Critical Lipid Species in the Hepatovirus Lifecycle
Critical Lipid Species in the Hepatovirus Lifecycle
批准号:
10530593
负责人:
Stanley M. Lemon
金额:
$51.68万
依托单位国家:
美国
项目类别:
财政年份:
2019
资助国家:
美国
项目状态:
已结题
起止时间:
2019-12-12 至 2024-11-30
关键词:
Acute HepatitisAddressAntibodiesBindingBiological AssayBiologyBloodCRISPR screenCapsidCapsid ProteinsCarbohydratesCarbonCellsCeramide glucosyltransferaseCessation of lifeClustered Regularly Interspaced Short Palindromic RepeatsComplexCytolysisDataDisease OutbreaksEndocytosisEnteralEnzymesFamily PicornaviridaeFatty AcidsGangliosidesGenesGeneticGenetic ScreeningGenetic TranscriptionGlucosylceramidesGlycoproteinsGlycosphingolipidsHepatitis AHepatitis A VirusHepatocyteHepatovirusHumanInfectionInfection preventionIntegration Host FactorsKnock-outLaboratoriesLife Cycle StagesLipidsMapsMeasuresMembraneOrganellesPersonsPlayProductionProductivityProteinsRNARepliconReproducibilityResearchResearch PersonnelRoleSialic AcidsSiteSphingolipidsStructureSurfaceTailTestingTransfectionUnited StatesVery Long Chain Fatty AcidViralViral hepatitisVirionVirusVirus DiseasesVirus Replicationdihydroceramide desaturaseexosomeexperimental studyextracellular vesiclesgenome-widehuman pathogenlate endosomelipid metabolismlipidomicsmemberneglectnovel strategiespathogenic viruspermissivenessthermostabilitytraffickingviral RNAviral transmission
中文摘要
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
英文摘要
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.
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Critical Lipid Species in the Hepatovirus Lifecycle
-
批准号:10306348
-
项目类别:
-
资助金额:$51.56万
-
财政年份:2019
-
负责人:Stanley M. Lemon
-
依托单位:
Critical Lipid Species in the Hepatovirus Lifecycle
-
批准号:9913862
-
项目类别:
-
资助金额:$52.83万
-
财政年份:2019
-
负责人:Stanley M. Lemon
-
依托单位:
Novel Pathogen Recognition Pathways and Control of Hepatitis A Virus
-
批准号:9233911
-
项目类别:
-
资助金额:$40.77万
-
财政年份:2014
-
负责人:Stanley M. Lemon
-
依托单位:
Membrane Hijacking: Biogenesis and Fate of Enveloped Hepatovirus
-
批准号:8549949
-
项目类别:
-
资助金额:$35.72万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Membrane Hijacking: Biogenesis and Fate of Quasi-Enveloped Hepatovirus
-
批准号:9764230
-
项目类别:
-
资助金额:$52.13万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Murine Model of HCV-Associated Human Liver Cancer
-
批准号:8625280
-
项目类别:
-
资助金额:$45.66万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Membrane Hijacking: Biogenesis and Fate of Quasi-Enveloped Hepatovirus
-
批准号:10223138
-
项目类别:
-
资助金额:$38.88万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Murine Model of HCV-Associated Human Liver Cancer
-
批准号:8219397
-
项目类别:
-
资助金额:$47.07万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Membrane Hijacking: Biogenesis and Fate of Enveloped Hepatovirus
-
批准号:8420039
-
项目类别:
-
资助金额:$38.0万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Murine Model of HCV-Associated Human Liver Cancer
-
批准号:8464678
-
项目类别:
-
资助金额:$44.25万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Membrane Hijacking: Biogenesis and Fate of Quasi-Enveloped Hepatovirus
-
批准号:9979729
-
项目类别:
-
资助金额:$38.88万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Membrane Hijacking: Biogenesis and Fate of Enveloped Hepatovirus
-
批准号:8711270
-
项目类别:
-
资助金额:$38.0万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Membrane Hijacking: Biogenesis and Fate of Enveloped Hepatovirus
-
批准号:8898710
-
项目类别:
-
资助金额:$38.0万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Membrane Hijacking: Biogenesis and Fate of Enveloped Hepatovirus
-
批准号:9115037
-
项目类别:
-
资助金额:$38.0万
-
财政年份:2012
-
负责人:Stanley M. Lemon
-
依托单位:
Micro-RNA 122 and Chronic Hepatitis C
-
批准号:8258235
-
项目类别:
-
资助金额:$36.84万
-
财政年份:2011
-
负责人:Stanley M. Lemon
-
依托单位:
Micro-RNA 122 and Chronic Hepatitis C
-
批准号:8163381
-
项目类别:
-
资助金额:$36.84万
-
财政年份:2011
-
负责人:Stanley M. Lemon
-
依托单位:
Micro-RNA 122 and Chronic Hepatitis C
-
批准号:8444519
-
项目类别:
-
资助金额:$34.63万
-
财政年份:2011
-
负责人:Stanley M. Lemon
-
依托单位:
Micro-RNA 122 and Chronic Hepatitis C
-
批准号:9889870
-
项目类别:
-
资助金额:$38.0万
-
财政年份:2011
-
负责人:Stanley M. Lemon
-
依托单位:
Micro-RNA 122 and Chronic Hepatitis C
-
批准号:8823725
-
项目类别:
-
资助金额:$36.84万
-
财政年份:2011
-
负责人:Stanley M. Lemon
-
依托单位:
Micro-RNA 122 and Chronic Hepatitis C
-
批准号:9121876
-
项目类别:
-
资助金额:$38.0万
-
财政年份:2011
-
负责人:Stanley M. Lemon
-
依托单位:
海外基金