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: Poulomi Biswas, Anirban Kundu, Ananta Kumar Ghosh
• Journal: The Journal of general virology (J Gen Virol) Vol. 96 Issue Pt 1 Pg. 95-105 (Jan 2015) ISSN: 1465-2099 [Electronic] England
• PMID: 25228490 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 The Authors.

Chemical References

• RNA Caps
• RNA, Viral
• Recombinant Proteins
• Viral Nonstructural Proteins
• S-Adenosylmethionine
• Methyltransferases
• Acid Anhydride Hydrolases
• RNA triphosphatase

Topics

• Acid Anhydride Hydrolases (genetics)
• Amino Acid Sequence
• Animals
• Cloning, Molecular
• Genome, Viral (genetics)
• Kinetics
• Methyltransferases (genetics)
• Molecular Sequence Data
• Moths (virology)
• Protein Structure, Tertiary (genetics)
• RNA Caps (genetics)
• RNA, Viral (genetics)
• Recombinant Proteins (genetics)
• Reoviridae (genetics)
• Reoviridae Infections (virology)
• S-Adenosylmethionine (genetics)
• Sequence Alignment
• Viral Nonstructural Proteins (genetics)