title: Suppressor mutations that allow sindbis virus RNA polymerase to function with nonaromatic amino acids at the N-terminus: evidence for interaction between nsP1 and nsP4 in minus-strand RNA synthesis.
abstract: The alphavirus RNA polymerase, nsP4, invariably has a Tyr residue at the N-terminus. Previously we reported that the N-terminal Tyr residue of nsP4 of Sindbis virus, the type species of the genus Alphavirus, can be substituted with Phe, Trp, or His without altering the wild-type phenotype in cultured cells but that other substitutions tested, except for Met, were lethal or quasilethal. Here we report the identification of two suppressor mutations in nsP4 (Glu-191 to Leu and Glu-315 to Gly, Val, or Lys) and one in nsP1 (Thr-349 to Lys) that allow nsP4 with nonaromatic amino acids at the N-terminus to function at 30 degrees C. The suppressor mutation at nsP4 Glu-315 occurred most frequently. All three suppressor mutations suppressed the effects of Ala, Arg, or Leu at the N-terminus of nsP4 with almost equal efficiency and thus the effect of the suppressing mutation is independent of the nsP4 N-terminal residue. Reconstructed mutants containing nsP1-T349K or nsP4-E315G combined with Ala-nsP4 had a defect in minus-strand RNA synthesis at 40 degrees C. A double mutant containing nsP4-Q191L combined with Ala-nsP4 was unstable and could not be tested for RNA synthesis because it reverted to temperature-independence too rapidly. Combinations of nsP1-T349K or nsP4-E315G with Leu, Arg, His, or any aromatic amino acid at the N-terminus of nsP4 also made the mutant viruses temperature sensitive. The results from this study and from a previous report on the shutoff of minus-strand RNA synthesis at 40 degrees C with the nsP1-A348T mutation in ts11 suggests that the N-terminus nsP4 interacts with nsP1 during initiation of minus-strand RNA synthesis.
authors.affiliation: Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA. shirako@ims.u-tokyo.ac.jp
authors.forename: Y
authors.initials: Y
authors.lastname: Shirako
authors.forename: E G
authors.initials: EG
authors.lastname: Strauss
authors.forename: J H
authors.initials: JH
authors.lastname: Strauss
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins; D002642:Chick Embryo; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis*; D029701:Saccharomyces cerevisiae Proteins; D012845:Sindbis Virus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D002135:Calcium-Binding Proteins; D005656:Fungal Proteins; C062748:NSP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D012367:RNA, Viral; D029701:Saccharomyces cerevisiae Proteins; D017361:Viral Nonstructural Proteins; D012321:DNA-Directed RNA Polymerases