title: Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities.
abstract: The Semliki Forest virus (SFV) replicase protein nsP1 has methyltransferase (MT) and guanylyltransferase-like (GT) activities, which are involved in the capping of viral mRNAs. MT catalyzes the transfer of the methyl group from S-adenosylmethionine (AdoMet) to position 7 of GTP, and this reaction is followed by GT-catalyzed formation of the covalent complex m7GMP-nsP1. These reactions are virus specific and thus potential targets for inhibitors of virus replication. We have mutated residues of SFV nsP1, which are conserved in related proteins of the large alphavirus-like superfamily. Mutations of D64, D90, R93, C135, C142, and Y249 to alanine destroyed or greatly reduced the MT activity of nsP1. All MT-negative mutants lost also the GT activity, confirming that methylation of GTP is an essential prerequisite for the synthesis of the covalent guanylate complex. Mutation of H38 prevented the GT reaction without destroying MT activity. Conservation of residues essential for both reactions in the alphavirus-like superfamily implies that they use a capping mechanism similar to that for the alphaviruses. Residues D64 and D90 were necessary for AdoMet binding, as measured by UV cross-linking. Secondary structure predictions of nsP1 and other proteins of the superfamily place these residues in positions corresponding to AdoMet-binding sites of cellular methyltransferases, suggesting that they all may be structurally related.
authors.affiliation: Institute of Biotechnology, University of Helsinki, Finland. tero.ahola@helsinki.fi
authors.forename: T
authors.initials: T
authors.lastname: Ahola
authors.forename: P
authors.initials: P
authors.lastname: Laakkonen
authors.forename: H
authors.initials: H
authors.lastname: Vihinen
authors.forename: L
authors.initials: L
authors.lastname: Kääriäinen
mesh_terms: D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D012314:RNA Cap Analogs / Q000378:metabolism; D012315:RNA Caps; D012367:RNA, Viral; D012436:S-Adenosylmethionine / Q000378:metabolism; D012672:Semliki forest virus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D000098556:mRNA Guanylyltransferases
chemical_list: D003432:Cross-Linking Reagents; D012314:RNA Cap Analogs; D012315:RNA Caps; D012367:RNA, Viral; D014764:Viral Proteins; C017516:7-methylguanosine triphosphate; D012436:S-Adenosylmethionine; D006160:Guanosine Triphosphate; D008780:Methyltransferases; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D000098556:mRNA Guanylyltransferases