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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）感染是全球肝脏疾病的主要原因，在美国影响超过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能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发病机制的核心，本项目旨在阐明HBV感染过程中hbx -宿主相互作用的细胞和分子机制。我们之前已经在体外和体内证明HBX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，它通过泛素依赖性和非依赖性途径降解细胞蛋白。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。