IDENTIFICATION AND CHARACTERIZATION OF THE ICP22 PROTEIN OF EQUINE HERPESVIRUS-1

The equine herpesvirus 1 (EHV-1) homolog of herpes simplex virus type 1 ICP22 is differently expressed from the fourth open reading frame of the inverted repeat (LR4) as a 1.4-kb early mRNA and a 1.7-kb late mR NA which are 3' coterminal (V. R. Holden, R. R Yalamanchili, R, N, Har ty, and D. J. O'Callaghan, J. Virol. 66:664-673, 1992). To extend the characterization of IR4 at the protein level, the synthesis and intrac ellular localization of the IR4 protein were investigated. Antiserum r aised against either a synthetic peptide corresponding to amino acids 270 to 286 or against a TrpE-IR4 fusion protein (IR4 residues 13 to 15 0) was used to identify the IR4 protein. Western immunoblot analysis r evealed that IR4 is expressed abundantly from an open reading frame co mposed of 293 codons as a family of proteins that migrate between 42 t o 47 kDa. The intracellular localization of IR4 was examined by cell f ractionation, indirect immunofluorescence, and laser-scanning confocal microscopy. These studies revealed that IR4 is localized predominantl y in the nucleus and is dispersed uniformly throughout the nucleus. In terestingly, when IR4 is expressed transiently in COS-l or LTK(-) cell s, a punctate staining pattern within the nucleus is observed by indir ect immunofluorescence. Cells transfected with an IR4 mutant construct that encodes a C-terminal truncated (19 amino acids) IR4 protein exhi bited greatly reduced intranuclear accumulation of the IR4 protein, in dicating that this domain possesses an important intranuclear localiza tion signal. Western blot analysis of EHV-1 virion proteins revealed t hat IR4 proteins are structural components of the virions. Surprisingl y, the 42-kDa species, which is the least abundant and the least modif ied form of the IR4 protein family in infected cell extracts, was the most abundant IR4 protein present in purified virions.