Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins.
The vaccinia virus B1 gene encodes a 34-kDa protein with homology to protein kinases. In L cells infected nonpermissively with mutants containing lesions in the B1 gene (ts2 and ts25), the infectious cycle arrests prior to DNA replication. In this report, we demonstrate that DNA synthesis ceases when cultures infected with these mutants at 32 degrees C are shifted to the nonpermissive temperature (39.5 degrees C) in the midst of DNA replication. We also show that B1 protein is synthesized transiently during the early phase of infection, even when the progression to later stages of gene expression is prevented. Although wild-type (wt) B1 is stable, the ts B1 proteins are markedly labile in both L and BSC40 cells at both permissive and nonpermissive temperatures. These results suggest that the ts phenotype of the mutants is complex and may in part reflect a temperature-dependent requirement for kinase activity, an induction of temperature sensitivity in B1 substrates under nonpermissive conditions, and/or ts complementation by host factors. To facilitate biochemical analyses, recombinant wt B1, ts2 B1, and ts25 B1 were produced in Escherichia coli. The wt protein was able to phosphorylate serine and threonine residues on several exogenous substrates in vitro. The activity of ts25 B1 was 3% that of the wt enzyme, and no detectable kinase activity was associated with ts2 B1. In light of the inactivity of the ts2 B1 protein in vitro and its extreme lability in vivo, we attempted to isolate a vaccinia virus B1 null mutant by targeted interruption of the B1 gene at 32 degrees C. No null mutants were isolated. These results indicate that the B1 protein kinase provides a vital function which cannot be supplied by the host or circumvented by incubation at 32 degrees C.
Rempel
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Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021.
Traktman
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P
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral; D004926:Escherichia coli / Q000235:genetics; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013912:Threonine / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D004279:DNA, Viral; D011994:Recombinant Proteins; C074058:VB1R protein, Vaccinia virus; D014764:Viral Proteins; D013912:Threonine; D012694:Serine