title: Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases.
abstract: 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 5' RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-l-methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2'-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 N(7)-and ribose 2'-O-MTase activities. A MTase assay using in vitro transcribed AmCPV S2 RNA having a 5' G*pppG end showed that guanine N(7) methylation occurred prior to the ribose 2'-O methylation to yield a m(7)GpppG/m(7)GpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished N(7)- and 2'-O-MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the Km values of N(7)-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 5' end of viral RNA.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India.
authors.forename: Poulomi
authors.initials: P
authors.lastname: Biswas
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India.
authors.forename: Anirban
authors.initials: A
authors.lastname: Kundu
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India.
authors.forename: Ananta Kumar
authors.initials: AK
authors.lastname: Ghosh
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D016679:Genome, Viral / Q000235:genetics*; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D009036:Moths / Q000821:virology*; D017434:Protein Structure, Tertiary / Q000235:genetics; D012315:RNA Caps / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D012087:Reoviridae / Q000235:genetics*; D012088:Reoviridae Infections / Q000821:virology; D012436:S-Adenosylmethionine / Q000235:genetics; D016415:Sequence Alignment; D017361:Viral Nonstructural Proteins / Q000235:genetics
chemical_list: D012315:RNA Caps; D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase