The roles of acidic autophagosomes in production of infectious poliovirus

项目摘要/摘要 到目前为止，所有研究的正链RNA病毒都重排了细胞膜，以促进它们自己的 复制。这些重排的原因之一是这些病毒将它们的基因组RNA复制到 与细胞膜结合。在脊髓灰质炎病毒(PV)中，一种具有重要医学意义的阳性病毒的模型 链RNA病毒，已被鉴定为两类截然不同的水泡。一级，单膜 囊泡类似于COPII分泌转运囊泡，与病毒RNA复制蛋白有关。 第二种囊泡是双膜的，也与病毒RNA复制蛋白有关 与自噬类似，自噬是一种细胞器，由细胞内稳态和应激途径诱导- 这种反应被称为自噬。自噬体样囊泡是由病毒特异性诱导的，并促进 光伏生产。该项目的长期目标是了解 小核糖核酸病毒引起的细胞膜重排。此应用程序的目标是确定角色 自噬小体在感染过程中的作用，并确定PV诱导自噬小体的机制 形态发生。我们的初步数据表明，感染病毒的最佳水平需要自噬。 病毒RNA复制需要自噬信号。然而，囊泡酸化，在这种情况下 自噬小体是与溶酶体融合所必需的，是切割病毒衣壳蛋白所必需的， 从新形成的病毒粒子产生传染性病毒的最后一步。这项提议的中心假设是 PV感染通过形态变化产生自噬小体，包括内陷 富含PIP4的分泌途径衍生的小泡，用于RNA复制。新形成的油层的酸化 自噬小体促进病毒粒子成熟。核心假设将通过追求两个具体的 目标。在目标I中，我们将研究囊泡环境的性质，以及病毒粒子成熟的要求。 在AIM II中，我们将分析感染过程中自噬小泡的发育，并定义 病毒诱导的自噬小体。这项研究的基本原理是了解微小核糖核酸病毒是如何颠覆 这通常是一种促进病毒粒子成熟的抗病原体途径。这将为我们提供信息 需要用疗法来瞄准病毒生产的这一后期步骤。我们的创新方法将确定 光伏病毒诱导自噬小体的机制及其如何促进感染性病毒的成熟和排出 病毒。这项拟议的研究意义重大，因为它将从根本上促进我们对 医学上重要的病毒家族颠覆了促进病毒复制的基本细胞途径。这部作品 将为病毒生命周期的后期阶段，特别是成熟和细胞出口提供新的见解， 并提供了确定治疗靶点的第一步，这些靶点最终可能导致对 多种病毒性疾病。这项工作也是理解现有的和 未来针对多种病毒疾病的治疗药物。

PROJECT SUMMARY/ABSTRACT All positive strand RNA viruses studied to date rearranged cellular membranes to promote their own replication. One reason for these rearrangements is that these viruses replicate their genomic RNA in association with cellular membranes. In poliovirus (PV), a model for a host of medically important positive strand RNA viruses, two distinct classes of vesicle have been identified. One class, a single-membraned vesicle which resembles a COPII secretory transport vesicle, associates with viral RNA replication proteins. The second type of vesicle, which is double-membraned, also associates with viral RNA replication proteins and resembles the autophagosome, an organelle induced by a pathway of cellular homeostasis and stress- response known as autophagy. Autophagosome-like vesicles are specifically induced by viruses and promote PV production. The long term goal of this project is to understand the mechanisms and consequences of cellular membrane rearrangements by picornaviruses. The objective of this application is to identify the roles played by autophagosomes during infection and define the mechanism by which PV induces autophagosome morphogenesis. Our preliminary data indicate that autophagy is required for optimal levels of infectious virus. Autophagic signaling is required for viral RNA replication. However, vesicle acidification, which in the case of autophagosomes is required for fusion with lysosomes, is required for cleavage of a viral capsid protein, the final step in generating infectious virus from newly formed virions. The central hypothesis of this proposal is that PV infection generates autophagosomes through morophological changes, including invagination, in the PIP4-rich secretory pathway-derived vesicles used for RNA replication. Acidification of the newly formed autophagosomes promotes virion maturation. The central hypothesis will be tested by pursuing two specific aims. In Aim I we will study the nature of the vesicle environment, and the requirements for virion maturation. In Aim II we will analyze the development of autophagic vesicles during infection and define the proteome of virus-induced autophagosomes. The rationale for this research is to understand how picornaviruses subvert what is often an anti-pathogen pathway to promote virion maturation. This will provide us with information needed to target this late step in virus production with therapeutics. Our innovative approaches will identify the mechanisms PV uses to induce autophagosomes, and how they promote maturation and egress of infectious virus. The proposed research is significant because it will fundamentally advance our understanding of how a medically important family of viruses subverts a basic cellular pathway to promote virus replication. This work will provide novel insights into the late stages of the viral life cycle, especially maturation and cellular egress, and provide the first steps in identifying therapeutic targets that may ultimately lead to treatments against multiple viral diseases. This work is also an important step in understanding the mechanisms of existing and future therapeutic agents against multiple viral diseases.