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Background: Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases., Methodology/principal Findings: Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i) inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii) reduction of minus-strand synthesis by the tombusvirus replicase; and (iii) inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication., Conclusion/significance: Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses.

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]

Abstract: 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. [Copyright &y& Elsevier]

Host factors are recruited into viral replicase complexes to aid replication of plus-strand RNA viruses. In this paper, we show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bγ) reduces Tomato bushy stunt virus (TBSV) replication in yeast host. Also, knock down of eEF1Bγ level in plant host decreases TBSV accumulation. eEF1Bγ binds to the viral RNA and is one of the resident host proteins in the tombusvirus replicase complex. Additional in vitro assays with whole cell extracts prepared from yeast strains lacking eEF1Bγ demonstrated its role in minus-strand synthesis by opening of the structured 3' end of the viral RNA and reducing the possibility of re-utilization of (+)-strand templates for repeated (-)-strand synthesis within the replicase. We also show that eEF1Bγ plays a synergistic role with eukaryotic translation elongation factor 1A in tombusvirus replication, possibly via stimulation of the proper positioning of the viral RNA-dependent RNA polymerase over the promoter region in the viral RNA template. These roles for translation factors during TBSV replication are separate from their canonical roles in host and viral protein translation. [ABSTRACT FROM AUTHOR]

Abstract: Replication of plus-strand RNA viruses depends on lipids present in cellular membranes. Recent genome-wide screens have revealed that eight phospholipid biosynthesis genes affected the replication of Tomato bushy stunt virus (TBSV) in yeast model host. To test the importance of phospholipids in TBSV replication, we studied one of the identified genes, namely INO2, which forms a heterodimer with Ino4, and is a transcription activator involved in regulation of phospholipid biosynthesis. Deletion of INO2, or double deletion of INO2/INO4, reduced TBSV replication and inhibited the activity of the viral replicase complex. In addition, the stability of the viral replication protein is decreased as well as the localization pattern of the viral protein changed dramatically in ino2∆ino4∆ yeast. Over-expression of Opi1, a repressor of Ino2 and phospholipid biosynthesis, also inhibited TBSV RNA accumulation. In contrast, over-expression of Ino2 stimulated TBSV RNA accumulation. We also observed an inhibitory effect on Flock house virus (FHV) replication and the reduced stability of the FHV replication protein in ino2∆ino4∆ yeast. These data are consistent with the important role of phospholipids in RNA virus replication. [Copyright &y& Elsevier]

Positive-stranded RNA (+RNA) viruses exploit host cell machinery by subverting host proteins and membranes and altering cellular pathways during infection. To achieve robust replication, some +RNA viruses, such as poliovirus (PV), build special intracellular compartments, called viral replication organelles. A recent work from the Altan-Bonnett laboratory [1] gave new insights into the formation of poliovirus replication organelles, which are unique subcellular structures containing many individual replication complexes as a result of dynamic cellular membrane remodeling. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, these viruses might use RNA chaperones as shown here for the essential p33 replication protein of Tomato bushy stunt virus (TBSV). In vitro experiments demonstrate that the purified recombinant p33 promotes strand separation of a DNA/RNA duplex. In addition, p33 renders dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNAs. We also demonstrate that the RNA chaperone activity of p33 facilitates self-cleavage by a ribozyme in vitro. In addition, purified p33 facilitates in vitro RNA synthesis on double-stranded (ds)RNA templates up to 5-fold by a viral RNA-dependent RNA polymerase. We propose that the RNA chaperone activity of p33 facilitates the initiation of plus-strand synthesis as well as affects RNA recombination. Altogether, the TBSV RNA chaperone might perform similar biological functions to the helicases of other RNA viruses with much larger coding capacity. [Copyright &y& Elsevier]