Phosphorylation of the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein IE62 by the VZV Open Reading Frame 66 Protein Kinase

Abstract

IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is nuclear at early times of VZV infection but then becomes predominantly cytoplasmic as a result of expression of the protein kinase encoded by open reading frame 66 (ORF66). Cytoplasmic forms of IE62 are required for its inclusion as an abundant VZV virion tegument protein. Here we show that ORF66 directly phosphorylates IE62 at two residues, with phosphorylation at S686 being sufficient to regulate IE62 nuclear import. Phosphotryptic peptide analyses established an ORF66 kinase-mediated phosphorylation of the complete IE62 protein in transfected and VZV-infected cells. Using truncated and point-mutated IE62 peptides, ORF66-directed phosphorylation was mapped to residues S686 and S722, immediately downstream of the IE62 nuclear localization signal. An IE62 protein with an S686A mutation retained efficient nuclear import activity, even in the presence of functional ORF66 protein kinase, but an IE62 protein containing an S686D alteration was imported into the nucleus inefficiently. In contrast, the nuclear import of IE62 carrying an S722A mutation was still modulated by ORF66 expression, and IE62 with an S722D mutation was imported efficiently into the nucleus. An in vitro phosphorylation assay was developed using bacterially expressed IE62-maltose binding protein fusions as substrates for immunopurified ORF66 protein kinase from recombinant baculovirus-infected insect cells. ORF66 kinase phosphorylated the IE62 peptides, with similar specificities for residues S686 and S722. These results indicate that IE62 nuclear import is modulated as a result of direct phosphorylation of IE62 by ORF66 kinase. This represents an interaction that is, so far, unique among the alphaherpesviruses.

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.…”

“…ORF47p (a homolog of HSV-1 U L 13, Epstein-Barr virus BGLF4, and cytomegalovirus U L 97) is related to the CKII family of cellular proteins, targeting Ser/Thr residues preceded by acidic residues, and is conserved in all Herpesvirinae (25,45,47). ORF66p (an HSV-1 U S 3 homolog) is present only in Alphaherpesvirinae and is a basophilic kinase that phosphorylates Ser/Thr residues with a target motif similar to that recognized by protein kinase A (PKA) (9,32,47). Both kinases carry out important functions that are required for efficient viral replication in a cell-type-dependent manner (3,11,24).…”

“…To differentiate between these possibilities, we asked if ORF66p could directly phosphorylate HDAC1 and HDAC2. To test this, we developed an improved in vitro kinase assay, based on that previously described to characterize ORF66p phosphorylation of ORF62p (9). In this system, kinase substrates are fused with MBP and protein kinases are fused with GST; each is then purified by affinity binding.…”

“…MBP substrates were purified as detailed previously (9) except that inductions were carried out in Escherichia coli BL21 cells at 30°C for 3 h.…”

“…Plasmids expressing maltose binding protein (MBP) and MBP-IE62 fusion proteins containing IE62 residues 571 to 733 were described previously (9). MBPs containing wild-type murine HDAC2 and a mutant form of protein containing five Ala substitutions at Ser 394, 407, 411, 422, and 424 were expressed from plasmid DNAs derived from murine HDAC2 cDNAs (gifts from Ed Seto, University of South Florida, Tampa, FL) in frame with MBP in pMalC2 (New England Biolabs, Inc., Beverly, MA).…”