title: Mutagenesis of the Sindbis virus nsP1 protein: effects on methyltransferase activity and viral infectivity.
abstract: It has been suggested that four amino acids which are absolutely conserved in th nsP1 nonstructural proteins encoded by togaviruses and in the homologous proteins encoded by plant viruses in the Sindbis virus (SV) superfamily may constitute a "methyltransferase motif." In the Sindbis virus nsP1 protein (540 amino acids) these four amino acids are represented by His39, Arg91, Asp94, and Tyr249. Earlier, in assays of methyltransferase (MTase) activity generated in SV-infected cells, we had shown that amino acid changes at positions 87 and 88 of SV nsP1 resulted in a 10-fold lower Km for S-adenosyl methionine, the methyl donor in MTase reactions. Using site-directed mutagenesis we now report the expression of nsP1 in Escherichia coli, and in the infectious clone of Sindbis virus, Toto/1101, in which His39, Arg91, Asp94, and Tyr249 were changed one at a time to Ala. We also expressed nsP1 with C-terminal deletions of varying size, as well as with internal deletions in the C-terminal portion of the protein, in E. coli. Changing His39, Arg91, Asp94, or Tyr249 to Ala led to a loss of both MTase activity and viral infectivity; however, changing Ile369 to Val, a conservative change in the carboxy-terminal half of nsP1, had no effect on either MTase activity or viral infectivity. With respect to the deleted forms of nsP1, a carboxy-terminal deletion of 48 amino acids was still compatible with MTase activity in vitro. However, larger deletions including those in which the amino acids between positions 442 and 492 were deleted abolished MTase activity.
authors.affiliation: Department of Molecular Genetics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854-5635, USA.
authors.forename: H L
authors.initials: HL
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: O'Rear
authors.forename: V
authors.initials: V
authors.lastname: Stollar
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D008780:Methyltransferases / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D012845:Sindbis Virus / Q000201:enzymology* / Q000472:pathogenicity; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases