Role of microRNAs in Coronavirus-induced lethal encephalitis

描述（由申请人提供）：MicroRNAs （miRNAs）是一种小rna，通过与目标mRNA的3'UTR结合，通过翻译抑制和/或mRNA降解来调节基因表达。越来越多的证据表明，mirna在基本的细胞过程、免疫系统的调节以及许多疾病的发生和发展中发挥着重要作用。尽管mirna在病毒-宿主相互作用中的作用正在出现，但很少有证据表明病毒对细胞mirna的调节实际上会影响体内的神经炎症反应。揭示mirna在人类病毒性脑炎中的作用是具有挑战性的。病毒性脑炎动物模型的研究是我们理解病毒如何驱动中枢神经系统疾病的关键。病毒性脑炎严重程度的差异可能反映了病毒在中枢神经系统内刺激先天免疫反应的不同能力。冠状病毒（cov）是包膜的、正义的、单链的、多腺苷化的RNA病毒。不同的小鼠冠状病毒（小鼠肝炎病毒，MHV）分离物可诱导急性致死性脑炎和脑脊髓炎，并伴有急性和慢性脱髓鞘。在这里，我们将比较两种在神经毒力上明显不同的菌株：MHV-JHM，一种产生致死性脑炎的高度神经毒力菌株；MHV-A59是一种轻度神经毒性毒株，可诱发急性脑膜脑炎和慢性脑炎。在初步研究中，我们利用miRNA微阵列分析和实时RT-PCR验证，提供了MHV-A59和MHV-JHM体外感染巨噬细胞中选定细胞miRNA表达差异的证据。小鼠冠状病毒感染后巨噬细胞miRNAs谱的变化重叠但不同，并且在感染后早期在缺乏I型IFN， IFN-？促炎细胞因子分泌。我们的数据表明MHV感染可以改变细胞miRNA的表达模式。我们已经鉴定出两个mirna被高神经毒性MHV-JHM显著上调（但不被MHV-A59上调）。有趣的是，我们还发现这些jhm上调的mirna可能靶向IL-1受体拮抗剂（IL-1Ra）的3' UTR， IL-1Ra是一种参与神经保护和神经发病机制的细胞因子，以及IFN？我们假设，冠状病毒诱导的不同中枢神经系统疾病结果促进巨噬细胞和小胶质细胞中不同的miRNAs表达谱，并且miRNAs表达的病毒特异性变化通过调节炎症反应导致脑炎结果的差异。我们建议：1)系统地鉴定感染脑中与致死性脑炎进展相关的mirna和mrna调控网络；2)确定那些jhm上调的mirna（在初步研究和Specific Aim 1中发现）在IL-1Ra和IFN中的作用？巨噬细胞和胶质细胞体外诱导，以及体内使用安他哥米慢病毒载体诱导。总的来说，拟议的研究具有重要的转化潜力，因为它们将促进治疗方法的发展，以预防和/或治疗病毒病因的神经炎症性疾病。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are small RNAs that regulate gene expression by translational repression and/or mRNA degradation through binding to the 3'UTR of target mRNAs. There is increasing evidence indicating that miRNAs play a major role in fundamental cellular processes, the regulation of the immune system, as well as in the onset and progression of many diseases. Although the role of miRNAs in virus-host interactions is emerging, there is little evidence that viral modulation of cellular miRNAs actually impacts neuro-inflammatory responses in vivo. Unraveling the roles of miRNAs in viral encephalitis in humans is challenging. The study of animal models of viral encephalitis is key to our understanding of how viruses drive disease in the CNS. Differences in the severity of viral encephalitis may reflect the differential ability of viruses to stimulate innate immune responses within the CNS. Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded, polyadenylated RNA viruses. Different murine CoVs (Mouse Hepatitis Virus, MHV) isolates induce acute lethal encephalitis and encephalomyelitis associated with acute and chronic demyelination. Here, we will compare two strains differing markedly in their neurovirulence: MHV-JHM, a highly neurovirulent strain that produces lethal encephalitis; and MHV-A59, a mildly neurovirulent strain that induces acute meningoencephalitis and chronic. In preliminary studies, we have used miRNA microarray analysis and validation by real-time RT-PCR to provide evidence of the differential expression of selected cellular miRNAs in macrophages upon in vitro infection with MHV-A59 and MHV-JHM. Changes in the macrophage miRNAs profiles after murine CoVs infection were overlapping but distinct, and were observed early after infection in the absence of type I IFNs, IFN-?, and pro-inflammatory cytokine secretion. Our data suggest that MHV infection can modify the pattern of cellular miRNA expression. We have identified two miRNAs that are significantly up-regulated by the highly neurovirulent MHV-JHM (but not by MHV-A59). Interestingly, we also found that these JHM-up-regulated miRNAs potentially target the 3' UTR of IL-1 receptor antagonist (IL-1Ra), a cytokine involved in both neuroprotection and neuropathogenesis, as well as IFN?. We hypothesize that CoVs inducing different CNS disease outcome promote distinct miRNAs expression profiles in macrophages and microglia, and that virus specific changes in miRNAs expression contribute to the differences in encephalitis outcome by modulation of the inflammatory response. We propose to: 1) systematically identify miRNAs and mRNAs regulatory networks that correlate with lethal encephalitis progression in the infected brain; 2) Define the role of those JHM-up-regulated miRNAs (identified in preliminary studies and in Specific Aim 1) in IL-1Ra and IFN? induction by macrophages and glial cells ex vivo, as well as in vivo using antagomir-lentivirus vectors. Overall, there is significant translational potential in the proposed investigations since they will facilitate the development of therapeutic approaches to prevent and/or treat neuro-inflammatory diseases of viral etiology. PUBLIC HEALTH RELEVANCE: The relative contribution of microRNAs to virus-host interactions in the context of the CNS, and to neuroinflammatory responses and/or neuropathogenesis is poorly understood. Our experimental model will provide a distinctive opportunity to define the role of selected microRNAs in neuro-inflammatory responses that result in viral-induced lethal encephalitis, and could potentially lead to novel therapeutic approaches to prevent and/or treat acute neuro-inflammatory diseases of viral etiology.