Members of a novel family of mammalian protein kinases complement the DNA-negative phenotype of a vaccinia virus ts mutant defective in the B1 kinase.
Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking sequence similarity between B1 and the VRK enzymes, we proposed that they might share overlapping substrate specificity. We therefore sought to determine whether the human and mouse VRK1 enzymes (hVRK1 and mVRK1, respectively) could complement a B1 deficiency in vivo. Recombinant ts2 viruses expressing hVRK1, mVRK1, or wild-type B1 were able to synthesize viral DNA at high temperature, but those expressing the more distantly related human casein kinase 1 alpha 2 could not. Complementation required the enzymatic activity of hVRK1, since a catalytically inactive allele of hVRK1 was unable to confer a temperature-insensitive phenotype. Interestingly, rescue of viral DNA synthesis was not coupled to the ability to phosphorylate H5, the only virus-encoded protein shown to be a B1 substrate in vivo. Expression of hVRK1 during nonpermissive ts2 infections restored virus production and plaque formation, whereas expression of mVRK1 resulted in an intermediate level of rescue. Taken together, these observations indicate that enzymatically active cellular VRK1 kinases can perform the function(s) of B1 required for genome replication, most likely due to overlapping specificity for cellular and/or viral substrates.
Boyle
Kathleen A
KA
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Traktman
Paula
P
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D016415:Sequence Alignment; D013696:Temperature; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D004279:DNA, Viral; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C074058:VB1R protein, Vaccinia virus; D014764:Viral Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C109890:VRK1 protein, human; C484518:Vrk1 protein, mouse