Molecular Mechanisms Of Hepatitis B Viral Pathogenesis And Persistence

感染B型肝炎病毒（HBV）是世界范围内肝病的主要原因，在美国影响超过100万人。由B型肝炎病毒感染引起的肝炎是一个复杂而复杂的过程，涉及多种宿主因子与病毒和/或病毒基因产物的相互作用。HBV X（HBX）基因在HBV的生命周期和致癌潜力中起着至关重要的作用。由于病毒-宿主相互作用是病毒感染和宿主损伤的发病机制的核心，因此本项目旨在阐明HBV感染期间HBX-宿主相互作用的细胞和分子机制。我们以前已经表明，HBX与蛋白酶体复合物在体外和体内相互作用。26 S蛋白酶体复合物是主要的细胞机制，其在泛素依赖性和非依赖性途径中降解细胞蛋白。它涉及多种转录和细胞周期因子、细胞应激反应和抗原呈递的调节。 为了进一步研究蛋白酶体在HBX生物学中的作用，我们先前分析了蛋白酶体抑制剂对细胞培养物中嗜肝DNA病毒复制的影响。产生表达复制型HBV或WHV基因组的重组腺病毒或杆状病毒以研究培养物中的病毒复制。在HepG 2细胞感染腺病毒HBV或bv-WHV，复制水平的X-阴性病毒的野生型病毒的约10%。在蛋白酶体抑制剂的存在下，野生型病毒的复制不受影响，而HBV或WHV的X阴性病毒的复制增强并恢复到野生型水平。最近，我们将研究扩展到体内，在6至8周龄时静脉注射蛋白酶体抑制剂MLN-273（Millennium Pharmaceuticals）对表达复制野生型或X阴性HBV的HBV转基因小鼠进行研究。一般来说，在这个年龄段，X阴性小鼠血清中的HBV DNA水平和肝脏中的复制水平远低于野生型小鼠。在注射后0、1和4周收集血清和肝脏。用定量PCR检测血清中HBV DNA，并分析肝脏中的复制中间体。在注射蛋白酶体抑制剂MLN-273的野生型HBV小鼠中，血清中的HBV DNA水平和肝脏中的复制水平未受到显著影响。在注射蛋白酶体抑制剂MLN-273后第1周，X阴性小鼠血清中的HBV DNA水平增加至第0周水平的100倍以上。这种增加也反映在肝脏中复制中间体的显著更高水平上。注射后第4周，血清和肝脏中的HBV DNA水平恢复到基线水平。这些数据表明，HBX的功能在嗜肝DNA病毒复制通过蛋白酶体依赖性途径在组织培养和HBV转基因小鼠模型。 由于HBX在HBV生命周期中的重要性，我们试图通过使用随机组合方法靶向HBX的功能来开发潜在的抗HBV药物。我们开发了一种改进的酵母双杂交干扰系统，以细菌蛋白TrxA为平台，筛选随机合成的肽适体文库。将破坏HBX-PSMA 7（蛋白酶体亚基）相互作用的肽适体克隆到CMV表达载体中用于转染研究。在HepG 2细胞中表征了这些肽适体对HBX交易、HBV复制、转录和抗原表达的影响。通过以HBX和PSMA 7作为相互作用对以及随机肽适体库作为破坏剂筛选1.5 x 10 E7酵母菌落，分离出367个酵母转化体。在二次筛选中，确认21个菌落显示出HBX-PSMA 7相互作用的特异性破坏。将来自这些酵母菌落的肽适体分离、测序并克隆到CMV驱动的构建体中用于在HepG 2细胞中转染。反式激活实验表明，这些肽适配子可以通过增加或降低荧光素酶活性来干扰HBX对RSV-Luc报告基因的反式激活作用。当与HBV复制能力构建体共转染时，许多抑制HBX反式激活的肽适体可以抑制HBV DNA复制约50至60%。我们目前正在HBV转基因小鼠中进行研究，以测试这些肽适体对HBV复制的功效。我们的研究结果表明，选择随机肽适体的基础上中断的HBX-蛋白酶体相互作用在一个修改的酵母双杂交系统可能会发现潜在的治疗药物的HBV感染。

Infection with hepatitis B virus (HBV) is a major cause of liver disease worldwide and affects more than 1 million people in the United States. Hepatitis caused by hepatitis B virus infection is a complex and intricate process involving interaction of multiple host factors with the virus andor the viral gene products. The HBV X (HBX) gene plays a crucial role in the life cycle and oncogenic potential of HBV. Since virus-host interactions are central to the pathogenesis of viral infection and host injury, this project aims to elucidate the cellular and molecular mechanisms of HBX-host interactions during HBV infection. We have previously shown that HBX interacts with the proteasome complex in vitro and in vivo. The 26S proteasome complex is the predominant cellular machinery, which degrades cellular proteins in both ubiquitin-dependent and -independent pathways. It has been implicated in the regulation of a variety of transcriptional and cell cycle factors, cellular stress response, and antigen presentation. To further study the role of the proteasome in the biology of HBX, we previously analyzed the effects of the proteasome inhibitors on the replication of hepadnaviruses in cell culture. Recombinant adenovirus or baculovirus expressing replicating HBV or WHV genome were generated to study viral replication in culture. In HepG2 cells infected with either the adeno-HBV or bv-WHV, the replication level of the X-negative virus was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Recently we extended the study to in