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: 9764230
• 负责人: Stanley M. Lemon
• 金额: $ 52.13万
• 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/9764230
• 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 甲型肝炎病毒（HAV）是小核糖核酸病毒科（肝病毒属）的成员，是一种古老的人类病原体 也是全球肠道传播病毒性肝炎的常见原因。长期以来被认为是“非包络”的，我们 发现 HAV 在体外和受感染人体体内均以非溶细胞方式从受感染细胞中释放 在大小和浮力密度与外泌体相似的细胞外囊泡内。 HAV衣壳是 完全被这些小囊泡的膜覆盖，但它们仍然具有特定的感染性 与裸露的 HAV 病毒颗粒无法区分。这笔赠款的目的是阐明潜在的机制 这些“准包膜”HAV (eHAV) 病毒粒子的生物发生及其在甲型肝炎发病机制中的作用。 细胞培养中产生的 eHAV 的广泛蛋白质组学、病毒学和生物物理特征表明 通过高度特异性的分选过程，选择病毒衣壳用于 eHAV 囊泡中的输出 取决于 VP1 衣壳蛋白的 pX 结构域中的序列。我们假设 (1) 准 eHAV 的包封是由含有 VP1pX 的衣壳组装成内体而产生的 （多泡体）在由衣壳蛋白的特定相互作用介导的 ESCRT 依赖性过程中 具有 ESCRT 复合物并由 NEDD4 家族 E3 泛素连接酶促进； (2) eHAV 进入细胞 与裸露的病毒体不同，它发生在晚期内体-溶酶体中，其中膜被水解 溶酶体酶，使衣壳能够接触未知的细胞受体。在具体目标 1 中，我们将绘制 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 敲除细胞中的成分。具体目标 2 将确定催化活性是否 NEDD4 家族连接酶是 eHAV 生物发生所必需的，有助于 pX 或其他衣壳的泛素化 蛋白质结构域，并确定非经典“分泌性自噬”的核心成分的程度 参与非裂解性 eHAV 流出。具体目标 3 将对比 eHAV 与 HAV 进入机制，包括 内吞作用和内吞转运，并定义 eHAV 与 HAV 的组织向性。体外研究结果 将使用新型人类甲型肝炎小鼠模型进行体内验证。eHAV 的发现已 深刻改变了我们对这种嗜肝人类病毒发病机制的理解，并具有重大意义 对其他非细胞病变“无包膜”病毒的生物学影响，使这项研究具有广泛的意义 与病毒学领域的相关性。