THE HERPES-SIMPLEX VIRUS-1 PROTEIN-KINASE ENCODED BY THE US3 GENE MEDIATES POSTTRANSLATIONAL MODIFICATION OF THE PHOSPHOPROTEIN ENCODED BY THE UL34 GENE

Earlier studies have shown that a herpes simplex virus 1 (HSV-1) open reading frame, U(S)3, encodes a novel protein kinase and have characterized the cognate amino acid sequence which is phosphorylated by this enzyme. This report identifies an apparently essential viral phosphoprotein whose posttranslational processing involves the viral protein kinase. Analyses of viral proteins phosphorylated in the course of productive infection revealed a phosphoprotein whose mobility was viral protein kinase and serotype dependent. Thus, the corresponding HSV-1 and HSV-2 phosphoproteins differ in their electrophoretic mobilities, and the phosphoprotein specified by the HSV-1 mutant deleted in U(S)3 (R7041) differs from that of the corresponding HSV-1 and HSV-2 proteins. Analyses of HSV-1 x HSV-2 recombinants mapped the phosphoprotein between 0.42 and 0.47 map units on the prototype HSV-1 DNA map. Within this region, the U(L)34 open reading frame was predicted to encode a protein of appropriate molecular weight which would also contain the consensus target site for phosphorylation by the viral protein kinase as previously defined with synthetic peptides. Replacement of the native U(L)34 gene with a U(L)34 gene tagged with a 17-amino-acid epitope from the alpha-4 protein identified this gene as encoding the phosphoprotein. Finally, mutagenesis of the predicted phosphorylation site on U(L)34 in the viral genome, and specifically the substitution of threonine or serine with alanine in the product of the U(L)34 gene, yielded phosphoproteins whose electrophoretic mobilities could not be differentiated from that of the U(S)3- mutant. We conclude that the posttranslational processing of the U(L)34 gene product to its wild-type phenotype requires the participation of the viral protein kinase. While the viral protein kinase is not essential for viral replication in cells in culture, the U(L)34 gene product itself may not be dispensable.