Membrane Hijacking: Biogenesis and Fate of Quasi-Enveloped Hepatovirus

膜劫持：准包膜肝病毒的生物发生和命运

• 批准号: 9979729
• 负责人: Stanley M. Lemon
• 金额: $ 38.88万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-24 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979729
• 关键词: ARHGEF5 gene; Autophagocytosis; Biogenesis; Biological Assay; Biology; C-terminal; CRISPR/Cas technology; Capsid; Capsid Proteins; Cell Culture Techniques; Cell membrane; Cells; Complement; Complex; Data; Endocytosis; Endosomes; Enteral; Enterovirus; Enzymes; Extracellular Space; Family; Family Picornaviridae; Galactose Binding Lectin; Glycoproteins; Goals; Grant; Hepatitis A; Hepatitis A Virus; Hepatovirus; Human; Image; In Vitro; Infection; Knock-out; Laboratories; Lectin; Ligase; Lysosomes; Maps; Mediating; Membrane; Molecular; Molecular Genetics; Multivesicular Body; Mus; Nonlytic; Outcome; Pathogenesis; Pathway interactions; Phospholipase; Phospholipases A; Process; Proteins; Proteomics; Reporter; Research; Role; Sampling; Signal Transduction; Site; Sorting - Cell Movement; TSG101 gene; Tertiary Protein Structure; Tissues; Vesicle; Viral; Viral hepatitis; Virion; Virus; Virus Diseases; Work; base; biophysical properties; density; exosome; extracellular vesicles; hepatitis A virus receptor; human model; human pathogen; in vivo; late endosome; lysosomal proteins; lysosome membrane; member; mouse model; novel; receptor; response; tissue tropism; trafficking; ubiquitin-protein ligase; uptake; virology; virus envelope

PROJECT ABSTRACT Hepatitis A virus (HAV), a member of the Picornaviridae (genus Hepatovirus), is an ancient human pathogen and a common cause of enterically-transmitted viral hepatitis globally. Long considered `nonenveloped', we discovered that HAV is released noncytolytically from infected cells both in vitro and in infected humans in vivo within extracellular vesicles that are similar in size and buoyant density to exosomes. The HAV capsid is completely cloaked by the membranes of these small vesicles, which nonetheless possess specific infectivity indistinguishable from naked HAV virions. The goal of this grant is to elucidate mechanisms underlying the biogenesis of these `quasi-enveloped' HAV (eHAV) virions and their role in the pathogenesis of hepatitis A. Extensive proteomics, virologic, and biophysical characterization of eHAV produced in cell culture demonstrate that viral capsids are selected for export in eHAV vesicles via a highly specific sorting process that is dependent on sequence in the pX domain of the VP1 capsid protein. We hypothesize (1) that the quasi- envelopment of eHAV results from budding of assembled VP1pX-containing capsids into endosomes (multivesicular bodies) in an ESCRT-dependent process mediated by specific interactions of capsid proteins with ESCRT complexes and facilitated by NEDD4-family E3 ubiquitin ligases; and (2) that cell entry by eHAV differs from naked virions and occurs within late endosome-lysosomes where membranes are hydrolyzed by lysosomal enzymes, providing capsid access to an unknown cellular receptor. In Specific Aim 1, we will map interactions of ESCRT components with VP1pX and ascertain the functional importance of ESCRT-0 and -I components in CRISPR/Cas9 knockout cells. Specific Aim 2 will determine whether catalytic activity of NEDD4-family ligases is required for eHAV biogenesis and contributes to ubiquitylation of pX or other capsid protein domains, and ascertain the extent to which core components of noncanonical `secretory autophagy' are involved in nonlytic eHAV egress. Specific Aim 3 will contrast mechanisms of eHAV vs. HAV entry, including endocytosis and endocytic transport, and define tissue tropisms of eHAV vs. HAV. Results from in vitro studies will be validated in vivo using a novel murine model of human hepatitis A. The discovery of eHAV has profoundly altered our understanding of the pathogenesis of this hepatotropic human virus and has substantial ramifications for the biology of other noncytopathic `nonenveloped' viruses, making this research of broad relevance to the field of virology.

项目摘要 肝炎病毒（HAV）是Picornaviridae（肝病属）的成员，是一种古老的人类病原体 这是全球进入肠道传播病毒肝炎的常见原因。长期以来被认为是“不发达的”，我们 发现HAV在体外和感染的人体中都从感染细胞中释放出非溶液释放 在大小和浮力密度与外泌体相似的细胞外囊泡中。 Hav Capsid是 这些小囊泡的膜完全掩盖，尽管如此 与裸露的Hav病毒体无法区分。这笔赠款的目的是阐明该机制 这些“准发达” HAV（EHAV）病毒体的生物发生及其在肝炎发病机理中的作用。 在细胞培养中产生的EHAV的广泛蛋白质组学，病毒学和生物物理表征证明了 该病毒式衣壳是通过高度特定的分类过程在EHAV囊泡中选择的 取决于VP1衣壳蛋白的PX结构域中的序列。我们假设（1）准 EHAV的包裹是由组装的含VP1PX的衣壳萌芽到内体 （多囊体）在由衣壳蛋白的特定相互作用介导的ESCRT依赖性过程中 用ESCRT复合物，并由NEDD4-家庭E3泛素连接酶促进； （2）EHAV的细胞进入 与裸毒素的不同，发生在晚期内体溶质体中，其中膜被水解。 溶酶体酶，为未知的细胞受体提供衣壳的访问。在特定目标1中，我们将映射 ESCRT组件与VP1PX的相互作用，并确定ESCRT -0和-i的功能重要性 CRISPR/CAS9基因敲除细胞中的组件。具体目标2将确定是否催化活性是否 EHAV生物发生需要NEDD4家族连接酶，并有助于PX或其他衣壳的泛素化 蛋白质领域，并确定非规范性“分泌自噬”的核心成分在多大程度上是 参与了非散发EHAV出口。具体目标3将对比EHAV与HAV进入的机制，包括 内吞作用和内吞转运，并定义EHAV与HAV的组织偏向主义。体外研究的结果 将使用人类肝炎A的新型鼠模型在体内进行验证。 深刻地改变了我们对这种肝脏人类病毒的发病机理的理解，并具有实质性 其他非肿瘤病毒的生物学的影响，使得这项广泛的研究 与病毒学领域有关。