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Co-opted oxysterol-binding ORP and VAP proteins channel sterols to RNA virus replication sites via membrane contact sites.

Barajas D, Xu K, de Castro Martín IF, Sasvari Z, Brandizzi F, Risco C, Nagy PD - PLoS Pathog. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
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.

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Model on the role of ORP and VAP proteins and MCSs in tombusvirus replicase assembly.The ORP proteins, such as the yeast Osh6p, are recruited via binding to the tombusviral p33 replication protein and the ER-bound Scs2p VAP to MCS, formed between the ER and peroxisomal membranes. The ORPs then facilitate the enrichment of sterols in the peroxisomal membrane (or mitochondrial membrane in case of CIRV), forming sterol-rich microdomains needed for the formation of virus-induced spherules. These spherules contain the VRCs performing viral RNA synthesis. Note that the MCSs are not part of the VRCs based on the difference in BiFC localization of p33:Ssa1p Hsp70 complex and p33:Scs2p, which suggests that different subpopulations of p33 are involved in each interaction. We have previously shown that the Ssa1p is present in the active VRCs.
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ppat-1004388-g008: Model on the role of ORP and VAP proteins and MCSs in tombusvirus replicase assembly.The ORP proteins, such as the yeast Osh6p, are recruited via binding to the tombusviral p33 replication protein and the ER-bound Scs2p VAP to MCS, formed between the ER and peroxisomal membranes. The ORPs then facilitate the enrichment of sterols in the peroxisomal membrane (or mitochondrial membrane in case of CIRV), forming sterol-rich microdomains needed for the formation of virus-induced spherules. These spherules contain the VRCs performing viral RNA synthesis. Note that the MCSs are not part of the VRCs based on the difference in BiFC localization of p33:Ssa1p Hsp70 complex and p33:Scs2p, which suggests that different subpopulations of p33 are involved in each interaction. We have previously shown that the Ssa1p is present in the active VRCs.

Mentions: In this paper, we show that members of lipid transfer proteins, namely the oxysterol-binding ORP proteins are recruited by tombusviruses to the sites of viral replication. The direct binding of Osh3/5/6/7 to the tombusvirus p33 replication protein leads to the enrichment of these ORPs in peroxisomal (or in mitochondrial in case of CIRV) and ER subcompartments. This also leads to enrichment of sterols at the site of viral replication. We propose that the subverted ORP proteins likely facilitate the re-distribution of sterols to viral sites (Fig. 8) at the expense of their natural plasma membrane localization. The enrichment of sterols in the membranes around the replicase complex likely promotes viral RNA replication as we observed more efficient TBSV repRNA replication in artificial PE vesicles containing cholesterol or ergosterol.


Co-opted oxysterol-binding ORP and VAP proteins channel sterols to RNA virus replication sites via membrane contact sites.

Barajas D, Xu K, de Castro Martín IF, Sasvari Z, Brandizzi F, Risco C, Nagy PD - PLoS Pathog. (2014)

Model on the role of ORP and VAP proteins and MCSs in tombusvirus replicase assembly.The ORP proteins, such as the yeast Osh6p, are recruited via binding to the tombusviral p33 replication protein and the ER-bound Scs2p VAP to MCS, formed between the ER and peroxisomal membranes. The ORPs then facilitate the enrichment of sterols in the peroxisomal membrane (or mitochondrial membrane in case of CIRV), forming sterol-rich microdomains needed for the formation of virus-induced spherules. These spherules contain the VRCs performing viral RNA synthesis. Note that the MCSs are not part of the VRCs based on the difference in BiFC localization of p33:Ssa1p Hsp70 complex and p33:Scs2p, which suggests that different subpopulations of p33 are involved in each interaction. We have previously shown that the Ssa1p is present in the active VRCs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199759&req=5

ppat-1004388-g008: Model on the role of ORP and VAP proteins and MCSs in tombusvirus replicase assembly.The ORP proteins, such as the yeast Osh6p, are recruited via binding to the tombusviral p33 replication protein and the ER-bound Scs2p VAP to MCS, formed between the ER and peroxisomal membranes. The ORPs then facilitate the enrichment of sterols in the peroxisomal membrane (or mitochondrial membrane in case of CIRV), forming sterol-rich microdomains needed for the formation of virus-induced spherules. These spherules contain the VRCs performing viral RNA synthesis. Note that the MCSs are not part of the VRCs based on the difference in BiFC localization of p33:Ssa1p Hsp70 complex and p33:Scs2p, which suggests that different subpopulations of p33 are involved in each interaction. We have previously shown that the Ssa1p is present in the active VRCs.
Mentions: In this paper, we show that members of lipid transfer proteins, namely the oxysterol-binding ORP proteins are recruited by tombusviruses to the sites of viral replication. The direct binding of Osh3/5/6/7 to the tombusvirus p33 replication protein leads to the enrichment of these ORPs in peroxisomal (or in mitochondrial in case of CIRV) and ER subcompartments. This also leads to enrichment of sterols at the site of viral replication. We propose that the subverted ORP proteins likely facilitate the re-distribution of sterols to viral sites (Fig. 8) at the expense of their natural plasma membrane localization. The enrichment of sterols in the membranes around the replicase complex likely promotes viral RNA replication as we observed more efficient TBSV repRNA replication in artificial PE vesicles containing cholesterol or ergosterol.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus