HBx BH3结构域在HBV复制和肝癌转化中的作用及机制研究

HBx protein plays pivotal roles in the processes of HBV infection, replication and has been implicated in the hepatocarcinogenesis. However, the essential functional domain the HBx is still not very clear. Our previous study demonstrated that the HBx interacted directly with two Bcl-2 family members, Bcl-2 and Bcl-xL through its BH3 domain to increase intracellular calcium level, thereby induced cell death and HBV replication. Recently, we determined the structural details of the complex of HBx BH3 domain and Bcl-xL by X-ray crystallography and identified the crucial amino acids which supported the interaction between BH3 domain and Bcl-xL. Based on these interesting findings, we firstly propose that the BH3 domain in the interaction of HBx and Bcl-2 is the potential target for modulating HBV replication and HCC transformation, thus we would like to prove this hypothesis. In this proposal, we plan to investigate the roles of these amino acids and their variants in the HBx-Bcl-2/xL binding, induction of hepatic cell toxicity, viral infectivity and replication in HepaRG cell model and primary human hepatocyte chimeric FRG mice which both support HBV infection. Moreover, we will establish an HBx induced HCC model in Fah-/- mice by virtue of the sleeping beauty (SB) transposon system, aiming to investigate the function and signature of the HBx BH3 domain on inducing hepatic inflammation and hepatocarcinogenesis. Our expected results will clarify the role and molecular mechanisms of HBx BH3 domain on HBV replication and hepatocarcinogenesis, which will lay solid foundations for further development of new intervening drugs targeting HBx BH3 domain for HBV therapy.

HBx在HBV感染复制及致癌过程中起重要作用。我们之前的研究揭示了HBx通过其BH3结构域直接结合Bcl-2/xL，上调细胞内钙离子水平来促进HBV复制和细胞死亡。我们新近解析了HBx BH3结构域与Bcl-xL的复合物结构，并鉴定出与之相关的关键氨基酸位点。我们提出“HBx BH3结构域作为影响HBV复制及肝癌转化的潜在靶点”的假设并拟以验证。本项目拟在上述研究基础上，利用基于HepaRG细胞的HBV感染复制模型和支持HBV感染的人肝嵌合鼠模型验证这些位点及其突变体对HBx与Bcl2/xL间相互作用、诱导肝细胞毒性反应、病毒感染和复制能力的影响，并进一步基于SB转座子系统和Fah基因缺陷鼠建立HBx致肝癌模型以探讨BH3结构域在肝癌转化的作用和机制。项目预期成果将阐明HBx BH3结构域在HBV复制和肝癌转化中的作用及机制，为未来进一步发展以HBx BH3为靶点的乙肝治疗新药物奠定基础。

HBx是乙型肝炎病毒HBV的一个多功能非结构蛋白，在HBV的感染、复制和致癌过程中起重要作用。我们之前的研究揭示了HBx通过其BH3结构域直接结合Bcl-2/xL，上调细胞内钙离子水平来促进HBV复制和细胞死亡。在此基础上我们进一步解析了乙肝病毒（HBV）X蛋白（HBx）BH3-like基序（aa113-135）与宿主抗凋亡蛋白Bcl-xL的复合物晶体结构，在体内外模型中证实了该基序对HBV复制的重要性。基于此发现设计的HBx（aa113-135）短肽可恢复HBx缺失的HBV病毒的复制，HBx（aa118-127）短肽可抑制HBV复制。该研究为靶向HBx的抗HBV新药设计提供了结构和功能方面的创新见解。BRF-1作为特异性调控RNA 聚合酶III转录的重要转录因子，在肿瘤发生发展中有重要作用，肝癌中BRF-1的高表达与肝癌患者的生存期呈负相关。在此基础上我们发现HBx能在转录水平上调RNA聚合酶III转录起始因子BRF-1表达，从而促进肝癌细胞的生长和增殖。我们还发现肝癌中BRF-1与HBx高表达成正相关。PP2A蛋白磷酸酶2a (PP2a) 是一种主要的丝氨酸-苏氨酸磷酸酶, 参与细胞内磷酸化信号转导过程, 对调节细胞周期和细胞凋亡至关重要。HBx能够通过靶向PP2a-B56γ促进肝癌细胞发生p53/p21依赖的细胞周期阻滞和凋亡。此外，我们还鉴定出HBx BH3结构域相互作用的新分子，它们可能成为乙肝及肝癌干预和治疗的新靶标。我们在HBx转基因鼠和乙肝相关肝癌的临床样本中初步揭示了HBx通过调控BRF-1和PP2a-B56γ引起肝细胞转化及肝癌发生的实验证据，研究结果将为我们揭示HBx在肝癌中致癌作用，为我们研究乙肝相关肝癌的靶向药物提供思路和指导。