Modulation of transcriptional machinery by hepatitis B virus X protein

HBx在肝癌的发生发展过程中具有积极的作用。以前报道RNA聚合酶II亚基5（RPB 5）是HBx的靶点之一，提示HBx可能调节转录机制。我们的主要结果如下。1). HBx、RPB 5和TFIIB之间的三聚体相互作用是HBx反式激活所必需的，因为与TFIIB或RPB 5结合缺陷的取代突变体的反式激活能力受损（J. Biol. Chem.，272：317（1997））。2）通过体内和体外转录测定，GaIDB融合的HBx不能充当转录激活因子，但HBx增强Gal-VP 16激活的转录，表明HBx可以充当共激活因子（J. Biol. Chem.，（1998））。3）通过Western克隆技术，获得了一个新的RPB 5介导蛋白（RMP）。在体外和体内，RMP强烈结合RPB 5，但既不结合HBx也不结合TBP。RMP以剂量依赖性方式抵消HBx反式激活。此外，RMP充当共阻遏物，因为它抑制Gal-VP 16的转录激活。这些结果表明，RMP在RPB 5和TFIIB之间的相互作用步骤负调节转录过程，这可能是HBx的靶过程（Mol. Cell.生物学，（1998））。4)HBx与p53相互作用并干扰p53依赖性反式激活。然而，HBx对p53功能的干扰不同于HBx的反式激活（Cancer Res.，（1997））。

HBx has been suspected to have positive roles in hepatocarcinogenesis. Previously e reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of HBx, suggesting a possibility that HBx may modulate transcription machinery. Our main results are following. 1).The trimeric interaction among HBx, RPB5 and TFIIB is necessary for HBx transactivation since the substitution mutants defective in binding either to TFIIB or RPB5 are impaired in transacting ability (J.Biol. Chem., 272 : 317 (1997)). 2)By in vivo and in vitro transcription assays, GaIDB-fused HBx can not act as transcriptional activator, but HBx augmented activated transcription by Gal-VP16, indicating that HBx can act as a coactivator (J.Biol. Chem., (1998)). 3)We isolated a novel RPB5-mediating protein (RMP) by a far Western cloning. RMP strongly bound RPB5 but neither HBx nor TBP in vitro and in vivo. RMP counteracts HBx transactivation in a dose dependent manner. Furthermore, RMP acts as a co-repressor since it inhibits transcriptional activation by Gal-VPl6. These RMP functions requires its RPB5-binding region, suggesting that these functional interference is due to competition between HBx and RMP to bind RMP.These results suggest that RMP negatively modulates transcription process at the interaction step between RPB5 and TFIIB which might be the target process of HBx (Mol. Cell. Biol., (1998)). 4)HBx interacts with p53 and interferes p53-dependent transactivation. However, the interference of the p53 function by HBx is distinct from the transactivation of HBx (Cancer Res., (1997)).