Functional development of picornavirus-induced membranes throughout infection

肠道病毒是人类疾病最严重的原因之一，最近爆发的新兴肠道病毒，如D 68和71型，突出了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-D 68的比较研究表明，这些病毒的生命周期有许多共同点。我们的研究重点是肠道病毒属的成员如何触发称为自噬的细胞途径，以促进感染性病毒的产生。自噬是一种组成性的降解细胞过程，需要周转受损的囊泡，聚集的蛋白质，和其他用过的细胞成分。在应激期间，包括氨基酸饥饿，生物体发育和感染，自噬被上调。自噬囊泡，被认为是来自病毒RNA复制膜的复杂网络，可以在中期至晚期感染期间观察到。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，通常包裹在这些细胞衍生的膜中。在这个提议中，我们概述了一个全面的计划，以了解在感染过程中产生的膜，从最初的RNA复制膜的自噬体促进成熟的病毒粒子的单膜病毒囊泡被释放的细胞。了解这一整个途径，其中后半部分仅在过去几年中被发现，将有助于了解这些病毒如何复制，成熟，从细胞中释放并逃避免疫检测。在具体目标I中，我们将分析特定的病毒非结构蛋白如何在感染的早期阶段启动自噬体形成，并调节自噬体形成，酸性成熟和病毒释放的下游步骤。在特定目标II中，我们将研究病毒和宿主对病毒RNA复制膜的发展的要求，以及在感染过程中从这些卷曲的膜结构形成自噬体的机制。这些实验可以在不存在自噬途径和其他细胞途径的宿主蛋白质的情况下进行。此外，我们已经表明，酸性自噬体通过诱导衣壳蛋白VP 0裂解成VP 2和VP 4来促进病毒粒子的成熟。我们提出了一种生物化学方法来鉴定病毒衣壳感染性成熟所需的自噬体内所含的因子。在理解肠道病毒诱导的膜的发展方面存在重大的研究空白，从病毒蛋白在感染开始时发出的最初信号到含病毒囊泡的释放。该提案将通过研究该途径中从开始-RNA复制膜的起始-到结束-含病毒囊泡的释放的每一步来解决这一需求。

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.