Jia Baosen, Guo Minggao, Li Gaiyun, Yu Demin, Zhang Xinxin, Lan Ke, Deng Qiang
Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Department of General Surgery, Sixth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
J Virol. 2015 Feb;89(4):2041-51. doi: 10.1128/JVI.03106-14. Epub 2014 Nov 26.
Hepatitis B, which caused by hepatitis B virus (HBV) infection, remains a major health threat worldwide. Hepatic injury and regeneration from chronic inflammation are the main driving factors of liver fibrosis and cirrhosis in chronic hepatitis B. Proinflammatory tumor necrosis factor alpha (TNF-α) has been implicated as a major inducer of liver cell death during viral hepatitis. Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes, expression of hepatitis B virus core (HBc) protein made cells susceptible to TNF-α-induced apoptosis. We found by tandem affinity purification and mass spectrometry that receptor of activated protein kinase C 1 (RACK1) interacted with HBc. RACK1 was recently reported as a scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating TNF-α-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase (JNK). In line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to TNF-α-induced apoptosis, while overexpression of RACK1 counteracted the proapoptotic activity of HBc. Capsid particle formation was not obligatory for HBc proapoptotic activity, as analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc sensitized hepatocytes to TNF-α-induced apoptosis by disrupting the interaction between MKK7 and RACK1. Our study is thus the first indication of the pathogenic effects of HBc in liver injury during hepatitis B.
Our study revealed a previously unappreciated role of HBc in TNF-α-mediated apoptosis. The proapoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In particular, HBV variants associated with severe hepatitis may upregulate apoptosis of hepatocytes through enhanced HBc expression. Our study also found that MKK7 is centrally involved in TNF-α-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this process.
由乙型肝炎病毒（HBV）感染引起的乙型肝炎仍然是全球主要的健康威胁。慢性炎症导致的肝损伤和再生是慢性乙型肝炎中肝纤维化和肝硬化的主要驱动因素。促炎细胞因子肿瘤坏死因子α（TNF-α）被认为是病毒性肝炎期间肝细胞死亡的主要诱导因子。在此，我们报告在肝癌细胞系以及原代小鼠和人肝细胞中，乙型肝炎病毒核心（HBc）蛋白的表达使细胞易受TNF-α诱导的凋亡影响。我们通过串联亲和纯化和质谱分析发现，活化蛋白激酶C 1受体（RACK1）与HBc相互作用。RACK1最近被报道为一种支架蛋白，可促进其上游激活剂对丝裂原活化蛋白激酶激酶7（MKK7）的磷酸化。我们的研究表明，HBc通过竞争性结合RACK1消除了MKK7与RACK1之间的相互作用，从而下调TNF-α诱导的MKK7磷酸化以及c-Jun氨基末端激酶（JNK）的激活。与此发现一致，特异性敲低MKK7增加了肝细胞对TNF-α诱导凋亡的敏感性，而RACK1的过表达则抵消了HBc的促凋亡活性。使用组装缺陷型HBc突变体分析表明，衣壳颗粒形成对于HBc的促凋亡活性并非必需。总之，HBc的表达通过破坏MKK7与RACK1之间的相互作用使肝细胞对TNF-α诱导的凋亡敏感。因此，我们的研究首次表明了HBc在乙型肝炎期间肝损伤中的致病作用。
我们的研究揭示了HBc在TNF-α介导的凋亡中以前未被认识的作用。HBc的促凋亡活性对于理解乙型肝炎发病机制很重要。特别是，与严重肝炎相关的HBV变异体可能通过增强HBc表达上调肝细胞凋亡。我们的研究还发现MKK7在TNF-α诱导的肝细胞凋亡中起核心作用，并揭示了JNK信号在此过程中的多方面作用。
