title: Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.
abstract: Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work withTomato bushy stunt tombusvirus(TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterolsin vitroThe sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication.IMPORTANCEOne intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, we demonstrate that TBSV p33 and p92 replication proteins can bind to sterolin vitroMutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol binding shows the important connection between the abilities of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Together, the results further strengthen the model that cellular sterols are essential as proviral lipids during viral replication.
authors.affiliation: Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Xu
authors.affiliation: Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, USA pdnagy2@uky.edu.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Nagy
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002784:Cholesterol / Q000737:chemistry* / Q000502:physiology; D021962:Membrane Microdomains / Q000378:metabolism; D011485:Protein Binding; D011523:Protoplasts / Q000821:virology*; D012441:Saccharomyces cerevisiae / Q000821:virology*; D014026:Nicotiana / Q000821:virology; D017864:Tombusvirus / Q000502:physiology*; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology; D014779:Virus Replication
chemical_list: D014764:Viral Proteins; D002784:Cholesterol
keywords: RNA replication; TBSV; lipids; membrane binding; plant; sterol; tombusvirus; virus-host interactions; yeast