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Hepatitis B virus replication and DDB1

乙型肝炎病毒复制和 DDB1

基本信息

批准号：
8685918
负责人：
BETTY L SLAGLE
金额：
$ 16.51万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8685918
关键词：
Acute Adaptor Signaling Protein Affect Animal Model Atlases Binding Binding Proteins Biological Models Cell Cycle Cell physiology Cells Chromosomes Chronic Chronic Hepatitis B Cirrhosis Complex Cullin Proteins DNA Damage DNA Repair DNA biosynthesis DNA-Binding Proteins Data Data Set Development Disease Progression Environment Fatty Liver Future Hepatitis B Hepatitis B Virus Hepatocyte Human Immune Immune system Immunodeficient Mouse Immunoprecipitation In Vitro Interferons Knowledge Laboratories Laboratory Study Lead Liver Liver diseases Mass Spectrum Analysis Mediating Metabolic syndrome Mining Mus Natural Immunity Pan Genus Pathogenesis Pathway interactions Physiology Primary carcinoma of the liver cells Proteins Recruitment Activity Relative (related person)Research Personnel Risk Factors Role Signal Transduction Tissues Transgenic Mice Ubiquitination Validation Viral Virus Virus Replication Western Blotting carcinogenesis cellular targeting cofactor genetic regulatory protein human H2AX protein in vivo innovation insight multicatalytic endopeptidase complex mutant prevent public health relevance receptor research study response response marker tumorigenesis ubiquitin-protein ligase viral DNA

项目摘要

DESCRIPTION (provided by applicant): Chronic hepatitis B virus (HBV) infection affects approximately 350 million people worldwide and is a significant risk factor for severe liver disease, including hepatocellular carcinoma (HCC). The role of HBV in mediating carcinogenesis is complex and includes integration of viral DNA into host chromosomes, the host immune system, and expression of the viral regulatory HBx protein. Our laboratory studies HBx, a small protein required for virus replication in vivo that is also involved in carcinogenesis We and others have shown that the interaction of HBx with cellular damaged DNA binding protein (DDB1) is critical for HBV replication. However, the functional implications of this interaction to viral replication and pathogenesis remain elusive. DDB1 is an adaptor protein for the Cul4-DDB1 E3 ligase complex, and acts in part by recruiting cellular DDB1- Cullin Accessory Factors (DCAFs) that act as substrate receptors to recruit additional cellular targets for ubiquitination and proteasome degradation. In this manner, DDB1 regulates diverse cellular functions such as DNA replication, cell cycle, innate immunity, and damaged DNA repair. We hypothesize that HBx acts as a viral DCAF, and displaces one or more cellular DCAFs from the Cul4-DDB1 complex, thereby modifying the cellular environment to favor virus replication. Human liver chimeric mice, in which human hepatocytes are engrafted into immunodeficient mice, provide the best, most biologically relevant model system to investigate the impact of HBV replication on Cul4-DDB1-DCAF complexes. In Specific Aim 1, an immunoprecipitation/mass spectrometry (MS) approach will be used to identify the Cul4-DDB1-DCAF profile of uninfected human liver, providing new insights into DDB1 pathways important to normal liver physiology. This will be compared to the Cul4-DDB1-DCAF profile from HBV-infected hepatocytes originating from the same donor liver, using quantitative MS analyses to compare relative binding levels of DCAFs to DDB1. In Specific Aim 2, the importance of HBx-DDB1 binding to DDB1 pathways will be investigated. To determine whether HBV activates the DNA damage response (DDR) as part of its acute replication strategy, uninfected and HBV- infected human liver tissue from chimeric mice will be examined by western blot for evidence of the DDR marker, phosphorylated histone H2AX. To determine whether HBx-inactivation of interferon signaling occurs through HBx-DDB1 binding, the ability of HBx mutants that cannot bind DDB1 to inhibit interferon signaling will be evaluated in vitro. Additionally, MS data will be mined for evidence that HBV replication induces recruitment of immune regulatory proteins to the Cul4-DDB1 complex. In summary, viruses are adept at targeting cellular pathways that benefit viral replication. The proposed studies will elucidate the impact of HBV replication on the cellular ubiquitination and degradation machinery using the innovative human liver chimeric animal model. This important new knowledge will reveal virus-induced changes at the post-translational level, and is predicted to provide mechanistic insight into new strategies through which to treat chronic HBV infection and prevent liver disease.

描述（由申请方提供）：慢性B型肝炎病毒（HBV）感染影响全球约3.5亿人，是严重肝病（包括肝细胞癌（HCC））的重要风险因素。HBV在介导癌发生中的作用是复杂的，包括病毒DNA整合到宿主染色体、宿主免疫系统和病毒调节HBx蛋白的表达中。我们的实验室研究HBx，一种体内病毒复制所需的小蛋白，也参与致癌作用。我们和其他人已经表明，HBx与细胞受损DNA结合蛋白（DDB 1）的相互作用对HBV复制至关重要。然而，这种相互作用对病毒复制和发病机制的功能影响仍然难以捉摸。DDB 1是Cul 4-DDB 1 E3连接酶复合物的衔接蛋白，并且部分地通过募集细胞DDB 1- Cullin辅助因子（DCAF）来起作用，所述细胞DDB 1- Cullin辅助因子（DCAF）充当底物受体以募集用于泛素化和蛋白酶体降解的额外细胞靶标。以这种方式，DDB 1调节多种细胞功能，如DNA复制，细胞周期，先天免疫和受损DNA修复。我们假设HBx充当病毒DCAF，并从Cul 4-DDB 1复合物中置换一个或多个细胞DCAF，从而改变细胞环境以有利于病毒复制。将人肝细胞移植到免疫缺陷小鼠中的人肝嵌合小鼠提供了最佳、最具生物学相关性的模型系统，以研究HBV复制对Cul 4-DDB 1-DCAF复合物的影响。在特定目标1中，免疫沉淀/质谱（MS）方法将用于识别未感染人类肝脏的Cul 4-DDB 1-DCAF谱，为正常肝脏生理学重要的DDB 1途径提供新的见解。将其与来自相同供体肝脏的HBV感染肝细胞的Cul 4-DDB 1-DCAF谱进行比较，使用定量MS分析比较DCAF与DDB 1的相对结合水平。在具体目标2中，将研究HBx-DDB 1与DDB 1途径结合的重要性。为了确定HBV是否激活DNA损伤应答（DDR）作为其急性复制策略的一部分，将通过蛋白质印迹检查来自嵌合小鼠的未感染和HBV感染的人肝组织中DDR标志物磷酸化组蛋白H2 AX的证据。为了确定干扰素信号传导的HBx失活是否通过HBx-DDB 1结合发生，将在体外评估无法结合DDB 1的HBx突变体抑制干扰素信号传导的能力。此外，MS数据将被挖掘， HBV复制诱导免疫调节蛋白募集到Cul 4-DDB 1复合物的证据。总之，病毒擅长靶向有利于病毒复制的细胞途径。拟议的研究将阐明HBV复制对细胞泛素化和降解机制的影响，使用创新的人肝嵌合动物模型。这一重要的新知识将揭示病毒诱导的翻译后水平的变化，并预计将为治疗慢性HBV感染和预防肝脏疾病的新策略提供机制性见解。