Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus.
Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123 kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123 kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37 degrees C at pH 6.0 in the presence of 3 mM MgCl(2). Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.
Ghorai
Suvankar
S
Department of Biotechnology, Indian Institute of Technology, Kharagpur, Kharagpur 721302, West Bengal, India.
Chakrabarti
Mrinmay
M
Roy
Sobhan
S
Chavali
Venkata Ramana Murthy
VR
Bagchi
Abhisek
A
Ghosh
Ananta Kumar
AK
D000818:Animals; D003001:Cloning, Molecular; D016000:Cluster Analysis; D003067:Coenzymes / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000821:virology*; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012087:Reoviridae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D003067:Coenzymes; D018076:DNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; D015636:Magnesium Chloride; D012324:RNA-Dependent RNA Polymerase