The varicella-zoster virus ORF66 protein induces kinase activity and is dispensable for viral replication

Varicella-zoster virus (VZV) open reading frames (ORFs) 47 and 66 encode proteins that are homologous to a family of eukaryotic serine-threonine kinases, Prior studies shelved that the VZV ORF47 protein has kinase activity in vitro and is dispensable for replication in cultured cells, To examine the role of the ORF66 protein during infection, we constructed VZV recombinants that are unable to express either the ORF66 protein (ROka 66S) or both the ORF47 and ORF66 proteins (ROka 47S/66S). VZV unable to express ORF66 grew to titers similar to those of the parental VZV (ROka) in vitro; however, VZV lacking both ORF66 and ORF47 grew to titers lower than those of ROka, Nuclear extracts from ROka 66S- or ROka 47S-infected cells showed a 48-kDa phosphoprotein(s); a phosphoprotein dth a similar size was not present in nuclear extracts from ROka 47S/66S-infected cells, To determine the role of the ORF66 protein in the phosphorylation of specific VZV-encoded proteins, we immunoprecipitated known VZV phosphoproteins (ORF4, ORP62, ORP63, and ORF68 proteins) from nuclear extracts of phosphate-labeled cells infected with ROka, ROka 66S, or ROka 47S/66S. Each of the VZV phosphoproteins was phosphorylated to a similar extent in the presence or absence of either the ORF66 protein or both the ORF66 and ORF47 proteins, From these studies we conclude (i) neither ORF66 alone nor ORF66 and ORF47 in combination are essential for VZV growth in cultured cells, (ii) ORF66 either is a protein kinase or induces protein kinase activity during infection, and (iii) the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 do not require either ORF66 alone or ORF66 and ORF47 for phosphorylation in vitro.