Phosphorylation of the Nuclear Form of Varicella-Zoster Virus Immediate-Early Protein 63 by Casein Kinase II at Serine 186

Abstract

Varicella-zoster virus (VZV) open reading frame (ORF) 63 is abundantly transcribed in latently infectedhuman ganglia and encodes a 278-amino-acid protein, IE63, with immediate-early kinetics. IE63 is expressed in the cytoplasm of neurons during VZV latency and in both the cytoplasm and the nucleus during productive infection; however, the mechanism(s) involved in IE63 nuclear import and retention has remained unclear. We constructed and identified a recombinant monoclonal antibody to detect a posttranslationally modified form of IE63. Analysis of a series of IE63 truncation and substitution mutants showed that amino acids 186 to 195 are required for antibody binding. Synthetic peptides corresponding to this region identified IE63 S186 as a target for casein kinase II phosphorylation. In addition, acidic charges supplied by E194 and E195 were required for antibody binding. Immunofluorescence analysis of VZV-infected MeWo cells using the recombinant monoclonal antibody detected IE63 exclusively in the nuclei of infected cells, indicating that casein kinase II phosphorylation of S186 occurs in the nucleus and possibly identifying an initial molecular event operative in VZV reactivation.Primary infection with the human varicella-zoster virus (VZV) typically causes varicella (chickenpox), after which the virus becomes latent in ganglionic neurons along the entire neuraxis. Reactivation decades later usually produces zoster (shingles) (15). During latency, transcripts mapping to VZV open reading frames (ORFs) 62, 63, and 66 and proteins corresponding to ORFs 21, 29, 62, 63, and 66 have been detected in human ganglia obtained at autopsy (7,9,12,16,19,20,25,26,36). Quantitative PCR has shown that ORF 63 is the most prevalent and abundant VZV transcript detected during latency (8).VZV ORF 63, which is located within the terminal and internal repeat regions of the virus genome and is present as two copies, encodes the immediate-early (IE) protein IE63. This 278-amino-acid protein is present in the cytoplasm during latency but is located predominantly in the nucleus during productive infection (13,26).During productive infection in tissue culture cells, VZV IE63 is phosphorylated by casein kinase I (CKI), casein kinase II (CKII), and cyclin-dependent kinase 1 (CDK1) (2,4,17,35,37). In vitro phosphorylation assays have shown that IE63 is phosphorylated by CKII, CDK1, and a protein kinase encoded by ORF 47 (2,4,17,22). Sequence analysis has suggested 19 potential phosphorylation sites (2, 4). IE63 amino acid S224 is phosphorylated by CDK1 (17), while S165, S173, and S185 are phosphorylated in vitro by serine/threonine kinase, as indicated by alanine substitution mutation of IE63 (2).Previous studies have shown that phosphorylation of IE63 is required for efficient VZV replication in cell culture (2, 6).Using an antibody that recognizes a posttranslationally modified (PTM) form of IE63, we found that IE63 S186 is phosphorylated by CKII and that phosphorylated S186 is present only in the nuclei of VZV-infected cells. MATERIALS...

Identification of phosphorylated residues on varicella-zoster virus immediate-early protein ORF63

“…Our identification of phosphorylation at Ser224 supports results that demonstrated that Ser224 was a target for CDK1 and that a Ser224Ala mutation altered localization of ORF63p and its transcriptional regulatory properties . Recently, we demonstrated that Ser186 was phosphorylated by CKII (Mueller et al, 2009), which is consistent with this study. Also, our identification of possible phosphorylation at Thr171 and/ or Ser173 (and to a lesser extent Ser12) confirms the work of Baiker et al (2004) who demonstrated that Ala substitution at these residues reduced overall phosphorylation of ORF63p.…”

“…The protein is extensively phosphorylated both in vitro and in vivo by viral and cellular kinases (Baiker et al, 2004;Bontems et al, 2002;Debrus et al, 1995;Habran et al, 2005;Heineman & Cohen, 1995;Kenyon et al, 2001;Mueller et al, 2009;Stevenson et al, 1996;Walters et al, 2008). Computerassisted analysis (http://www.cbs.dtu.dk/services/NetPhos) of ORF63p identified 29 putative Ser, Thr and Tyr phosphorylation sites, including consensus target sequences for casein kinase I (CKI), CKII and cyclin-dependent kinase 1 (CDK1).…”

Varicella-Zoster Virus Transcriptome in Latently Infected Human Ganglia

“…VZV ORFs 4 and 62 encode regulatory proteins involved in activation of early and late gene transcription (20,24). VZV ORF 63 encodes a highly phosphorylated protein (32) and regulates VZV gene transcription (25), most likely through epigenetic modification of the virus genome (1,15,19). VZV ORF 18 encodes the small subunit of ribonucleotide reductase, an enzyme critical to synthesis of DNA precursors, and also protects neurons from apoptotic cell death (34).…”

A comparison of herpes simplex virus type 1 and varicella-zoster virus latency and reactivation

“…VZV IE63 is a 278 aa protein present in the virus tegument (Kinchington et al, 1995;Sadzot-Delvaux et al, 1998). During productive infection, the highly phosphorylated protein (Mueller et al, 2010) is predominantly located in the nucleus (Mueller et al, 2009). In tissue culture cells, VZV IE63 represses transcription (Di Valentin et al, 2005), most likely through histone modification (Ambagala et al, 2009), as well as reducing translation in response to IFN induction (Ambagala & Cohen, 2007); however, in human ganglia the presence of VZV IE63 is yet to be confirmed, but may function to inhibit VZV-induced apoptosis (Hood et al, 2006;Pugazhenthi et al, 2011).…”