Viral use and mimicry of autophagy pathway and components

这项工作的长期目标是确定 自噬途径促进病毒复制和传播，并确定发展的目标 无毒的抗病毒化合物。典型的自噬(自噬)途径，由 最近的诺贝尔奖获得者大滨义典被营养剥夺刺激并达到顶峰 在细胞质内容物的降解，从而滋养饥饿的细胞。为此， 戏剧性的细胞事件，如大量的脂质清除，新的细胞膜的生长 隔室，通过凹面和凸面膜曲率对细胞质的包裹； 与溶酶体的融合在几分钟内完成。大量的微生物，包括 脊髓灰质炎病毒和登革热病毒，已经进化到颠覆细胞自噬的片段 途径或其单个成分，以促进其感染周期。精准的个体 自噬途径中的基因和蛋白质的贡献尚未被确定为 这些病毒。在这里提出的实验中，CRISPR/Cas9技术将被用于 产生匹配的细胞系，用于个别步骤，如自噬启动、扩增 以及限制膜的曲率，以及将关键的融合蛋白LC3放置在 新生自噬小体的表面。测试单个基因缺失对基因突变的影响 脊髓灰质炎病毒和登革热病毒的进入、翻译、RNA复制和形态发生将揭示 自噬途径的哪些组成部分被这两个具有代表性的阳性- 链RNA病毒。 根据初步结果，中心假设是脊髓灰质炎病毒和登革热病毒使用 不同目的的细胞自噬途径的不同步骤和组成部分：RNA 脊髓灰质炎病毒的复制和非裂解传播，登革热病毒的病毒粒子组装和成熟。 拟议研究的基本原理是，一旦这些成分和机制 如果确定了颠覆性事件，则将抗病毒化合物作为特定目标是可行的 分子和过程。毫不奇怪，考虑到这些病毒对自噬的依赖 机制，短期禁食大大加剧了这两种动物模型的发病 脊髓灰质炎病毒和登革热病毒感染。这种恶化也可以观察到作为对 已知的刺激自噬的常用药物。将使用严格的基因测试 为了确定这种增加的发病机制是否真的依赖于细胞自噬 路径。

The long-term goals of this work are to determine the mechanisms by which components of the autophagy pathway promote viral replication and spread and to identify targets for development of non-toxic antiviral compounds. The canonical autophagy (self-eating) pathway, described by recent Nobel Prize winner Yoshinori Ohsumi, is stimulated by nutrient deprivation and culminates in the degradation of cytoplasmic contents, thus nourishing the starving cell. To this end, dramatic cellular events such as massive lipid scavenging, growth of novel membranous compartments, entrapment of cytoplasm by both concave and convex membrane curvature, and fusion with lysosomes are accomplished within minutes. Numerous microbes, including poliovirus and Dengue virus, have evolved to subvert segments of the cellular autophagy pathway or its individual constituents to promote their infectious cycles. The precise individual contributions of genes and proteins from autophagy pathways have not been identified for any of these viruses. In the experiments proposed here, CRISPR/Cas9 technology will be used to generate matched cell lines oblated for individual steps such as autophagy initiation, expansion and curvature of the limiting membrane, and placement of crucial fusion protein LC3 on the surface of the nascent autophagosome. Testing the effects of individual gene deletions on the entry, translation, RNA replication and morphogenesis of poliovirus and dengue virus will reveal which components of the autophagy pathway are usurped by these two representative positive- strand RNA viruses. The central hypothesis, based on preliminary results, is that poliovirus and dengue virus use distinct steps and components of the cellular autophagy pathway for disparate purposes: RNA replication and nonlytic spread for poliovirus, virion assembly and maturation for dengue virus. The rationale of the proposed research is that, once the components and mechanisms of these subversive events are identified, it will be feasible to target antiviral compounds to particular molecules and processes. Not surprisingly, given the dependence of these viruses on autophagy machinery, short periods of fasting greatly exacerbate pathogenesis in mouse models of both poliovirus and dengue virus infection. This exacerbation is also observed in response to commonly used medications known to stimulate autophagy. Rigorous genetic tests will be used to determine whether this increased pathogenesis is indeed dependent on the cellular autophagy pathway.