Department of Global Health, University of Washington, Seattle, WA, United States of America.
PLoS Pathog. 2018 Apr 17;14(4):e1006977. doi: 10.1371/journal.ppat.1006977. eCollection 2018 Apr.
During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.
在不成熟衣壳组装过程中，HIV-1 基因组包装是通过一个尚未完全了解的过程启动的，该过程中 Gag 首先与未剪接的 HIV-1 RNA 结合。此前，我们定义了 HIV-1 衣壳组装中间产物的顺序细胞内途径；在这里，我们试图确定 HIV-1 Gag 首先与未剪接的 HIV-1 RNA 结合的中间产物。在表达前病毒的细胞中，未剪接的 HIV-1 RNA 不在细胞质的可溶部分中，而是主要存在于≥30S 的复合物中。我们没有在由可溶性 Gag 组成的第一个组装中间产物中检测到与未剪接的 HIV-1 RNA 结合的 HIV-1 Gag。相反，我们检测到的与未剪接的 HIV-1 RNA 结合的最早的组装中间产物是第二个组装中间产物（80S 中间产物），它来自于一个含有两种组装辅助因子 ABCE1 和 RNA 颗粒蛋白 DDX6 的宿主 RNA 颗粒。在稳定状态下，无论裂解物中是否含有完整或破坏的核糖体，或者是否表达 WT 或组装缺陷型 Gag，这个源自 RNA 颗粒的80S 复合物都是含有与未剪接病毒 RNA 结合的 Gag 的最小组装中间产物。在 HIV-1 感染的 T 细胞中也鉴定到了类似的复合物。在原位检测到 RNA 颗粒衍生的组装中间产物，Gag 与 ABCE1 和 DDX6 共定位；此外，这些颗粒比研究较多的 P 体 RNA 颗粒更为丰富和更小。最后，我们确定了导致组装中的 Gag 与未剪接的 HIV-1 RNA 结合的两个步骤。独立于病毒 RNA 结合，Gag 与一大类主要缺乏未剪接病毒 RNA 的 RNA 颗粒结合（步骤 1）。如果存在病毒 RNA 结合域，Gag 进一步定位于包含未剪接病毒 RNA 的这些颗粒的子集（步骤 2）。因此，我们的数据提出了这样一种可能性，即 HIV-1 包装不是由可溶性 Gag 启动，而是由靶向含有未剪接 HIV-1 RNA 的宿主 RNA 颗粒的 Gag 启动。
