Hepatitis B virus replication and DDB1

乙型肝炎病毒复制和 DDB1

• 批准号: 8564763
• 负责人: BETTY L SLAGLE
• 金额: $ 20.42万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8564763
• 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

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(申请人提供)：慢性乙肝病毒感染影响全球约3.5亿人，是包括肝细胞癌在内的严重肝病的重要风险因素。乙肝病毒在介导肿瘤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。我们的实验室研究HBx，这是一种在体内复制病毒所需的小蛋白，也参与了致癌过程。我们和其他人已经证明，HBx与细胞受损DNA结合蛋白(DDB1)的相互作用对乙肝病毒的复制至关重要。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。DDB1是CUL4-DDB1 E3连接酶复合体的适配蛋白，部分作用是通过招募细胞DDB1-Cullin辅助因子(DCAF)作为底物受体来招募泛素化和蛋白酶体降解的额外细胞靶点。通过这种方式，DDB1调节多种细胞功能，如DNA复制、细胞周期、先天免疫和受损的DNA修复。我们假设HBx充当病毒DCAF，并取代CUL4-DDB1复合体中的一个或多个细胞DCAF，从而改变细胞环境以利于病毒复制。人肝嵌合小鼠将人肝细胞移植到免疫缺陷小鼠体内，为研究乙肝病毒复制对CUL4-DDB1-DCAF复合体的影响提供了最好的、最具生物学相关性的模型系统。在具体目标1中，将使用免疫沉淀/质谱仪(MS)方法来鉴定未感染的人肝脏的CUL4-DDB1-DCAF谱，为了解对正常肝脏生理重要的DDB1通路提供新的见解。这将与来自同一供体肝脏的乙肝病毒感染的肝细胞的CUL4-DDB1-DCAF图谱进行比较，使用定量的MS分析来比较DCAF与DDB1的相对结合水平。在特定目标2中，将研究HBx-DDB1与DDB1途径结合的重要性。为了确定作为其急性复制策略的一部分，乙肝病毒是否激活了DNA损伤反应(DDR)，来自嵌合小鼠的未感染和乙肝病毒感染的人肝组织将通过蛋白质印迹法检测DDR标记磷酸化组蛋白H2AX的证据。为了确定干扰素信号的HBx失活是否通过HBx-DDB1结合发生，将在体外评估不能结合DDB1的HBx突变体抑制干扰素信号的能力。此外，还将挖掘MS数据以获取 乙肝病毒复制诱导免疫调节蛋白重新聚集到CUL4-DDB1复合体的证据。总而言之，病毒擅长瞄准有利于病毒复制的细胞途径。拟议的研究将利用创新的人类肝脏嵌合动物模型阐明乙肝病毒复制对细胞泛素化和降解机制的影响。这一重要的新知识将揭示病毒在翻译后水平上引起的变化，并预计将为治疗慢性乙肝感染和预防肝病的新策略提供机械性见解。