Co-opted Cellular Sac1 Lipid Phosphatase and PI(4)P Phosphoinositide Are Key Host Factors during the Biogenesis of the Tombusvirus Replication Compartment.

Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication.IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.

Methylation of translation elongation factor 1A by the METTL10-like See1 methyltransferase facilitates tombusvirus replication in yeast and plants.

Replication of tombusviruses and other plus-strand RNA viruses depends on several host factors that are recruited into viral replicase complexes. Previous studies have shown that eukaryotic translation elongation factor 1A (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. In this paper, we show that methylation of eEF1A by the METTL10-like See1p methyltransferase is required for tombusvirus and unrelated nodavirus RNA replication in yeast model host. Similar to the effect of SEE1 deletion, yeast expressing only a mutant form of eEF1A lacking the 4 known lysines subjected to methylation supported reduced TBSV accumulation. We show that the half-life of several viral replication proteins is decreased in see1Δ yeast or when a mutated eEF1A was expressed as a sole source for eEF1A. Silencing of the plant ortholog of See1 methyltransferase also decreased tombusvirus RNA accumulation in Nicotiana benthamiana.

Tombusvirus replication depends on Sec39p endoplasmic reticulum-associated transport protein.

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.

The GEF1 proton-chloride exchanger affects tombusvirus replication via regulation of copper metabolism in yeast.

Replication of plus-strand RNA viruses [(+)RNA viruses] is performed by viral replicases, whose function is affected by many cellular factors in infected cells. In this paper, we demonstrate a surprising role for Gef1p proton-chloride exchanger in replication of Tomato bushy stunt virus (TBSV) model (+)RNA virus. A genetic approach revealed that Gef1p, which is the only proton-chloride exchanger in Saccharomyces cerevisiae, is required for TBSV replication in the yeast model host. We also show that the in vitro activity of the purified tombusvirus replicase from gef1Δ yeast was low and that the in vitro assembly of the viral replicase in a cell extract was inhibited by the cytosolic fraction obtained from gef1Δ yeast. Altogether, our data reveal that Gef1p modulates TBSV replication via regulating Cu(2+) metabolism in the cell. This conclusion is supported by several lines of evidence, including the direct inhibitory effect of Cu(2+) ions on the in vitro assembly of the viral replicase, on the activity of the viral RNA-dependent RNA polymerase, and an inhibitory effect of deletion of CCC2 copper pump on TBSV replication in yeast, while altered iron metabolism did not reduce TBSV replication. In addition, applying a chloride channel blocker impeded TBSV replication in Nicotiana benthamiana protoplasts or in whole plants. Overall, blocking Gef1p function seems to inhibit TBSV replication through altering Cu(2+) ion metabolism in the cytosol, which then inhibits the normal functions of the viral replicase.

Proteome-wide overexpression of host proteins for identification of factors affecting tombusvirus RNA replication: an inhibitory role of protein kinase C.

To identify host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model positive-stranded RNA virus, we overexpressed 5,500 yeast proteins individually in Saccharomyces cerevisiae, which supports TBSV replication. In total, we identified 141 host proteins, and overexpression of 40 of those increased and the remainder decreased the accumulation of a TBSV replicon RNA. Interestingly, 36 yeast proteins were identified previously by various screens, greatly strengthening the relevance of these host proteins in TBSV replication. To validate the results from the screen, we studied the effect of protein kinase C1 (Pkc1), a conserved host kinase involved in many cellular processes, which inhibited TBSV replication when overexpressed. Using a temperature-sensitive mutant of Pkc1p revealed a high level of TBSV replication at a semipermissive temperature, further supporting the idea that Pkc1p is an inhibitor of TBSV RNA replication. A direct inhibitory effect of Pkc1p was shown in a cell-free yeast extract-based TBSV replication assay, in which Pkc1p likely phosphorylates viral replication proteins, decreasing their abilities to bind to the viral RNA. We also show that cercosporamide, a specific inhibitor of Pkc-like kinases, leads to increased TBSV replication in yeast, in plant single cells, and in whole plants, suggesting that Pkc-related pathways are potent inhibitors of TBSV in several hosts.

Inhibition of sterol biosynthesis reduces tombusvirus replication in yeast and plants.

The replication of plus-strand RNA viruses depends on subcellular membranes. Recent genome-wide screens have revealed that the sterol biosynthesis genes ERG25 and ERG4 affected the replication of Tomato bushy stunt virus (TBSV) in a yeast model host. To further our understanding of the role of sterols in TBSV replication, we demonstrate that the downregulation of ERG25 or the inhibition of the activity of Erg25p with an inhibitor (6-amino-2-n-pentylthiobenzothiazole; APB) leads to a 3- to 5-fold reduction in TBSV replication in yeast. In addition, the sterol biosynthesis inhibitor lovastatin reduced TBSV replication by 4-fold, confirming the importance of sterols in viral replication. We also show reduced stability for the p92(pol) viral replication protein as well as a decrease in the in vitro activity of the tombusvirus replicase when isolated from APB-treated yeast. Moreover, APB treatment inhibits TBSV RNA accumulation in plant protoplasts and in Nicotiana benthamiana leaves. The inhibitory effect of APB on TBSV replication can be complemented by exogenous stigmasterol, the main plant sterol, suggesting that sterols are required for TBSV replication. The silencing of SMO1 and SMO2 genes, which are orthologs of ERG25, in N. benthamiana reduced TBSV RNA accumulation but had a lesser inhibitory effect on the unrelated Tobacco mosaic virus, suggesting that various viruses show different levels of dependence on sterol biosynthesis for their replication.

Inhibition of RNA recruitment and replication of an RNA virus by acridine derivatives with known anti-prion activities.

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.