Purification and Biochemical Characterization of the Protein Kinase Encoded by the US3 Gene of Herpes Simplex Virus Type 2

Abstract

Post-ribosomal cytoplasmic fractions from Vero cells mock-infected or infected with wild-type herpes simplex virus type 2 (HSV-2) or a US3 gene-disrupted mutant of HSV-2 were fractionated with DEAE-cellulose chromatography, and the peak fraction of the protein kinase which was detectable only in the extract of wild-type virus-infected cells was subjected to succesive chromatography. The enzyme was purified more than 1000-fold from the post-ribosomal supernatant, and the final preparation contained one major protein of apparent molecular weight 66 kilodalton (K), which was phosphorylated in the autophosphorylation reaction. Western blotting analysis showed that antibodies to an synthetic peptide corresponding to the 15 amino acids of the predicted HSV-2 US3 protein sequence strongly reacted with a 66 K protein in the enzyme fractions. On Superose 12 HR chromatography, the protein kinase activity was eluted as a single major peak at a position corresponding to an apparent molecular mass of approximately 60 K. These results suggest that the 66 K protein is the protein kinase encoded by the US3 gene of HSV-2 and that it acts as a monomer. The HSV-2 protein kinase was relatively resistant to high concentrations of salt, but KCl above 400 mM exerted a significant inhibitory effect. When the substrate specificity was investigated using synthetic oligopeptides, the peptides containing arginyl residues on the amino-terminal side of the target seryl residue were found to be the best substrates for the protein kinase. However, the replacement of the seryl residue to threonine markedly reduced the rate of phosphorylation by this enzyme, suggesting that threonine is a poor phosphate acceptor of the protein kinase. The enzyme was resistant to heparin, a potent inhibitor of casein kinase II, but was moderately sensitive to H-9 (N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride), a potent inhibitor of cyclic nucleotide-dependent protein kinases and protein kinase C. Quercetin, a bioflavonoid, also inhibited the protein kinase and the inhibitory effect was competitive towards ATP (Ki = 10 μM). The results indicate that the biochemical properties of the HSV-2 US3 protein kinase are very similar to those of the HSV-1 counterpart and pseudorabies virus-encoded 38-kDa protein kinase, but are different from those in several respects.