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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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Enrichment of sterols at the sites of tombusvirus replication in yeast.(A) Re-localization of ergosterols to internal punctate structures in yeast replicating CIRV or TBSV. Fluorescent microscopic images of yeast cells stained with filipin dye. Note that filipin stains ergosterols present mostly at the plasma membrane in virus-free cells. (B) Co-localization of ergosterols and CIRV RFP-p36 and CNV RFP-p33 is shown by confocal laser microscopy. Yeasts were stained with filipin dye. (C) Enrichment of fluorescently-labeled sterol at the sites of tombusvirus replication in yeast deficient in ergosterol synthesis (erg9Δ). The BODIPY-cholesterol was taken up by yeast from the culture media. The bottom two rows represent images from yeast not expressing tombusvirus proteins and serve as control. (D) Sterols stimulate in vitro TBSV replication in artificial vesicles. Artificial PE vesicles (liposomes) were made in the presence of increasing concentrations of cholesterol or ergosterol, followed by in vitro TBSV replication assay as shown schematically. The amount of TBSV repRNA synthesized in the in vitro assay is shown in PAGE images. PE and PI(4)P were used as controls. Note that addition of extra PE did not change TBSV replication (second panel from the bottom), while PI(4)P inhibited it when used at higher concentrations (bottom image).
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ppat-1004388-g003: Enrichment of sterols at the sites of tombusvirus replication in yeast.(A) Re-localization of ergosterols to internal punctate structures in yeast replicating CIRV or TBSV. Fluorescent microscopic images of yeast cells stained with filipin dye. Note that filipin stains ergosterols present mostly at the plasma membrane in virus-free cells. (B) Co-localization of ergosterols and CIRV RFP-p36 and CNV RFP-p33 is shown by confocal laser microscopy. Yeasts were stained with filipin dye. (C) Enrichment of fluorescently-labeled sterol at the sites of tombusvirus replication in yeast deficient in ergosterol synthesis (erg9Δ). The BODIPY-cholesterol was taken up by yeast from the culture media. The bottom two rows represent images from yeast not expressing tombusvirus proteins and serve as control. (D) Sterols stimulate in vitro TBSV replication in artificial vesicles. Artificial PE vesicles (liposomes) were made in the presence of increasing concentrations of cholesterol or ergosterol, followed by in vitro TBSV replication assay as shown schematically. The amount of TBSV repRNA synthesized in the in vitro assay is shown in PAGE images. PE and PI(4)P were used as controls. Note that addition of extra PE did not change TBSV replication (second panel from the bottom), while PI(4)P inhibited it when used at higher concentrations (bottom image).

Mentions: Since the proposed major function of ORPs is to transfer sterols between organellar membranes inside cells [52], [64], we predicted that recruitment of Osh3/5/6/7 ORPs by tombusviruses to the sites of viral replication could result in enrichment of sterols in these viral subcompartments. Accordingly, testing the distribution of sterols in yeast using the fluorescent sterol probe filipin dye [61] revealed striking differences between yeast cells replicating or not replicating tombusviruses. Namely, the sterols, which are mostly enriched in the plasma membrane in yeast reaching ∼60% of total ergosterols (images without tombusviruses on the right, Fig. 3A) [53], were redistributed to internal punctate-like subcellular compartments in yeast replicating TBSV and CIRV tombusviruses (left and central images). The redistributed sterols were mostly present in punctate-like structures in yeast replicating the mitochondria-localized CIRV, while the punctate structures were also visible, but somewhat more diffused in yeast replicating the peroxisome-localized TBSV (Fig. 3A). The reduced localization of sterols in the plasma membrane was also noticeable in yeast replicating these tombusviruses.


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)

Enrichment of sterols at the sites of tombusvirus replication in yeast.(A) Re-localization of ergosterols to internal punctate structures in yeast replicating CIRV or TBSV. Fluorescent microscopic images of yeast cells stained with filipin dye. Note that filipin stains ergosterols present mostly at the plasma membrane in virus-free cells. (B) Co-localization of ergosterols and CIRV RFP-p36 and CNV RFP-p33 is shown by confocal laser microscopy. Yeasts were stained with filipin dye. (C) Enrichment of fluorescently-labeled sterol at the sites of tombusvirus replication in yeast deficient in ergosterol synthesis (erg9Δ). The BODIPY-cholesterol was taken up by yeast from the culture media. The bottom two rows represent images from yeast not expressing tombusvirus proteins and serve as control. (D) Sterols stimulate in vitro TBSV replication in artificial vesicles. Artificial PE vesicles (liposomes) were made in the presence of increasing concentrations of cholesterol or ergosterol, followed by in vitro TBSV replication assay as shown schematically. The amount of TBSV repRNA synthesized in the in vitro assay is shown in PAGE images. PE and PI(4)P were used as controls. Note that addition of extra PE did not change TBSV replication (second panel from the bottom), while PI(4)P inhibited it when used at higher concentrations (bottom image).
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Related In: Results  -  Collection

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ppat-1004388-g003: Enrichment of sterols at the sites of tombusvirus replication in yeast.(A) Re-localization of ergosterols to internal punctate structures in yeast replicating CIRV or TBSV. Fluorescent microscopic images of yeast cells stained with filipin dye. Note that filipin stains ergosterols present mostly at the plasma membrane in virus-free cells. (B) Co-localization of ergosterols and CIRV RFP-p36 and CNV RFP-p33 is shown by confocal laser microscopy. Yeasts were stained with filipin dye. (C) Enrichment of fluorescently-labeled sterol at the sites of tombusvirus replication in yeast deficient in ergosterol synthesis (erg9Δ). The BODIPY-cholesterol was taken up by yeast from the culture media. The bottom two rows represent images from yeast not expressing tombusvirus proteins and serve as control. (D) Sterols stimulate in vitro TBSV replication in artificial vesicles. Artificial PE vesicles (liposomes) were made in the presence of increasing concentrations of cholesterol or ergosterol, followed by in vitro TBSV replication assay as shown schematically. The amount of TBSV repRNA synthesized in the in vitro assay is shown in PAGE images. PE and PI(4)P were used as controls. Note that addition of extra PE did not change TBSV replication (second panel from the bottom), while PI(4)P inhibited it when used at higher concentrations (bottom image).
Mentions: Since the proposed major function of ORPs is to transfer sterols between organellar membranes inside cells [52], [64], we predicted that recruitment of Osh3/5/6/7 ORPs by tombusviruses to the sites of viral replication could result in enrichment of sterols in these viral subcompartments. Accordingly, testing the distribution of sterols in yeast using the fluorescent sterol probe filipin dye [61] revealed striking differences between yeast cells replicating or not replicating tombusviruses. Namely, the sterols, which are mostly enriched in the plasma membrane in yeast reaching ∼60% of total ergosterols (images without tombusviruses on the right, Fig. 3A) [53], were redistributed to internal punctate-like subcellular compartments in yeast replicating TBSV and CIRV tombusviruses (left and central images). The redistributed sterols were mostly present in punctate-like structures in yeast replicating the mitochondria-localized CIRV, while the punctate structures were also visible, but somewhat more diffused in yeast replicating the peroxisome-localized TBSV (Fig. 3A). The reduced localization of sterols in the plasma membrane was also noticeable in yeast replicating these tombusviruses.

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