title: Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity.
abstract: The replication complexes of all positive strand RNA viruses of eukaryotes are associated with membranes. In the case of Semliki Forest virus (SFV), the main determinant of membrane attachment seems to be the virus-encoded non-structural protein NSP1, the capping enzyme of the viral mRNAs, which has guanine-7-methyltransferase and guanylyltransferase activities. We show here that both enzymatic activities of SFV NSP1 are inactivated by detergents and reactivated by anionic phospholipids, especially phosphatidylserine. The region of NSP1 responsible for binding to membranes as well as to liposomes was mapped to a short segment, which is conserved in the large alphavirus-like superfamily of viruses. A synthetic peptide of 20 amino acids from the putative binding site competed with in vitro synthesized NSP1 for binding to liposomes containing phosphatidylserine. These findings suggest a molecular mechanism by which RNA virus replicases attach to intracellular membranes and why they depend on the membranous environment.
authors.affiliation: Institute of Biotechnology, Viikki Biocenter, PO Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland.
authors.forename: T
authors.initials: T
authors.lastname: Ahola
authors.forename: A
authors.initials: A
authors.lastname: Lampio
authors.forename: P
authors.initials: P
authors.lastname: Auvinen
authors.forename: L
authors.initials: L
authors.lastname: Kääriäinen
mesh_terms: D000595:Amino Acid Sequence; D000838:Anions / Q000378:metabolism; D001667:Binding, Competitive; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence / Q000235:genetics; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000037:antagonists & inhibitors / Q000378:metabolism; D012672:Semliki forest virus / Q000201:enzymology* / Q000235:genetics; D012995:Solubility / Q000187:drug effects; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000098556:mRNA Guanylyltransferases
chemical_list: D000838:Anions; D003902:Detergents; D008081:Liposomes; D010446:Peptide Fragments; D010718:Phosphatidylserines; D010743:Phospholipids; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012436:S-Adenosylmethionine; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases