Genetic and biochemical evidence that EBNA 2 interaction with a 63-kDa cellular GTG-binding protein is essential for B lymphocyte growth transformation by EBV.
Genetic and biochemical evidence that EBNA 2 interaction with a 63-kDa cellular GTG-binding protein is essential for B lymphocyte growth transformation by EBV.
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遗传和生化证据表明，EBNA 2 与 63 kDa 细胞 GTG 结合蛋白的相互作用对于 EBV 引起的 B 淋巴细胞生长转化至关重要。
作者：
Yalamanchili,R;Tong,X;Grossman,S;Johannsen,E;Mosialos,G;Kieff,E
EB病毒（EBV）核蛋白2（EBNA 2）是病毒和细胞基因表达的酸性转录反式激活因子，并且对于原代B淋巴细胞的生长转化是必需的。EBNA 2反式激活反应元件（E2 RE）可以通过与GTGGGAA特异性DNA结合因子的相互作用来介导。我们现在通过S-Sepharose和EBNA 2亲和层析纯化该因子，并将其鉴定为单一的63-kDa蛋白。该蛋白质显示与来自用EBNA 2 FLAG表达载体转染的淋巴母细胞的EBNA 2特异性共免疫沉淀。在Cp、LMP 2和LMP 1启动子共有的GTGGGAA基序中，GTG突变为TCT导致p63识别丧失。EBNA 2氨基酸310-336足以与p63结合。在该27个氨基酸序列中，与1型和2型EBV的EBNA 2基因共有的唯一基序是GPPWWPP（I/V）（C/R）DP，因此其可能介导p63相互作用。WW突变为SS或FF消除了与p63的相互作用，表明WW的疏水性和芳香性特征对于其与p63的“关键”相互作用是必不可少的。WW突变为SS的EBNA 2也不能从EBNA 2缺失的病毒感染的细胞中标记拯救原代B淋巴细胞转化病毒，在平行实验中，白色在其他方面同基因的野生型EBNA 2容易标记拯救转化病毒。通过Cp E2 RE的EBNA 2反式激活被WW至SS突变完全消除，而-234至+40 LMP 1 E2 RE的反式激活仅部分受到影响。这些遗传学和生物化学实验支持EBNA 2 WW与p63 GTGGGA结合蛋白的相互作用对于EBV介导的细胞生长转化是必需的这一假设，因为它特异性地将EBNA 2与其应答元件相关联。这使得EBNA 2酸性结构域能够转录反式激活特定基因。
Epstein-Barr virus (EBV) nuclear protein 2 (EBNA 2) is an acidic transcriptional transactivator of virus and cell gene expression and is essential for growth transformation of primary B lymphocytes. EBNA 2 transactivation of response elements (E2REs) can be mediated by interaction with a GTGGGAA-specific DNA-binding factor(s). We now purify the factor by S-Sepharose and EBNA 2 affinity chromatography and identify it as a single 63-kDa protein. The protein is shown to specifically coimmunoprecipitate with EBNA 2 from lymphoblasts transfected with an EBNA 2 FLAG expression vector. Mutation of GTG to TCT in a GTGGGAA motif common to the Cp, LMP2, and LMP1 promoters results in loss of recognition by p63. EBNA 2 amino acids 310-336 are sufficient for p63 binding. The only motif in this 27 amino acid sequence which is common to the EBNA 2 genes of EBV types 1 and 2 is GPPWWPP (I/V) (C/R) DP, which is therefore likely to mediate p63 interaction. Mutation of WW to SS or FF ablates interaction with p63, indicating that both the hydrophobic and aromatic characteristics of WW are essential for its "key" interaction with p63. EBNA 2 with a WW mutated to SS is also unable to marker rescue primary B lymphocyte transforming virus from cells infected with an EBNA 2-deleted virus, white otherwise isogeneic wild-type EBNA 2 readily marker rescues transforming virus in parallel experiments. EBNA 2 transactivation through the Cp E2RE is completely abolished by the WW to SS mutation while transactivation of -234 to +40 LMP1 E2RE is only partially affected. These genetic and biochemical experiments support the hypothesis that EBNA 2 WW interaction with a p63 GTGGGAA-binding protein is essential for EBV-mediated cell growth transformation because it specifically associates EBNA 2 with its response elements. This enables the EBNA 2 acidic domain to transcriptionally transactivate specific genes.