Critical Lipid Species in the Hepatovirus Lifecycle

肝病毒生命周期中的关键脂质种类

• 批准号: 10306348
• 负责人: Stanley M. Lemon
• 金额: $ 51.56万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-12 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10306348
• 关键词: Acute Hepatitis; Address; Antibodies; Binding; Biological Assay; Biology; Blood; CRISPR screen; Capsid; Capsid Proteins; Carbohydrates; Carbon; Cells; Ceramide glucosyltransferase; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytolysis; Data; Disease Outbreaks; Endocytosis; Enteral; Enzymes; Family Picornaviridae; Fatty Acids; Gangliosides; Genes; Genetic; Genetic Screening; Genetic Transcription; Glucosylceramides; Glycoproteins; Glycosphingolipids; Head; Hepatitis A; Hepatitis A Virus; Hepatocyte; Hepatovirus; Human; Infection; Infection prevention; Integration Host Factors; Knock-out; Laboratories; Lipids; Maps; Measures; Membrane; Organelles; Persons; Play; Production; Proteins; RNA; Replicon; Reproducibility; Research; Research Personnel; Role; Sialic Acids; Site; Sphingolipids; Structure; Surface; Tail; Testing; United States; Very Long Chain Fatty Acid; Viral; Viral hepatitis; Virion; Virus; Virus Diseases; Virus Replication; dihydroceramide desaturase; exosome; experimental study; extracellular vesicles; genome-wide; human pathogen; late endosome; lipid metabolism; lipidomics; member; neglect; novel strategies; pathogenic virus; permissiveness; thermostability; trafficking; viral RNA; virus envelope

PROJECT ABSTRACT Hepatitis A virus (HAV) is an unusual picornavirus (genus Hepatovirus) that is released without lysis from infected hepatocytes within small extracellular vesicles (EVs) resembling exosomes. These membrane-cloaked virions, or `quasi-enveloped' HAV (eHAV), lack virus-encoded glycoprotein peplomers on their surface, yet they are infectious and the only form of the virus found circulating in the blood of persons with acute hepatitis A. Numerous other `non-enveloped' viruses have been found to be released from cells as quasi-enveloped virions in EVs of varying size, making studies of eHAV relevant to a broad range of viral pathogens. This application proposes to investigate two aspects of lipid metabolism found to be essential for productive hepatovirus infection in a genome-wide forward genetic screen, and will test the following hypotheses: (i) that GD3 and possibly other gangliosides play a crucial role in cellular entry and the initiation of infection by both naked HAV and quasi- enveloped eHAV, by binding to and possibly triggering uncoating of the capsid within late endosomes or endolysosomes, and (ii) that HAV-infected cells and quasi-enveloped eHAV virions are highly enriched in sphingolipids with very long-chain fatty acid (VLCFA, C≥22) tails, and that VLCFA synthesis is required for efficient production and release of infectious eHAV. In Aim 1, exceptional lipidomics expertise will be brought to bear on the question of which ganglioside species are necessary and sufficient for HAV and eHAV to enter cells and initiate infection, and will define the carbohydrate headgroups and acyl tail structures that are optimal for restoring the capacity of virus to infect CRISPR-derived ceramide glucosyltransferase (UGCG) knockout cells. The proposed experiments will also determine the impact of UGCG knockout on endocytosis and intracellular trafficking of HAV and eHAV, and the subcellular localization of exogenous gangliosides that restore the capacity of virus to enter these cells and initiate infection. Aim 2 will identify which ganglioside classes bind specifically to the naked HAV capsid, and whether ganglioside binding destabilizes its structure, lessening its thermostability at neutral or acidic pH, as a surrogate measure of uncoating. Aim 3 will characterize the role played by VLCFA in the hepatovirus lifecycle, and determine the degree to which VLCFA are enriched in infected cells, and in eHAV virions versus non-viral exosomes, and whether VLCFA are required for the production and cellular release of infectious eHAV, or for assembly of replication organelles involved in the synthesis of viral RNA. Additional experiments will determine whether and how HAV infection upregulates VLCFA synthetic flux, and whether this is essential for productive infection. By studying the role of lipids in hepatovirus replication, the proposed research will address an unexplored and neglected facet of the pathobiology of this important human pathogen.

项目摘要 甲型肝炎病毒 (HAV) 是一种不寻常的小核糖核酸病毒（肝病毒属），感染后无需裂解即可释放 类似于外泌体的小细胞外囊泡（EV）内的肝细胞。这些膜包裹的病毒体， 或“准包膜”HAV (eHAV)，其表面缺乏病毒编码的糖蛋白聚合物，但它们 具有传染性，是在急性甲型肝炎患者血液中循环的唯一病毒形式。 已发现其他“无包膜”病毒以准包膜病毒粒子的形式从细胞中释放出来 大小各异，使得 eHAV 的研究与广泛的病毒病原体相关。本申请建议 研究脂质代谢的两个方面，发现它们对于肝炎病毒的生产性感染至关重要 全基因组正向遗传筛选，并将测试以下假设：(i) GD3 和可能的其他 神经节苷脂在裸 HAV 和准 HAV 的细胞进入和感染启动中发挥着至关重要的作用。 包膜的 eHAV，通过结合并可能触发晚期内涵体内衣壳的脱壳或 内溶酶体，以及 (ii) HAV 感染细胞和准包膜 eHAV 病毒颗粒高度富集 具有极长链脂肪酸 (VLCFA, C≥22) 尾部的鞘脂，并且需要合成 VLCFA 有效生产和释放传染性 eHAV。在目标 1 中，卓越的脂质组学专业知识将被引入 涉及哪些神经节苷脂种类对于 HAV 和 eHAV 进入细胞是必要和充分的问题 并引发感染，并将定义最适合的碳水化合物头基和酰基尾结构 恢复病毒感染 CRISPR 衍生的神经酰胺葡萄糖基转移酶 (UGCG) 敲除细胞的能力。 拟议的实验还将确定 UGCG 敲除对内吞作用和细胞内 HAV 和 eHAV 的运输，以及恢复能力的外源性神经节苷脂的亚细胞定位 病毒进入这些细胞并引发感染。目标 2 将确定哪些神经节苷脂类特异性结合 裸露的 HAV 衣壳，以及神经节苷脂结合是否会破坏其结构的稳定性，从而降低其热稳定性 在中性或酸性 pH 值下，作为脱涂层的替代指标。目标 3 将描述 VLCFA 所扮演的角色 肝病毒生命周期，并确定 VLCFA 在受感染细胞中的富集程度，以及 eHAV 病毒颗粒与非病毒外泌体，以及生产和细胞是否需要 VLCFA 释放感染性 eHAV，或组装参与病毒 RNA 合成的复制细胞器。 其他实验将确定 HAV 感染是否以及如何上调 VLCFA 合成通量，以及 这对于生产性感染是否至关重要。通过研究脂质在肝病毒复制中的作用， 拟议的研究将解决这一重要人类病理学中未经探索和被忽视的方面 病原。