乙肝病毒X蛋白(HBx)对HBV复制具有增强作用。我们的研究显示：HBx增强HBV复制的作用与其在基因转录水平对HBV C启动子的反式激活功能密切相关，且HBx反式激活功能区第52至65位和88至154位氨基酸是实现其增强HBV转录复制的两个重要功能区段。 本项目拟利用HBx反式激活功能区不同区段系列突变质粒，结合噬菌体展示技术和GST pull-down检测，通过筛选与 HBx反式激活功能区结合的蛋白分子，从而获得与有增强HBV复制功能的HBx相互作用的宿主蛋白；进一步利用HBV体内外复制模型与HBV启动子报导基因体系，观察HBx反式激活功能区结合蛋白在HBx激活HBV 启动子、增强HBV RNA转录和HBV DNA复制中的确切作用和机理。 研究HBx反式激活功能区结合蛋白在HBx增强HBV转录复制中的作用及分子机制，将有助于进一步阐明HBV的致病机理和寻找新的抗HBV作用靶点。

HBx has an important role in enhancing HBV replication. Our findings show that the augmentation effects of HBx on HBV replication are closely related to its trans-activation function to HBV C promoter, and the C-terminal two-thirds of HBx, which span aa 52 to 65 and 88 to 154 respectively, is responsible for its augmentation effect on HBV transcription and replication. In this project, by using a series of HBx clustered alanine substitution mutants in different sections of the trans-activation function domain, phage-display technology and GST pull-down assay, we plan to screen host binding proteins with HBx trans-activation function domain, and obtain candidate host proteins which can interact with C-terminal mutant HBx, and the mutant HBx has an augmentation role on HBV transcription and replication. Additionally, we would use in vitro and in vivo models of HBV replication as well as HBV promoter reporter gene system, and observe the exact role and mechanism of HBx trans-activation domain binding protein in HBV promoter activation, HBV RNA transcription and HBV DNA replication enhancement by HBx. Studying the role and molecular mechanism of HBx trans-activation domain binding protein in enhancing HBV transcription and replication, will help to further elucidate the pathogenic mechanism of HBV and looking for new targets for anti-HBV.

乙肝病毒X蛋白(HBx)对HBV复制具有增强作用。我们的研究显示：HBx增强HBV复制的作用与其在基因转录水平对HBV C启动子的反式激活功能密切相关。本项目利用反式激活功能区一系列突变HBx、噬菌体展示技术联合差异蛋白质组学筛选出可能与HBx增强HBV转录复制功能相关的差异宿主蛋白。通过综合分析，筛选出的可能与HBx增强HBV复制作用相关的结合蛋白包括GLUD1、USO1、SEC61A1、AHNAK、SMC4、LUC7L2、RPS6KA3、APOA1、TAGLN2、RIMKLB、ABCB4、PCK1和CYP2E1等。进一步利用HBV体外复制模型，验证了HBx与候选宿主蛋白RPS6KA3、TAGLN2、PCK1的相互作用及调控，阐明了这些宿主蛋白在HBx增强HBV转录复制中的作用及分子机制。本项目发现 ① HBx对HBV转录与复制的增强作用受宿主蛋白RPS6KA3表达水平的影响；② TAGLN2可能是HBx相互作用及调控的宿主蛋白，并在HBV转录复制调控中起着一定作用；③ HBx能够促进PCK1表达，上调的PCK1通过下调PGC-1α和PPARγ水平抑制HBV复制。本项目对宿主蛋白在HBx调控HBV复制中的作用和机制的研究结果将有助于进一步阐明HBV的致病机理，并为进一步寻找新的抗HBV作用靶点提供了线索。