乙型肝炎病毒X蛋白与翻译延长因子1A1（eEF1a1)相互作用研究

Hepatitis B virus (HBV)-encoded X protein (HBx protein) is a multi-functional regulatory protein. It can interact with a variety of cellular proteins thus affect its function and plays a pivotal role in the pathogenesis of HBV related diseases. We previously identified a cellular protein eukaryotic translation elongation factor 1 alpha 1 (eEF1a1) that interacts specifically with HBx, the interaction between HBx protein and eEF1a1 was further confirmed using a GST pull-down assay and co-immunoprecipitation, and in vitro translation experiments indicated that HBx can inhibit the translation of luciferase reporter mRNA by interaction with eEF1a1. In this project, the effects of HBx on eEF1a1-mediated protein translation will be further analyzed. At first, a mammalian two-hybrid assay and co-immunoprecipitation will be employed to mapping the regions of HBx and eEF1a1 required for the binding of these two proteins. Then to investigate the molecular mechanism involved in the influences of HBx on protein translation including whether HBx could interfere the eEF1a1 combined with the aminoacyl -tRNA and the impact of eEF1a1 phosphorylation levels regulated by the HBx protein. Finally, to screening for the target genes regulated by HBx in the protein translation level in hepatocellular carcinoma cells. This study will provide new insights into the molecular mechanisms involved in the functions of the HBx protein, also provide new ideas for the development of more effective means of prevention and treatment of HBV infection.

乙型肝炎病毒X蛋白（HBx）是乙型肝炎病毒（HBV）相关性肝细胞疾病重要的致病因子，具有多功能性，可与细胞内的多种蛋白质发生相互作用影响其功能。我们在前期研究证实HBx与翻译延长因子1A1(eEF1a1)相互作用，且通过体外翻译实验表明HBx同eEF1a1结合抑制了eEF1a1参与蛋白翻译的功能，本项目是在此研究基础上，进一步深入研究HBx与eEF1a1结合的功能区段，探讨HBx对eEF1a1同氨酰-tRNA结合的影响以及对eEF1a1磷酸化水平的影响，筛查在蛋白翻译水平上受HBx影响的肝细胞蛋白。本研究从全新角度揭示HBx参与HBV致病作用的机制，丰富HBx生物学功能的认识，也为发展更为有效的HBV防治手段提供新思路。

乙型肝炎病毒X蛋白（HBx）是乙型肝炎病毒（HBV）相关性肝细胞疾病重要的致病因子，具有多功能性，可与细胞内的多种蛋白质发生相互作用影响其功能。我们的研究证实HBx与翻译延长因子1A1（eEF1a1）存在相互作用，体外翻译实验证实HBx同eEF1a1结合抑制了eEF1a1参与蛋白翻译的功能。本项目中我们通过对HBx和eEF1a1的编码区进行分段克隆，免疫共沉淀（Co-IP）和细胞双杂交实验确定了二者结合的功能区段，即HBx蛋白末端121-154aa区段与eEF1a1蛋白C端333-462aa区段结合。合成14C同位素标记的氨酰-tRNA，同GST-eEF1a1融合蛋白以及纯化的HBx融合蛋白进行体外氨酰-tRNA结合实验，结果提示HBx同eEF1a1结合抑制后者与氨酰t-RNA结合。另外，采用磷酸化的丝/苏氨酸抗体进行Westen Blot实验，发现过表达HBx可促进肝癌细胞eEF1a1蛋白发生磷酸化。以上结果提示HBx同eEF1a1相互作用，抑制了eEF1a1与氨酰t-RNA结合，并促进eEF1a1蛋白磷酸化，从而抑制了eEF1a1参与蛋白翻译的功能。将HBx的重组腺病毒或者空对照病毒感染Huh7细胞，采用表达谱芯片联合定量蛋白质组学（iTRAQ蛋白质谱技术）检测，获得了92个候选的基因，其可能在蛋白翻译水平上受HBx调控。进一步通过RT-qPCR和Western Blot等实验最终验证了6个基因，包括PRR36、CNTRL、MYL1、MST161、PLA2G12B、KRT14 ，在蛋白翻译水平上受HBx调控，导致蛋白表达下调。