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: 8420039
• 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/8420039
• 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; 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; hepatitis A virus antibodies; insight; member; 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. PUBLIC HEALTH RELEVANCE: Hepatitis A virus (HAV) is a common cause of acute viral hepatitis. It is a member of the Picornaviridae family, a diverse group of non-enveloped RNA viruses that includes many human pathogens. We have made the surprising discovery that HAV particles released by infected liver-derived cells in vitro, or circulating in the blood during acue hepatitis A, are packaged within a single-membrane envelope and, although fully infectious, are resistant to antibody-mediated neutralization. This "membrane hijacking" by HAV has broad implications for other non-enveloped viruses, and for the mechanism by which hepatitis A vaccines protect against disease even when administered 2 weeks after infection.

描述（由申请人提供）：小核糖核酸病毒科是一个庞大且多样化的正链 RNA 病毒家族，包括许多重要的人类病原体，其中包括甲型肝炎病毒 (HAV)，一种嗜肝病毒，是急性甲型肝炎的病原体， 肝病毒属中唯一的物种。作为一个家族，小核糖核酸病毒是无包膜的。它们的基因组被包裹在二十面体衣壳内，衣壳由 3-4 种多肽各 60 个拷贝组成。几种致病性小核糖核酸病毒（但不是 HAV）的高分辨率结构模型已经开发出来，而且其结构仍不清楚。我们发现，培养的人肝癌 (Huh-7.5) 细胞释放的大多数 HAV 颗粒完全被宿主来源的细胞膜包裹。值得注意的是，生物物理学研究表明，这些包膜肝病毒 (eHAV) 也是急性甲型肝炎期间血液中循环的病毒的主要形式。eHAV 颗粒具有完全传染性，但对中和抗 HAV 抗体具有高度抵抗力。这些新的观察结果为甲型肝炎发病机制提供了新的观点，并提出了关于疫苗即使在感染后两周接种也能提供针对甲型肝炎的保护的机制的重要问题。具体目标 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蛋白，因为这些可能为 eHAV 的起源提供线索。具体目标 2 直接关注 eHAV 的生物发生，并将利用反向分子遗传学和多种宿主蛋白​​的 RNAi 敲低来探测 HAV 与参与内体分选的 ESCRT 复合物和参与自噬的蛋白质的相互作用。这两个细胞过程都可能与细胞中 eHAV 的释放相关。具体目标 3 解决了 eHAV 的命运，特别关注抗 HAV 抗体如何在 eHAV 进入允许的细胞后中和 eHAV，这一过程可能解释了感染病毒后接种疫苗对疾病的保护作用。另一个目标是开发甲型肝炎小鼠模型，可以在其中研究这些事件，并可以取代现有的 HAV 感染的非人类灵长类动物模型。尽管这是一个全新的发现，但“膜劫持”可能并非 HAV 所独有。因此，这些研究与其他无包膜病毒以及疫苗诱导的抗体如何提供针对疾病的保护广泛相关。 公共卫生相关性：甲型肝炎病毒 (HAV) 是急性病毒性肝炎的常见原因。它是小核糖核酸病毒科的成员，这是一组不同的无包膜 RNA 病毒，包括许多人类病原体。我们惊奇地发现，受感染的肝源性细胞在体外释放的 HAV 颗粒，或在急性甲型肝炎期间在血液中循环的 HAV 颗粒，被包装在单膜包膜内，尽管具有完全传染性，但对抗体介导的中和具有抵抗力。 HAV 的这种“膜劫持”对于其他无包膜病毒以及甲型肝炎疫苗即使在感染后 2 周内接种也能预防疾病的机制具有广泛的影响。