Viral use and mimicry of autophagy pathway and components

这项工作的长期目标是确定机制， 自噬途径促进病毒复制和传播，并确定发展目标 无毒的抗病毒化合物。经典的自噬（自食）途径，描述为 最近的诺贝尔奖赢家大隅良典，是由营养缺乏刺激和高潮 细胞质内容物的降解，从而滋养饥饿的细胞。为此目的， 戏剧性的细胞事件，如大量的脂质清除，新的膜 隔室，凹和凸膜曲率对细胞质的截留，以及 与溶酶体的融合在几分钟内完成。许多微生物，包括 脊髓灰质炎病毒和登革热病毒，已经进化到破坏细胞自噬的片段 途径或其个别成分，以促进其感染周期。精确的个体 来自自噬途径的基因和蛋白质的贡献还没有被确定为任何 这些病毒。在本文提出的实验中，CRISPR/Cas9技术将用于 产生匹配的细胞系，用于单个步骤，例如自噬起始、扩增 和曲率的限制膜，并放置关键的融合蛋白LC 3上， 新生自噬体的表面。测试单个基因缺失对 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 自噬途径的哪些成分被这两种代表性的阳性细胞所取代， 链RNA病毒。 基于初步结果的中心假设是，脊髓灰质炎病毒和登革热病毒使用 细胞自噬途径的不同步骤和组分用于不同目的：RNA 脊髓灰质炎病毒的复制和非裂解性传播，登革病毒的病毒体组装和成熟。 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，则将抗病毒化合物靶向特定的 分子和过程。鉴于这些病毒对自噬的依赖性， 机械，短期禁食大大加剧了小鼠模型的发病机制， 脊髓灰质炎病毒和登革热病毒感染。这种恶化也被观察到， 通常使用的药物已知刺激自噬。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确实依赖于细胞自噬， 通路

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.