近日，来自美国科罗拉多大学和中国厦门大学的两个研究小组在肝细胞中发现了乙型肝炎病毒(Hepatitis B virus, HBV)的两种主要靶标。这项发现可能导致人们开发出新的方法来治疗全世界当前大约感染这种病毒的4亿人中一些人所患的肝病。

科罗拉多大学教授Ding Xue说，科学家在过去三十多年一直都在寻找HBV的细胞靶标。HBV感染促进肝炎、肝硬化和肝癌产生，而且能够通过血液、体液、不安全性行为和未消毒的针头进行传播，此外被感染的母亲在分娩过程中也能够传染给子女。

Xue说，一段时间以来，科学家们就已知道HBV编码一种致病的促进肿瘤产生的蛋白HBx，但是它的作用机制何在很大程度上仍是个未知数。在两项新的研究中，Xue和他的同事们在人细胞中证实HBx的宿主靶标是两种小分子蛋白Bcl-2和 Bcl-xL。已知这两种蛋白是细胞死亡抑制剂，但是人们之前并不知道它们参与HBV感染。

HBx使用一种特殊的基序---它是一小段氨基酸序列，类似于在一些导致细胞死亡的蛋白中发现的序列---来与靶标Bcl-2和Bcl-xL相互作用，促进宿主细胞中钙离子水平提高。Xue说，钙离子水平提高随后触发病毒HBV复制和细胞死亡。

当研究人员让这种基序发生基因突变时，HBx结合到Bcl-2和 Bcl-xL蛋白上，并阻止病毒复制。类似地，当人肝细胞中Bcl-2或Bcl-xL蛋白被敲降或功能减弱时，HBx更不能够导致被感染的细胞内的钙离子水平增加和病毒复制。

在其中一项研究中，研究人员利用秀丽隐杆线虫(C. elegans)鉴定出HBx的宿主靶标。他们证实HBx通过一种被称作CED-9的蛋白能够诱导秀丽隐杆线虫中的细胞死亡，这就模拟了HBV感染人肝脏的早期阶段。

之前的研究已证实秀丽隐杆线虫蛋白CED-9是人蛋白Bcl-2的同源物---在不同动物中的一种不同蛋白，但有类似的功能。尽管线虫与人之间存在显著性的差别，但是科学家们估计35%的秀丽隐杆线虫基因拥有人的同源物。

这两项研究都证实如果让这个短的HBx基序发生两个突变，那么它就丧失结合到Bcl-2蛋白家族成员的能力。这就完全阻止病毒复制和HBx表达导致的宿主细胞死亡。

Xue说，尽管干扰素和抗病毒药物被用来治疗一些慢性HBV携带者，但是当前还没有有效的方法来治疗他们。在大多数HBV感染发生的发展中国家，这些治疗方法要么不能获得，要么过于昂贵。因此，这些新的研究发现可能在被HBV感染的病人治疗上产生深刻的临床意义和药物开发上的意义。

Xue教授领导的这两项研究于10月22日在线刊登在《美国国家科学院院刊》(Proceedings of the National Academy of Sciences, PNAS)上。

Hepatitis B virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca2+ increase and cell death in Caenorhabditis elegans

Xin Genga, Brian L. Harry, Qinghua Zhou, Riley Robert Skeen-Gaar, Xiao Ge, Eui Seung Lee, Shohei Mitani, and Ding Xue

HBx is a multifunctional hepatitis B virus (HBV) protein that is crucial for HBV infection and pathogenesis and a contributing cause of hepatocyte carcinogenesis. However, the host targets and mechanisms of action of HBx are poorly characterized. We show here that expression of HBx in Caenorhabditis elegans induces both necrotic and apoptotic cell death, mimicking an early event of liver infection by HBV. Genetic and biochemical analyses indicate that HBx interacts directly with the B-cell lymphoma 2 (Bcl-2) homolog CED-9 (cell death abnormal) through a Bcl-2 homology 3 (BH3)-like motif to trigger both cytosolic Ca2+ increase and cell death. Importantly, Bcl-2 can substitute for CED-9 in mediating HBx-induced cell killing in C. elegans, suggesting that CED-9 and Bcl-2 are conserved cellular targets of HBx. A genetic suppressor screen of HBx-induced cell death has produced many mutations, including mutations in key regulators from both apoptosis and necrosis pathways, indicating that this screen can identify new apoptosis and necrosis genes. Our results suggest that C. elegans could serve as an animal model for identifying crucial host factors and signaling pathways of HBx and aid in development of strategies to treat HBV-induced liver disorders.

Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction

Xin Genga,1, Chenghao Huangb,1, Yan Qinc, Janet E. McCombsc, Quan Yuanb, Brian L. Harrya, Amy E. Palmerc, Ning-Shao Xiab,2, and Ding Xue

Infection with the hepatitis B virus (HBV) promotes the development of hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) and is a leading cause of morbidity and mortality worldwide. HBV X protein (HBx) is an important effector for HBV pathogenesis, but its cellular targets and acting mechanisms remain elusive. We show here that HBx interacts with the anti-apoptotic proteins Bcl-2 and Bcl-xL through a Bcl-2 homology 3 (BH3)-like motif in mammalian cells. Importantly, mutations in the BH3-like motif that prevent HBx binding to Bcl-2 and Bcl-xL abrogate cytosolic calcium elevation and cell death induced by HBx expression in hepatocytes and severely impair HBV viral replication, which can be substantially rescued by restoring cytosolic calcium. These results suggest that HBx binding to Bcl-2 family members and subsequent elevation of cytosolic calcium are important for HBV viral replication. Consistently, RNAi knockdown of Bcl-2 or Bcl-xL results in reduced calcium elevation by HBx and decreased viral replication in hepatocytes. Our results suggest that HBx targets Bcl-2 proteins through its BH3-like motif to promote cytosolic calcium elevation, cell death, and viral replication during HBV pathogenesis, which presents an excellent therapeutic intervention point in treating patients with chronic HBV.