Cheong J H, Yi M, Lin Y, Murakami S
Department of Molecular Biology, Kanazawa University, Japan.
EMBO J. 1995 Jan 3;14(1):143-50. doi: 10.1002/j.1460-2075.1995.tb06984.x.
The X gene of human hepatitis B virus encodes the polypeptide HBx which transactivates viral and host genes through a variety of cis-acting enhancer elements present in RNA polymerases I, II and III promoters. To better understand the mechanism of X transactivation, we cloned cDNAs of proteins that bind HBx. Here we demonstrate that one of these cDNAs is a full-length cDNA of human RPB5, a subunit shared by RNA polymerases. The HBx transactivation domain and the central region of human RPB5 were necessary for the specific binding of the two proteins as shown by: (i) in vitro assays using deletion mutants of fusion proteins; (ii) in vivo assays which detect associated proteins by co-immunoprecipitation of the non-fused proteins from transfected HepG2 cells. Over-expressed HBx seemed to associate with assembled forms of endogenous human RPB5 in HBx-transfected cells, since the endogenous RPB5 co-immunoprecipitated with HBx. The HBx binding region of human RPB5 by itself stimulated chloramphenicol acetyltransferase activities from several different reporters having X-responsive element(s). Our results support the idea that the interaction of HBx and human RPB5 can facilitate HBx transactivation and that human RPB5 has a domain which can communicate with transcriptional regulators.
人类乙型肝炎病毒的X基因编码多肽HBx，该多肽通过RNA聚合酶I、II和III启动子中存在的多种顺式作用增强子元件反式激活病毒和宿主基因。为了更好地理解X反式激活的机制，我们克隆了与HBx结合的蛋白质的cDNA。在此我们证明，其中一个cDNA是人类RPB5的全长cDNA，RPB5是RNA聚合酶共有的一个亚基。如下所示，HBx反式激活结构域和人类RPB5的中央区域对于这两种蛋白质的特异性结合是必需的：(i) 使用融合蛋白缺失突变体的体外试验；(ii) 通过从转染的HepG2细胞中非融合蛋白的共免疫沉淀来检测相关蛋白的体内试验。在HBx转染的细胞中，过表达的HBx似乎与内源性人类RPB5的组装形式相关，因为内源性RPB5与HBx共免疫沉淀。人类RPB5的HBx结合区域自身可刺激来自几个具有X反应元件的不同报告基因的氯霉素乙酰转移酶活性。我们的结果支持这样一种观点，即HBx与人类RPB5的相互作用可促进HBx反式激活，并且人类RPB5具有一个可与转录调节因子进行交流的结构域。
