Membrane Hijacking: Biogenesis and Fate of Enveloped Hepatovirus

描述（由申请人提供）：小核糖核酸病毒科是一个大而多样的正链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。它们的基因组在二十面体衣壳内被包裹，所述二十面体衣壳由3-4种多肽中的每一种的60个拷贝组成。高分辨率的结构模型已经开发了几种致病性小核糖核酸病毒，但没有HAV，其结构仍然不明确。我们已经发现，大多数HAV颗粒释放培养的人肝癌细胞（Huh-7.5）完全包膜在宿主衍生的膜。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主要病毒形式。eHAV颗粒是完全感染性的，但对中和抗HAV抗体具有高度抗性。这些新的观察结果为甲型肝炎发病机制提供了一个新的视角，并提出了有关疫苗即使在感染后2周接种也能提供甲型肝炎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，因为这些可能提供eHAV起源的线索。具体目标2直接关注eHAV的生物发生，并将使用反向分子遗传学和多种宿主蛋白质的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。这两种细胞过程都可能与eHAV从细胞中释放有关。具体目标3解决了eHAV的命运，特别关注抗HAV抗体如何在eHAV进入允许细胞后中和eHAV，这一过程可能是病毒感染后接种疫苗预防疾病的原因。一个辅助的目的是开发一种小鼠模型的甲型肝炎，其中这些事件可以进行研究，并可以取代现有的非人灵长类动物模型的HAV感染。虽然这是一个全新的发现，但“膜劫持”可能不是HAV所独有的。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对疾病的保护广泛相关。

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.