Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase.
Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae. Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5' end of the viral genome. To identify and characterize the enzymatic activities associated with the N-terminal domain of the BaMV ORF1 protein, the intact replicase and two C-terminally truncated proteins were expressed in Saccharomyces cerevisiae. All three versions of BaMV ORF1 proteins could be radiolabeled by [alpha-(32)P]GTP, which is a characteristic of guanylyltransferase activity. The presence of S-adenosylmethionine (AdoMet) was essential for this enzymatic activity. Thin-layer chromatography analysis suggests that the radiolabeled moiety linked to the N-terminal domain of the BaMV ORF1 protein is m(7)GMP. The N-terminal domain also exhibited methyltransferase activity that catalyzes the transfer of the [(3)H]methyl group from AdoMet to GTP or guanylylimidodiphosphate. Therefore, during cap structure formation in BaMV, methylation of GTP may occur prior to transguanylation as for alphaviruses and brome mosaic virus. This study establishes the association of RNA capping activity with the N-terminal domain of the replicase of potexviruses and further supports the idea that the reaction sequence of RNA capping is conserved throughout the alphavirus-like superfamily of RNA viruses.
Li
Y I
YI
Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.
Chen
Y J
YJ
Hsu
Y H
YH
Meng
M
M
D000595:Amino Acid Sequence; D002855:Chromatography, Thin Layer / Q000379:methods; D006160:Guanosine Triphosphate / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D006109:Poaceae / Q000821:virology*; D017863:Potexvirus / Q000201:enzymology* / Q000235:genetics; D012315:RNA Caps / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D000098556:mRNA Guanylyltransferases
D012315:RNA Caps; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D006160:Guanosine Triphosphate; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D012324:RNA-Dependent RNA Polymerase; D000098556:mRNA Guanylyltransferases