Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites
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{"title"=>"Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites", "type"=>"journal", "authors"=>[{"first_name"=>"Daniel", "last_name"=>"Barajas", "scopus_author_id"=>"6505905651"}, {"first_name"=>"Kai", "last_name"=>"Xu", "scopus_author_id"=>"25931275600"}, {"first_name"=>"Isabel Fernández", "last_name"=>"de Castro Martín", "scopus_author_id"=>"56398497400"}, {"first_name"=>"Zsuzsanna", "last_name"=>"Sasvari", "scopus_author_id"=>"36862286300"}, {"first_name"=>"Federica", "last_name"=>"Brandizzi", "scopus_author_id"=>"6701684369"}, {"first_name"=>"Cristina", "last_name"=>"Risco", "scopus_author_id"=>"56251715300"}, {"first_name"=>"Peter D.", "last_name"=>"Nagy", "scopus_author_id"=>"7202146648"}], "year"=>2014, "source"=>"PLoS Pathogens", "identifiers"=>{"sgr"=>"84908344367", "doi"=>"10.1371/journal.ppat.1004388", "pui"=>"600311082", "pmid"=>"25329172", "scopus"=>"2-s2.0-84908344367", "issn"=>"15537374", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)"}, "id"=>"3f28c20b-0755-37ef-ba09-daa08f16cea7", "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.", "link"=>"http://www.mendeley.com/research/coopted-oxysterolbinding-orp-vap-proteins-channel-sterols-rna-virus-replication-sites-via-membrane-c", "reader_count"=>42, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>4, "Researcher"=>8, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>12, "Student > Postgraduate"=>2, "Student > Master"=>7, "Other"=>1, "Student > Bachelor"=>3, "Lecturer"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>4, "Researcher"=>8, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>12, "Student > Postgraduate"=>2, "Student > Master"=>7, "Other"=>1, "Student > Bachelor"=>3, "Lecturer"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>10, "Agricultural and Biological Sciences"=>26, "Neuroscience"=>1, "Chemistry"=>1, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>26}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>10}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"France"=>1}, "group_count"=>2}

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  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721602/Figure_1.tif"], "description"=>"<p>(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 His<sub>6</sub>-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 <i>osh3,5,6,7Δ</i> yeast. To launch TBSV repRNA replication, we expressed His<sub>6</sub>-p33 from the galactose-inducible <i>GAL1</i> promoter, His<sub>6</sub>-p92 from the copper-inducible <i>CUP1</i> promoter and DI-72(+) repRNA from the galactose-inducible <i>GAL10</i> promoter in the parental (SEY6210) and in <i>osh3,5,6,7Δ</i> yeast strains. His<sub>6</sub>-tagged Osh3, 5, 6, 7 were expressed from <i>GAL1</i> 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 CuSO<sub>4</sub>. 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 His<sub>6</sub>-tagged p33, His<sub>6</sub>-p92 and His<sub>6</sub>-Osh proteins using anti-His antibodies. Each experiment was performed three times. (C) Decreased accumulation of the mitochondrial CIRV in <i>osh3,5,6,7Δ</i> yeast. See further details in Panel B. (D) Scheme of the <i>in vitro</i> tombusvirus replicase assay based on yeast CFEs and purified recombinant TBSV replication proteins. (E) Reduced activity of the tombusvirus replicase assembled in CFE from <i>osh3,5,6,7Δ</i> yeast. Denaturing PAGE analysis of <i>in vitro</i> 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, <i>in vitro</i>. The CFEs contained the same amount of total yeast proteins (not shown). Each experiment was performed three times.</p>", "links"=>[], "tags"=>["RNA Virus Replication Sites", "Membrane Contact Sites Viruses", "Scs 2p VAP protein", "tombusvirus replication", "sterol", "yeast model host", "tombusvirus replication protein", "lipid", "membrane contact sites", "mcs", "orp", "VAP Proteins Channel Sterols"], "article_id"=>1207741, "categories"=>["Biological Sciences"], "users"=>["Daniel Barajas", "Kai Xu", "Isabel Fernández de Castro Martín", "Zsuzsanna Sasvari", "Federica Brandizzi", "Cristina Risco", "Peter D. Nagy"], "doi"=>["http://dx.doi.org/10.1371/journal.ppat.1004388.