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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 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.

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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Related in: MedlinePlus

Interaction between p33 replication protein and yeast oxysterol-binding Osh proteins.(A) Co-purification of the yeast Osh6p and Osh7p proteins with the p33 replication protein. Top panel: Western blot analysis of co-purified HA-tagged cellular proteins with Flag-affinity purified p33 from isolated membrane fraction of yeast cells. Osh6p and Osh7p were detected with anti-HA antibody. The negative control was His6-tagged p33 purified from yeast extracts using a FLAG-affinity column. Middle panel: Western blot of purified Flag-p33 detected with anti-FLAG antibody. Bottom panel: Western blot of HA-tagged Osh6p and Osh7p proteins in the total yeast extracts using anti-HA antibody. (B) Decreased TBSV repRNA accumulation in osh3,5,6,7Δ yeast. To launch TBSV repRNA replication, we expressed His6-p33 from the galactose-inducible GAL1 promoter, His6-p92 from the copper-inducible CUP1 promoter and DI-72(+) repRNA from the galactose-inducible GAL10 promoter in the parental (SEY6210) and in osh3,5,6,7Δ yeast strains. His6-tagged Osh3, 5, 6, 7 were expressed from GAL1 promoter. The yeast cells were pre-cultured for 24 hours at 23°C in 2% glucose SC minimal media, and then for 48 h at 23°C in 2% galactose SC minimal media supplemented with 50 µM CuSO4. Northern blot analysis was used to detect DI-72(+) repRNA accumulation. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA levels. Bottom panel: Western blot analysis of the accumulation level of His6-tagged p33, His6-p92 and His6-Osh proteins using anti-His antibodies. Each experiment was performed three times. (C) Decreased accumulation of the mitochondrial CIRV in osh3,5,6,7Δ yeast. See further details in Panel B. (D) Scheme of the in vitro tombusvirus replicase assay based on yeast CFEs and purified recombinant TBSV replication proteins. (E) Reduced activity of the tombusvirus replicase assembled in CFE from osh3,5,6,7Δ yeast. Denaturing PAGE analysis of in vitro tombusvirus replicase activity in the CFEs. Note that this image shows the repRNAs made by a full cycle of replicase activity, producing both (−) and (+)-strands, in vitro. The CFEs contained the same amount of total yeast proteins (not shown). Each experiment was performed three times.
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ppat-1004388-g001: Interaction between p33 replication protein and yeast oxysterol-binding Osh proteins.(A) Co-purification of the yeast Osh6p and Osh7p proteins with the p33 replication protein. Top panel: Western blot analysis of co-purified HA-tagged cellular proteins with Flag-affinity purified p33 from isolated membrane fraction of yeast cells. Osh6p and Osh7p were detected with anti-HA antibody. The negative control was His6-tagged p33 purified from yeast extracts using a FLAG-affinity column. Middle panel: Western blot of purified Flag-p33 detected with anti-FLAG antibody. Bottom panel: Western blot of HA-tagged Osh6p and Osh7p proteins in the total yeast extracts using anti-HA antibody. (B) Decreased TBSV repRNA accumulation in osh3,5,6,7Δ yeast. To launch TBSV repRNA replication, we expressed His6-p33 from the galactose-inducible GAL1 promoter, His6-p92 from the copper-inducible CUP1 promoter and DI-72(+) repRNA from the galactose-inducible GAL10 promoter in the parental (SEY6210) and in osh3,5,6,7Δ yeast strains. His6-tagged Osh3, 5, 6, 7 were expressed from GAL1 promoter. The yeast cells were pre-cultured for 24 hours at 23°C in 2% glucose SC minimal media, and then for 48 h at 23°C in 2% galactose SC minimal media supplemented with 50 µM CuSO4. Northern blot analysis was used to detect DI-72(+) repRNA accumulation. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA levels. Bottom panel: Western blot analysis of the accumulation level of His6-tagged p33, His6-p92 and His6-Osh proteins using anti-His antibodies. Each experiment was performed three times. (C) Decreased accumulation of the mitochondrial CIRV in osh3,5,6,7Δ yeast. See further details in Panel B. (D) Scheme of the in vitro tombusvirus replicase assay based on yeast CFEs and purified recombinant TBSV replication proteins. (E) Reduced activity of the tombusvirus replicase assembled in CFE from osh3,5,6,7Δ yeast. Denaturing PAGE analysis of in vitro tombusvirus replicase activity in the CFEs. Note that this image shows the repRNAs made by a full cycle of replicase activity, producing both (−) and (+)-strands, in vitro. The CFEs contained the same amount of total yeast proteins (not shown). Each experiment was performed three times.

Mentions: Tombusvirus replication greatly depends on sterols [47], which are distributed in cells via vesicle transfer and sterol-binding ORP proteins in a vesicle independent pathway [53], [55]. Therefore, we have tested if TBSV replication proteins interact with the yeast ORPs, named Osh1-7p, to facilitate sterol transfer in virus-infected cells. Our co-purification experiments with the affinity-purified tombusvirus p33 replication protein from isolated membranous fraction of yeast model host showed that four of the seven yeast Osh proteins, which are lipid-transfer proteins involved in oxysterol/sterol-binding, were associated with the membrane-bound p33 (Fig. 1A and S1A). These yeast proteins, namely Osh3p, Osh5p, Osh6p and Osh7p, were not only efficiently co-purified with p33 replication protein, but they also bound directly to p33 in a pull-down assay (Fig. S1B) and interacted with p33 in a membrane (split-ubiquitin-based) yeast two-hybrid assay (Fig. S1C). Co-purification of Osh4p was less robust with p33 from membranous fraction of yeast, while the co-purified Osh1p and Osh2p were close to the detection limit (Fig. S1A).


