Subversion of Autophagy Pathway and Constituents by RNA viruses

描述（由申请人提供）：感染真核细胞的正链RNA病毒都在胞膜内重新排列，并在细胞质表面复制其RNA基因组。其中一些病毒，包括脊髓灰质炎病毒和登革热病毒，模仿或破坏自噬的细胞途径，形成双膜细胞内区室。研究人员提出了实验来探索这些膜有助于病毒从感染细胞中退出的假设：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溶解性退出，对于登革热病毒，因为细胞自噬的途径或产物是感染性病毒粒子组装所必需的。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过程中的作用，利用活细胞延时显微镜、药物扰动、病毒和小鼠遗传学来区分裂解和非裂解，以及凋亡和非凋亡传播。这些原则也适用于甲型肝炎病毒的研究，这是一种小核糖核酸病毒，其通过肝脏传播而没有明显的细胞裂解的能力仍然是神秘的，可能是感染期间诱导的双膜囊泡的拓扑结构。发现单一脊髓灰质炎病毒蛋白3AB足以由单层脂质体形成双层膜，并验证了这种机制可能与细胞自噬蛋白共享的假设。通过确定细胞自噬被抑制时产生的非感染性病毒粒子的生化缺陷，将从机制上探索登革病毒传染性对功能性细胞自噬途径的新颖和戏剧性依赖。自噬的多用途细胞过程，虽然已知是先天免疫反应的一部分，提供了抗病毒控制的新靶点。

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.