Phosphorylation of Varicella-Zoster Virus IE63 Protein by Casein Kinases Influences Its Cellular Localization and Gene Regulation Activity

Abstract

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined mostly to the cytoplasm. Because phosphorylation is known to regulate many cellular events, we investigated the importance of this modification on the cellular localization of IE63 and on its regulatory properties. We demonstrate here that cellular casein kinases I and II are implicated in the in vitro and in vivo phosphorylation of IE63. A mutational approach also indicated that phosphorylation of the protein is important for its correct cellular localization in a cell type-dependent fashion. Using an activity test, we demonstrated that IE63 was able to repress the gene expression driven by two VZV promoters and that phosphorylation of the protein was required for its full repressive properties. Finally, we showed that IE63 was capable of exerting its repressive activity in the cytoplasm, as well as in the nucleus, suggesting a regulation at the transcriptional and/or posttranscriptional level.

The Immediate-Early 63 Protein of Varicella-Zoster Virus: Analysis of Functional Domains Required for Replication In Vitro and for T-Cell and Skin Tropism in the SCIDhu Model In Vivo

“…Four mutations were made in ORF63 to disrupt the putative NLS in the IE63 C terminus (5,46). The mutations were ⌬KRRR (deletion of amino acids 260 to 263), ⌬KRPQ (deletion of amino acids 226 to 229), ⌬KRRRϩKRPQ (deletion of amino acids 260 to 263 and amino acids 226 to 229), and a T244 frameshift that alters the C terminus of IE63 at amino acid 244 by deleting an adenosine at nucleotide 732.…”

“…Nuclear localization signal sequences in IE63. Intact IE63 shows a distinct nuclear localization in transiently transfected cells and at immediate-early times in VZV-infected cells (5,27,46). To assess whether mutations designed to disrupt the putative NLS resulted in altered cellular localization of IE63, HeLa cells were transfected with the pLXIN-IE63 mutant constructs and stained with polyclonal rabbit antiserum against IE63 (indocarbocyanine) and a murine monoclonal antibody against human nuclei (fluorescein isothiocyanate).…”

“…IE63 is heavily phosphorylated and is a substrate for phosphorylation by VZV ORF47 kinase and cellular kinases (5,18,34,46). The contributions of IE63 to VZV replication are poorly understood, although it does not seem to have direct transactivating properties (5,23,46).…”

“…IE63 is heavily phosphorylated and is a substrate for phosphorylation by VZV ORF47 kinase and cellular kinases (5,18,34,46). The contributions of IE63 to VZV replication are poorly understood, although it does not seem to have direct transactivating properties (5,23,46). IE63 binds to IE62, the major VZV transactivating protein, suggesting that IE63 might cooperate with IE62 to regulate viral gene expression, as indicated by the enhancement of IE62-induced transcription of the glycoprotein I (gI) promoter in the presence of IE63 (27,43).…”

“…To investigate IE63 nuclear localization, mutations were introduced to disrupt a potential nuclear localization signal (NLS), based upon previous mapping to the C terminus, amino acids 210 to 278 (46), and evidence that deletion of a KRRR sequence (amino acids 260 to 263) resulted in cytoplasmic localization of IE63 (5). When transfer of the ORF63 mutations into cosmids yielded infectious VZV, IE63 mutant viruses were evaluated for effects on VZV replication in vitro and virulence in skin and T-cell xenografts in vivo in the SCIDhu model of VZV pathogenesis (32,33,42).…”