Localization of the coding region for an Epstein-Barr virus early antigen and inducible expression of this 60-kilodalton nuclear protein in transfected fibroblast cell lines.
Localization of the coding region for an Epstein-Barr virus early antigen and inducible expression of this 60-kilodalton nuclear protein in transfected fibroblast cell lines.
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Epstein-Barr 病毒早期抗原编码区的定位以及这种 60 千道尔顿核蛋白在转染的成纤维细胞系中的诱导表达。
DOI：
10.1128/jvi.56.3.852-859.1985
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发表时间：
1985
影响因子：
5.4
通讯作者：
Hayward,SD
中科院分区：
文献类型：
--
作者：
Cho,MS;Jeang,KT;Hayward,SD
在用野生型和P3 HR-1缺陷DNA片段转染的成纤维细胞中研究了EB病毒早期抗原（EA）复合物组分的表达，所述DNA片段覆盖EB病毒基因组的BamHI-M-S区域。用BglII-J片段转染并用人血清染色的幼仓鼠肾（BHK）细胞在5%的细胞群中显示出阳性核染色，所述人血清在间接免疫荧光测定中对EA [EA（D）]的弥散组分呈阳性。在用限制性内切酶BglII、StuI、HindIII或PvuII转染前切割BglII-J不影响EA表达，而在用BamHI或EcoRI切割前减少或消除了EA的合成。通过使用单个克隆的亚片段证实了这些观察结果。其中HindIII和StuI位点被消除的Bal 31缺失克隆（pTS 1）保持活性，而其中缺失延伸至更接近EcoRI位点的克隆（pTS 5）活性大大降低。跨BglII-J的单个BamHI-M或BamHI-S片段的转染也导致很少或没有EA表达。由这些实验确定的2.1-丝氨酸蛋白酶生物活性区精确地对应于BMLF 1开放阅读框架。用P3 HR-1缺陷型DNA克隆或BglII-J野生型片段转染的BHK细胞的免疫印迹分析鉴定了该EA（D）编码区的产物是由主要的60千道尔顿产物和次要的45和50千道尔顿种类组成的多肽家族。在潜伏性EB病毒感染的淋巴细胞中，这些早期抗原不表达，但可以通过用丁酸钠或佛波醇酯处理培养物来诱导。使用在瞬时测定中呈阳性的BglII-J和pTS 6克隆，我们还建立了Neor共选择的BHK和Vero细胞系，其显示出类似的60千道尔顿EA（D）蛋白的调节表达。在这些细胞系中，EA（D）的组成型表达是有限的（通过间接免疫荧光检测为0.1%阳性，并且通过免疫印迹分析检测不到）。而丁酸钠处理可诱导EA（D）的表达。在诱导的培养物中，高达30%的细胞通过免疫荧光是EA（D）阳性的，并且伴随着60千道尔顿的EA（D）多肽的出现。
Expression of a component of the Epstein-Barr virus early antigen (EA) complex has been studied in fibroblast cells transfected with both wild-type and P3HR-1 defective DNA fragments covering the BamHI-M-S region of the Epstein-Barr virus genome. Baby hamster kidney (BHK) cells transfected with the BglII-J fragment and stained with human serum that was positive for the diffuse component of EA [EA(D)] in an indirect immunofluorescence assay exhibited positive nuclear staining in 5% of the cell population. Cleavage of BglII-J before transfection with the restriction enzyme BglII, StuI, HindIII, or PvuII did not affect EA expression, whereas prior cleavage with BamHI or EcoRI reduced or eliminated synthesis of EA. These observations were confirmed by using individual cloned subfragments. A Bal 31 deletion clone (pTS1) in which the HindIII and StuI sites were eliminated retained activity, whereas a clone (pTS5) in which the deletion extended closer to the EcoRI site had greatly reduced activity. Transfection of the individual BamHI-M or BamHI-S fragments, which span BglII-J, also resulted in little or no EA expression. The 2.1-kilobase biologically active region defined by these experiments corresponds precisely to the BMLF1 open reading frame. Immunoblot analyses of BHK cells transfected with either P3HR-1 defective DNA clones or the BglII-J wild-type fragment identified the product of this EA(D) coding region as a family of polypeptides consisting of a major 60-kilodalton product and minor 45- and 50-kilodalton species. In latently Epstein-Barr virus-infected lymphocytes these early antigens are not expressed, but can be induced by treatment of the cultures with sodium butyrate or phorbol esters. Using the BglII-J and pTS6 clones that were positive in transient assays, we also established Neor coselected BHK and Vero cell lines which showed similar regulated expression of the 60-kilodalton EA(D) protein. In these cell lines constitutive expression of EA(D) was limited (0.1% positive by indirect immunofluorescence and undetectable by immunoblot analysis). However, expression of EA(D) could be induced by treatment with sodium butyrate. In the induced cultures, up to 30% of the cells were EA(D) positive by immunofluorescence, and there was a concomitant appearance of the 60-kilodalton EA(D) polypeptide.