Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) 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 5ʹ RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-L-methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2ʹ-O-methyltransferase (MTase), which suggested that S4 may encode RTPase and MTase. The ORF of S4 was expressed in Escherichia coli 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 N7-and ribose 2ʹ-O-MTase activities. A MTase assay using in vitro transcribed AmCPV S2 RNA having a 5ʹ G*pppG end showed that guanine N7 methylation occurred prior to the ribose 2ʹ-O methylation to yield a m7GpppG/m7GpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished N7-and 2ʹ-O-MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the Km values of N7-MTase for SAM and RNA were calculated as 4.41 and 0.39 μM, respectively. These results suggested that AmCPV S4-encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5ʹ end of viral RNA.