Expression and RNA dependent RNA polymerase activity of birnavirus VP1 protein in bacteria and yeast.
Birnaviruses typically encode a polyprotein that is the precursor to the structural proteins of the virus and a protein of rare abundance, VP1, that is the putative dsRNA replicase and/or transcriptase. We have reconstructed the VP1 gene of IBDV from a library of cDNA clones and expressed the gene in Escherichia coli and in Saccharomyces cerevisiae. We could not detect an RNA polymerase activity associated with E. coli-derived VP1, and neither could we promote the yeast-derived VP1 to replicate exogenous IBDV dsRNA. However, the yeast-derived VP1 was shown to have an actinomycin-insensitive RNA polymerase activity that can recognise an endogenous template in S. cerevisiae. Our work suggests that further studies on birnavirus replication may be best addressed using an S. cerevisiae expression system.
Macreadie
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CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Azad
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D001345:Autoradiography; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007243:Infectious bursal disease virus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012367:RNA, Viral / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D018076:DNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase