Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus.
The core of bluetongue virus (BTV) is a multienzyme complex composed of two major proteins (VP7 and VP3) and three minor proteins (VP1, VP4, and VP6) in addition to the viral genome. The core is transcriptionally active and produces capped mRNA from which all BTV proteins are translated, but the relative role of each core component in the overall reaction process remains unclear. Previously we showed that the 76-kDa VP4 protein possesses guanylyltransferase activity, a necessary part of the RNA capping reaction. Here, through the use of highly purified (>95%) VP4 and synthetic core-like particles containing VP4, we have investigated the extent to which this protein is also responsible for other activities associated with cap formation. We show that VP4 catalyzes the conversion of unmethylated GpppG or in vitro-produced uncapped BTV RNA transcripts to m7GpppGm in the presence of S-adenosyl-L-methionine. Analysis of the methylated products of the reaction by HPLC identified both methyltransferase type 1 and type 2 activities associated with VP4, demonstrating that the complete BTV capping reaction is associated with this one protein.
Ramadevi
N
N
Natural Environment Research Council Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, United Kingdom.
Burroughs
N J
NJ
Mertens
P P
PP
Jones
I M
IM
Roy
P
P
D001820:Bluetongue virus / Q000378:metabolism*; D002213:Capsid / Q000235:genetics* / Q000378:metabolism*; D036022:Capsid Proteins; D008745:Methylation; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D036022:Capsid Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C060339:VP4 protein, Rotavirus