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.

项目摘要 病原体已演变为同事的细胞功能，以支持其复制和扩散，同时灭活 限制其生长的先天免疫机制。发现和表征细胞成分 调节发病机理有望揭示治疗传染病的新方法。肠病毒 （EV）包括一个阳性极性的单链RNA病毒的大属，其成员引起A 许多重要的人类疾病，例如脊髓灰质炎，心肌炎，急性松弛性瘫痪和常见 寒冷的。电动汽车对促进复制和引起发病机理的相互作用函数如何不完全 理解。通过坚固，公正的淘汰筛选方法，我们发现了蛋白质 甲基转移酶SETD3是由广泛的人类电动汽车感染所必需的。我们证明了肠病毒 在缺乏setD3的人类细胞中，复制受到严重阻碍，并且该块发生在RNA期间 复制步骤。 setD3是一种单麦甲基肌动蛋白的甲基转移酶，从而控制肌动蛋白功能。 但是，我们发现setD3的甲基转移酶活性在病毒复制中的作用不需要 表明肠病毒对SETD3的缓解独立于肌动蛋白甲基化。我们进一步表明 SETD3与几种肠病毒的病毒非结构2A蛋白相互作用。 setd3对 体内发病机理，因为我们表明setd3 - / - 小鼠完全免受致命的颅内接种 在新生儿模型中使用EV-A71。这些发现表明，setD3控制着大类的发病机理 对人类健康的影响很大的病毒，包括可能导致严重神经系统的非polio电动汽车 症状（EV-A71，EV-D68）。在此应用中，我们将确定setD3在病毒RNA中的特定作用 复制，结构表征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一种新型分子机制 细胞机制可实现基因组扩增。此外，研究setD3在小鼠中的体内作用 模型更忠实地概括肠病毒的传输周期和发病机理，我们将 在免疫能力小鼠中开发并应用EV-A71的口腔感染模型。我们的结果将提供细节 关于宿主因子促进肠病毒RNA复制的分子机制，揭示了非 - 甲基转移酶的催化功能作用于微生物发病机理，并发现setD3在体内作用 促进参与初始复制，全身传播的潜水细胞类型中的EV-A71复制，最终 在神经病发生中。