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

Abstract

Efficient replication of varicella-zoster virus (VZV) in cell culture requires expression of protein encoded by VZV open reading frame 63 (ORF63p). Two-dimensional gel analysis demonstrates that ORF63p is extensively modified. Mass spectroscopy analysis of ORF63p isolated from transiently transfected HEK 293 and stably transfected MeWo cells identified 10 phosphorylated residues. In VZV-infected MeWo cells, only six phosphorylated residues were detected. This report identifies phosphorylation of two previously uncharacterized residues (Ser5 and Ser31) in ORF63p extracted from cells infected with VZV or transfected with an ORF63p expression plasmid. Computational analysis of ORF63p for known kinase substrates did not identify Ser5 or Ser31 as candidate phosphorylation sites, suggesting that either atypical recognition sequences or novel cellular kinases are involved in ORF63p post-translational modification.

Fig. 1.

2D gel analysis of ORF63p from VZV-infected MeWo cells. Two days post-infection, cell lysates from VZV-infected MeWo cells were first resolved by isoelectric focusing (pH 3.9–5.1) followed by Western blot analysis as described in the text. Arrows indicate major ORF63p species detected by Western blot analysis.

Fig. 2.

MS analysis of ORF63p in vivo phosphorylation. Peptides corresponding to 98 % of the protein were identified from autonomously expressed ORF63p and protein isolated from VZV-infected cells. Lower case, italicized, bold letters indicate residues not covered by MS. All phosphorylated residues are identified with a box, the amino acid position, and stars indicate ORF63p protein source from which phosphorylated residues were identified. The four sources of ORF63p protein are indicated: HEK 293, ORF63p purified from transfected HEK 293 cells; MeWo+VZV, ORF63p purified from VZV-infected MeWo cells; MeWo-ECD63, ORF63p purified from MeWo-ECD63 following ecdyosone induction; MeWo-ECD63+VZV, ORF63p purified from MeWo-ECD63 following ecdyosone induction and VZV infection.