Molecular Mechanisms Of Hepatitis B Viral Pathogenesis And Persistence

乙肝病毒感染是全球肝病的主要原因，在美国有100多万人受到影响。乙型肝炎病毒感染引起的肝炎是一个复杂而复杂的过程，涉及多种宿主因素与病毒和/或病毒基因产物的相互作用。HBx基因在乙肝病毒的生命周期和致癌潜能中起着至关重要的作用。由于病毒-宿主相互作用在病毒感染和宿主损伤的发病机制中处于核心地位，本项目旨在阐明HBx-宿主相互作用在乙肝病毒感染过程中的细胞和分子机制。我们之前已经证明，HBX在体外和体内都与蛋白酶体复合体相互作用。26S蛋白酶体复合体是主要的细胞机制，通过泛素依赖和非依赖途径降解细胞蛋白质。它与多种转录和细胞周期因子的调节、细胞应激反应和抗原提呈有关。 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中肝病毒复制的影响。用重组腺病毒或杆状病毒表达复制的乙肝病毒或WHV基因组，以研究病毒在培养上的复制。在腺病毒或BV-WHV感染的HepG2细胞中，X-阴性病毒的复制水平约为野生型病毒的10%。在蛋白酶体抑制剂存在的情况下，野生型病毒的复制不受影响，而X阴性病毒的复制被促进，并恢复到野生型水平。最近，我们将研究扩展到体内，表达复制野生型或X-阴性乙肝病毒的转基因小鼠在6-8周龄时静脉注射蛋白酶体抑制剂MLN-273(Millennium PharmPharmticals)。总的来说，在这个年龄，X阴性小鼠血清中的HBVDNA水平和肝脏中的复制水平都比野生型小鼠低得多。分别于注射后0周、1周和4周采集血清和肝脏。用定量聚合酶链式反应检测血清中的HBVDNA，并分析肝脏中的复制中间体。在注射蛋白酶体抑制剂mln-273的野生型乙肝小鼠中，血清中的HBVDNA水平和肝脏中的复制水平均未受到明显影响。在注射蛋白酶体抑制剂mln-273后1周，X阴性小鼠血清中HBVDNA水平升高到0周水平的100倍以上。这种增加还反映在肝脏中复制中间体的水平显著提高。在注射后第4周，血清和肝脏中的HBVDNA水平恢复到基线水平。用表达复制乙肝病毒基因组的重组腺病毒感染C57BL/6小鼠也得到了类似的结果，无论是否有功能性HBx。我们的数据表明，在体内，乙肝病毒的复制受到细胞蛋白酶体和HBx功能的调节，通过抑制蛋白酶体的活性来增强乙肝病毒的复制。 鉴于HBx在乙肝病毒生命周期中的重要性，我们试图通过随机组合的方法靶向HBx的功能来开发潜在的抗HBx药物。我们建立了一种改良的酵母双杂交干扰物系统来筛选随机多肽适配子文库，该文库以细菌蛋白TrxA为平台展示随机合成的多肽适体。将破坏HBX-PSMA7(蛋白酶体亚单位)相互作用的多肽适配子克隆到CMV表达载体中进行转染研究。研究了这些肽适配子对HepG2细胞HBx转运、乙肝病毒复制、转录和抗原表达的影响。以HBx和PSMA7为相互作用对，以随机多肽适配子文库为干扰物，筛选1.5×10E7酵母菌落，共分离到367株酵母转化子。在二次筛选中，21个菌落被证实显示HBx-PSMA7相互作用的特异性中断。这些酵母菌落的多肽适配子被分离、测序并克隆到CMV驱动的构建体中，用于在HepG2细胞中转染。反式激活实验表明，这些多肽适配子可通过增加或降低荧光素酶活性来干扰HBx反式激活RSV-Luc报告基因的作用。当与具有复制能力的构建体共转染时，许多抑制HBx反式激活的多肽适配子可以抑制大约50-60%的HBVDNA复制。我们目前正在进行乙肝转基因小鼠和流体动力注射模型的研究，以测试这些肽适配子对乙肝病毒复制的效果。我们的结果表明，在改进的酵母双杂交系统中，基于HBx-蛋白酶体相互作用的破坏来选择随机的多肽适配子，可能会发现潜在的治疗乙肝病毒感染的药物。

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. Similar results were obtained by using C57BL/6 mice infected with recombinant adenoviruses expressing replicating HBV genome with or without functional HBX. Our data suggest that HBV replication is subjected to regulation by cellular proteasome and HBX functions through the inhibition of proteasome activities to enhance HBV replication in vivo. 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 interactingpair 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 and hydrodynamic injection models 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.