Molecular Mechanisms Of Hepatitis B Viral Pathogenesis And Persistence

乙型肝炎病毒发病机制和持久性的分子机制

基本信息

批准号：
7734190
负责人：
T. Jake Liang
金额：
$ 46.61万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734190
关键词：
26S proteasome Adenoviruses Affect Age Antigen Presentation Antigens Antiviral Agents Bacterial Proteins Baculoviruses Biological Assay Biology Cell Cycle Cells Cellular Stress Response Complex Cultured Cells Cytomegalovirus DNA biosynthesis Data Development Disruption Genes Genetic Transcription Genome Hepadnaviridae Hepatitis Hepatitis B Virus Hepatitis B X-Protein In Vitro Infection Injection of therapeutic agent Injury Integration Host Factors Libraries Life Cycle Stages Liver Liver diseases Luciferases Molecular Mus Oncogenic Pathogenesis Pathway interactions Peptide aptamers Pharmaceutical Preparations Pharmacologic Substance Play Polymerase Chain Reaction Process Proteasome Binding Proteasome Inhibitor Proteins Random Allocation Recombinants Regulation Reporter Role Screening procedure Serum Simian B disease System Testing Therapeutic Transactivation Transfection Transgenic Mice Transgenic Organisms Ubiquitin United States Viral Viral Pathogenesis Viral Proteins Virus Virus Diseases Virus Replication Week Wild Type Mouse Yeasts anti-hepatitis B base combinatorial in vivo mouse model multicatalytic endopeptidase complex tissue culture transfection/expression vector viral DNA virus host interaction yeast two hybrid system

项目摘要

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.

乙型肝炎病毒 (HBV) 感染是全世界肝病的主要原因，在美国影响着超过 100 万人。乙型肝炎病毒感染引起的肝炎是一个复杂的过程，涉及多种宿主因素与病毒和/或病毒基因产物的相互作用。 HBV X (HBX) 基因在 HBV 的生命周期和致癌潜力中发挥着至关重要的作用。由于病毒-宿主相互作用是病毒感染和宿主损伤发病机制的核心，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。我们之前已经证明 HBX 在体外和体内与蛋白酶体复合物相互作用。 26S 蛋白酶体复合物是主要的细胞机制，它以泛素依赖性和非依赖性途径降解细胞蛋白质。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，我们之前分析了蛋白酶体抑制剂对细胞培养中嗜肝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。在感染腺-HBV或bv-WHV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下，野生型病毒的复制不受影响，而X阴性HBV或WHV病毒的复制得到增强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 HBV 的 HBV 转基因小鼠在 6 至 8 周龄时静脉注射蛋白酶体抑制剂 MLN-273（Millennium Pharmaceuticals）。总体而言，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该年龄的野生型小鼠。注射后0、1和4周收集血清和肝脏。通过定量 PCR 检测血清中的 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没有受到显着影响。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水平增强至第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水平显着升高。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，在组织培养和 HBV 转基因小鼠模型中，HBX 通过蛋白酶体依赖性途径在肝炎病毒复制中发挥作用。由于 HBX 在 HBV 生命周期中的重要性，我们尝试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。我们开发了一种改良的酵母双杂交干扰系统来筛选随机肽适体库，该库使用细菌蛋白TrxA作为展示随机合成的肽适体的平台。将破坏 HBX-PSMA7（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 HepG2 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 PSMA7 作为相互作用对以及随机肽适体库作为干扰子筛选 1.5 x 10E7 酵母菌落，分离出 367 个酵母转化体。在二次筛选中，21 个菌落被证实显示出 HBX-PSMA7 相互作用的特异性破坏。来自这些酵母菌落的肽适体被分离、测序并克隆到 CMV 驱动的构建体中，用于转染 HepG2 细胞。反式激活测定表明，这些肽适体可以通过增加或减少荧光素酶活性来干扰 HBX 反式激活对 RSV-Luc 报告基因的影响。当与具有 HBV 复制能力的构建体共转染时，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。我们的结果表明，基于修饰的酵母双杂交系统中 HBX-蛋白酶体相互作用的破坏来选择随机肽适体可能会识别出治疗 HBV 感染的潜在治疗药物。