MECHANISM OF HEPATITIS VIRUS-MEDIATED LIVER CARCINOGENESIS

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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,