Role of microRNAs in Coronavirus-induced lethal encephalitis

描述（由申请人提供）：微小RNA（miRNA）是通过结合靶mRNA的3 'UTR，通过翻译抑制和/或mRNA降解来调节基因表达的小RNA。越来越多的证据表明，miRNAs在基本细胞过程、免疫系统的调节以及许多疾病的发生和发展中起着重要作用。尽管miRNA在病毒-宿主相互作用中的作用正在显现，但几乎没有证据表明病毒对细胞miRNA的调节实际上影响体内神经炎症反应。揭示miRNAs在人类病毒性脑炎中的作用具有挑战性。病毒性脑炎动物模型的研究是我们理解病毒如何驱动CNS疾病的关键。病毒性脑炎严重程度的差异可能反映了病毒刺激CNS内先天免疫反应的能力差异。冠状病毒（CoV）是有包膜的、正义的、单链的、多腺苷酸化的RNA病毒。不同的小鼠CoV（小鼠肝炎病毒，MHV）分离株诱导与急性和慢性脱髓鞘相关的急性致死性脑炎和脑脊髓炎。在这里，我们将比较两种在神经毒力方面明显不同的菌株：MHV-JHM，一种产生致命脑炎的高度神经毒力菌株;和MHV-A59，一种诱导急性脑膜脑炎和慢性脑膜脑炎的轻度神经毒力菌株。在初步研究中，我们使用了miRNA微阵列分析和实时RT-PCR验证，以提供证据表明，在体外感染MHV-A59和MHV-JHM后，巨噬细胞中选定的细胞miRNA的差异表达。小鼠冠状病毒感染后巨噬细胞miRNAs谱的变化是重叠的，但不同，并且在缺乏I型IFN，IFN-β，和促炎细胞因子分泌。我们的数据表明，MHV感染可以改变细胞miRNA表达的模式。我们已经鉴定了两种被高度神经毒性的MHV-JHM显著上调的miRNA（但不是被MHV-A59）。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-1 Ra）的3' UTR，IL-1 Ra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，冠状病毒诱导不同的中枢神经系统疾病的结果，促进不同的miRNA表达谱在巨噬细胞和小胶质细胞，和病毒特异性的变化，miRNA表达的脑炎结果的差异，通过调节炎症反应。我们建议：1）系统地鉴定与感染脑中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（在初步研究和特异性目标1中鉴定）在IL-1 Ra和IFN？通过巨噬细胞和神经胶质细胞的离体诱导，以及在体内使用曲马多米尔-慢病毒载体。总的来说，在拟议的研究中存在显著的转化潜力，因为它们将促进预防和/或治疗病毒病因学的神经炎性疾病的治疗方法的开发。

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.