Expression and characterization of RNA-dependent RNA polymerase of Dendrolimus punctatus tetravirus.
Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis virus (NomegaV). To establish the function of DpTV RNA genome and to better understand the mechanism of viral replication, the putative RNA-dependent RNA polymerase (RdRp) domain has been cloned and expressed in Escherichia coli. The recombinant protein was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate viral RNA synthesis in a primer-independent manner but not by terminal nucleotidyle transferase activity in the presence of Mg2+ and RNA template. Mutation of the GDD to GAA interferes with the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.
Zhou
Liang
L
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, P R China.
Zhang
Jiamin
J
Wang
Xiaochun
X
Jiang
Hong
H
Yi
Fuming
F
Hu
Yuanyang
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D018076:DNA, Complementary; D016679:Genome, Viral; D007304:Insect Viruses / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D012328:RNA Viruses / Q000201:enzymology*; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014779:Virus Replication
D018076:DNA, Complementary; D012367:RNA, Viral; D011994:Recombinant Proteins; D012324:RNA-Dependent RNA Polymerase