Virion Association of IE62, the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein, Requires Expression of the VZV Open Reading Frame 66 Protein Kinase

Abstract

IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is associated with the tegument of gradient-purified virions. Here, we show that most, if not all, of the association requires the expression of open reading frame 66 (ORF66), a protein kinase. The association of IE62 with wild-type VZV virions was confirmed using immunoelectron microscopy with IE62-specific antibodies, which reacted with virions in ultrathin sections of VZV-infected cells. Fractionated purified virions from cells infected with recombinant VZV ROka contained substantial levels of the 175-kDa virion IE62 protein and also contained the ORF66 protein. However, virions from cells infected with recombinant VZV ROka66S, in which ORF66 is disrupted, lacked not only the ORF66 protein but also most of the virion 175-kDa IE62 polypeptide. The virion-associated protein kinase activity was still present in ROka66S virions, although the 175-kDa protein substrate for the virion kinase was absent, implying that the virion protein kinase is encoded by genes other than ORF66. The very low levels of IE62 in ROka66S virions indicate that ORF66 protein mediates the redistribution of IE62 to sites of tegument assembly. IE62 was resolved into several species from VZV-infected cells which showed mobility differences between ROka and ROka66S, and a specific form of IE62 was detected in ROka virions. These results are consistent with a role for the ORF66-mediated phosphorylation of IE62 that results in cytoplasmic distribution of the regulatory protein for tegument inclusion. They support a model in which VZV tegument acquisition occurs in the cytoplasm. As such, two unusual features of VZV IE62, namely, its virion inclusion and its phosphorylation and nuclear exclusion by the ORF66 protein kinase, are functionally linked.Varicella-zoster virus (VZV) is the ubiquitous human alphaherpesvirus that causes chickenpox upon primary infection and herpes zoster following reactivation from a long period of latency (reviewed in reference 1). In lytically infected cells, VZV gene expression occurs in a sequential cascade (34) and is likely regulated predominantly at the transcriptional level like that seen in cells infected with herpes simplex virus type 1 (HSV-1) (15). Viral genes are subdivided into immediateearly, early, and late, depending upon the requirements for their transcription and the timing of their synthesis. In transfected cells, transcription of VZV promoter-reporter constructs is influenced by a subset of VZV proteins including those encoded by open reading frames (ORFs) 4, 61, 62, 63, 10, and 29, and it is thus likely that these are the predominant regulatory proteins in VZV-infected cells (reviewed in reference 19).The major transactivator of viral genes is the product of the ORF62 gene, which stimulates transcription from all VZV promoters studied to date, including its own in certain cells (16,24,28,31,32). In VZV-infected cells, ORF62 is expressed as an immediate-early gene (10) and encodes a 1,310-residue protein desi...

Phosphorylation by the Varicella-Zoster Virus ORF47 Protein Serine Kinase Determines whether Endocytosed Viral gE Traffics to the trans- Golgi Network or Recycles to the Cell Membrane

“…Besides our results, a separate line of evidence supports the second cell-cell spread mechanism in the absence of virion assembly. Namely, in an ORF66-null VZV mutant, where IE62 is not incorporated into the virion, infectivity continued to spread through the cell culture (17,22). As also shown in Fig.…”

“…The gE protein is the major mediator of this property in alphaherpesviruses (18,24,26,38,46). The two mutually exclusive paradigms are (i) that fully assembled and enveloped capsids pass across plasma membranes and start a new cycle of infection in adjacent cells, and that infection follows the temporal classes of viral protein production, namely, regulatory proteins such as IE62, followed by early proteins such as ORF47, and finally late proteins such as the glycoproteins (18), and (ii) that syncytium formation or other cell-cell spread is not bound by virion assembly processes, but rather that VZV infection proceeds by viral glycoproteins entering uninfected cells and fusing the plasma membranes into syncytia before the appearance of intact virions (22,24).…”

Histone Deacetylases 1 and 2 Are Phosphorylated at Novel Sites during Varicella-Zoster Virus Infection

“…To date, the only well-characterized target of ORF66p is ORF62p, the major transcriptional regulatory protein of VZV. ORF66p directly phosphorylates ORF62p and modulates its cellular distribution during viral infection (9,(26)(27)(28). Based on their homology, it is suspected that ORF66p and U S 3 phosphorylate similar viral and cellular targets, although there are few data to substantiate this hypothesis.…”

Herpesvirus Assembly and Egress

“…It is interesting in this context that many tegument proteins are phosphorylated. Moreover, two virus-encoded protein kinases, the product of the UL13 gene, which is conserved in all herpesviruses, and the product of the US3 gene, which is present only in alphaherpesviruses, have been shown to represent virion components, presumably located in the tegument (16,42,56,62,99). The HSV-1 UL13 protein complexes with and phosphorylates gE (54), phosphorylates UL49 (15), and modulates UL41 function (57).…”