A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.
Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.
Stuart
D T
DT
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Upton
C
C
Higman
M A
MA
Niles
E G
EG
McFadden
G
G
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D005470:Fluorometry; D005814:Genes, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009699:N-Glycosyl Hydrolases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D014498:Uracil / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication
D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D014498:Uracil; D011189:Potassium Chloride; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase