Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
J Virol. 2018 Jun 13;92(13). doi: 10.1128/JVI.00368-18. Print 2018 Jul 1.
Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using and assays. Purified CPSF6-358 protein formed oligomers and bound -assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our biochemical and transmission electron microscopy data and intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358. After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.
剪接因子 6（CPSF6）是一种结合 HIV-1 衣壳并介导 HIV-1 前整合复合物核转运和整合靶向的人类蛋白。该蛋白在其 C 端核靶向精氨酸/丝氨酸富含（RS）结构域处的截断产生一种蛋白，CPSF6-358，通过靶向衣壳并抑制核进入，有力地抑制 HIV-1 感染。为了了解这种限制的分子机制，使用 和 测定法表征了 CPSF6-358 与 HIV-1 衣壳之间的相互作用。纯化的 CPSF6-358 蛋白形成寡聚体并结合 组装的野生型（WT）衣壳蛋白（CA）管，但不结合衣壳中存在 CPSF6 结合位点突变的 CA 管。有趣的是，CPSF6-358 寡聚体与 WT CA 管的结合物理上破坏了管状组装体成小片段。此外，固定和活细胞成像显示，稳定表达的 CPSF6-358 在 WT HIV-1 感染时形成细胞质斑点，并导致衣壳通透。当 HIV-1 衣壳包含已知可防止 CPSF6 结合的突变时，这些事件不会发生，当存在衣壳与 CPSF6-358 结合的小分子抑制剂时，这些事件也不会发生。总之，我们的 生化和透射电子显微镜数据和 细胞内成像结果为 CPSF6-358 的寡聚性质提供了第一个直接证据，并提出了 CPSF6-358 限制 HIV-1 感染的合理机制。进入细胞后，含有病毒基因组的 HIV-1 衣壳与许多宿主细胞因子相互作用，以促进复制所需的关键事件，包括脱壳。一种这样的宿主细胞因子，称为 CPSF6，主要位于细胞核内并与 HIV-1 衣壳相互作用。CA 与 CPSF6 之间的相互作用在 HIV-1 复制过程中至关重要。CPSF6 的截断导致其定位到细胞质并抑制 HIV-1 感染。在这里，我们确定截断的 CPSF6 蛋白形成直接结合 HIV-1 衣壳的大高级复合物，导致其破坏。细胞中截断的 CPSF6 表达导致过早的衣壳脱壳，这对 HIV-1 感染有害。我们的研究为截断的 CPSF6 的寡聚性质提供了第一个直接证据，并深入了解了 HIV-1 衣壳脱壳的高度调控过程。
