Department of Plant Pathology, University of Kentucky, Lexington, KY, United States of America.
PLoS Pathog. 2018 May 10;14(5):e1007028. doi: 10.1371/journal.ppat.1007028. eCollection 2018 May.
Positive-strand RNA viruses assemble numerous membrane-bound viral replicase complexes within large replication compartments to support their replication in infected cells. Yet the detailed mechanism of how given subcellular compartments are subverted by viruses is incompletely understood. Although, Tomato bushy stunt virus (TBSV) uses peroxisomal membranes for replication, in this paper, we show evidence that the ER-resident SNARE (soluble NSF attachment protein receptor) proteins play critical roles in the formation of active replicase complexes in yeast model host and in plants. Depletion of the syntaxin 18-like Ufe1 and Use1, which are components of the ER SNARE complex in the ERAS (ER arrival site) subdomain, in yeast resulted in greatly reduced tombusvirus accumulation. Over-expression of a dominant-negative mutant of either the yeast Ufe1 or the orthologous plant Syp81 syntaxin greatly interferes with tombusvirus replication in yeast and plants, thus further supporting the role of this host protein in tombusvirus replication. Moreover, tombusvirus RNA replication was low in cell-free extracts from yeast with repressed Ufe1 or Use1 expression. We also present evidence for the mislocalization of the tombusviral p33 replication protein to the ER membrane in Ufe1p-depleted yeast cells. The viral p33 replication protein interacts with both Ufe1p and Use1p and co-opts them into the TBSV replication compartment in yeast and plant cells. The co-opted Ufe1 affects the virus-driven membrane contact site formation, sterol-enrichment at replication sites, recruitment of several pro-viral host factors and subversion of the Rab5-positive PE-rich endosomes needed for robust TBSV replication. In summary, we demonstrate a critical role for Ufe1 and Use1 SNARE proteins in TBSV replication and propose that the pro-viral functions of Ufe1 and Use1 are to serve as assembly hubs for the formation of the extensive TBSV replication compartments in cells. Altogether, these findings point clearly at the ERAS subdomain of ER as a critical site for the biogenesis of the TBSV replication compartment.
正链 RNA 病毒在大型复制腔内组装大量膜结合的病毒复制酶复合物，以支持其在感染细胞中的复制。然而，关于特定亚细胞区室如何被病毒颠覆的详细机制尚不完全清楚。尽管番茄丛矮病毒 (TBSV) 使用过氧化物酶体膜进行复制，但在本文中，我们提供的证据表明内质网驻留 SNARE（可溶性 NSF 附着蛋白受体）蛋白在酵母模型宿主和植物中对于活性复制酶复合物的形成起着关键作用。在酵母中，耗尽内质网 SNARE 复合物的成分 syntaxin 18 样 Ufe1 和 Use1，即内质网到达部位 (ERAS) 亚域中的内质网 SNARE 复合物的组成部分，会导致 TBSV 积累大大减少。在酵母和植物中，过表达酵母 Ufe1 或同源植物 Syp81 yntaxin 的显性负突变体，会极大地干扰 TBSV 的复制，从而进一步支持该宿主蛋白在 TBSV 复制中的作用。此外，在 Ufe1 或 Use1 表达受抑制的酵母细胞无细胞提取物中，TBSV RNA 复制水平较低。我们还提供了证据，证明 TBSV 复制蛋白 p33 在 Ufe1p 耗尽的酵母细胞中错误定位到内质网膜上。病毒 p33 复制蛋白与 Ufe1p 和 Use1p 相互作用，并将它们募集到酵母和植物细胞中的 TBSV 复制腔内。被募集的 Ufe1 影响病毒驱动的膜接触位点形成、复制部位的固醇富集、几个促病毒宿主因子的招募以及用于稳健 TBSV 复制的 Rab5 阳性 PE 丰富内体的颠覆。总之，我们证明了 Ufe1 和 Use1 SNARE 蛋白在 TBSV 复制中的关键作用，并提出 Ufe1 和 Use1 的促病毒功能是作为形成细胞中广泛的 TBSV 复制腔的组装中心。总的来说，这些发现清楚地表明内质网 AS 亚域是 TBSV 复制腔生物发生的关键部位。