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Interaction_between_p33_replication_protein_and_yeast_oxysterol_binding_Osh_proteins_/1207741", "title"=>"Interaction between p33 replication protein and yeast oxysterol-binding Osh proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-16 04:34:03"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721615/Figure_2.tif"], "description"=>"<p>(A–B) Left panels: BiFC analysis of interaction between Osh6p and p33 in yeast cells. Confocal laser microscopy images also show the peroxisomal localization of Pex13-CFP or ER-localized Pho86-CFP. The merged images demonstrate the interaction between p33 and Osh6p (detected via BiFC) and their partial co-localization with Pex13-CFP and Pho86-CFP. DIC (differential interference contrast) images are shown on the right. (C) Control BiFC experiments with yeast lacking the p33 sequence. Yeast was grown under similar conditions and images were taken as in panel A–B. (D–E) BiFC analysis of interaction between Osh6p and the CIRV p36 in yeast cells. Confocal laser microscopy images show the mitochondrial localization of Ilv6-CFP or ER-localized Pho86-CFP. The merged images demonstrate the interaction between p36 and Osh6p and their partial co-localization with Ilv6-CFP and Pho86-CFP.</p>", "links"=>[], "tags"=>["RNA Virus Replication Sites", "Membrane Contact Sites Viruses", "Scs 2p VAP protein", "tombusvirus replication", "sterol", "yeast model host", "tombusvirus replication protein", "lipid", "membrane contact sites", "mcs", "orp", "VAP Proteins Channel Sterols"], "article_id"=>1207747, "categories"=>["Biological Sciences"], "users"=>["Daniel Barajas", "Kai Xu", "Isabel Fernández de Castro Martín", "Zsuzsanna Sasvari", "Federica Brandizzi", "Cristina Risco", "Peter D. Nagy"], "doi"=>["http://dx.doi.org/10.1371/journal.ppat.1004388.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_The_tombusvirus_p33_replication_protein_interacts_with_the_yeast_Osh6p_protein_in_the_ER_/1207747", "title"=>"The tombusvirus p33 replication protein interacts with the yeast Osh6p protein in the ER.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-16 04:34:03"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721646/Figure_3.tif"], "description"=>"<p>(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 (<i>erg9Δ</i>). 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 <i>in vitro</i> TBSV replication in artificial vesicles. Artificial PE vesicles (liposomes) were made in the presence of increasing concentrations of cholesterol or ergosterol, followed by <i>in vitro</i> TBSV replication assay as shown schematically. The amount of TBSV repRNA synthesized in the <i>in vitro</i> 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).</p>", "links"=>[], "tags"=>["RNA Virus Replication Sites", "Membrane Contact Sites Viruses", "Scs 2p VAP protein", "tombusvirus replication", "sterol", "yeast model host", "tombusvirus replication protein", "lipid", "membrane contact sites", "mcs", "orp", "VAP Proteins Channel Sterols"], "article_id"=>1207759, "categories"=>["Biological Sciences"], "users"=>["Daniel Barajas", "Kai Xu", "Isabel Fernández de Castro Martín", "Zsuzsanna Sasvari", "Federica Brandizzi", "Cristina Risco", "Peter D. Nagy"], "doi"=>["http://dx.doi.org/10.1371/journal.ppat.1004388.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Enrichment_of_sterols_at_the_sites_of_tombusvirus_replication_in_yeast_/1207759", "title"=>"Enrichment of sterols at the sites of tombusvirus replication in yeast.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-16 04:34:03"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721665/Figure_4.tif"], "description"=>"<p>(A) The split ubiquitin assay was used to test binding between p33 and Scs2p in wt (NMY51) yeast. The bait p33 was co-expressed with the shown prey proteins. <i>SSA1</i> (HSP70 chaperone), and the empty prey vector (NubG) were used as positive and negative controls, respectively. (B) Co-purification of the Scs2p protein with the tombusvirus p33 replication protein. The FLAG-tagged p33 was purified from the membrane fractions of yeast extracts using a FLAG-affinity column. Top panel: Western blot analysis of co-purified 6xHA-tagged Scs2p using anti-HA antibody. Middle panel: Western blot of purified p33 (either His<sub>6</sub>- or Flag-tagged, as shown) detected