A DNA ligase gene in the Copenhagen strain of vaccinia virus is nonessential for viral replication and recombination.
Biochemical and genetic analyses have been conducted to determine whether a vaccinia virus open reading frame (orf) with extensive homology to the Saccharomyces cerevisiae DNA ligase gene encodes a functional ligase activity. This orf in HindIII A, designated A50R, is capable of encoding a 552-amino-acid, 63.4-kDa polypeptide. Full-length A50R mRNA produced in vitro directed the synthesis of a polypeptide with an apparent molecular weight of 57 kDa. Significantly, translation reactions programmed with A50R mRNA were capable of ligating a 3-kb Notl restriction fragment into multimers. DNA ligase activity was not detectable when either truncated sense or full-length antisense mRNA was translated in vitro. In extracts prepared from cells infected with wt vaccinia virus, DNA ligase activity was detected as assayed by the formation of a 57 kDa ligase-AMP adduct which was expressed early in the viral replication cycle. In cells infected with a DNA ligase deletion mutant no equivalent AMP-labeled adduct was detected. Relative to wt virus, the DNA ligase deletion mutant exhibited no significant differences in homologous recombination. These results indicate that the vaccinia orf A50R encodes a functional DNA ligase expressed early in infection, but this DNA ligase is nonessential for either recombination or viral replication.
Colinas
R J
RJ
Department of Microbiology and Immunology, Albany Medical College, New York 12208.
Goebel
S J
SJ
Davis
S W
SW
Johnson
G P
GP
Norton
E K
EK
Paoletti
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002872:Chromosome Deletion; D011088:DNA Ligases / Q000235:genetics* / Q000378:metabolism; D003718:Denmark; D005814:Genes, Viral; D008969:Molecular Sequence Data; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D014709:Vero Cells; D015678:Viral Structural Proteins / Q000235:genetics*; D014779:Virus Replication
D015678:Viral Structural Proteins; D011088:DNA Ligases