Identification of a novel function of the alphavirus capping apparatus. RNA 5'-triphosphatase activity of Nsp2.
Both genomic and subgenomic RNAs of the Alphavirus have m(7)G(5')ppp(5')N (cap0 structure) at their 5' end. Previously it has been shown that Alphavirus-specific nonstructural protein Nsp1 has guanine-7N-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. During normal cap synthesis the 5' gamma-phosphate of the nascent viral RNA chain is removed by a specific RNA 5'-triphosphatase before condensation with GMP, delivered by the guanylyltransferase. Using a novel RNA triphosphatase assay, we show here that nonstructural protein Nsp2 (799 amino acids) of Semliki Forest virus specifically cleaves the gamma,beta-triphosphate bond at the 5' end of RNA. The same activity was demonstrated for Nsp2 of Sindbis virus, as well as for the amino-terminal fragment of Semliki Forest virus Nsp2-N (residues 1-470). The carboxyl-terminal part of Semliki Forest virus Nsp2-C (residues 471-799) had no RNA triphosphatase activity. Replacement of Lys-192 by Asn in the nucleotide-binding site completely abolished RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N. Here we provide biochemical characterization of the newly found function of Nsp2 and discuss the unique properties of the entire Alphavirus-capping apparatus.
Vasiljeva
L
L
Program in Cellular Biotechnology, Institute of Biotechnology, Biocenter Viikki, University of Helsinki, FIN-00014, Helsinki, Finland.
Merits
A
A
Auvinen
P
P
Kääriäinen
L
L
D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D000524:Alphavirus / Q000201:enzymology*; D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D012315:RNA Caps / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012672:Semliki forest virus / Q000201:enzymology*; D013379:Substrate Specificity
D012315:RNA Caps; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase; D017766:Acid Anhydride Hydrolases; D020558:GTP Phosphohydrolases; C023090:RNA triphosphatase