with anti-FLAG antibody. Bottom panel: Western blot of 6xHA-tagged Scs2p in the total yeast extract using anti-HA antibody. (C) BiFC analysis of interactions between Scs2p and p33 and between Hsp70 (Ssa1p) and p33. Confocal laser microscopy images also show the peroxisomal localization of Pex13p marker protein (left panels) or Pho86-CFP ER-marker protein (right panels). The merged images at the top show the interaction between p33 and Scs2p and their partial co-localization with Pex13p-CFP or Pho86-CFP ER-marker, while the merged images at the bottom demonstrate the interaction between p33 and Ssa1 and their co-localization with Pex13p. DIC (differential interference contrast) images are shown on the right. Each row represents a separate yeast cell. Note that the Venus-(N-terminal portion) tag was fused to the N-terminus of p33 and the Venus-C tag was fused to host proteins, which are all N-terminal tags. Both p33 and Scs2p have cytosolic N-terminal regions. (D) Control BiFC experiments. Yeast was grown under similar conditions and images were taken as in panel C. (E) Decreased TBSV repRNA accumulation in <i>scs2Δ</i> yeast. To launch TBSV repRNA replication, we expressed His<sub>6</sub>-p33 and FLAG-tagged p92 from the copper-inducible <i>CUP1</i> promoter and DI-72(+) repRNA from the galactose-inducible <i>GAL1</i> promoter in the parental (BY4741) and in <i>scs2Δ</i> yeast strains. The yeast cells were cultured for 16 hours at 23°C on 2% galactose SC minimal media, and then for 24 h at 23°C on 2% galactose SC minimal media supplemented with 50 µM CuSO<sub>4</sub>. 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 panels: Western blot analysis of the accumulation level of His<sub>6</sub>-tagged p33 and FLAG-tagged p92 proteins using anti-His or anti-FLAG antibodies. Each experiment was performed three times. (F) Reduced activity of the tombusvirus replicase assembled in <i>scs2Δ</i> yeast. Top: Scheme of the experimental design. Denaturing PAGE analysis of <i>in vitro</i> replicase activity in the membrane-enriched fraction from wt and <i>scs2Δ</i> yeasts using the co-purified repRNA. The yeast cells were harvested for analysis at 24 h time point after launching TBSV replication. Note that this image shows the repRNAs made by the replicase <i>in vitro</i>. Each experiment was performed three times.</p>", "links"=>[], "tags"=>["RNA Virus Replication Sites", "Membrane Contact Sites Viruses", "Scs 2p VAP protein", "tombusvirus replication", "sterol", "yeast model host", "tombusvirus replication protein", "lipid", "membrane contact sites", "mcs", "orp", "VAP Proteins Channel Sterols"], "article_id"=>1207778, "categories"=>["Biological Sciences"], "users"=>["Daniel Barajas", "Kai Xu", "Isabel Fernández de Castro Martín", "Zsuzsanna Sasvari", "Federica Brandizzi", "Cristina Risco", "Peter D. Nagy"], "doi"=>["http://dx.doi.org/10.1371/journal.ppat.1004388.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_The_tombusvirus_p33_replication_protein_binds_to_the_yeast_Scs2p_VAP_protein_in_the_ER_/1207778", "title"=>"The tombusvirus p33 replication protein binds to the yeast Scs2p VAP protein in the ER.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-16 04:34:03"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721685/Figure_5.tif"], "description"=>"<p>(A) The split ubiquitin assay was used to test binding between p33 and six <i>Arabidopsis</i> VAP proteins in <i>scs2Δ</i> yeast. The bait p33 was co-expressed with the shown prey proteins. <i>SCS2</i> and the empty prey vector (NubG) were used as positive and negative controls, respectively. The left panel shows p33: VAP interactions, the right panel demonstrates that comparable amounts of yeasts were used for these experiments. (B) Expression of <i>Arabidopsis</i> VAP proteins can complement the defect in TBSV repRNA accumulation in <i>scs2Δ</i> yeast. Northern blot analysis of DI-72(+) repRNA accumulation in <i>scs2Δ</i> yeast expressing <i>Arabidopsis</i> VAP proteins from the native <i>SCS2</i> promoter. Note that pYC expresses a short peptide and used as a negative control. (C) Stimulation of tombusvirus RNA accumulation in plants by expression of two <i>Arabidopsis</i> VAP proteins. Expression of the <i>Arabidopsis</i> VAP proteins was done in <i>N. benthamiana</i> leaves, which were co-infiltrated with <i>Agrobacterium</i> carrying a plasmid to launch CNV replication from the 35S promoter. The control samples were obtained from leaves expressing no VAP proteins (35S, lanes 1–2 and 7–8). Total RNA was extracted from leaves 3 days after agroinfiltration. The accumulation of CNV RNAs in <i>N. benthamiana</i> leaves was analyzed by Northern blot. The ribosomal RNA (rRNA), visualized by ethidium-bromide staining, was used as a loading control.