乙型肝炎病毒X蛋白与RNA聚合酶Ⅱ亚单位竞争结合转录因子ⅡB的研究

Study of HBV X protein and RMP, an RPB5 mediate protein competitively interacting with general transcription factor TF Ⅱ B

目的探讨人ＲＮＡ聚合酶 Ⅱ亚单位ＲＰＢ５介导蛋白（ＲＰＢ５ ｍｅｄｉａｔｅ ｐｒｏｔｅｉｎ， ＲＭＰ）抑制乙型肝炎病毒Ｘ蛋白（ＨＢＸ）的反式转录激活作用的机制。方法应用基因重组技术、体外蛋白和蛋白结合试验（Ｐｕｌｌ—Ｄｏｗｎ试验）、氯霉素乙酰转移酶转化率分析（ ＣＡＴ试验）等。结果ＲＭＰ能与普通转录因子（ｔｒａｎｓｃｒｉｐｔｉｏｎ ｆａｃｔｏｒｓ， ＴＦ） Ⅱ Ｂ结合。ＴＦⅡＢ ｄ１０（１２－２９５）则是ＴＦⅡＢ结台ＲＭＰ的部位，与ＴＦⅡＢ结合ＨＢＸ的基因一致。蛋白质结台竞争试验提示 ＲＭＰ能特异地抑制 ＴＦ ⅡＢ与 ＨＢＸ的结合，同样 ＨＢＸ能抑制 ＴＦⅡＢ与 ＲＭＰ的结合。 ＲＭＰ特异地抑制 ＨＢＸ对ＨＢＸ反应元件（ ＨＢＸ ｒｏｐｎｓｉｖｅ ｅｌｅｍｅｎｔ， ＸＲＥ）的转录激活作用，此作用能被过量表达的 ＴＦ ⅡＢ取消。结论 ＲＭＰ是与 ＨＢＸ竞争结合普通转录因子 ＴＦⅡ Ｂ而抑制 ＨＢＸ的反式转录激活作用的。

Objective HBX is an essential viral protein that is important for HBV replication and might be a cofactor in the development of hepatocellular carcinoma. So many researchers are trying to find out the antagonistic factors of HBX. RMP, a newly cloned RNA polymerase II subunit RPB5 mediate protein, represses the transcription transactivation of HBV X protein. The purpose of this study was to elucidate the mechanism how RMP represses the transactivation of HBX. Methods in this report, DNA recombinant technique in vitro protein-protein binding assay(PullDown assay) and chloramphenicol acetyltransferase assay were used. Results The d10 domain of TF II B are responsible for binding to RMP, which is same as TF II B binding to the HBx protein. RMP specifically disrupts the binding between TF n B and HBX protein, vice versa HBX protein also disrupt the interaction of TF II B-RMP as demonstrated by the in vitro protein binding competition assay. Overexpression of RMP represses HBX transactivation on the reporters with HBX responsive ets-elements in transtient transfected cos- I cells. The repression can be rescued by overexpression of TF II B. Conclusion The results clearly suggested that transcription co-activator HBX protein and corepressor RMP competitively associated with TF fi B. Disrupting the interaction between transcription co-repressor RMP with TFllB is probably one of the mechanisms to account for the transcriptional transactivation of HBX protein.