Critical Lipid Species in the Hepatovirus Lifecycle

项目摘要 甲型肝炎病毒（HAV）是一种不常见的小核糖核酸病毒（肝病毒属）， 类似于外来体的小细胞外囊泡（EV）内的肝细胞。这些披着膜的病毒体 或“准包膜”HAV（eHAV），在其表面上缺乏病毒编码的糖蛋白peplomer，但它们是 传染性和唯一的形式的病毒发现循环的人与急性甲型肝炎。许多 已经发现其它“无包膜”病毒在以下EV中作为准包膜病毒体从细胞释放： 不同的大小，使eHAV的研究与广泛的病毒病原体有关。本申请建议 研究了在一个人中发现对生产性肝病毒感染至关重要的脂质代谢的两个方面， 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的细胞进入和感染起始中起关键作用， 包膜eHAV，通过结合并可能触发晚期内体内衣壳的脱壳，或 内溶酶体，和（ii）HAV感染的细胞和准包膜eHAV病毒粒子高度富集 具有极长链脂肪酸（VLCFA，C≥22）尾鞘脂，VLCFA合成是 有效生产和释放传染性eHAV。在目标1中，将把卓越的脂质组学专业知识带到 关于HAV和eHAV进入细胞所必需和充分的神经节苷脂种类的问题 并启动感染，并将确定碳水化合物头部和酰基尾部结构，这是最佳的 恢复病毒感染CRISPR衍生的神经酰胺葡糖基转移酶（UGCG）敲除细胞的能力。 所提出的实验还将确定UGCG敲除对内吞作用和细胞内分泌的影响。 HAV和eHAV的运输，以及恢复HAV和eHAV的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开始感染。目的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使其结构不稳定，降低其热稳定性 在中性或酸性pH下，作为脱涂层的替代测量。目标3将描述VLCFA所发挥的作用 在肝病毒生命周期中，并确定VLCFA在感染细胞中富集的程度， eHAV病毒粒子与非病毒外泌体，以及VLCFA是否是生产和细胞增殖所必需的。 感染性eHAV的释放，或参与病毒RNA合成的复制细胞器的组装。 另外的实验将确定HAV感染是否以及如何上调VLCFA合成通量， 这是否是生产性感染所必需的。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疾病病理生物学的一个未探索和被忽视的方面。 病原体

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.