1887

Abstract

Cloning and sequencing of cytoplasmic polyhedrosis virus (CPV) genome segment S4 showed that it consists of 3410 nt with a single ORF of 1110 aa which could encode a protein of ~127 kDa (p127). Bioinformatics analysis showed the presence of a 5′ RNA triphosphatase (RTPase) domain (LRDR), a -adenosyl--methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2′--methyltransferase (MTase), which suggested that S4 may encode RTPase and MTase. The ORF of S4 was expressed in as a His-tagged fusion protein and purified by nickel-nitrilotriacetic acid affinity chromatography. Biochemical analysis of recombinant p127 showed its RTPase as well as SAM-dependent guanine -and ribose 2′--MTase activities. A MTase assay using transcribed CPV S2 RNA having a 5′ G*pppG end showed that guanine methylation occurred prior to the ribose 2′- methylation to yield a mGpppG/mGpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished - and 2′--MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the values of -MTase for SAM and RNA were calculated as 4.41 and 0.39 µM, respectively. These results suggested that CPV S4-encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5′ end of viral RNA.

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2015-01-01
2020-04-09
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