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Function of Hepatitis B Virus X protein

乙型肝炎病毒X蛋白的功能

基本信息

批准号：
12213050
负责人：
MURAKAMI Seishi
金额：
$ 27.14万
依托单位：
KANAZAWA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12213050/
关键词：
HBv X protein RNA polymerase RPB5 RMP HBV replicon Transcriptional modulation Telomerase HVBレプリコン HBVX蛋白 TFIIF RAP30 テロメラーゼ活性 RAP40

项目摘要

For better understanding molecular mechanism of transcriptional modulation by HBV X protein (HBx), we studied structure and function of RNA polymerase subunit 5 (RPB5) as a nuclear target of HBx, and contribution of HBx on immortalization and/or transformation process of human cells. In addition, subcellular localization and nuclear function of RMP which is a functional antagonist of HBx. The followings are main results of the project in this fiscal year.1) By analyzing clustered alanine substitution mutant (Cm) and point mutant (Pm) libraries of the middle part of RPB5, we pinpointed 6 resideus critical for HBx-binding and 6 residues for TFIIF subunit RAP30-binding.Among them, 4 residues・F76, 1104, T111 and S113, are critical both for the bindings. The former two residues are not solvent exposed and probably contributing to the structural integrity. T111 and S113 are exposed and is in near position to DNA in light of the Pol II crystal models. The 4 residues are also critical or impor … More tant for DNA-binding ability of RPB5 (in preparation). Taken together, DNA-binding ability of RPB5 may be the target of HBx and RAP30.2) Using the Cm library of HBx, we addressed the critical region(s) of HBx for augmentation ability on HBV replication in a HBV replicon system. which is defective in X-ORF. Two discontinuous regions in the coactivation domain of HBx are indispensable for the augmentation effect on HBV replication. In the same experiment, the same regions were required not only for increase in HBV DNA but also for increase in pregenomic (pg) RNA. The same regions were also critical for the coactivation function of HBx, suggesting that HBx coactivates pgRNA synthesis that resulted in increase in HBV DNA synthesis.3) Recently it was found that RMP/URI, a functional antagonist of HBx, is localized with RPB5 in cytoplasm. Subcellular localization of RMP/URI can be modulated in the presence of DMAP1 and nuclear RMP/URI acts as a corepressor. From these results, RMP/URI is a regulatory protein in cytoplasm as well as nucleus.4) We addressed whether HBx acts positively in immortalization and/or transformation process of human cells. In our preliminary results, immortalization of human primary cells is barely affected by HBx, but transformation frequency of immortalized human cells seems to be augmented by HBx in the presence of activated oncogenes. This facilitating role of HBx requires the coactivation domain of HBx. Less

为了更好地了解HBVX蛋白（HBx）转录调控的分子机制，我们研究了HBx核靶RNA聚合酶亚基5（RPB 5）的结构和功能，以及HBx在人细胞永生化和/或转化过程中的作用。此外，还对HBx的功能性拮抗剂RMP的亚细胞定位和核功能进行了研究。本财年该项目的主要成果如下：1）通过分析RPB 5中部的聚集性丙氨酸取代突变体（Cm）和点突变体（Pm）库，我们找到了6个对HBx结合至关重要的残基和6个对TFIIF亚基RAP 30结合至关重要的残基，其中4个残基·F76、1104、T111和S113对结合都至关重要。前两种残留物未暴露于溶剂，可能有助于结构完整性。T111和S113是暴露的，并且根据Pol II晶体模型处于与DNA接近的位置。这4个残基也是关键或重要的。 ...更多信息 RPB 5的DNA结合能力的突变体（制备中）。综上所述，RPB 5的DNA结合能力可能是HBx和RAP 30的靶点。2）使用HBx的Cm文库，我们解决了HBx在HBV复制子系统中增强HBV复制能力的关键区域。在X-ORF中有缺陷。HBx共激活结构域中的两个不连续区域对于增强HBV复制的作用是必不可少的。在相同的实验中，相同的区域不仅需要HBV DNA的增加，而且也需要前基因组（pg）RNA的增加。这些区域对HBx的共激活功能也很关键，提示HBx共激活pgRNA的合成，导致HBV DNA合成增加。3）最近发现，HBx的功能性拮抗剂RMP/URI与RPB 5定位于细胞质中。RMP/URI的亚细胞定位可以在DMAP 1的存在下调节，并且核RMP/URI充当辅阻遏物。从这些结果来看，RMP/URI是一种细胞质和细胞核中的调节蛋白。4）我们探讨了HBx是否在人类细胞的永生化和/或转化过程中起积极作用。在我们的初步结果中，人原代细胞的永生化几乎不受HBx的影响，但在激活的癌基因存在下，永生化人细胞的转化频率似乎被HBx增强。HBx的这种促进作用需要HBx的共激活结构域。少