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Positive-stranded RNA viruses subvert subcellular membranes to built viral replicases complexes (VRCs) in infected cells. Tombusviruses use peroxisomal membranes for the assembly of their VRCs and they can efficiently switch to the endoplasmic reticulum membrane in the absence of peroxisomes. In this paper, we show that the ER-resident Sec39p vesicular transport protein is critical for the formation of active VRCs in yeast model host. Repression of Sec39p expression in yeast or in plants resulted in greatly reduced tombusvirus accumulation. Moreover, the purified tombusvirus replicase from Sec39p-depleted yeast cells showed low in vitro activity. Also, tombusvirus RNA replication was poor in cell-free extracts or in isolated ER membranes from yeast with repressed Sec39p expression. The tombusvirus p33 replication protein was mislocalized to the ER when Sec39p was depleted in yeast. Overall, Sec39p is the first peroxisomal biogenesis protein characterized that is critical for tombusvirus replication in yeast and plants., (© 2013 Elsevier Inc. All rights reserved.)

Replication of Tomato bushy stunt virus (TBSV) RNA takes place on the cytosolic membrane surface of peroxisomes in plants and in yeast, a model host. To identify the host proteins involved in assisting the peroxisomal localization of the tombusvirus p33 replication protein, we tested if p33 could bind directly to yeast proteins involved in peroxisomal transport in vitro. This work has led to the demonstration of Pex19p-p33 interaction via pull-down and co-purification experiments. Pex19p was also detected in the tombusvirus replicase after protein cross-linking, suggesting that Pex19p transiently binds to the replicase as could be expected from a transporter. To validate the importance of Pex19p-p33 interaction in TBSV replication in yeast, we re-targeted Pex19p to the mitochondria, which resulted in the re-distribution of a large fraction of p33 to the mitochondria. The expression of the mitochondrial-targeted Pex19p inhibited TBSV RNA accumulation by 2-4-fold in vivo and reduced the in vitro activity of the tombusvirus replicase by 80%. These data support the model that Pex19p is a cellular transporter for localization of p33 replication protein to the host peroxisomal membranes.

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 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. [ABSTRACT FROM AUTHOR]

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. [ABSTRACT FROM AUTHOR]

Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication. [ABSTRACT FROM AUTHOR]