Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.
Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.
复制标题
DOI：
10.1371/journal.ppat.1006977
复制
发表时间：
2018-04
期刊：
影响因子：
6.7
通讯作者：
Lingappa JR
中科院分区：
文献类型：
--
作者：
Barajas BC;Tanaka M;Robinson BA;Phuong DJ;Chutiraka K;Reed JC;Lingappa JR
在不成熟的衣壳组装过程中，当Gag首先通过一个知之甚少的过程与未剪接的HIV-1 RNA结合时，HIV-1基因组包装开始。以前，我们定义了一个连续的细胞内HIV-1衣壳组装中间体的途径;在这里，我们试图确定中间体，其中HIV-1 Gag首先与未剪接的HIV-1 RNA相关联。在表达前病毒的细胞中，未剪接的HIV-1 RNA在胞质溶胶的可溶性部分中未发现，而是大部分在≥ 30 S的复合物中。我们在第一个组装中间体中没有检测到与Gag相关的未剪接的HIV-1 RNA，该中间体由可溶性Gag组成。相反，我们检测到与未剪接的HIV-1 RNA相关的Gag的最早组装中间体是第二个组装中间体（~ 80 S中间体），它来自含有两个细胞组装促进剂ABCE 1和RNA颗粒蛋白DDX 6的宿主RNA颗粒。在稳态下，这种RNA颗粒衍生的~ 80 S复合物是含有与未剪接病毒RNA相关的Gag的最小组装中间体，无论裂解物是否含有完整或破坏的核糖体，或表达WT或组装缺陷型Gag。在HIV-1感染的T细胞中发现了类似的复合物。RNA颗粒衍生的组装中间体被检测到在原位的加格与ABCE 1和DDX 6共定位的网站，此外，这些颗粒是更多的和更小的比充分研究的RNA颗粒称为P机构。最后，我们确定了两个步骤，导致组装Gag与未剪接的HIV-1 RNA的关联。与病毒RNA结合无关，Gag与大量缺乏未剪接病毒RNA的RNA颗粒结合（步骤1）。如果存在病毒RNA结合结构域，Gag进一步定位于含有未剪接病毒RNA的这些颗粒的子集（步骤2）。因此，我们的数据提出了这样的可能性，即HIV-1包装不是由可溶性Gag启动的，而是由Gag靶向含有未剪接HIV-1 RNA的宿主RNA颗粒的子集。在HIV-1未成熟衣壳组装期间，当HIV-1衣壳蛋白Gag首先与未剪接的HIV-1 RNA结合时，病毒基因组的包装开始。虽然这种结合最初发生的复合物对于感染性病毒的形成至关重要，但这种复合物形成的身份、组成和机制仍然未知。为了解决这个问题，我们利用了先前描述的HIV-1不成熟衣壳组装中间体的时间途径。该途径中的晚期中间产物来源于宿主RNA颗粒，其是用于细胞RNA储存和降解的不同复合物。在这里，我们试图确定细胞内衣壳装配中间体，其中HIV-1 Gag最初与未剪接的HIV-1 RNA相关联。我们未能检测到含有可溶性Gag的第一个组装中间体与未剪接的HIV-1 RNA之间的关联。相反，Gag和未剪接的HIV-1 RNA之间的关联仅在对应于RNA颗粒衍生的组装中间体的复合物中观察到。我们还发现，Gag使用两个决定簇来形成含有未剪接的HIV-1 RNA的RNA颗粒衍生的中间体。总之，这些研究支持HIV-1基因组包装的新模型，其中HIV-1 Gag和未剪接的HIV-1 RNA之间的第一个关联发生在宿主RNA颗粒内。
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. During HIV-1 immature capsid assembly, packaging of the viral genome is initiated when the HIV-1 capsid protein, Gag, first associates with unspliced HIV-1 RNA. Although the complex in which this association initially occurs is critical for formation of infectious virus, the identity, composition, and the mechanism by which this complex forms remain unknown. To address this question, we utilized a previously described temporal pathway of intermediates in HIV-1 immature capsid assembly. The late intermediates in this pathway are derived from host RNA granules, which are diverse complexes utilized for cellular RNA storage and degradation. Here we sought to identify the intracellular capsid assembly intermediate in which HIV-1 Gag initially associates with unspliced HIV-1 RNA. We failed to detect an association between the first assembly intermediate, which contains soluble Gag, and unspliced HIV-1 RNA. Instead, the association between Gag and unspliced HIV-1 RNA was observed only in complexes corresponding to the RNA-granule-derived assembly intermediates. We also showed that Gag uses two determinants to form RNA-granule-derived intermediates that contain unspliced HIV-1 RNA. Together, these studies support a novel model for HIV-1 genome packaging, in which the first association between HIV-1 Gag and unspliced HIV-1 RNA occurs within a host RNA granule.
登录
查看更多内容
影响因子：
5.4
作者：
Cimarelli, A;Luban, J
通讯作者：
Luban, J
影响因子：
5.4
作者：
Anderson, Emma C.;Lever, Andrew M. L.
通讯作者：
Lever, Andrew M. L.
影响因子：
5.4
作者：
Burdick, Ryan;Smith, Jessica L.;Pathak, Vinay K.
通讯作者：
Pathak, Vinay K.
宿主 ABCE1 位于质膜 HIV 组装位点，其与 Gag 的解离与随后的病毒产生事件相关。
影响因子：
4.5
作者：
Dooher, Julia E.;Schneider, Bobbie L.;Reed, Jonathan C.;Lingappa, Jaisri R.
通讯作者：
Lingappa, Jaisri R.
影响因子：
3.3
作者：
Chatel-Chaix, Laurent;Boulay, Karine;Mouland, Andrew J.;DesGroseillers, Luc
通讯作者：
DesGroseillers, Luc