title: Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity.
abstract: During mRNA synthesis, the polymerase of vesicular stomatitis virus (VSV) copies the genomic RNA to produce five capped and polyadenylated mRNAs with the 5'-terminal structure 7mGpppA(m)pApCpApGpNpNpApUpCp. The 5' mRNA processing events are poorly understood but presumably require triphosphatase, guanylyltransferase, [guanine-N-7]- and [ribose-2'-O]-methyltransferase (MTase) activities. Consistent with a role in mRNA methylation, conserved domain VI of the 241-kDa large (L) polymerase protein shares sequence homology with a bacterial [ribose-2'-O]-MTase, FtsJ/RrmJ. In this report, we generated six L gene mutations to test this homology. Individual substitutions to the predicted MTase active-site residues K1651, D1762, K1795, and E1833 yielded viruses with pinpoint plaque morphologies and 10- to 1,000-fold replication defects in single-step growth assays. Consistent with these defects, viral RNA and protein synthesis was diminished. In contrast, alteration of residue G1674 predicted to bind the methyl donor S-adenosylmethionine did not significantly perturb viral growth and gene expression. Analysis of the mRNA cap structure revealed that alterations to the predicted active site residues decreased [guanine-N-7]- and [ribose-2'-O]-MTase activity below the limit of detection of our assay. In contrast, the alanine substitution at G1674 had no apparent consequence. These data show that the predicted MTase active-site residues K1651, D1762, K1795, and E1833 within domain VI of the VSV L protein are essential for mRNA cap methylation. A model of mRNA processing consistent with these data is presented.
authors.affiliation: HMS-Microbiology, 200 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Jianrong
authors.initials: J
authors.lastname: Li
authors.forename: Errin C
authors.initials: EC
authors.lastname: Fontaine-Rodriguez
authors.forename: Sean P J
authors.initials: SP
authors.lastname: Whelan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D006147:Guanine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012266:Ribose / Q000378:metabolism; D016415:Sequence Alignment; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism / Q000502:physiology*; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D012359:tRNA Methyltransferases / Q000378:metabolism*
chemical_list: D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D006147:Guanine; D012266:Ribose; D012359:tRNA Methyltransferases; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase