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: 10530593
• 负责人: Stanley M. Lemon
• 金额: $ 51.68万
• 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/10530593
• 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; Hepatitis A; Hepatitis A Virus; Hepatocyte; Hepatovirus; Human; Infection; Infection prevention; Integration Host Factors; Knock-out; Laboratories; Life Cycle Stages; Lipids; Maps; Measures; Membrane; Organelles; Persons; Play; Production; Productivity; Proteins; RNA; Replicon; Reproducibility; Research; Research Personnel; Role; Sialic Acids; Site; Sphingolipids; Structure; Surface; Tail; Testing; Transfection; 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; viral transmission

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)是一种罕见的微小核糖核酸病毒(肝炎病毒属)，在感染后释放而不溶解 小的细胞外小泡(EVS)内的肝细胞，类似于外体。这些被膜包裹的病毒粒子， 或‘准包膜’甲型肝炎病毒(EHAV)，其表面缺乏病毒编码的糖蛋白聚体，但它们是 传染性的，也是在急性甲型肝炎患者的血液中发现的唯一的病毒形式 另一些无包膜的病毒已被发现以准包膜病毒粒子的形式从细胞中释放出来。 不同的大小，使对甲型肝炎病毒的研究与广泛的病毒病原体相关。此应用程序建议 研究脂代谢的两个方面，发现在生产性肝病毒感染中是必不可少的 全基因组正向遗传筛选，并将检验以下假设：(I)GD3和可能的其他 神经节苷脂在裸露甲型肝炎病毒和准甲型肝炎病毒进入细胞和启动感染过程中发挥关键作用。 包膜的eHAV，通过结合并可能触发晚期内体内衣壳的脱衣或 内溶酶体，以及(Ii)甲型肝炎病毒感染细胞和半包膜eHAV病毒粒子高度浓缩 含有超长链脂肪酸(VLCFA，C≥22)尾巴的鞘磷脂，合成VLCFA是必需的 高效生产和释放传染性甲型肝炎病毒。在AIM 1中，杰出的脂质组学专业知识将被带到 关于哪些神经节苷脂物种是甲型和乙型肝炎病毒进入细胞的必要条件和充分条件的问题 并引发感染，并将定义碳水化合物头部基团和酰基尾部结构，这些结构对 恢复病毒感染CRISPR衍生神经酰胺葡萄糖转移酶(UGCG)基因敲除细胞的能力。 拟议的实验还将确定UGCG基因敲除对内吞作用和细胞内的影响 甲型肝炎病毒和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定结合 裸露的甲型肝炎病毒衣壳，以及神经节苷脂结合是否破坏其结构的稳定性，降低其热稳定性 在中性或酸性的pH下，作为脱涂层的替代措施。目标3将描述VLCFA所扮演的角色 在肝病毒生命周期中，并确定VLCFA在感染细胞中的富集度，以及在 EHAV病毒粒子与非病毒外切体的比较，以及VLCFA是否需要生产和细胞 释放传染性的eHAV，或组装参与合成病毒RNA的复制细胞器。 其他实验将确定甲型肝炎病毒感染是否以及如何上调VLCFA合成通量，以及 这是否对生产性感染是必要的。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的病理生物学中一个未被探索和忽视的方面 病原体。