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)

Interaction between p33 replication protein and yeast oxysterol-binding Osh proteins.(A) Co-purification of the yeast Osh6p and Osh7p proteins with the p33 replication protein. Top panel: Western blot analysis of co-purified HA-tagged cellular proteins with Flag-affinity purified p33 from isolated membrane fraction of yeast cells. Osh6p and Osh7p were detected with anti-HA antibody. The negative control was His6-tagged p33 purified from yeast extracts using a FLAG-affinity column. Middle panel: Western blot of purified Flag-p33 detected with anti-FLAG antibody. Bottom panel: Western blot of HA-tagged Osh6p and Osh7p proteins in the total yeast extracts using anti-HA antibody. (B) Decreased TBSV repRNA accumulation in osh3,5,6,7Δ yeast. To launch TBSV repRNA replication, we expressed His6-p33 from the galactose-inducible GAL1 promoter, His6-p92 from the copper-inducible CUP1 promoter and DI-72(+) repRNA from the galactose-inducible GAL10 promoter in the parental (SEY6210) and in osh3,5,6,7Δ yeast strains. His6-tagged Osh3, 5, 6, 7 were expressed from GAL1 promoter. The yeast cells were pre-cultured for 24 hours at 23°C in 2% glucose SC minimal media, and then for 48 h at 23°C in 2% galactose SC minimal media supplemented with 50 µM CuSO4. Northern blot analysis was used to detect DI-72(+) repRNA accumulation. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA levels. Bottom panel: Western blot analysis of the accumulation level of His6-tagged p33, His6-p92 and His6-Osh proteins using anti-His antibodies. Each experiment was performed three times. (C) Decreased accumulation of the mitochondrial CIRV in osh3,5,6,7Δ yeast. See further details in Panel B. (D) Scheme of the in vitro tombusvirus replicase assay based on yeast CFEs and purified recombinant TBSV replication proteins. (E) Reduced activity of the tombusvirus replicase assembled in CFE from osh3,5,6,7Δ yeast. Denaturing PAGE analysis of in vitro tombusvirus replicase activity in the CFEs. Note that this image shows the repRNAs made by a full cycle of replicase activity, producing both (−) and (+)-strands, in vitro. The CFEs contained the same amount of total yeast proteins (not shown). Each experiment was performed three times.
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Related In: Results  -  Collection

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ppat-1004388-g001: Interaction between p33 replication protein and yeast oxysterol-binding Osh proteins.(A) Co-purification of the yeast Osh6p and Osh7p proteins with the p33 replication protein. Top panel: Western blot analysis of co-purified HA-tagged cellular proteins with Flag-affinity purified p33 from isolated membrane fraction of yeast cells. Osh6p and Osh7p were detected with anti-HA antibody. The negative control was His6-tagged p33 purified from yeast extracts using a FLAG-affinity column. Middle panel: Western blot of purified Flag-p33 detected with anti-FLAG antibody. Bottom panel: Western blot of HA-tagged Osh6p and Osh7p proteins in the total yeast extracts using anti-HA antibody. (B) Decreased TBSV repRNA accumulation in osh3,5,6,7Δ yeast. To launch TBSV repRNA replication, we expressed His6-p33 from the galactose-inducible GAL1 promoter, His6-p92 from the copper-inducible CUP1 promoter and DI-72(+) repRNA from the galactose-inducible GAL10 promoter in the parental (SEY6210) and in osh3,5,6,7Δ yeast strains. His6-tagged Osh3, 5, 6, 7 were expressed from GAL1 promoter. The yeast cells were pre-cultured for 24 hours at 23°C in 2% glucose SC minimal media, and then for 48 h at 23°C in 2% galactose SC minimal media supplemented with 50 µM CuSO4. Northern blot analysis was used to detect DI-72(+) repRNA accumulation. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA levels. Bottom panel: Western blot analysis of the accumulation level of His6-tagged p33, His6-p92 and His6-Osh proteins using anti-His antibodies. Each experiment was performed three times. (C) Decreased accumulation of the mitochondrial CIRV in osh3,5,6,7Δ yeast. See further details in Panel B. (D) Scheme of the in vitro tombusvirus replicase assay based on yeast CFEs and purified recombinant TBSV replication proteins. (E) Reduced activity of the tombusvirus replicase assembled in CFE from osh3,5,6,7Δ yeast. Denaturing PAGE analysis of in vitro tombusvirus replicase activity in the CFEs. Note that this image shows the repRNAs made by a full cycle of replicase activity, producing both (−) and (+)-strands, in vitro. The CFEs contained the same amount of total yeast proteins (not shown). Each experiment was performed three times.
Mentions: Tombusvirus replication greatly depends on sterols [47], which are distributed in cells via vesicle transfer and sterol-binding ORP proteins in a vesicle independent pathway [53], [55]. Therefore, we have tested if TBSV replication proteins interact with the yeast ORPs, named Osh1-7p, to facilitate sterol transfer in virus-infected cells. Our co-purification experiments with the affinity-purified tombusvirus p33 replication protein from isolated membranous fraction of yeast model host showed that four of the seven yeast Osh proteins, which are lipid-transfer proteins involved in oxysterol/sterol-binding, were associated with the membrane-bound p33 (Fig. 1A and S1A). These yeast proteins, namely Osh3p, Osh5p, Osh6p and Osh7p, were not only efficiently co-purified with p33 replication protein, but they also bound directly to p33 in a pull-down assay (Fig. S1B) and interacted with p33 in a membrane (split-ubiquitin-based) yeast two-hybrid assay (Fig. S1C). Co-purification of Osh4p was less robust with p33 from membranous fraction of yeast, while the co-purified Osh1p and Osh2p were close to the detection limit (Fig. S1A).

Bottom Line: 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.

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