CHARACTERIZATION OF THE NOVEL PROTEIN-KINASE ACTIVITY PRESENT IN THE R1 SUBUNIT OF HERPES-SIMPLEX VIRUS RIBONUCLEOTIDE REDUCTASE

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide red uctase following expression in Escherichia coli. Autophosphorylation a ctivity was observed when kinase assays were performed with immunoprec ipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of his tones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of th ese two proteins are similar. We further characterized the protein kin ase activity of HSV-1 R1 by producing insertion and deletion mutants c onstructed with a plasmid expressing R1 amino acids 1 to 449. C-termin al deletion analysis identified the catalytic core of the enzyme as co mprising residues 1 to 292, and this polypeptide will be useful for st ructural determinations by X-ray crystallography. Insertion of a 4-ami no-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 comp letely inactivated activity, and an insertion at residue 136 reduced a ctivity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoylade nosine, which covalently modifies conventional eukaryotic kinases at a n essential lysine residue within the active site, did label HSV R1, b ut this labelling occurred outside the N-terminal domain. These data i ndicate that the HSV R1 kinase is novel and distinct hom other eukaryo tic protein kinases.