Membrane Hijacking: Biogenesis and Fate of Enveloped Hepatovirus

描述(申请人提供)：短链核糖核酸病毒是一个庞大和多样化的正链RNA病毒家族，包括一些重要的人类病原体，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和 这是肝炎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个多肽的每个60个拷贝组成。针对几种致病的微小核糖核酸病毒而不是甲型肝炎病毒，已经建立了高分辨率的结构模型，其结构仍然定义不清。我们发现，培养的人肝癌细胞(Huh-7.5)释放的大部分甲型肝炎病毒颗粒被完全包裹在宿主来源的膜中。值得注意的是，生物物理研究表明，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的发病机制提供了一个新的视角，并提出了一些重要的问题，即疫苗即使在感染后2周接种也能提供预防甲型肝炎的机制。特殊目的1将使用定量蛋白质组学和免疫电子显微镜来表征存在于eHAV颗粒中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。具体目的2直接关注eHAV的生物发生，并将利用反向分子遗传学和多宿主蛋白的RNAi击倒来探讨HAV与参与内体分选的ESCRT复合体和参与自噬的蛋白质之间的相互作用。这两个细胞过程都可能与eHAV从细胞中释放有关。具体目标3涉及甲型肝炎病毒的命运，特别侧重于抗甲型肝炎病毒抗体在eHAV进入允许细胞后如何中和，这一过程可能解释了疫苗在感染病毒后接种时对疾病提供的保护。一个辅助目标是开发一种甲型肝炎的小鼠模型，在该模型中可以研究这些事件，并可以取代现有的非人灵长类甲型肝炎病毒感染模型。尽管这是一个全新的发现，但“膜劫持”可能并不是甲型肝炎病毒独有的。因此，这些研究与其他非包膜病毒以及疫苗诱导的抗体如何提供疾病保护具有广泛的相关性。

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.