The roles of acidic autophagosomes in production of infectious poliovirus

描述(申请人提供)：到目前为止所研究的所有正链RNA病毒都重排了细胞膜以促进自身复制。这些重排的一个原因是这些病毒与细胞膜一起复制它们的基因组RNA。在脊髓灰质炎病毒(PV)中，一种具有重要医学意义的正链RNA病毒的模型已经被识别出两种不同类型的水泡。一类是类似于COPII分泌转运囊泡的单膜囊泡，与病毒RNA复制蛋白有关。第二种类型的囊泡是双层的，也与病毒RNA复制蛋白联系在一起，类似于自噬，自噬是一种由细胞内稳态和应激反应途径诱导的细胞器，称为自噬。自噬体样囊泡是由病毒特异性诱导并促进光伏生产的。该项目的长期目标是了解微小核糖核酸病毒引起的细胞膜重排的机制和后果。此应用程序的目标是确定所扮演的角色 在感染过程中通过自噬小体，并确定PV诱导自噬小体形态发生的机制。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，病毒衣壳蛋白的裂解需要囊泡酸化，在自噬小体的情况下，这是与溶酶体融合所必需的，这是从新形成的病毒粒子产生传染性病毒的最后一步。这一建议的中心假设是，PV感染通过形态变化，包括内陷，在富含PIP4的分泌途径衍生的小泡中产生自噬小体，用于RNA复制。新形成的自噬小体的酸化促进了病毒粒子的成熟。核心假设将通过追求两个具体目标来检验。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的蛋白质组。这项研究的基本原理是了解微小核糖核酸病毒如何颠覆通常是促进病毒粒子成熟的抗病原体途径。这将为我们提供所需的信息，以针对病毒生产的这一后期阶段进行治疗。我们的创新方法将确定光伏病毒用来诱导自噬小体的机制，以及它们如何促进传染性病毒的成熟和排出。这项拟议的研究意义重大，因为它将从根本上促进我们对一个医学上重要的病毒家族如何颠覆促进病毒复制的基本细胞途径的理解。这项工作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，并为确定最终可能导致治疗多种病毒疾病的治疗靶点提供第一步。这项工作也是了解现有和未来治疗药物对抗多种病毒疾病的机制的重要一步。

DESCRIPTION (provided by applicant): 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 morphological 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.