Regulation of positive-stranded RNA virus infection by host factors of the endomembrane system

项目摘要 正链RNA病毒，包括肠道病毒和黄病毒，是导致严重疾病的原因 世界各地的表现。肠道病毒，如肠道病毒71（EV 71）和柯萨奇病毒B3（CVB），进入 因此，必须首先穿过肠上皮细胞以引起严重的 疾病，包括急性弛缓性麻痹和脑膜炎。相反地，黄病毒通过免疫球蛋白进入宿主。 被感染节肢动物咬伤寨卡病毒（ZIKV）和登革热病毒（DENV）是由蚊子传播的， 亚热带和热带地区。重要的是，已知几种黄病毒引起严重的 神经系统疾病，包括先天性寨卡综合征，这是在2015年巴西 爆发要引起神经系统疾病，大多数病毒必须穿过血脑屏障。因此， 了解调节病毒感染屏障细胞的细胞过程可以促进 新的广泛的抗病毒策略和治疗方法。有趣的是，所有的正链RNA病毒 需要操纵宿主膜以将病毒和宿主因子集中在病毒复制位点。 在感染期间，肠道病毒和黄病毒操纵内膜系统的膜，从而 连接核膜和细胞外空间通过囊泡运输之间的内质网 内质网（ER）和高尔基复合体。因此，我试图更好地理解共享的细胞过程， 与病毒感染期间操纵的内膜系统相关。我直接比较~50 宿主内膜因子对于它们调节肠道病毒（EV 71和CVB）和黄病毒（ZIKV和 DENV）感染。我的初步结果确定了网状吞噬调节蛋白（RETREG）的成员 家族和几种可溶性N-乙基马来酰亚胺敏感相关受体（SNARE）蛋白，包括囊泡 相关膜蛋白7（VAMP 7）。有趣的是，这些蛋白质都与自噬有关， 这是一种细胞应激反应途径， 感染因此，我假设内膜系统的选择成分调节病毒 通过促进自噬过程感染。为了解决病毒操纵的机制， 内膜系统，我已经开发了质粒为基础的报告，将用于监测（1）ER和 高尔基体形态和（2）感染和宿主因子耗竭过程中自噬的诱导。这些新颖 报道分子将用于血脑屏障细胞模型中的长期延时成像。 此外，我还将提供RETREG蛋白在肠道病毒感染过程中的作用机制， 黄病毒感染此外，我将使用血脑屏障表征肠道病毒诱导的自噬 细胞模型和高度相关的原代人肠上皮模型，我们已经建立了我们的 实验室从我们的结果中获得的信息将为共享的细胞过程提供重要的见解 被这些病毒操纵以在细胞屏障中有效地复制。

PROJECT SUMMARY Positive-stranded RNA viruses, including enteroviruses and flaviviruses, are responsible for severe disease manifestations worldwide. Enteroviruses, such as enterovirus 71 (EV71) and coxsackievirus B3 (CVB), enter the host via the fecal-oral route and, therefore, must initially cross the intestinal epithelium to cause severe disease, including acute flaccid paralysis and meningitis. Conversely, flaviviruses enter the host through the bite of an infect arthropod. Zika virus (ZIKV) and dengue virus (DENV) are transmitted by mosquitos in subtropical and tropical regions of the world. Importantly, several flaviviruses are known to cause severe neurological disease, including congenital Zika syndrome, which was first observed during a 2015 Brazilian outbreak. To cause neurological disease, the majority of viruses must cross the blood brain barrier. Thus, an understanding of cellular processes that regulate virus infection of barrier cells can facilitate the development of novel broad-range antiviral strategies and therapeutics. Interestingly, all positive-stranded RNA viruses require the manipulation of host membranes to concentrate viral and host factors at sites of viral replication. During infection enteroviruses and flaviviruses manipulate membranes of the endomembrane system, which connects the nuclear membrane to the extracellular space via vesicle trafficking between the endoplasmic reticulum (ER) and Golgi complex. Thus, I sought to better understand the shared cellular processes associated with the endomembrane system that are manipulated during virus infection. I directly compared ~50 host endomembrane factors for their ability to regulate enterovirus (EV71 and CVB) and flavivirus (ZIKV and DENV) infection. My preliminary results identified members of the reticulophagy regulator (RETREG) protein family and several soluble N-ethylmaleimide-sensitive associated receptor (SNARE) proteins, including vesicle associated membrane protein 7 (VAMP7). Interestingly, these proteins are all associated with autophagy, which is a cellular stress response pathway that is manipulated by enteroviruses and flaviviruses during infection. Thus, I hypothesize that select components of the endomembrane system regulate virus infection through facilitation of autophagic processes. To address the mechanisms of viral manipulation of the endomembrane system, I have developed plasmid-based reporters that will be used to monitor (1) ER and Golgi morphology and (2) induction of autophagy during infection and host factor depletion. These novel reporters will be used for long-term time-lapse imaging in a cellular model of the blood brain barrier. Furthermore, I will provide mechanistic insight into the role of RETREG proteins during enterovirus and flavivirus infection. Additionally, I will characterize enterovirus-induced autophagy using a blood brain barrier cell model and a highly relevant primary human intestinal epithelium model, that we have established in our laboratory. Information derived from our results will provide significant insight into the shared cellular processes manipulated by these viruses to efficiently replicate in cellular barriers.