O'Callaghan D J, Colle C F, Flowers C C, Smith R H, Benoit J N, Bigger C A
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130.
J Virol. 1994 Sep;68(9):5351-64. doi: 10.1128/JVI.68.9.5351-5364.1994.
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.
马疱疹病毒1型（EHV-1）的IR6基因是一个新基因，定位于每个反向重复序列（IR）内，编码一种含272个氨基酸的潜在蛋白，并表达为一种1.2 kb的RNA，其合成在感染后很早的时间（1.5小时）开始，并在整个感染周期持续进行（C.A. Breeden、R.R. Yalamanchili、C.F. Colle和D.J. O'Callaghan，《病毒学》191:649 - 660，1992）。为了鉴定IR6蛋白并确定其特性，我们针对一种包含IR6蛋白129个氨基酸（第134至262位残基）的TrpE/IR6融合蛋白制备了IR6特异性多克隆抗血清。该抗血清免疫沉淀了由体外翻译从包含整个IR6开放阅读框的pGEM构建体（IR6/pGEM - 3Z）转录的mRNA所产生的一种33 kDa蛋白。抗IR6抗体还识别一种感染细胞蛋白，其大小约为33 kDa，在感染后1至2小时就开始表达，并在整个感染周期持续合成。包括用寡糖前体对IR6蛋白进行放射性标记、在犬胰腺微粒体存在下翻译IR6 mRNA、在衣霉素存在下对IR6蛋白进行放射性标记以及脉冲追踪标记实验在内的各种生化分析表明，N - 连接糖基化的两个潜在位点未被利用，并且IR6蛋白不进入分泌途径。为了探究独特的IR6基因是否编码一种新型调节蛋白，我们将一个IR6表达构建体单独或与一个立即早期基因表达构建体以及一个代表性的EHV - 1立即早期、早期或晚期启动子 - 氯霉素乙酰转移酶报告构建体一起瞬时转染到L - M成纤维细胞中。结果表明，IR6蛋白不影响这些代表性启动子构建体的表达。有趣的是，IR6蛋白被证明可被磷酸化，并与纯化的EHV - 1病毒粒子和核衣壳相关联。最后，免疫荧光和激光扫描共聚焦显微镜分析显示，IR6蛋白在感染后早期分布于整个细胞质中，到4至6小时时，它呈现为定位于核周区域的“短划线状”结构。在感染后期（8至12小时），这些结构围绕细胞核聚集，三维图像分析显示，IR6蛋白形成一个围绕细胞核的冠状结构，作为一个核周网络。
