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: 9913862
• 负责人: Stanley M. Lemon
• 金额: $ 52.83万
• 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/9913862
• 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; 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 丙型肝炎病毒（HAV）是一种不寻常的picornavirus（肝病毒属），没有受到感染的裂解而释放 小细胞外蔬菜（EV）中类似外泌体的肝细胞。这些膜状病毒， 或“ Quasi Gevaled” Hav（EHAV），表面缺乏病毒编码的糖蛋白肽蛋白，但它们是 感染性和病毒的唯一形式发现在急性肝炎患者的血液中循环。 发现其他“非发育”病毒被发现是从细胞中释放的，作为准发育病毒 变化的大小，使对EHAV的研究与广泛的病毒病原体有关。此申请建议 研究脂质代谢的两个方面，发现对生产性肝病病毒感染至关重要 全基因组的正向遗传筛选，并将检验以下假设：（i）GD3和其他可能 神经节苷脂在细胞进入中起着至关重要的作用，而赤裸裸的Hav和Quasi则是感染的倡议 包裹的eHav，通过与后期内体内的衣壳结合并可能触发capsID的脱涂或 内溶性，（ii）HAV感染的细胞和准发育的EHAV病毒在高度富集 具有非常长链脂肪酸（VLCFA，C≥22）尾巴的鞘脂，并且需要VLCFA合成 感染性EHAV的有效生产和释放。在AIM 1中，将带来卓越的脂肪组学专业知识 在哪种神经节苷脂物种是必要的，足以让HAV和EHAV进入细胞的问题 并启动感染，并将定义最佳的碳水合头组和酰基尾巴结构 恢复病毒感染CRISPR衍生的神经酰胺葡萄糖基转移酶（UGCG）基因敲除细胞的能力。 提出的实验还将确定UGCG敲除对内吞和细胞内的影响 贩运HAV和EHAV，以及恢复容量的外源性神经节的亚细胞定位 进入这些细胞并引发感染的病毒。 AIM 2将确定哪种神经节类别与 裸帽帽，以及神经节结合是否破坏了其结构，降低了其热稳定性 在中性或酸性pH下，作为替代涂层的替代测量。 AIM 3将描述VLCFA扮演的角色 在肝病毒病毒生命周期中，并确定VLCFA富含感染细胞的程度 EHAV病毒与非病毒外泌体，以及生产和细胞是否需要VLCFA 传染性EHAV的释放，或用于组合病毒RNA的复制细胞器的组装。 其他实验将确定是否以及如何上调VLCFA合成通量，以及如何上调 这是否对于产品感染至关重要。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个重要人类病理生物学的意外而被忽视的方面 病原。