Cho M S, Milman G, Hayward S D
J Virol. 1985 Dec;56(3):860-6. doi: 10.1128/JVI.56.3.860-866.1985.
We used antiserum raised against the bacterially synthesized product of one of the open reading frames in Epstein-Barr virus (EBV) BamHI fragment M to demonstrate that this reading frame (BMRF1) codes for a nuclear protein of the diffuse early antigen (EA) class. In indirect immunofluorescence assays, the rabbit anti-BMRF1 antiserum gave nuclear staining in approximately 5% of Raji cells which had been treated with sodium butyrate, and positive fluorescence was observed in both acetone- and methanol-fixed cells. Uninduced Raji cultures contained less than 0.1% positive cells regardless of whether indirect immunofluorescence or anti-complement immunofluorescence was used. In immunoblot analyses, the rabbit serum identified a family of polypeptides of 46 to 55 kilodaltons (kDa) in total protein extracts from B95-8 cells or from butyrate-induced Raji cells. In both cell types, the dominant polypeptides were the 48- and 50-kDa species. This same family of polypeptides was identified when the immunoblots were reacted with the R3 monoclonal antibody, and we concluded that this antibody also recognized the product of the BMRF1 open reading frame. Fibroblast cell lines containing EBV BamHI fragment M were established by cotransfection of baby hamster kidney cells with BamHI-M and the gene for neomycin resistance. Aminoglycoside G418-resistant colonies which showed evidence for EBV antigen expression in immunofluorescence assays were selected, and clonal cell lines were established. After 3 to 4 months of passaging, constitutive synthesis of EA was no longer detectable in these cell lines either by immunofluorescence or by immunoblot analysis. However, in the one cell line examined, synthesis of the 48- to 50-kDa EA was induced by treatment of the culture with sodium butyrate. Thus, the regulation of expression of this EA in transfected fibroblasts is analogous to that seen in Raji lymphoblasts. We showed previously that BamHI fragment M also contains the coding sequences for a 60-kDa nuclear EA, and hence BamHI-M encodes two separate components of the diffuse EA complex.
我们使用针对爱泼斯坦 - 巴尔病毒（EBV）BamHI片段M中一个开放阅读框的细菌合成产物产生的抗血清，来证明这个阅读框（BMRF1）编码一种弥漫性早期抗原（EA）类的核蛋白。在间接免疫荧光试验中，兔抗BMRF1抗血清在约5%经丁酸钠处理的Raji细胞中产生核染色，并且在丙酮固定和甲醇固定的细胞中均观察到阳性荧光。无论使用间接免疫荧光还是抗补体免疫荧光，未诱导的Raji培养物中阳性细胞均少于0.1%。在免疫印迹分析中，兔血清在来自B95 - 8细胞或丁酸钠诱导的Raji细胞的总蛋白提取物中鉴定出一族46至55千道尔顿（kDa）的多肽。在这两种细胞类型中，主要的多肽是48 kDa和50 kDa的种类。当免疫印迹与R3单克隆抗体反应时，鉴定出了同一族多肽，并且我们得出结论，该抗体也识别BMRF1开放阅读框的产物。通过将幼仓鼠肾细胞与BamHI - M和新霉素抗性基因共转染，建立了含有EBV BamHI片段M的成纤维细胞系。选择在免疫荧光试验中显示出EBV抗原表达证据的氨基糖苷G418抗性菌落，并建立克隆细胞系。传代3至4个月后，通过免疫荧光或免疫印迹分析在这些细胞系中均不再可检测到EA的组成型合成。然而，在所检测的一个细胞系中，用丁酸钠处理培养物可诱导48至50 kDa EA的合成。因此，这种EA在转染的成纤维细胞中的表达调控与在Raji淋巴母细胞中所见的类似。我们先前表明，BamHI片段M还包含一种60 kDa核EA的编码序列，因此BamHI - M编码弥漫性EA复合物的两个独立成分。
