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.
Document Type
Article
Publication Date
10-16-2014
Digital Object Identifier (DOI)
http://dx.doi.org/10.1371/journal.ppat.1004388
Funding Information
This research is supported by NSF (MCB 1122039) for PDN, and an FPI program fellowship for IFdCM and research grants BIO2009-07255 and BIO2012-33314 from the Spanish Ministry of Economy and Competitiveness for CR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Repository Citation
Barajas, Daniel; Xu, Kai; de Castro Martín, Isabel Fernández; Sasvari, Zsuzsanna; Brandizzi, Federica; Risco, Cristina; and Nagy, Peter D., "Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites" (2014). Plant Pathology Faculty Publications. 43.
https://uknowledge.uky.edu/plantpath_facpub/43
Figure S1. Identification of the yeast Osh proteins interacting with the tombusvirus p33 replication protein.
Figure_S2.eps (1562 kB)
Figure S2. Co-purification of the Arabidopsis Orp3A and other ORP proteins with the tombusvirus p33 replication protein from yeast cells.
Figure_S3.eps (820 kB)
Figure S3. Expression of Scs2p or its MSP/TM domains in yeast complement TBSV repRNA accumulation in scs2Δ yeast.
Figure_S4.eps (3539 kB)
Figure S4. Tombusvirus replication and expression of p33, p92 and DI-72 repRNA induces MCS-like structures in the vicinity of tombusvirus-induced spherules in plant cells.
Figure_S5.eps (4293 kB)
Figure S5. Detection of MT-tagged p33 replication protein in wild type yeast.
Materials_and_Methods_S1.doc (118 kB)
Materials and Methods S1.
journal.ppat.1004388.g001.png (3339 kB)
Figure 1 (PNG). Interaction between p33 replication protein and yeast oxysterol-binding Osh proteins.
journal.ppat.1004388.g001.ppt (195 kB)
Figure 1 (PPT). Interaction between p33 replication protein and yeast oxysterol-binding Osh proteins.
journal.ppat.1004388.g001.TIF (4377 kB)
Figure 1 (TIFF). Interaction between p33 replication protein and yeast oxysterol-binding Osh proteins.
journal.ppat.1004388.g002.png (6855 kB)
Figure 2 (PNG). The tombusvirus p33 replication protein interacts with the yeast Osh6p protein in the ER.
journal.ppat.1004388.g002.ppt (186 kB)
Figure 2 (PPT). The tombusvirus p33 replication protein interacts with the yeast Osh6p protein in the ER.
journal.ppat.1004388.g002.TIF (6744 kB)
Figure 2 (TIFF). The tombusvirus p33 replication protein interacts with the yeast Osh6p protein in the ER.
journal.ppat.1004388.g003.png (7957 kB)
Figure 3 (PNG). Enrichment of sterols at the sites of tombusvirus replication in yeast.
journal.ppat.1004388.g003.ppt (343 kB)
Figure 3 (PPT). Enrichment of sterols at the sites of tombusvirus replication in yeast.
journal.ppat.1004388.g003.TIF (7885 kB)
Figure 3 (TIFF). Enrichment of sterols at the sites of tombusvirus replication in yeast.
journal.ppat.1004388.g004.png (5832 kB)
Figure 4 (PNG). The tombusvirus p33 replication protein binds to the yeast Scs2p VAP protein in the ER.
journal.ppat.1004388.g004.ppt (200 kB)
Figure 4 (PPT). The tombusvirus p33 replication protein binds to the yeast Scs2p VAP protein in the ER.
journal.ppat.1004388.g004.TIF (6799 kB)
Figure 4 (TIFF). The tombusvirus p33 replication protein binds to the yeast Scs2p VAP protein in the ER.
journal.ppat.1004388.g005.png (2907 kB)
Figure 5 (PNG). Six Arabidopsis VAP proteins interact with the TBSV p33 replication protein in yeast.
journal.ppat.1004388.g005.ppt (167 kB)
Figure 5 (PPT). Six Arabidopsis VAP proteins interact with the TBSV p33 replication protein in yeast.
journal.ppat.1004388.g005.TIF (4037 kB)
Figure 5 (TIFF). Six Arabidopsis VAP proteins interact with the TBSV p33 replication protein in yeast.
journal.ppat.1004388.g006.png (4000 kB)
Figure 6 (PNG). The tombusvirus p33 replication protein co-localizes with AtOrp3A and AtPVA12 proteins in Nicotiana benthamiana.
journal.ppat.1004388.g006.ppt (185 kB)
Figure 6 (PPT). The tombusvirus p33 replication protein co-localizes with AtOrp3A and AtPVA12 proteins in Nicotiana benthamiana.
journal.ppat.1004388.g006.TIF (5048 kB)
Figure 6 (TIFF). The tombusvirus p33 replication protein co-localizes with AtOrp3A and AtPVA12 proteins in Nicotiana benthamiana.
journal.ppat.1004388.g007.png (11984 kB)
Figure 7 (PNG). 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.
journal.ppat.1004388.g007.ppt (253 kB)
Figure 7 (PPT). 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.
journal.ppat.1004388.g007.TIF (12849 kB)
Figure 7 (TIFF). 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.
journal.ppat.1004388.g008.png (2183 kB)
Figure 8 (PNG). Model on the role of ORP and VAP proteins and MCSs in tombusvirus replicase assembly.
journal.ppat.1004388.g008.ppt (217 kB)
Figure 8 (PPT). Model on the role of ORP and VAP proteins and MCSs in tombusvirus replicase assembly.
journal.ppat.1004388.g008.TIF (2844 kB)
Figure 8 (TIFF). Model on the role of ORP and VAP proteins and MCSs in tombusvirus replicase assembly.
Notes/Citation Information
Published in PLOS Pathogens, v. 10, no. 10, article e1004388, p. 1-18.
© 2014 Barajas et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.