Subversion of Autophagy Pathway and Constituents by RNA viruses

描述(申请人提供)：感染真核细胞的正链RNA病毒都会重新排列细胞内膜，并在拓扑学上的细胞质表面复制它们的RNA基因组。其中几种病毒，包括脊髓灰质炎病毒和登革病毒，模仿或颠覆细胞自噬的途径，形成细胞内的双膜室。建议进行实验，以探索这样的假设：对于脊髓灰质炎病毒，因为双膜拓扑结构可以允许病毒和其他细胞质成分非裂解地退出；对于登革病毒，因为细胞自噬的途径或产物是传染性病毒粒子组装所必需的，所以这些膜有助于病毒从受感染的细胞中退出。为了研究双膜囊泡在脊髓灰质炎病毒感染组织培养细胞和小鼠中的作用，采用活细胞时间推移显微镜、药物扰动以及病毒和小鼠遗传学来区分裂解和非裂解以及凋亡性和非凋亡性传播。这些原理也被应用于甲型肝炎病毒的研究，甲型肝炎病毒是一种微小核糖核酸病毒，其通过肝脏传播而没有明显的细胞溶解的能力仍然是个谜，可以通过感染期间诱导的双膜泡的拓扑结构来解释。一种单一的脊髓灰质炎病毒蛋白3AB被发现足以从单膜脂质体产生双膜，并对这种机制可能与细胞自噬蛋白相同的假设进行了测试。通过确定当细胞自噬被抑制时产生的非传染性病毒粒子中的生化缺陷，将从机制上探索登革病毒感染性对正常细胞自噬途径的戏剧性依赖。尽管已知自噬是先天免疫反应的一部分，但自噬的多种细胞过程为抗病毒控制提供了一个新的靶点。

DESCRIPTION (provided by applicant): Positive-strand RNA viruses that infect eukaryotic cells all rearrange intracellular membranes and replicate their RNA genomes on the topologically cytoplasmic surfaces. Several of these viruses, including poliovirus and Dengue virus, mimic or subvert the cellular pathway of autophagy to form double-membraned intracellular compartments. Experiments are proposed to explore the hypothesis that these membranes facilitate viral exit from infected cells: for poliovirus, because the double-membraned topology can allow non-lytic exit of virus and other cytoplasmic constituents, and for Dengue virus, because the pathway or products of cellular autophagy are required for infectious virion assembly. To investigate the contribution of double-membraned vesicles during poliovirus infection of tissue-culture cells and mice, live-cell time-lapse microscopy, pharmaceutical perturbations, and viral and mouse genetics are employed to distinguish between lytic and non-lytic, and between apoptotic and non-apoptotic spread. These principles are also applied to the study of hepatitis A virus, a picornavirus whose ability to spread through the liver with no apparent cell lysis remains mysterious and may be explained by the topology of the double-membraned vesicles induced during infection. A single poliovirus protein, 3AB, was found to be sufficient to create double-membraned from single-membraned liposomes, and the hypothesis that this mechanism might be shared with cellular autophagy proteins tested. The novel and dramatic dependence of Dengue virus infectivity on a functioning cellular autophagy pathway will be explored mechanistically by determining the biochemical defect in the non-infectious virions produced when cellular autophagy is inhibited. The versatile cellular process of autophagy, although known to be part of the innate immune response, provides a new target for antiviral control.