title: Dual roles for an arginine-rich motif in specific genome recognition and localization of viral coat protein to RNA replication sites in flock house virus-infected cells.
abstract: Assembly of many RNA viruses entails the encapsidation of multiple genome segments into a single virion, and underlying mechanisms for this process are still poorly understood. In the case of the nodavirus Flock House virus (FHV), a bipartite positive-strand RNA genome consisting of RNA1 and RNA2 is copackaged into progeny virions. In this study, we investigated whether the specific packaging of FHV RNA is dependent on an arginine-rich motif (ARM) located in the N terminus of the coat protein. Our results demonstrate that the replacement of all arginine residues within this motif with alanines rendered the resultant coat protein unable to package RNA1, suggesting that the ARM represents an important determinant for the encapsidation of this genome segment. In contrast, replacement of all arginines with lysines had no effect on RNA1 packaging. Interestingly, confocal microscopic analysis demonstrated that the RNA1 packaging-deficient mutant did not localize to mitochondrial sites of FHV RNA replication as efficiently as wild-type coat protein. In addition, gain-of-function analyses showed that the ARM by itself was sufficient to target green fluorescent protein to RNA replication sites. These data suggest that the packaging of RNA1 is dependent on trafficking of coat protein to mitochondria, the presumed site of FHV assembly, and that this trafficking requires a high density of positive charge in the N terminus. Our results are compatible with a model in which recognition of RNA1 and RNA2 for encapsidation occurs sequentially and in distinct cellular microenvironments.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. paventer@scripps.edu
authors.forename: P Arno
authors.initials: PA
authors.lastname: Venter
authors.forename: Dawn
authors.initials: D
authors.lastname: Marshall
authors.forename: Anette
authors.initials: A
authors.lastname: Schneemann
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D030381:Nodaviridae / Q000502:physiology*; D011485:Protein Binding; D021381:Protein Transport; D012367:RNA, Viral / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins; D012367:RNA, Viral; D001120:Arginine