Genome segment 5 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes a bona fide guanylyltransferase

Abstract

BackgroundAntheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects non mulberry Indian silk worm, Antheraea mylitta, and contains eleven segmented double stranded RNA in its genome (S1-S11). Some of its genome segments (S1-S3, and S6-S11) have been previously characterized but genome segment encoding the viral guanylyltransferase which helps in RNA capping has not been characterized.ResultsIn this study genome segment 5 (S5) of AmCPV was converted to cDNA, cloned and sequenced. S5 consisted of 2180 nucleotides, with one long ORF of 1818 nucleotides and could encode a protein of 606 amino acids with molecular mass of ~65 kDa (p65). Bioinformatics analysis showed presence of KLRS and HxnH motifs as observed in some other reoviral guanylyltransferase and suggests that S5 may encodes viral guanylyltransferase. The ORF of S5 was expressed in E. coli as 65 kDa his tagged fusion protein, purified through Ni-NTA chromatography and polyclonal antibody was raised. Immunoblot analysis of virion particles with the purified antibody showed specific immunoreactive band and suggests p65 as a viral structural protein. Functional analysis showed that recombinant p65 possesses guanylyltransferase activity, and transfers GMP moiety to the 5' diphosphate (A/G) ended viral RNA after the formation of p65-GMP complex for capping. Kinetic analysis showed Km of this enzyme for GTP and RNA was 34.24 uM and 98.35 nM, respectively. Site directed mutagenesis at K21A in KLRS motif, and H93A or H105A in HxnH motif completely abolished the autoguanylylation activity and indicates importance of these residues at these sites. Thermodynamic analysis showed p65-GTP interaction was primarily driven by enthalpy (ΔH = -399.1 ± 4.1 kJ/mol) whereas the p65-RNA interaction by favorable entropy (0.043 ± 0.0049 kJ/ mol).ConclusionViral capping enzymes play a critical role in the post transcriptional or post replication modification in case of RNA virus. Our results of cloning, sequencing and functional analysis of AmCPV S5 indicates that S5 encoded p65 through its guanylyltransferase activity can transfer guanine residue to the 5' end of viral RNA for capping. Further studies will help to understand complete capping process of cypoviral RNA during viral replication within the viral capsid.

Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases

“…In several viruses, such as BmCPV, BTV and rotavirus, GTase and MTase activities are provided by one protein; however, in the insect orbivirus JKT-7400, these two enzyme activities are provided by two different proteins: VP4 provides MTase activity and VP6 provides GTase activity (Liao & Stollar, 1997a, b). In this respect AmCPV seems to be different from BmCPV, BTV and rotavirus, but similar to orbivirus, as it has been reported recently that AmCPV S5-encoded p65 provides GTase without any MTase activity (Biswas et al, 2014). Therefore, for the completion of the capping reaction, MTase activity must be provided by proteins encoded by separate genome segments.…”

“…S1 and S3 code for viral capsid proteins (Chakrabarti et al, 2010), S2 codes for RdRp (Ghorai et al, 2010), S6 codes for a protein having ATP-binding and ATPase activity (Chavali et al, 2008), S7 and S8 code for viral structural proteins (Chavali & Ghosh, 2007;Jangam et al, 2006), S9 codes for a non-structural protein having RNA-binding properties (Qanungo et al, 2002), S10 codes for viral polyhedrin (Sinha-Datta et al, 2005), and S11 does not contain any ORF (Jangam et al, 2006). We have reported recently that AmCPV S5 encodes a 65 kDa viral GTase without any RTPase activity and catalyses the formation of a RNA cap structure using GTP as a substrate through initial formation of an enzyme-GMP complex via phosphoamide linkage and then transferring the GMP moiety to the diphosphate-ended viral RNA (Biswas et al, 2014). As sequences of AmCPV do not show any homology with BmCPV and the mechanism of capping has not been fully elucidated, especially the methylation of the cap structure, molecular and biochemical characterization of the capping enzymes is necessary.…”

“…32 P]GTP and 5 mg S5-encoded recombinant p65 (GTase) at 37 uC for 30 min to transfer the GMP moiety to the 59-diphosphate-ended RNA creating G*pppG-RNA (Biswas et al, 2014). RNA was extracted with phenol/ chloroform (1 : 1, v/v), precipitated with ethanol and resuspended in DEPC-treated water.…”