PHOSPHORYLATION OF VARICELLA-ZOSTER VIRUS OPEN READING FRAME (ORF) 62REGULATORY PRODUCT BY VIRAL ORF 47-ASSOCIATED PROTEIN-KINASE

Varicella-zoster virus (VZV) encodes within its unique long region a g ene product with protein kinase motifs. In a previous study, we demons trated that immunoprecipitated VZV open reading frame (ORF) 47 protein was associated with a functional protein kinase activity, on the basi s of its ability to both autophosphorylate and phosphorylate artificia l substrates. To further define potential substrates of ORF 47-associa ted protein kinase, we analyzed individual viral phosphoproteins to de termine whether any were modified by the viral protein kinase. These c andidates included gene products of VZV ORFs 4, 61, 62, and 63, which are homologs of herpes simplex virus type 1 (HSV-1) immediate-early pr oteins. Each of the above VZV proteins was coimmunoprecipitated with O RF 47 kinase, and the immune complex was incubated in a protein kinase assay. Under these conditions, only the VZV immediate-early ORF 62 pr otein was phosphorylated by ORF 47-associated protein kinase. The spec ificity of this phosphorylation event was analyzed by a competition as say in which a recombinant ORF 47 protein lacking enzymatic activity w as able to reduce the amount of phosphorylation of ORF 62 protein by V ZV ORF 47-associated kinase. To provide an additional evaluation of sp ecificity, the experiment was repeated with [P-32]GTP instead of [P-32 ]ATP, because the VZV ORF 47 kinase has the distinctive property of us ing GTP as a phosphate donor. Again the ORF 62 substrate was phosphory lated. In summary, the VZV ORF 47-associated protein kinase (the HSV-1 UL13 homolog) catalyzed the in vitro phosphorylation of the VZV ORF 6 2 protein, the homolog of the HSV-1 ICP4 regulatory protein.