available to HIV-1. In addition, the results imply that locations of genes in cells are not fixed and differ from cell to cell. ___After fusion, the complex of HIV-1 viral capsid core, reverse transcriptase, and RNA carry out reverse transcription. The viral core must disassemble (uncoat) before integration of the viral DNA into the host genome, but where and when viral core uncoating occurs are not well understood. Studies of uncoating have been hampered by the inability to directly label viral capsid protein (CA) and quantify the amount of CA that remains associated with viral complexes at various stages after infection. We have developed a new method to directly label CA with green fluorescent protein (GFP) in infectious viral capsids with minimal loss of infectivity. We have characterized this labeling method and observed that when only 5-10% of the CA proteins are GFP labeled, the viral complexes retain most of their infectivity, have normal core stability, and bind to cyclophilin A. This robust GFP-CA-labeling method will facilitate future studies of the early stage of HIV-1 replication. ___We have used the direct GFP-CA-labeling method to gain insights into viral core uncoating in infected cells by live-cell microscopy. We observed that, contrary to current models of cytoplasmic or NE-associated viral core uncoating, most viral CA remains associated with nuclear viral complexes. The nuclear viral complexes undergo a rapid nuclear uncoating event just prior to integration. We also found that the nuclear viral complexes remain sensitive to capsid inhibitor PF74, indicating that the nuclear complexes retain CA hexamers. These groundbreaking studies have transformed our view of the early stage of viral replication and viral core uncoating. Our publication describing this work in the Proceedings of the National Academy of the United States of America (Burdick et al., PNAS 117: 5486-5493, 2020) was selected by Editors of PNAS to receive the Cozzarelli Prize as the most important publication from 300 papers published in the category of Biomedical Sciences. PNAS featured interviews with lead author Vinay Pathak about this award-winning publication in a video at https://youtu.be/6X3f-1vDeC8 and podcast at https://traffic.libsyn.com/secure/pnas-science-sessions-podcast/vinayPathakPodcast.mp3. ___Our previous studies showed that viral complexes exhibit long NE residence times, indicating that translocation of the viral complexes through the nuclear pore is a difficult and time-consuming step during viral replication. However, very little is known about the molecular interactions between viral complexes and host proteins that are essential for nuclear import. To gain insights into this essential yet poorly understood step in viral replication, we have identified CA mutants that exhibit increased NE residence time and a delay in nuclear import. Previous studies have indicated that viral complexes that are not bound to host factor cyclophilin A use a different nuclear import pathway compared with normal viral complexes. Our results indicate that the CA mutants that exhibit a delay in nuclear import involve cyclophilin A binding to the viral complexes. These studies provide novel insights into the mechanism of viral complex translocation into the nucleus.