Lafemina R L, Pizzorno M C, Mosca J D, Hayward G S
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Virology. 1989 Oct;172(2):584-600. doi: 10.1016/0042-6822(89)90201-8.
Stable DNA-transfected Vero cell lines that express the major immediate-early nuclear antigen (IE68) of HCMV-(Towne) have been established. Immunofluorescence staining with monoclonal antibodies revealed that the protein was distributed either in a uniform diffuse nuclear pattern or as punctate nuclear granules in up to 80% of the cells in these cultures. In addition, 1 to 2% of the positive nuclei gave a distinctive staining pattern suggesting an association with the chromosomes of mitotic cells. Colcemid-blocking studies confirmed that most of the IE antigen was localized in the vicinity of condensed chromosomes in all metaphase cells after methanol fixation. In contrast, the SV40 large T-antigen protein was found to be preferentially excluded from metaphase chromosomes in a similar colcemid-treated human cell line. In transient expression assays, 1 to 2% of IE antigen-positive Vero, 293, or Balb/c3T3 cells also displayed a metaphase chromosome association pattern. Mapping studies using deletion and truncation mutants revealed that the monoclonal antibodies recognized epitopes encoded within the small NH2-terminal exons that are common to both the IE1 and IE2 gene products. However, an intact exon-4 (IE1) region, but not the exon-5 (IE2) region of the HCMV IE gene complex, was required for conferring both the normal diffuse nuclear localization pattern and the chromosome-association properties. Furthermore, removal of the glutamic acid-rich COOH-terminal coding portions of exon-4 resulted in aberrant staining patterns with production of large, phase-dense nuclear globules in all positive cells. An association between the IE68 IE1 protein and metaphase chromosomes was also detected after HCMV-(Towne) infection in a small proportion of both nonpermissive Balb/c3T3 cells and permissive HF cells. We conclude that the IE1 acidic nuclear phosphoprotein displays some properties similar to those of the EBNA-1 protein of Epstein-Barr virus and suggest that it may potentially play a role in maintenance of the latent state of HCMV DNA.
已建立稳定转染 DNA 的 Vero 细胞系，其表达人巨细胞病毒（HCMV）-（Towne 株）的主要即刻早期核抗原（IE68）。用单克隆抗体进行免疫荧光染色显示，在这些培养物中，高达 80%的细胞中该蛋白呈均匀弥散的核模式分布或呈点状核颗粒分布。此外，1%至 2%的阳性细胞核呈现出独特的染色模式，提示与有丝分裂细胞的染色体相关。秋水仙酰胺阻断研究证实，在甲醇固定后的所有中期细胞中，大多数 IE 抗原定位于浓缩染色体附近。相比之下，在类似秋水仙酰胺处理的人细胞系中，发现 SV40 大 T 抗原蛋白优先排除在中期染色体之外。在瞬时表达试验中，1%至 2%的 IE 抗原阳性的 Vero 细胞、293 细胞或 Balb/c3T3 细胞也呈现中期染色体相关模式。使用缺失和截短突变体的定位研究表明，单克隆抗体识别 IE1 和 IE2 基因产物共有的小 NH2 末端外显子内编码的表位。然而，人巨细胞病毒 IE 基因复合体的完整外显子 4（IE1）区域而非外显子 5（IE2）区域，对于赋予正常的弥散核定位模式和染色体相关特性是必需的。此外，去除外显子 4 富含谷氨酸的 COOH 末端编码部分会导致异常染色模式，在所有阳性细胞中产生大的、相位密集的核球。在一小部分非允许性 Balb/c3T3 细胞和允许性 HF 细胞经 HCMV-（Towne）感染后，也检测到 IE68 IE1 蛋白与中期染色体之间的关联。我们得出结论，IE1 酸性核磷蛋白表现出一些与爱泼斯坦-巴尔病毒的 EBNA-1 蛋白相似的特性，并提示它可能在维持 HCMV DNA 的潜伏状态中发挥潜在作用。
