Lin Y, Nomura T, Cheong J, Dorjsuren D, Iida K, Murakami S
Department of Molecular Biology, Cancer Research Institute, Kanazawa University, Takara-machi 13-1, Kanazawa 920, Japan.
J Biol Chem. 1997 Mar 14;272(11):7132-9. doi: 10.1074/jbc.272.11.7132.
Hepatitis B virus X protein (HBx) transactivates viral and cellular genes through a wide variety of cis-elements. However, the mechanism is still obscure. Our finding that HBx directly interacts with RNA polymerase II subunit 5 (RPB5), a common subunit of RNA polymerases, implies that HBx directly modulates the function of RNA polymerase (Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 142-150). In this context, we examined the possibility that HBx and RPB5 interact with other general transcription factors. HBx and RPB5 specifically bound to transcription factor IIB (TFIIB) in vitro, both of which were detected by either far-Western blotting or the glutathione S-transferase-resin pull-down assay. Delineation of the binding regions of these three proteins revealed that HBx, RPB5, and TFIIB each has two binding regions for the other two proteins. Co-immunoprecipitation using HepG2 cell lysates that express HBx demonstrated trimeric interaction in vivo. Some HBx substitution mutants, which had severely impaired transacting activity, exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner, suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB. These results indicated that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB.
乙型肝炎病毒X蛋白（HBx）通过多种顺式元件反式激活病毒和细胞基因。然而，其机制仍不清楚。我们发现HBx直接与RNA聚合酶II亚基5（RPB5）相互作用，RPB5是RNA聚合酶的一个共同亚基，这意味着HBx直接调节RNA聚合酶的功能（Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 142 - 150）。在此背景下，我们研究了HBx和RPB5与其他一般转录因子相互作用的可能性。HBx和RPB5在体外特异性结合转录因子IIB（TFIIB），这两种蛋白均可通过远缘Western印迹法或谷胱甘肽S - 转移酶树脂下拉试验检测到。对这三种蛋白结合区域的描绘显示，HBx、RPB5和TFIIB各自具有与其他两种蛋白的两个结合区域。使用表达HBx的HepG2细胞裂解物进行的共免疫沉淀证明了体内三聚体相互作用。一些反式作用活性严重受损的HBx替代突变体，以互斥的方式表现出与TFIIB或RPB5的结合亲和力降低，这表明HBx反式激活需要RPB5和TFIIB两者的相互作用。这些结果表明，HBx是一种新型病毒调节剂，通过与RPB5和TFIIB相互作用来稳定RNA聚合酶与TFIIB之间的结合，从而促进转录起始。
