Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.Varicella-zoster virus (VZV) contains two genes, the ORF47 gene and the ORF66 gene, with classical serine/threonine kinase motifs (26). Two VZV immediate-early proteins, ORF62 and ORF63, are functional homologs of herpes simplex virus (HSV) regulatory proteins ICP4 and ICP22, respectively.

The varicella zoster virus (VZV) IE63 protein is required for growth of the virus in cell culture and is expressed during both lytic and latent phases of VZV infection. We have investigated the physical and functional interaction of this protein with the major VZV transactivating protein IE62. The region of the IE63 protein required for interaction with the IE62 protein has been identified and encompasses the N-terminal 142 amino acids. We have found that the interaction is stable at physiological ionic strength. We have also shown that a portion of the IE63 and IE62 proteins colocalize in VZV-infected cells at both 15 and 48 h postinfection. IE63 was found to have no transcriptional activating or repressing activity within the context of a minimal VZV glycoprotein promoter. The presence of the IE63, however, upmodulated the IE62 transactivation of this promoter. Finally, we show that the IE63 protein can be coimmunoprecipitated with the cellular RNA polymerase II from infected cell extracts, indicating that it is present in a complex with that enzyme.

Like all alphaherpesviruses, varicella-zoster virus (VZV) infection proceeds by both cell-cell spread and virion production. Virions are enveloped within vacuoles located near the trans-Golgi network (TGN), while in cell-cell spread, surface glycoproteins fuse cells into syncytia. In this report, we delineate a potential role for serine/threonine phosphorylation of the cytoplasmic tail of the predominant VZV glycoprotein, gE, in these processes. The fact that VZV gE (formerly called gpI) is phosphorylated has been documented (E. A. Montalvo and C. Grose, Proc. Natl. Acad. Sci. USA 83:8967-8971, 1986), although respective roles of viral and cellular protein kinases have never been delineated. VZV ORF47 is a viral serine protein kinase that recognized a consensus sequence similar to that of casein kinase II (CKII). During open reading frame 47 (ORF47)-specific in vitro kinase assays, ORF47 phosphorylated four residues in the cytoplasmic tail of VZV gE (S593, S595, T596, and T598), thus modifying the known phosphofurin acidic cluster sorting protein 1 domain. CKII phosphorylated gE predominantly on the two threonine residues. In wild-type-virus-infected cells, where ORF47-mediated phosphorylation predominated, gE endocytosed and relocalized to the TGN. In cells infected with a VZV ORF47-null mutant, internalized VZV gE recycled to the plasma membrane and did not localize to the TGN. The mutant virus also formed larger syncytia than the wild-type virus, linking CKII-mediated gE phosphorylation with increased cell-cell spread. Thus, ORF47 and CKII behaved as "team players" in the phosphorylation of VZV gE. Taken together, the results showed that phosphorylation of VZV gE by ORF47 or CKII determined whether VZV infection proceeded toward a pathway likely involved with either virion production or cell-cell spread.Varicella-zoster virus (VZV), an alphaherpesvirus, displays a highly cell-associated phenotype in tissue culture; that is, VZV infection spreads from VZV-infected cells to uninfected cells with little or no extracellular virion production. Other alphaherpesviruses do not share this tissue culture phenotype (45). Because VZV is highly cell associated, viral mutant creation requires a cosmid system containing the entire VZV Oka genome, which can then be manipulated by molecular biological methods (2). Using this cosmid system, called rOka, several VZV-null mutants have been constructed.One of the mutants was open reading frame 47 (ORF47)-null VZV, designated VZV rOka-47S, in which stop codons replaced codons 166 and 167 of ORF47, the VZV UL protein serine kinase (15). In cell culture, the ORF47-null VZV displayed no distinguishing phenotype with regard to either plaque morphology or growth kinetics. However, VZV rOka-47S could not replicate in fetal skin or thymus implants in SCID-hu mice and replicated less efficiently in human T lymphocytes derived from fetal cord blood, a puzzling development since no tissue culture phenotype was previously observed (27, 40). Thus, ORF47 was required for efficient replication...

ORF47, a serine/threonine protein kinase encoded by varicella-zoster virus (VZV), has often been compared to the ubiquitous cellular kinase, casein kinase II (CKII). However, no direct comparison of the two protein kinases has been carried out. Herein, we show that the ORF47 kinase was resistant to heparin, while CKII activity is profoundly inhibited by the acidic molecule in vitro. ORF47 required the presence of polyamines (aliphatic, positively-charged molecules) for in vitro activity. When polyamines were depleted from MeWo cells prior to VZV infection by pretreatment with D,L-alpha-difluoromethylornithine, VZV replication was reduced by 80%. Finally, the substrate specificity of the ORF47 kinase was defined using an in vitro assay. The ORF47 kinase phosphorylated maltose-binding protein, the mouse IgG2A heavy chain, the rabbit IgG heavy chain, casein, VZV ORF62, and VZV ORF63. The ORF47 kinase failed to phosphorylate an ORF62 truncation mutant, glutathione-S-transferase, or VZV gB. In contrast, CKII weakly phosphorylated VZV gB in vitro. By analyzing the sequences of these substrates, the minimal ORF47 consensus sequence was deduced to be the following motif: S/T-X-D/E-D/E, with a marked preference for additional acidic amino acids in the -1 and +1 position.

ORF47, a serine protein kinase of varicella-zoster virus (VZV) and homolog of herpes simplex virus UL13, is an interesting modulator of VZV pathogenesis. This chapter summarizes research showing that ORF47 protein kinase activity, by virtue of phosphorylation of or binding to various viral substrates, regulates VZV proteins during all phases of viral infection and has a pronounced effect on the trafficking of gE, the predominant VZV glycoprotein, which in turn is critical for cell-to-cell spread of the virus. Casein kinase II, an ubiquitous cellular protein kinase, recognizes a similar but less stringent phosphorylation consensus sequence and can partially compensate for lack of ORF47 activity in VZV-infected cells. Differences between the phosphorylation consensus sites of the viral and cellular kinases are outlined in detail.