title: Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli.
abstract: We have expressed the Semliki Forest virus (SFV)-specific nonstructural protein nsP1 both in insect cells and in Escherichia coli in the absence of other viral proteins. A substantial amount of nsP1 was synthesized in Sf9 cells infected with the recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) AcNPV-nsP1. These cells had a high level of guanine-7-methyltransferase activity compared with that of wild-type AcNPV-infected cells. The methyltransferase activity and nsP1 were mostly in the mitochondrial pellet fraction (P15). The enzymatic activity was increased by treatment with deoxycholate (DOC), as in the case of SFV-infected BHK cells. The material released by DOC treatment from P15 of the AcNPV-nsP1-infected cells was analyzed by gel filtration and sucrose gradient centrifugation. Both the methyltransferase activity and nsP1 were in aggregates. nsP1 expressed in E. coli at 37 degrees C sedimented at 15,000 x g, whereas after expression at 15 degrees C, both nsP1 and methyltransferase activity were in the supernatant fraction. Paradoxically, the activity from E. coli was completely inhibited by Triton X-100 and DOC. Sucrose gradient analysis showed that even the "soluble" nsP1-methyltransferase was in aggregates. The methyltransferase activities in the P15 fractions of SFV-infected BHK cells and AcNPV-nsP1-infected Sf9 cells and in E. coli catalyzed linear incorporation of the [3H]methyl group from S-adenosylmethionine to GTP for a 60-min period. The enzymes from the three sources had similar substrate specificities and Km values for S-adenosylmethionine. In addition to GTP, they all methylated dGTP and GpppG, but not m7GTP or GpppA, or in vitro-transcribed RNAs with GpppA and GpppG caps. The unique properties of SFV-specific nsP1 methyltransferase are discussed.
authors.affiliation: Institute of Biotechnology, University of Helsinki, Finland.
authors.forename: P
authors.initials: P
authors.lastname: Laakkonen
authors.forename: M
authors.initials: M
authors.lastname: Hyvönen
authors.forename: J
authors.initials: J
authors.lastname: Peränen
authors.forename: L
authors.initials: L
authors.lastname: Kääriäinen
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D008780:Methyltransferases / Q000096:biosynthesis* / Q000378:metabolism; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012672:Semliki forest virus / Q000201:enzymology*; D018411:Spodoptera / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis*
chemical_list: D017361:Viral Nonstructural Proteins; D006160:Guanosine Triphosphate; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase