MECHANISM OF HEPATITIS VIRUS-MEDIATED LIVER CARCINOGENESIS

细胞动态平衡受到多种细胞蛋白活性的严格调控。这些蛋白质的亚细胞定位和稳定性对它们的活性至关重要。例如，特定受体通过识别核定位信号(NLS)和/或核输出信号将蛋白质运入或运出细胞核。最近发现的一个NES是一个短的、疏水的、富含亮氨酸的基序，它是介导大载体蛋白和mRNAs核输出所必需的，也是足够的。据报道，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受其NES的空间调控。有趣的是，许多病毒癌蛋白也利用CRM1介导的途径来调节细胞蛋白和mRNAs的输出。然而，其中一些病毒蛋白被认为足够小，可以被动地通过核孔复合体扩散。这些致癌病毒蛋白已获得NES活性和修饰的核输出，这一事实暗示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，我们验证了一种假设，即乙肝病毒(乙肝病毒)的X蛋白(HBx)和丙型肝炎病毒(丙型肝炎病毒)的核心蛋白(HC-core)是肝细胞癌的两个主要危险因素，可能通过破坏CRM1介导的核输出途径而诱导肿瘤转化。对HBx和HC-core序列的仔细检查发现，这两种蛋白质中都存在疏水性的富含亮氨酸的NES。一个可能的NES位于HBx的中心区，它经常被保留在肝细胞癌中，是其反式激活所必需的。P21core的C末端也有两个Ness，而p19core和p16core分别缺失一个或两个。其活性受到核输出严格调控的细胞蛋白包括：核转录因子-AT/肿瘤坏死因子-α诱导通路、IkappaB/NFkappaB级联通路、MAP激酶级联通路和MDM2/P53通路。NFkappaB通过IkappaB在细胞质中的隔离失活，其细胞质定位受NES调控。转录因子核因子-AT在抑制其NES后被激活，从而诱导诸如肿瘤坏死因子-α等基因的产生。P53也可以通过其自身的NES或MDM2介导的核输出在细胞质中被隔离而失活。HBx和HC-core可以激活NFkappaB、NF-AT和MAP激活级联反应，而HBx可以抑制P53介导的活性。在p21core中包含两个可能的Ness的区域对于NFkappaB的激活是必不可少的。P21core主要定位于细胞质，p19core和p16core定位于胞核。因此，可以通过阻断核出口来实现对NF-AT和NFkappaB活性的调节。我们的研究表明，软霉素B可以阻止HBx从细胞核向细胞质的迁移。此外，当HBX NES与绿色荧光蛋白融合时，它位于细胞质中，但如果发生突变，就会使这种能力失活。此外，HBx还调节P53的定位。HBx还与CRM1在细胞质中共存，这表明HBx可能通过与CRM1结合并限制细胞核内的CRM1来阻止其他正常利用这一途径的细胞蛋白的输出。同样，全长的HC核心主要位于细胞质中，而NES突变的HC核心位于细胞核中。目前，我们正在检测这些NES突变体激活NFkappaB和NF-AT途径的能力。此外，我们还在测试这些突变体联合H-ras转化大鼠胚胎成纤维细胞的能力。这项研究旨在测试一种新的机制，即持续的核输出中断可能是乙肝病毒和丙型肝炎诱导的慢性肝炎的常见事件，从而最终导致肝细胞癌。这样的机制也可以解释与这些病毒蛋白相关的类似的多效性效应。-乙肝病毒，丙型肝炎病毒，肝癌变，CRM1/Exportin，p，NFkappaB，核因子-AT，核出口，HBX，HC-core，

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,