Abstract
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.
Highlights
Positive-strand (+)RNA viruses assemble numerous membrane-bound viral replicase complexes (VRCs) within large replication compartments/organelles to support their replication in infected cells [1,2,3,4,5]
The authors show that the syntaxin 18-like Ufe1 and Use1 endoplasmic reticulum (ER) SNARE proteins, which are present in ER subdomains called ERAS (ER arrival site), are necessary for the formation of the viral replication organelles
By binding to the p33 replication protein of Tomato bushy stunt virus (TBSV), Ufe1 and Use1 serve as an assembly hub for biogenesis of the replication compartment and facilitating the transfer of phospholipids and sterols to the growing sites of viral replication
Summary
Positive-strand (+)RNA viruses assemble numerous membrane-bound viral replicase complexes (VRCs) within large replication compartments/organelles to support their replication in infected cells [1,2,3,4,5]. In addition to the viral-coded replication proteins and the viral RNA (s), the VRCs consist of co-opted host proteins and subcellular membranes. Various (+)RNA viruses subvert or modify different subcellular compartments, such as endoplasmic reticulum (ER), mitochondria, peroxisomes, lysosomes/tonoplasts, chloroplasts or plasma membrane [4,6,7,8]. The detailed mechanism of how given subcellular compartments are subverted by (+)RNA viruses is incompletely understood. Tomato bushy stunt virus (TBSV) and other tombusviruses code for two viral replication proteins, termed p33 and p92pol, which are essential for virus replication [9,10,11]. TBSV recruits numerous host proteins, whose functions have been studied in some details [15,16,17]
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