Membrane Hijacking: Biogenesis and Fate of Enveloped Hepatovirus

膜劫持：包膜肝病毒的生物发生和命运

• 批准号: 9115037
• 负责人: Stanley M. Lemon
• 金额: $ 38万
• 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 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9115037
• 关键词: Accounting; Acute; Acute Hepatitis; Address; Antibodies; Autophagocytosis; Autophagosome; Bile fluid; Biogenesis; Biological Assay; Blood; C-terminal; Capsid; Capsid Proteins; Cell Culture Techniques; Cell membrane; Cell physiology; Cells; Complex; Data; Detergents; Development; Disease; Elements; Event; Family; Family Picornaviridae; Genome; Goals; Growth; Health; Hepatitis A; Hepatitis A Vaccines; Hepatitis A Virus; Hepatovirus; Human; Immunoelectron Microscopy; Immunoglobulins; In Vitro; Infection; Interferons; Liver; Mediating; Membrane; Modeling; Molecular Genetics; Multivesicular Body; Mus; Oral; Pan Genus; Pathogenesis; Pathway interactions; Play; Primary carcinoma of the liver cells; Process; Production; Prophylactic treatment; Proteins; Proteomics; RNA Interference; RNA Viruses; Resistance; Resolution; Role; Serum; Signal Transduction; Sorting - Cell Movement; Structural Models; Structure; Study models; Vaccines; Viral; Viral hepatitis; Virion; Virus; Virus Diseases; anti-hepatitis A; base; biophysical analysis; exosome; hepatitis A virus antibodies; insight; knock-down; member; mouse model; neonatal Fc receptor; neutralizing antibody; nonhuman primate; novel; particle; pathogen; polarized cell; polypeptide; protective effect; protein expression; viral resistance; virus pathogenesis

DESCRIPTION (provided by applicant): The Picornaviridae are a large and diverse family of positive-strand RNA viruses that include a number of important human pathogens, among them hepatitis A virus (HAV), an hepatotropic virus that is the causative agent of acute hepatitis A and the only species in the genus Hepatovirus. As a family, picornaviruses are non-enveloped. Their genomes are encapsidated within icosahedral capsids comprised of 60 copies of each of 3-4 polypeptides. High-resolution structural models have been developed for several pathogenic picornaviruses but not HAV, and its structure remains poorly defined. We have discovered that most HAV particles released by cultured human hepatoma (Huh-7.5) cells are fully enveloped in host-derived membranes. Remarkably, biophysical studies indicate that these enveloped hepatoviruses (eHAV) are also the dominant form of virus circulating in the blood during acute hepatitis A. eHAV particles are fully infectious, yet highly resistant to neutralizing anti-HAV antibodies. These novel observations provide a new view of hepatitis A pathogenesis, and raise important questions about the mechanism by which vaccines provide protection against hepatitis A even when administered 2 weeks after infection. Specific Aim 1 will use quantitative proteomics and immunoelectron microscopy to characterize viral and host-derived proteins present in eHAV particles, as these may provide clues to the origin of eHAV. Specific Aim 2 focuses directly on the biogenesis of eHAV, and will use reverse molecular genetics and RNAi knockdown of multiple host proteins to probe interactions of HAV with ESCRT complexes involved in endosomal sorting and proteins involved in autophagy. Both cellular processes are potentially relevant to eHAV release from cells. Specific Aim 3 addresses the fate of eHAV, focusing particularly on how anti-HAV antibodies neutralize eHAV after its entry into permissive cells, a process that is likely to account for the protection vaccines afford against disease when administered after infection with the virus. A subsidiary aim is to develop a murine model of hepatitis A in which these events can be studied and which can replace existing nonhuman primate models of HAV infection. Although a completely novel finding, "membrane hijacking" may not be unique to HAV. These studies are thus broadly relevant to other non-enveloped viruses and how vaccine-induced antibodies provide protection against disease.

描述（由申请人提供）：picornaviridae是一个大而多样化的阳性RNA病毒家族，其中包括许多重要的人类病原体，其中包括肝炎病毒（HAV），这是一种肝炎病毒，是肝炎的病毒 肝病病毒属的唯一物种。作为一家人，picornaviruses是非开发的。它们的基因组被封装在二十面体皮膜中，由3-4个多肽中的每本副本组成。高分辨率的结构模型已经开发了几种致病性丘脑病毒，但没有开发出HAV，其结构的定义仍然很差。我们发现，培养的人肝癌（HuH-7.5）细胞释放的大多数HAV颗粒都完全包裹在宿主衍生的膜上。值得注意的是，生物物理研究表明，这些包膜的肝病病毒（EHAV）也是急性丙型肝炎时血液中循环的病毒的主要形式。EHAV颗粒完全具有感染性，但对中和中和抗HAV抗体具有极大的抵抗力。这些新颖的观察结果提供了肝炎的一种发病机理的新观点，并提出了有关疫苗在感染后2周进行保护的机制的重要问题。特定的目标1将使用定量蛋白质组学和免疫电子显微镜来表征EHAV颗粒中存在的病毒和宿主衍生的蛋白质，因为这些蛋白质可能为EHAV起源提供了线索。特定的目标2直接集中在EHAV的生物发生上，并将使用多种宿主蛋白​​的反向分子遗传学和RNAi敲低来探测HAV与参与内体分类的ESCRT复合物的相互作用，并探究与自噬相关的内体分类和蛋白质的相互作用。两种细胞过程都可能与EHAV从细胞释放有关。特定的目标3解决了EHAV的命运，特别关注抗HAV抗体在进入允许细胞后如何中和EHAV，这一过程可能会说明在感染病毒后给予疾病的保护疫苗。子公司的目的是开发一种可以研究这些事件的肝炎A模型，并可以取代现有的非人类灵长类动物模型。尽管一个完全新颖的发现，但“膜劫持”可能并不是HAV独有的。因此，这些研究与其他非发达病毒广泛相关，以及疫苗诱导的抗体如何提供疾病的保护。