vivo, HBV transgenic mice expressing either replicating wild-type or X-negative HBV were injected intravenously with proteasome inhibitor MLN-273 (Millennium Pharmaceuticals) at the age of 6 to 8 weeks. In general, the HBV DNA levels in the sera and the replication levels in the livers of the X-negative mice were much lower than those of the wild-type mice at this age. The sera and livers were collected at 0, 1, and 4 weeks post-injection. The sera were tested for HBV DNA by quantitative PCR and the livers were analyzed for replicative intermediates. In the wild-type HBV mice injected with proteasome inhibitor MLN-273, the HBV DNA level in the sera and the replication level in the livers were not significantly affected. At week 1 post-injection of proteasome inhibitor MLN-273, the level of HBV DNA in the serum of the X-negative mice was enhanced to more than 100-fold of the week 0 level. This increase was also reflected in a significant higher level of replicative intermediates in the liver. At week 4 post-injection, the HBV DNA levels in the sera and livers returned to the baseline level. These data suggest that HBX functions in hepadnaviral replication through a proteasome-dependent pathway in both tissue culture and HBV transgenic mouse model. Because of the importance of HBX in HBV life cycle, we attempted to develop potential anti-HBV agents by targeting the functions of HBX using a random combinatorial approach. We developed a modified yeast two-hybrid disruptor system to screen a random peptide aptamer library which uses the bacterial protein TrxA as a platform to display the randomly synthesized peptide aptamers. The peptide aptamers which disrupted HBX-PSMA7 (a proteasome subunit) interaction were cloned into CMV expression vector for transfection studies. The effects of these peptide aptamers on HBX transaction, HBV replication, transcription, and antigen expression were characterized in HepG2 cells. By screening 1.5 x 10E7 yeast colonies with HBX and PSMA7 as interacting pair and a random peptide aptamer library as disruptors, 367 yeast tranformants were isolated. On secondary screening, 21 colonies were confirmed to show specific disruption of the HBX-PSMA7 interaction. The peptide aptamers from these yeast colonies were isolated, sequenced, and cloned into a CMV-driven construct for transfection in HepG2 cells. Transactivation assays showed that these peptide aptamers could interfere with the effect of HBX transactivation on RSV-Luc reporter by increasing or decreasing the luciferase activities. When co-transfected with a HBV replication competent construct, many of the peptide aptamers which inhibited the HBX transactivation could suppress HBV DNA replication by about 50 to 60%. We are currently conducting studies in HBV transgenic mice to test the efficacy of these peptide aptamers on HBV replication. Our results demonstrate that selection of random peptide aptamers based on disruption of the HBX-proteasome interaction in a modified yeast two-hybrid system may identify potential therapeutic drugs for HBV infection.