</p>", "links"=>[], "tags"=>["RNA Virus Replication Sites", "Membrane Contact Sites Viruses", "Scs 2p VAP protein", "tombusvirus replication", "sterol", "yeast model host", "tombusvirus replication protein", "lipid", "membrane contact sites", "mcs", "orp", "VAP Proteins Channel Sterols"], "article_id"=>1207786, "categories"=>["Biological Sciences"], "users"=>["Daniel Barajas", "Kai Xu", "Isabel Fernández de Castro Martín", "Zsuzsanna Sasvari", "Federica Brandizzi", "Cristina Risco", "Peter D. Nagy"], "doi"=>["http://dx.doi.org/10.1371/journal.ppat.1004388.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Six_Arabidopsis_VAP_proteins_interact_with_the_TBSV_p33_replication_protein_in_yeast_/1207786", "title"=>"Six <i>Arabidopsis</i> VAP proteins interact with the TBSV p33 replication protein in yeast.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-16 04:34:03"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721700/Figure_6.tif"], "description"=>"<p>(A) Partial co-localization of TBSV BFP-tagged p33 replication protein with the YFP-AtPVA12 VAP protein in <i>N. benthamiana</i> cells. Expression of the above proteins from the 35S promoter was done after agro-infiltration into <i>N. benthamiana</i> leaves. Note the altered membrane structure in the subcellular area showing co-localization of BFP-p33 and YFP-AtPVA12 (portion of the image was enlarged at the bottom panel), which might be due to local membrane proliferation. (B) Co-localization of TBSV BFP-tagged p33 replication protein with the nYFP-AtPVA12 VAP and AtOrp3A-cYFP protein complexes in <i>N. benthamiana</i> cells. Expression of the above proteins from the 35S promoter was done after agro-infiltration into <i>N. benthamiana</i> leaves. Note that nYFP-AtPVA12 VAP and AtOrp3A-cYFP proteins were detected by BiFC. The subcellular areas (likely representing the ER membranes) where one viral and two cellular proteins are co-localized are marked with arrows. Control BiFC experiments were as in a previous paper (not shown) <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004388#ppat.1004388-Saravanan1\" target=\"_blank\">[56]</a>.</p>", "links"=>[], "tags"=>["RNA Virus Replication Sites", "Membrane Contact Sites Viruses", "Scs 2p VAP protein", "tombusvirus replication", "sterol", "yeast model host", "tombusvirus replication protein", "lipid", "membrane contact sites", "mcs", "orp", "VAP Proteins Channel Sterols"], "article_id"=>1207801, "categories"=>["Biological Sciences"], "users"=>["Daniel Barajas", "Kai Xu", "Isabel Fernández de Castro Martín", "Zsuzsanna Sasvari", "Federica Brandizzi", "Cristina Risco", "Peter D. Nagy"], "doi"=>["http://dx.doi.org/10.1371/journal.ppat.1004388.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_The_tombusvirus_p33_replication_protein_co_localizes_with_AtOrp3A_and_AtPVA12_proteins_in_Nicotiana_benthamiana_/1207801", "title"=>"The tombusvirus p33 replication protein co-localizes with AtOrp3A and AtPVA12 proteins in <i>Nicotiana benthamiana</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-16 04:34:03"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721735/Figure_7.tif"], "description"=>"<p>(A) Representative electron microscopic images of portions of <i>N. benthamiana</i> cells. Several characteristic virus-induced spherules are marked with arrowheads and the MCS-like structures are indicated by arrows. These spherules are formed via membrane invagination into peroxisome-derived membranes. (B) Close up view of spherules and MCS-like structures in plant cells infected with CNV. The ER and the associated ribosomes are indicated with white arrowheads. See further details in panel A. (C) TEM of stained ultra-thin sections in yeast cells replicating TBSV repRNA. Characteristic spherule-like vesicles are assembled in a peripheral membranous compartment (arrows) in the vicinity of MCS-like structures. (D) METTEM of ultra-thin sections of yeasts replicating TBSV repRNA. The MT-tagged p33 was visualized by ∼1 nm gold nano-clusters associated to MT. Black arrowhead points at a spherule-like structure, while black arrows show possible MCS with p33 present in opposing membranes. Bars in panels C-D represent 100 nm.