Role of microRNAs in Coronavirus-induced lethal encephalitis

描述(申请人提供)：microRNAs(MiRNAs)是一种通过翻译抑制和/或通过与目标mRNAs的3‘UTR结合来调节基因表达的小RNA。越来越多的证据表明，miRNAs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。虽然miRNAs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miRNAs的调节实际上影响体内的神经炎性反应。解开miRNAs在人类病毒性脑炎中的作用是具有挑战性的。对病毒性脑炎动物模型的研究是我们理解病毒如何在中枢神经系统中驱动疾病的关键。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)是一种包膜、阳性、单链、多腺化的RNA病毒。不同的小鼠冠状病毒(小鼠肝炎病毒，MHV)分离株可引起急性致死性脑炎和与急性和慢性脱髓鞘相关的脑脊髓炎。在这里，我们将比较两个在神经毒力方面有显著差异的毒株：MHV-JHM，一种高度神经毒力的毒株，会导致致命性脑炎；以及MHV-A59，一种轻度神经毒力的毒株，会导致急性脑膜脑炎和慢性脑炎。在初步研究中，我们使用miRNA微阵列分析和实时RT-PCR验证，以提供体外感染MHV-A59和MHV-JHM时选定的细胞miRNAs在巨噬细胞中差异表达的证据。小鼠冠状病毒感染后巨噬细胞miRNAs谱的变化是重叠但明显的，并且在感染后早期观察到没有I型IFN、干扰素-β和促炎细胞因子的分泌。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miRNAs，它们被高度神经毒力的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miRNAs潜在地针对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的细胞因子，以及干扰素？我们假设，CoV诱导不同的CNS疾病结局促进巨噬细胞和小胶质细胞中不同的miRNAs表达谱，并且病毒特异性的miRNAs表达变化通过调节炎症反应而导致脑炎结局的差异。我们建议：1)系统地识别与感染脑中致死性脑炎进展相关的miRNAs和mRNAs调控网络；2)确定那些JHM上调的miRNAs(在初步研究中确定并在特定的目标1中)在IL-1ra和干扰素中的作用？巨噬细胞和神经胶质细胞的体外诱导，以及体内使用慢病毒载体的诱导。总体而言，拟议的研究具有很大的翻译潜力，因为它们将促进开发治疗方法，以预防和/或治疗病毒病原学的神经炎性疾病。

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.