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.

项目概要 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 世界范围内的表现。肠道病毒，例如肠道病毒 71 (EV71) 和柯萨奇病毒 B3 (CVB)，进入 宿主通过粪口途径传播，因此必须首先穿过肠上皮才能引起严重的感染 疾病，包括急性弛缓性麻痹和脑膜炎。相反，黄病毒通过 被感染节肢动物咬伤。寨卡病毒（ZIKV）和登革热病毒（DENV）通过蚊子传播 世界亚热带和热带地区。重要的是，已知几种黄病毒会导致严重的 神经系统疾病，包括先天性寨卡综合症，该病首次在 2015 年巴西一次观察到 暴发。为了引起神经系统疾病，大多数病毒必须穿过血脑屏障。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育 新型广泛的抗病毒策略和治疗方法。有趣的是，所有正链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。 在感染过程中，肠道病毒和黄病毒会操纵内膜系统的膜，从而 通过内质膜之间的囊泡运输将核膜与细胞外空间连接起来 网状结构（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有关。我直接比较了~50 宿主内膜因子具有调节肠道病毒（EV71 和 CVB）和黄病毒（ZIKV 和 登革热病毒）感染。我的初步结果确定了网状噬菌调节蛋白 (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 (VAMP7)。有趣的是，这些蛋白质都与自噬有关， 这是一种由肠道病毒和黄病毒在细胞应激过程中操纵的细胞应激反应途径 感染。因此，我假设内膜系统的选择成分调节病毒 通过促进自噬过程进行感染。解决病毒操纵的机制 在内膜系统中，我开发了基于质粒的报告基因，将用于监测 (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 记者将用于血脑屏障细胞模型的长期延时成像。 此外，我还将提供有关 RETREG 蛋白在肠道病毒和病毒感染过程中的作用的机制见解。 黄病毒感染。此外，我将使用血脑屏障来表征肠道病毒诱导的自噬 我们在我们的研究中建立了细胞模型和高度相关的原代人肠上皮模型 实验室。从我们的结果中获得的信息将为共享细胞过程提供重要的见解 由这些病毒操纵，在细胞屏障中有效复制。