Identification and initial characterization of the IR6 protein of equine herpesvirus 1

The IR6 gene of equine herpesvirus 1 (EHV-1) is a novel gene that maps within each inverted repeat (IR), encodes a potential protein of 272 amino acids, and is expressed as a 1.2-kb RNA whose synthesis begins at very early times (1.5 h) after infection and continues throughout the infection cycle (C. A. Breeden, R. R. Yalamanchili, C.F. Colle, and D.J. O'Callaghan, Virology 191:649-660,1992). To identify the IR6 protein and ascertain its properties, we generated an IR6-specific polyclonal antiserum to a TrpE/IR6 fusion protein containing 129 amino acids (residues 134 to 262) of the IR6 protein. This antiserum immunoprecipitated a 33-kDa protein generated by in vitro translation of mRNA transcribed from a pGEM construct (IR6/pGEM-3Z) that contains the entire IR6 open reading frame. The anti-IR6 antibody also recognized an infected-cell protein of approximately 33 kDa that was expressed as early as 1 to 2 h postinfection and was synthesized throughout the infection cycle. A variety of biochemical analyses including radiolabeling the IR6 protein with oligosaccharide precursors, translation of IR6 mRNA in the presence of canine pancreatic microsomes, radiolabeling the IR6 protein in the presence of tunicamycin, and pulse-chase labeling experiments indicated that the two potential sites for N-linked glycosylation were not used and that the IR6 protein does not enter the secretory pathway. To address the possibility that the unique IR6 gene encodes a novel regulatory protein, we transiently transfected an IR6 expression construct into L-M fibroblasts alone or with an immediate-early gene expression construct along with a representative EHV-1 immediate-early, early, or late promoter-chloramphenicol acetyltransferase reporter construct. The results indicated that the IR6 protein does not affect the expression of these representative promoter constructs. Interestingly, the IR6 protein was shown to be phosphorylated and to associate with purified EHV-1 virions and nucleocapsids. Lastly, immunofluorescence and laser-scanning confocal microscopic analyses revealed that the IR6 protein is distributed throughout the cytoplasm at early times postinfection and that by 4 to 6 h it appears as "dash-shaped" structures that localize to the perinuclear region. At late times after infection (8 to 12 h), these structures assemble around the nucleus, and three-dimensional image analyses reveal that the IR6 protein forms a crown-like structure that surrounds the nucleus as a perinuclear network.