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.
Highlights
Antheraea 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)
This is somehow unusual for Cytoplasmic polyhedrosis virus (CPV) Reuter et al [21] and Graham et al [22] have reported presence of 171 bp and 141 nt long 5′UTR sequence in segment 6 of a novel seadornavirus related to Banna virus in Europe and in segment 5 of Choristoneura occidentalis CPV16, respectively
This type of analysis showed that p65 could be aligned with GTase domain (Figure 1A) containing a conserved Kx(I/V/L)S and HxnH motifs of Chuzan virus (CHV), blue tongue virus (BTV), Figure 1 Amino acid sequence alignment of AmCPV segment 5 (S5) protein with the guanylyltransferases of other reoviruses. (A) in the vicinity of putative Kx(I/V/L/R) S motif. [CHV, Banna virus (BAV); Kadipiro virus (KDV); BTV; avian orthoreovirus (ARV); mammalian reovirus (MRV); AmCPV S5; BmCPV] and (B) in the vicinity of putative (HxnH) motif
Summary
Antheraea 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). Cytoplasmic polyhedrosis virus (CPV) belongs to the genus Cypovirus of the family Reoviridae [1] and contains 10 segmented double stranded (ds) RNA [2]. The capping of nascent viral RNA strand is mediated by a guanylyltransferase (GTase) that uses GTP to form a covalently bound enzyme-substrate intermediate (autoguanylylation) before the GMP residue is transferred to 5′ end of mRNA. In case of CPV infecting mulberry silk worm, Bombyx mori (BmCPV), capsid is formed by three major proteins: VP1 (capsid shell protein), VP3 (turret protein) and VP5 (spike like protein) encoded by its genome segment 1, 4 and 7, respectively. The most recent structural comparison via cryo-EM of transcribing and non transcribing BmCPV showed that transfer of GMP moiety occurs to the 5′ end of the di phosphate ended RNA after its binding to the Lys234 residue of GTase pocket via phosphoamide linkage [9]
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