RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus.
Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L-P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of gamma-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5' sequence. The L protein forms a covalent enzyme-guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717-2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.
Gopinath
M
M
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Shaila
M S
MS
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D009713:Nucleotidyltransferases / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012302:Rinderpest virus / Q000201:enzymology*; D014764:Viral Proteins / Q000378:metabolism*
D012315:RNA Caps; D012367:RNA, Viral; D014764:Viral Proteins; D006157:Guanosine Monophosphate; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D012321:DNA-Directed RNA Polymerases; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase