Membrane Hijacking: Biogenesis and Fate of Quasi-Enveloped Hepatovirus

项目摘要 甲型肝炎病毒（Hepatitis A virus，HAV）是一种古老的人类病原体，属于细小核糖核酸病毒科（Picornaviridae，Hepatovirus）属 也是全球肠道传播病毒性肝炎的常见原因。长期以来被认为是“不受欢迎的”，我们 发现HAV在体外和体内均从受感染的细胞中以非细胞溶解的方式释放 在大小和浮力密度与外来体相似的细胞外囊泡内。HAV衣壳是 完全被这些小泡的膜所掩盖，尽管如此，这些小泡仍然具有特定的感染性 与裸露的HAV病毒体难以区分。这项资助的目标是阐明 这些“准包膜”HAV（eHAV）病毒粒子的生物发生及其在甲型肝炎发病机制中的作用。 细胞培养中产生的eHAV的广泛的蛋白质组学、病毒学和生物物理学特征表明， 通过高度特异性的分选过程选择病毒衣壳以输出到eHAV囊泡中， 这取决于VP 1衣壳蛋白pX结构域中的序列。我们假设（1）准- eHAV的复制是由组装的含VP 1 pX的衣壳出芽到核内体中引起的 （多泡体）在由衣壳蛋白特异性相互作用介导的ESCRT依赖性过程中 ESCRT复合物和促进NEDD 4家族E3泛素连接酶;和（2）细胞进入由eHAV 与裸病毒体不同，发生在晚期内体-溶酶体中，在那里膜被 溶酶体酶，提供衣壳进入未知细胞受体。在具体目标1中，我们将绘制 ESCRT组分与VP 1 pX的相互作用，并确定ESCRT-0和-I的功能重要性 CRISPR/Cas9敲除细胞中的组分。具体目标2将决定是否催化活性的 NEDD 4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，并确定在何种程度上的核心组成部分的非典型的“分泌性自噬”， 参与了非裂解性甲型肝炎病毒的逃逸。具体目标3将对比eHAV与HAV进入的机制，包括 内吞作用和内吞转运，并定义了eHAV与HAV的组织嗜性。体外研究的结果 将在体内使用人甲型肝炎的新型鼠模型进行验证。eHAV的发现 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， 其他非致细胞病变的“无包膜”病毒的生物学分支，使这项研究的广泛 与病毒学领域的关系。

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.