Functional development of picornavirus-induced membranes throughout infection

肠病毒是人类疾病的最严重原因之一，最近爆发的新出现的肠病毒，如D68型和71型，突出表明需要更多地了解这些病原体。我们对脊髓灰质炎病毒（PV）、柯萨奇病毒B3和现在的EV-D68的比较研究表明，这些病毒在生命周期中有许多共性。我们的研究重点是肠病毒属的成员如何触发被称为自噬的细胞途径来促进感染性病毒的产生。自噬是一种基本的细胞降解过程，用于修复受损的囊泡、聚集的蛋白质和其他消耗的细胞成分。在应激时期，包括氨基酸饥饿、生物体发育和感染，自噬被上调。自噬囊泡，被认为来源于病毒RNA复制膜的复杂网络，可以在感染中后期观察到。我们和其他人已经证明病毒蛋白特异性诱导自噬信号和自噬体的形成。这些自噬体酸化，促进病毒衣壳的成熟和新形成的感染性病毒的释放，这些病毒通常被包裹在这些来源于细胞的膜中。在这个建议中，我们概述了一个全面的计划，以了解感染过程中膜的产生，从最初的RNA复制膜到促进病毒粒子成熟的自噬体，再到从细胞释放的单膜含病毒囊泡。了解整个途径（后一部分在过去几年中才被确定）将有助于了解这些病毒如何复制、成熟、从细胞中释放和逃避免疫检测。在Specific Aim I中，我们将分析特异性病毒非结构蛋白如何在感染的早期阶段启动自噬体的形成，并调节自噬体形成、酸性成熟和病毒释放的下游步骤。在Specific Aim II中，我们将研究病毒和宿主对病毒RNA复制膜发育的需求，以及在感染过程中这些卷曲的膜结构形成自噬体的机制。这些实验可以在自噬途径和其他细胞途径缺乏宿主蛋白的情况下进行。此外，我们已经证明酸性自噬体通过诱导衣壳蛋白VP0裂解成VP2和VP4来促进病毒粒子的成熟。我们提出了一种生化方法来识别病毒衣壳感染性成熟所需的自噬体中包含的因子。从感染开始时病毒蛋白发送的初始信号到含病毒囊泡的释放，在理解肠病毒诱导膜的发育方面存在重大的研究空白。该提案将通过研究这一途径的每一步来解决这一需求，从开始- RNA复制膜的起始-到结束-含病毒囊泡的释放。

Enteroviruses are among the most severe causes of human disease, and recent outbreaks of emerging enteroviruses such as types D68 and 71 have highlighted the need to understand more about these pathogens. Our comparative studies of poliovirus (PV), coxsackievirus B3, and now EV-D68 have shown many commonalities in the life-cycles of these viruses. We have focused our studies on how members of the Enterovirus genus trigger a cellular pathway known as autophagy to promote production of infectious virus. Autophagy is a constitutive degradative cellular process required for turnover of damaged vesicles, aggregated proteins, and other spent cellular components. During times of stress, including amino acid starvation, organismal development, and infection, autophagy is up-regulated. Autophagic vesicles, thought to be derived from the complex web of viral RNA replication membranes, can be observed during mid-to-late infection. We and others have demonstrated that viral proteins specifically induce autophagic signaling and autophagosome formation. These autophagosomes acidify, promoting maturation of the viral capsid and release of newly formed infectious viruses, often encased in these cell-derived membranes, from the cell. In this proposal, we outline a comprehensive plan to understand generation of membranes during infection, from the initial RNA replication membranes to the autophagosomes promoting maturation of virions to the single-membraned virus- containing vesicles being released from the cell. Understanding this entire pathway, the latter parts of which have only been identified within the past few years, will provide an understanding of how these viruses replicate, mature, release from cells, and evade immune detection. In Specific Aim I, we will analyze how specific viral non-structural proteins initiate autophagosome formation at the early stages of infection and regulate downstream steps in autophagosome formation, acidic maturation, and virus release. In Specific Aim II, we will investigate the viral and host requirements for development of viral RNA replication membranes, and the mechanism of formation of autophagosomes from these convoluted membrane structures, during infection. These experiments can be carried out in the absence of host proteins from the autophagic pathway and other cellular pathways. In addition, we have shown that acidic autophagosomes promote maturation of the virion by inducing cleavage of the capsid protein VP0 into VP2 and VP4. We propose a biochemical approach to identifying the factor(s) contained within autophagosomes required for infectious maturation of viral capsids. There is a major research gap in understanding development of enterovirus-induced membranes, from the initial signals sent by virus proteins at the beginning of infection, to the release of virus-containing vesicles. This proposal will address that need by investigating each step in this pathway from beginning - initiation of RNA replication membranes - to end - the release of virus-containing vesicles.