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.