Guanylyltransferase and RNA 5'-triphosphatase activities of the purified expressed VP4 protein of bluetongue virus.
We have examined the RNA-capping enzyme activities of bluetongue virus (BTV) minor core protein, VP4. Recombinant BTV VP4 protein was purified to homogeneity from insect cell culture infected with a baculovirus VP4 of BTV serotype 10. We demonstrate that the purified protein, and VP4 encapsidated in core-like particles, react with GTP and covalently bind GMP via a phosphoamide linkage, a characteristic feature of guanylyltransferase enzyme. VP4 also catalyses a GTP-PPi exchange reaction indicating that the protein is the guanylyltransferase of the virus. In addition, VP4 possesses an RNA 5'-triphosphatase activity which catalyses the first step in the RNA-capping sequence. Further, an inorganic pyrophosphatase activity was identified which may aid the transcription activity within the virus by removing inorganic pyrophosphate which is an inhibitor of the polymerization reaction. Finally, the direct evidence of VP4 capping activity has been obtained by demonstrating in vitro transfer of GMP to the 5' end of in vitro synthesized BTV ssRNA transcripts to form a cap structure.
NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford, OX1 3SR, UK.
J
J
Martinez-Costas
G
G
Sutton
N
N
Ramadevi
P
P
Roy
D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D001820:Bluetongue virus / Q000201:enzymology / Q000378:metabolism*; D002213:Capsid / Q000302:isolation & purification / Q000378:metabolism*; D036022:Capsid Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism
D036022:Capsid Proteins; C060339:VP4 protein, Rotavirus; D006160:Guanosine Triphosphate; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D017766:Acid Anhydride Hydrolases; D011755:Pyrophosphatases; C023090:RNA triphosphatase