Membrane Hijacking: Biogenesis and Fate of Quasi-Enveloped Hepatovirus

项目摘要 甲型肝炎病毒(HAV)是微小病毒科(肝病毒属)的一员，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，我们一直被认为是不受限制的 发现甲型肝炎病毒在体外和体内从感染的细胞中以非细胞溶解的方式释放 在大小和浮力密度上与外体相似的细胞外小泡内。甲型肝炎病毒衣壳是 完全被这些小泡的膜所遮盖，这些小泡仍然具有特定的传染性 与裸露的甲型肝炎病毒粒子难以区分。这笔赠款的目的是阐明 这些“准包膜”甲型肝炎病毒粒子的生物发生及其在甲型肝炎发病机制中的作用 广泛的蛋白质组学、病毒学和生物物理特征表明，在细胞培养中产生的eHAV 通过高度特异的分选过程，选择病毒衣壳在eHAV囊泡中出口，这是 依赖于VP1衣壳蛋白Px区的序列。我们假设(1)准- 含有VP1pX的组装衣壳萌发成内体导致eHAV的包膜 (多泡小体)在衣壳蛋白特异性相互作用介导的ESCRT依赖过程中 与ESCRT复合体结合，并由NEDD4家族E3泛素连接酶促进；以及(2)通过eHAV进入细胞 不同于裸露的病毒粒子，出现在晚期的内体-溶酶体中，膜被 溶酶体酶，为未知的细胞受体提供衣壳。在具体目标1中，我们将绘制 ESCRT组分与VP1pX的相互作用及ESCRT-0和-I的功能重要性 CRISPR/Cas9基因敲除细胞中的成分。特定目标2将决定是否具有催化活性 NEDD4家族连接酶是eHAV生物发生所必需的，并有助于PX或其他衣壳的泛素化 蛋白质结构域，并确定非规范性“分泌性自噬”的核心成分的程度 牵涉到非溶血性甲型肝炎病毒的出口。具体目标3将对比eHAV和HAV进入的机制，包括 内吞作用和内吞转运，并定义eHAV与HAV的组织趋向性。体外研究的结果 将使用一种新的甲型肝炎小鼠模型在体内进行验证。 深刻地改变了我们对这种嗜肝性人类病毒发病机制的理解，并具有实质性的 对其他非细胞病变的“无包膜”病毒的生物学影响，使这项研究具有广泛的 与病毒学领域相关。

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.