MECHANISM OF HEPATITIS VIRUS-MEDIATED LIVER CARCINOGENESIS

细胞内稳态受到许多细胞蛋白质活性的严格调节。这些蛋白质的亚细胞定位和稳定性对其活性至关重要。实例包括通过识别核定位信号（NLS）和/或核输出信号由特异性受体转运入或转运出核的蛋白质。最近鉴定的一种内斯是一种短的、疏水的、富含亮氨酸的基序，其对于介导大载体蛋白和mRNA的核输出是必需的且足够的。许多细胞转录因子、癌蛋白、细胞周期调节因子和肿瘤抑制蛋白的活性已被报道由它们的内斯在空间上控制。有趣的是，许多病毒癌蛋白也利用Crm 1介导的途径来调节细胞蛋白和mRNA的输出。然而，这些病毒蛋白中的一些被认为是足够小的，以被动地通过核孔复合物扩散。这些致癌病毒蛋白具有内斯活性和修饰的核输出的事实意味着核输出可能是病毒介导的肿瘤发生的重要靶点。在这项研究中，我们正在研究的假设，即X蛋白（HBx）的B型肝炎病毒（HBV）和核心蛋白（HC-core）的丙型肝炎病毒（HCV），肝细胞癌的两个主要危险因素，可能会导致肿瘤转化的Crm 1介导的核输出途径失调。对HBx和HC核心序列的仔细检查揭示了这两种蛋白质中假定的疏水性富含亮氨酸的内斯。推测的内斯位于HBx的中心区域，HBx在HCC中经常保留，并且对于其反式激活是必需的。两个推定的NES也位于p21核心的C-末端，而p19核心和p16核心分别缺乏一个或两个。其活性受核输出严格调节的细胞蛋白的实例包括NF-AT/TNF-α诱导途径、IkappaB/NF κ B级联、MAP激酶级联和mdm 2/p53途径中的那些。NF κ B通过IkappaB在细胞质中的隔离而失活，IkappaB的细胞质定位由内斯调节。转录因子NF-AT在其内斯被抑制时被激活，从而诱导基因如TNF-α。p53也可以通过其自身的内斯或Mdm 2介导的核输出在细胞质中隔离而失活。已知HBx和HC-核心激活NF κ B、NF-AT和MAP激酶级联，而HBx可以抑制p53介导的活性。在p21核心中含有两个推定的NES的区域对于NF κ B的活化是必需的。p21 core主要定位于细胞质，p19 core和p16 core定位于细胞核。因此，有可能通过阻断核输出来实现NF-AT和NF κ B活性的调节。我们的研究表明，来普霉素B可以阻断HBx从细胞核向细胞质的迁移。此外，HBx内斯，当融合到一个绿色荧光蛋白，位于细胞质中，但如果突变，失活这种能力。此外，HBx调节p53的定位。HBx还与Crm 1共定位在细胞质中，这表明HBx可以通过结合和限制细胞核中的Crm 1来阻止通常利用该途径的其他细胞蛋白的输出。类似地，虽然全长HC核心主要位于细胞质中，但NES突变的HC核心位于细胞核中。目前，我们正在研究这些内斯突变体激活NF κ B和NF-AT通路的能力。此外，我们还测试了这些突变体与H-ras结合转化大鼠胚胎成纤维细胞的能力。本研究旨在测试一种新的机制，即持续破坏核输出可能是HBV和HCV诱导的慢性肝炎的常见事件，从而最终导致HCC。这种机制也可以解释与这些病毒蛋白相关的类似多效性效应。- B型肝炎病毒，丙型肝炎病毒，肝癌发生，Crm 1/Exportin，p，，NF κ B B，NF-AT，核输出，HBx，HC-核心，

Cellular homeostasis is tightly regulated by the activities of many cellular proteins. The subcellular localization and stability of these proteins are critical for their activities. Examples include proteins that are transported into or out of the nucleus by specific receptors through the recognition of nuclear-localization signals (NLS) and/or nuclear export signals. One recently identified NES is a short, hydrophobic, leucine-rich motif that is necessary and sufficient to mediate nuclear export of large carrier proteins and mRNAs. The activity of many cellular transcription factors, oncoproteins, cell cycle regulators and tumor suppressor proteins has been reported to be spatially controlled by their NES. Interestingly, many viral oncoproteins also utilize the Crm1-mediated pathway to regulate the export of cellular proteins and mRNAs. However, some of these viral proteins are thought to be small enough to passively diffuse through the nuclear pore complex. The fact that these oncogenic viral proteins have acquired NES activity and modified nuclear export implies that nuclear export may be an important target for viral-mediated oncogenesis. In this study, we are examining the hypothesis that the X protein (HBx) of hepatitis B virus (HBV) and core protein (HC-core) of hepatitis C virus (HCV), two major risk factors for hepatocellular carcinoma, may induce neoplastic transformation by disregulation of the Crm1-mediated nuclear export pathway. Close inspection of the HBx and HC-core sequences revealed putative hydrophobic leucine-rich NES in both proteins. A putative NES is located at the center region of HBx, which is frequently retained in HCC and is essential for its transactivation. Two putative NESs are also located at the C-terminus of p21core, while p19core and p16core lack one or both, respectively. Examples of cellular proteins whose activities are tightly regulated by nuclear export include those in the NF-AT/TNF-alpha induction pathway, the IkappaB/NFkappaB cascade, the MAP kinase cascade and the mdm2/p53 pathway. NFkappaB is inactivated through sequestration at the cytoplasm by IkappaB, whose cytoplasmic localization is regulated by NES. The transcription factor NF-AT is activated upon inhibition of its NES, thereby inducing genes such as TNF-alpha. p53 also can be inactivated through sequestration at the cytoplasm by either its own NES or Mdm2- mediated nuclear export. HBx and HC-core are known to activate NFkappaB, NF-AT and MAP kinase cascades, whereas HBx can inactivate p53-mediated activity. The region that contains the two putative NESs in p21core is essential for the activation of NFkappaB. While p21core is mainly located in cytoplasm, p19core and p16core are located in the nucleus. Thus, it is possible that the modulation of the activity of NF-AT and NFkappaB can be achieved by blocking nuclear export. Our studies indicate that leptomycin B can block HBx migration from the nucleus to the cytoplasm. In addition, the HBx NES, when fused to a green fluorescent protein, is located in the cytoplasm, but if mutated, inactivates this ability. Furthermore, HBx modulates the localization of p53. HBx also colocalizes with Crm1 in the cytoplasm, suggesting that HBx may prevent the export of other cellular proteins that normally utilize this pathway by binding to and limiting Crm1 in the nucleus. Similarly, while the full length HC-core is mainly located in the cytoplasm, the NES-mutated HC-core is located in the nucleus. Currently, we are examining the activity of these NES mutants for their ability to activate NFkappaB and NF-AT pathways. In addition, we are also testing the ability of these mutants to transform rat embryo fibroblasts in combination with H-ras. This study is designed to test a novel mechanism whereby a persistent disruption of nuclear export may be a common event for HBV- and HCV-induced chronic hepatitis, thus ultimately leading to HCC. Such a mechanism could also explain the similar pleiotropic effect associated with these viral proteins. - Hepatitis B virus, Hepatitis C virus, Liver Carcinogenesis, Crm1/Exportin, p, , NFkappaB, NF-AT, Nuclear Export, HBx, HC-core,