Characterization of the Shope fibroma virus DNA ligase gene.
The Shope fibroma virus (SFV) DNA ligase gene has been cloned and sequenced, and the biochemical requirements of the gene product have been determined in vitro. The SFV ligase gene maps to the BamHI L1/L2 boundary and spans 1.7 kb. The gene is predicted to encode a 559-amino-acid protein of M(r) = 63,139 which shares 45% amino acid identity with Orthopoxvirus ligases. The C-terminal two-thirds of the protein appears to encode the catalytic domain and shares distant homology with many ligases. The N-terminal homology is shared between only Orthopoxviruses and Leporipoxviruses and suggests that DNA ligases may be composite structures consisting of two independently evolved protein domains. Although the the gene encodes features characteristic of both early and late poxviral genes, Northern analysis showed that SFV ligase is expressed as a late gene product. In order to prove the identity of the protein it was expressed as a glutathione S-transferase fusion in Escherichia coli, affinity purified, and shown to be a Mg2+.ATP-dependent ligase in vitro. The recombinant protein can also form a covalent ligase.AMP complex characteristic of ATP-dependent DNA ligases. The SFV ligase gene can be disrupted and is thus not essential for viral growth in culture. This was shown by recombining a PCR product, encoding a P7.5 promoter and E. coli guanine phosphoribosyltransferase gene (gpt) into the open reading frame, and selecting for gpt+ viruses. This work provides insights into the evolution of Orthopoxviruses and Leporipoxviruses and strains suitable for a detailed analysis of the role DNA ligases play in poxviral recombination.
Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
R J
RJ
Parks
B D
BD
Lichty
C
C
Karakis
D H
DH
Evans
D000595:Amino Acid Sequence; D001483:Base Sequence; D011088:DNA Ligases / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D012776:Fibroma Virus, Rabbit / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015678:Viral Structural Proteins / Q000235:genetics*
D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D011088:DNA Ligases