title: Characterization of dominant-negative and temperature-sensitive mutants of tombusvirus replication proteins affecting replicase assembly.
abstract: The assembly of the viral replicase complex (VRC) on subcellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have identified lethal and temperature sensitive (ts) point mutations within the essential p33:p33/p92 interaction domain of p33 and p92 replication proteins of Cucumber necrosis virus, a tombusvirus. Mutations within the p33:p33/p92 interaction domain also affected viral RNA recombination in yeast model host. An in vitro approach based on yeast cell free extract demonstrated that several p33 and p92 mutants behaved as dominant-negative during VRC assembly, and they showed reduced binding to the viral (+)RNA and affected activation of the p92 RdRp protein, while they did not directly influence (-) or (+)-strand synthesis. Overall, the presented data provide direct evidence that the p33:p33/p92 interaction domains in p33 and p92 are needed for the early stage of virus replication and also influence viral recombination.
authors: Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA.
authors: Kunj B
authors: KB
authors: Pathak
authors: Zuodong
authors: Z
authors: Jiang
authors: Verena
authors: V
authors: Ochanine
authors: Monika
authors: M
authors: Sharma
authors: Judit
authors: J
authors: Pogany
authors: Peter D
authors: PD
authors: Nagy
mesh_terms: D017354:Point Mutation; D054730:Protein Interaction Domains and Motifs; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae; D013696:Temperature; D017864:Tombusvirus / Q000235:genetics* / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics*
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase