Host determinants of enterovirus RNA replication and in vivo neuropathogenesis

肠道病毒RNA复制和体内神经发病机制的宿主决定因素

• 批准号: 10209690
• 负责人: Jan E Carette
• 金额: $ 54.72万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10209690
• 关键词: Actins; Acute; Address; Biology; Cell physiology; Cells; Child; Common Cold; Communicable Diseases; Complex; Coxsackie Viruses; Cryoelectron Microscopy; Crystallography; Data; Enteral; Enterovirus; Enterovirus 68; Enterovirus Infections; Family Picornaviridae; Genes; Genome; Goals; Growth; Health; Human; Human poliovirus; Immunity; Immunocompetent; In Vitro; Infection; Injections; Integration Host Factors; Invaded; Knock-out; Knowledge; Medical; Methods; Methylation; Methyltransferase; Modeling; Molecular; Mus; Mutation; Myocarditis; Neurologic Symptoms; Neuropathogenesis; Oral; Paralysed; Pathogenesis; Play; Poliomyelitis; Polyproteins; Protein Methylation; Protein Methyltransferases; Proteins; Proteomics; RNA Viruses; RNA replication; Regulation; Rhinovirus; Rhinovirus infection; Role; Site; Structure; Testing; Therapeutic Intervention; Tissues; Translations; Veterinary Medicine; Viral; Viral Nonstructural Proteins; Viral Pathogenesis; Viremia; Virus; Virus Diseases; Virus Replication; acute flaccid myelitis; asthma exacerbation; cell type; human disease; in vivo; innate immune mechanisms; insight; member; microbial; mouse model; neonate; new therapeutic target; novel; novel strategies; oral infection; pathogen; relating to nervous system; screening; transmission process; viral RNA; virus host interaction

PROJECT SUMMARY Pathogens have evolved to co-opt cellular functions to support their replication and spread while inactivating innate immune mechanisms that restrict their growth. Discovery and characterization of cellular components that regulate pathogenesis hold promise for revealing new approaches to treat infectious diseases. Enteroviruses (EVs) comprise a large genus of single-stranded RNA viruses of positive polarity whose members cause a number of important human diseases such as poliomyelitis, myocarditis, acute flaccid paralysis and the common cold. How EVs co-opt cellular functions to promote replication and cause pathogenesis is incompletely understood. Through robust, unbiased knockout screening approaches, we have discovered that the protein methyltransferase SETD3 is required for infection by a broad range of human EVs. We showed that enterovirus replication is severely hampered in human cells lacking SETD3 and that the block occurs during the RNA replication step. SETD3 is a methyltransferase that mono-methylates actin, thereby regulating actin function. However, we found that methyltransferase activity of SETD3 is not required for its role in viral replication indicating that enteroviruses’ reliance on SETD3 is independent of actin methylation. We further showed that SETD3 interacts with the viral nonstructural 2A protein of several enteroviruses. SETD3 is critically important for in vivo pathogenesis as we show that Setd3-/- mice are completely protected from lethal intracranial inoculation with EV-A71 in a neonate model. These findings demonstrate that SETD3 controls pathogenesis for a large class of viruses with a strong impact on human health including non-polio EVs that can cause severe neurological symptoms (EV-A71, EV-D68). In this application, we will determine the specific role of SETD3 in viral RNA replication, structurally characterize the interaction between SETD3 and 2A, and test the hypothesis that SETD3’s interactions with viral nonstructural proteins are a novel molecular mechanism by which EVs hijack cellular machinery to enable genome amplification. Furthermore, to study the in vivo role of SETD3 in a mouse model that recapitulates more faithfully the transmission cycle and pathogenesis of enteric enteroviruses, we will develop and apply an oral infection model of EV-A71 in immune-competent mice. Our results will provide details on the molecular mechanisms by which host factors promote enteroviral RNA replication, reveal how non- catalytic functions of methyltransferases act in microbial pathogenesis and uncover the in vivo role of SETD3 in promoting EV-A71 replication in diverse cell types involved in initial replication, systemic spread and ultimately in neuropathogenesis.

项目概要 病原体已经进化到可以选择细胞功能来支持其复制和传播，同时灭活 限制其生长的先天免疫机制。细胞成分的发现和表征 调节发病机制有望揭示治疗传染病的新方法。肠道病毒 （EV）由一大类正极性单链 RNA 病毒组成，其成员会引起 一些重要的人类疾病，如脊髓灰质炎、心肌炎、急性弛缓性麻痹和常见的 寒冷的。 EV如何利用细胞功能来促进复制并引起发病机制尚不完全 明白了。通过稳健、公正的敲除筛选方法，我们发现该蛋白质 广泛的人类 EV 感染需要甲基转移酶 SETD3。我们证明肠道病毒 在缺乏 SETD3 的人类细胞中，复制受到严重阻碍，并且这种阻断发生在 RNA 复制过程中。 复制步骤。 SETD3 是一种甲基转移酶，可单甲基化肌动蛋白，从而调节肌动蛋白功能。 然而，我们发现 SETD3 的甲基转移酶活性并不是其在病毒复制中发挥作用所必需的 表明肠道病毒对 SETD3 的依赖与肌动蛋白甲基化无关。我们进一步表明 SETD3 与多种肠道病毒的病毒非结构 2A 蛋白相互作用。 SETD3 对于以下方面至关重要 体内发病机制，我们表明 Setd3-/- 小鼠完全免受致命性颅内接种的影响 新生儿模型中使用 EV-A71。这些发现表明 SETD3 控制一大类疾病的发病机制 对人类健康产生严重影响的病毒，包括可导致严重神经系统疾病的非脊髓灰质炎病毒 症状（EV-A71、EV-D68）。在此应用中，我们将确定SETD3在病毒RNA中的具体作用 复制，从结构上表征 SETD3 和 2A 之间的相互作用，并检验以下假设： SETD3 与病毒非结构蛋白的相互作用是 EV 劫持的一种新颖的分子机制 实现基因组扩增的细胞机制。此外，研究 SETD3 在小鼠体内的作用 模型更忠实地概括了肠道病毒的传播周期和发病机制，我们将 在免疫功能正常的小鼠中开发并应用 EV-A71 口腔感染模型。我们的结果将提供详细信息 宿主因子促进肠道病毒 RNA 复制的分子机制，揭示非 甲基转移酶的催化功能在微生物发病机制中发挥作用，并揭示了SETD3在体内的作用 促进 EV-A71 在不同细胞类型中的复制，参与初始复制、系统性传播和最终 在神经发病机制中。