Host determinants of enterovirus RNA replication and in vivo neuropathogenesis

项目总结 病原体已经进化到利用细胞功能来支持它们的复制和传播，同时灭活 限制它们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 (EVS)包括一大类正极性的单链RNA病毒，其成员引起 一些重要的人类疾病，如脊髓灰质炎、心肌炎、急性迟缓性麻痹和常见的 冷的。EVS如何利用细胞功能促进复制并导致致病机制尚不完全 明白了。通过稳健、无偏见的基因敲除筛选方法，我们发现这种蛋白质 甲基化转移酶SETD3是感染多种人类肠道病毒所必需的。我们证明了肠道病毒 在缺乏SETD3的人类细胞中，复制受到严重阻碍，并且阻断发生在RNA 复制步骤。SETD3是一种甲基化转移酶，使肌动蛋白单甲基化，从而调节肌动蛋白的功能。 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所必需的 这表明肠道病毒对SETD3的依赖与肌动蛋白甲基化无关。我们进一步表明， SETD3与几种肠道病毒的病毒非结构蛋白2A相互作用。SETD3对于 体内发病机制，因为我们表明，Setd3-/-小鼠完全免受致命的颅内接种 EV-A71病毒在新生儿模型中。这些发现表明，SETD3控制了一大类人的发病 对人类健康有重大影响的病毒，包括可能导致严重神经疾病的非脊髓灰质炎EV 症状(EV-A71、EV-D68)。在这个应用中，我们将确定SETD3在病毒RNA中的特定作用 复制，从结构上表征SETD3和2A之间的相互作用，并检验假设 SETD3‘S与病毒非结构蛋白相互作用是EV劫持的一种新的分子机制 使基因组扩增成为可能的细胞机制。此外，为了研究SETD3在小鼠体内的作用 模型更真实地概括了肠道病毒的传播周期和发病机制，我们将 建立和应用免疫活性小鼠EV-A71口服感染模型。我们的结果将提供详细信息 关于宿主因素促进肠道病毒RNA复制的分子机制，揭示了非 甲基转移酶的催化作用在微生物发病中的作用和揭示SETD3在体内的作用 促进EV-A71在不同细胞类型中的复制，参与最初的复制、全身传播和最终 在神经发病机制方面。

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.