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

内膜系统宿主因子对正链RNA病毒感染的调节

• 批准号: 9720399
• 负责人: Nicholas J Lennemann
• 金额: $ 16.2万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9720399
• 关键词: Acute; Address; Antiviral Agents; Arthropods; Autophagocytosis; Biological; Bite; Blood - brain barrier anatomy; Cell Nucleus; Cell model; Cell physiology; Cells; Cellular Stress; Cellular Stress Response; Coxsackie B Viruses; Coxsackie Viruses; Culicidae; Data; Dengue Infection; Dengue Virus; Development; Disease; Disease Outbreaks; Distant; Encephalitis; Endoplasmic Reticulum; Endothelial Cells; Enterovirus; Enterovirus 71; Ethylmaleimide; Event; Extracellular Space; Family; Flavivirus; Flavivirus Infections; Foundations; Genome; Golgi Apparatus; Human; Image; Infection; Integration Host Factors; Intracellular Membranes; Invaded; Laboratories; Life Cycle Stages; Lipids; Mammalian Cell; Mammals; Membrane; Meningitis; Microcephaly; Modeling; Monitor; Morphology; Movement; N-ethylmaleimide-sensitive protein; Neuraxis; Nuclear Envelope; Oral; Organelles; Orthologous Gene; Paralysed; Pathway interactions; Physiological; Plasmids; Process; Protein Family; Protein Glycosylation; Proteins; Public Health; RNA Virus Infections; RNA Viruses; Regulation; Reporter; Research; Role; Route; Site; System; Therapeutic; Time; Vesicle; Viral; Viral Hemorrhagic Fevers; Virus; Virus Diseases; Virus Replication; Yeasts; Zika Virus; base; career; cell type; combat; congenital zika syndrome; global health; human stem cells; imaging approach; innovation; insight; intestinal epithelium; lipid transport; live cell imaging; member; neonate; nervous system disorder; neurotropic virus; new therapeutic target; novel; receptor; trafficking; treatment strategy; vesicle-associated membrane protein; virus host interaction

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.

项目摘要 阳性链RNA病毒，包括肠病毒和黄素病毒，导致严重疾病 全球表现。肠病毒，例如肠病毒71（EV71）和Coxsackievivirus B3（CVB），请输入 通过粪便途径的宿主，因此最初必须越过肠上皮才能引起严重 疾病，包括急性松弛麻痹和脑膜炎。相反，黄病毒通过 咬伤感染节肢动物。 Zika病毒（ZIKV）和登革热病毒（DENV）由蚊子传播 世界亚热带和热带地区。重要的是，已知几种黄素病毒会导致严重 神经疾病，包括先天性寨卡综合症，该综合征是在2015年巴西人首次观察到的 暴发。为了引起神经疾病，大多数病毒必须越过血脑屏障。因此，一个 了解调节屏障细胞病毒感染的细胞过程可以促进发展 新型的宽范围抗病毒策略和治疗剂。有趣的是，所有阳性RNA病毒 需要操纵宿主膜以在病毒复制部位浓缩病毒和宿主因子。 在感染期间，肠病毒和黄病毒操纵膜的膜，该膜系统的膜，该系统 通过内质之间的囊泡运输将核膜连接到细胞外空间 网状（ER）和高尔基体复合物。因此，我试图更好地理解共享的蜂窝过程 与在病毒感染过程中操纵的内膜系统相关。我直接比较了〜50 宿主内膜因素的调节能力（EV71和CVB）和Flavivivirus（ZIKV和 DENV）感染。我的初步结果确定了网状调节剂（Retreg）蛋白的成员 家族和几种可溶性N-乙基米酰胺敏感的相关受体（SNARE）蛋白，包括囊泡 相关的膜蛋白7（VAMP7）。有趣的是，这些蛋白质都与自噬有关， 这是一种细胞应力反应途径，在肠病毒和黄病毒中操纵 感染。因此，我假设内膜系统的某些成分调节病毒 通过促进自噬过程感染。解决病毒操纵的机制 我已经开发了基于质粒的记者的内膜系统，这些记者将用于监视（1）ER和 高尔基形态和（2）感染和宿主因子耗竭期间自噬的诱导。这些小说 记者将在血脑屏障的细胞模型中用于长期延时成像。 此外，我将提供机械洞察力的洞察力 黄病毒感染。此外，我将使用血脑屏障来表征肠病毒诱导的自噬 细胞模型和高度相关的原代人肠上皮模型，我们已经在我们的 实验室。从我们的结果中得出的信息将为共享蜂窝过程提供重大洞察力 这些病毒操纵以在细胞屏障中有效复制。