</p>", "links"=>[], "tags"=>["RNA Virus Replication Sites", "Membrane Contact Sites Viruses", "Scs 2p VAP protein", "tombusvirus replication", "sterol", "yeast model host", "tombusvirus replication protein", "lipid", "membrane contact sites", "mcs", "orp", "VAP Proteins Channel Sterols"], "article_id"=>1207827, "categories"=>["Biological Sciences"], "users"=>["Daniel Barajas", "Kai Xu", "Isabel Fernández de Castro Martín", "Zsuzsanna Sasvari", "Federica Brandizzi", "Cristina Risco", "Peter D. Nagy"], "doi"=>["http://dx.doi.org/10.1371/journal.ppat.1004388.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_The_presence_of_MCS_like_structures_in_the_vicinity_of_tombusvirus_induced_spherules_in_plant_cells_infected_with_CNV_or_in_yeast_cells_replicating_TBSV_repRNA_/1207827", "title"=>"The presence of MCS-like structures in the vicinity of tombusvirus-induced spherules in plant cells infected with CNV or in yeast cells replicating TBSV repRNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-16 04:34:03"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721746/Figure_8.tif"], "description"=>"<p>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.</p>", "links"=>[], "tags"=>["RNA Virus Replication Sites", "Membrane Contact Sites Viruses", "Scs 2p VAP protein", "tombusvirus replication", "sterol", "yeast model host", "tombusvirus replication protein", "lipid", "membrane contact sites", "mcs", "orp", "VAP Proteins Channel Sterols"], "article_id"=>1207838, "categories"=>["Biological Sciences"], "users"=>["Daniel Barajas", "Kai Xu", "Isabel Fernández de Castro Martín", "Zsuzsanna Sasvari", "Federica Brandizzi", "Cristina Risco", "Peter D. Nagy"], "doi"=>["http://dx.doi.org/10.1371/journal.ppat.1004388.g008"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Model_on_the_role_of_ORP_and_VAP_proteins_and_MCSs_in_tombusvirus_replicase_assembly_/1207838", "title"=>"Model on the role of ORP and VAP proteins and MCSs in tombusvirus replicase assembly.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-16 04:34:03"}
  • {"files"=>["https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721800/Figure_S1.eps", "https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721801/Figure_S2.eps", "https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721802/Figure_S3.eps", "https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721803/Figure_S4.eps", "https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721804/Figure_S5.eps", "https://s3-eu-west-1.amazonaws.com/pstorage-plos-3567654/1721805/Materials_and_Methods_S1.doc"], "description"=>"<div><p>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 <i>in vitro</i> 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.</p></div>", "links"=>[], "tags"=>["RNA Virus Replication Sites", "Membrane Contact Sites Viruses", "Scs 2p VAP protein", "tombusvirus replication", "sterol", "yeast model host", "tombusvirus replication protein", "lipid", "membrane contact sites", "mcs", "orp", "VAP Proteins Channel Sterols"], "article_id"=>1207858, "categories"=>["Biological Sciences"], "users"=>["Daniel Barajas", "Kai Xu", "Isabel Fernández de Castro Martín", "Zsuzsanna Sasvari", "Federica Brandizzi", "Cristina Risco", "Peter D. Nagy"], "doi"=>["http://dx.doi.org/10.1371/journal.ppat.1004388.s001", "http://dx.doi.org/10.1371/journal.ppat.1004388.s002", "http://dx.doi.org/10.1371/journal.ppat.1004388.s003", "http://dx.doi.org/10.1371/journal.ppat.1004388.s004", "http://dx.doi.org/10.1371/journal.ppat.1004388.s005", "http://dx.doi.org/10.1371/journal.ppat.1004388.s006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"http://figshare.com/articles/_Co_opted_Oxysterol_Binding_ORP_and_VAP_Proteins_Channel_Sterols_to_RNA_Virus_Replication_Sites_via_Membrane_Contact_Sites_/1207858", "title"=>"Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-10-16 04:34:03"}

PMC Usage Stats | Further Information

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