title: Analysis of a temperature-sensitive mutant rotavirus indicates that NSP2 octamers are the functional form of the protein.
abstract: Evidence that NSP2 plays a role in packaging and replication comes from studies on tsE(1400), a rotavirus mutant with a temperature-sensitive (ts) lesion in the NSP2 gene. Cells infected with tsE and maintained at nonpermissive temperature contain few replication-assembly factories (viroplasms) or replication intermediates and produce virus particles that are mostly empty. Sequence analysis has indicated that an A152V mutation in NSP2 is responsible for the ts phenotype of tsE. To gain insight into the effect of the mutation on the octameric structure and biochemical activities of tsE NSP2, the protein was expressed in bacteria and purified to homogeneity. Analytical ultracentrifugation showed that tsE NSP2 formed octamers which, like those formed by wild-type (wt) NSP2, undergo conformational change into more compact structures upon binding of nucleotides. However, exposure to Mg(2+) and the nonpermissive temperature caused disruption of the tsE octamers and yielded the formation of polydisperse NSP2 aggregates, events not observed with wt octamers. Biochemical analysis showed that the RNA-binding, helix-destabilizing and NTPase activities of tsE NSP2 were significantly less at the nonpermissive temperature than at the permissive temperature. In contrast, these activities for wt NSP2 were higher at the nonpermissive temperature. Our results indicate that the octamer is the fully functional form of NSP2 and the form required for productive virus replication. The propensity of tsE NSP2 to form large aggregates provides a possible explanation for the inability of the protein to support packaging and/or replication in the infected cell at the nonpermissive temperature.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Zenobia F
authors.initials: ZF
authors.lastname: Taraporewala
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
authors.forename: Robert F
authors.initials: RF
authors.lastname: Ramig
authors.forename: Lynn
authors.initials: L
authors.lastname: Silvestri
authors.forename: John T
authors.initials: JT
authors.lastname: Patton
mesh_terms: D002460:Cell Line; D009154:Mutation; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D013696:Temperature; D014461:Ultracentrifugation; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; C079234:NS35 protein, rotavirus