水疱性口炎病毒mRNA Guanine-N-7和Ribose 2'-O位点甲基化修饰在先天免疫逃逸中的分子标签机制

The induction of antiviral responses in hosts and the evasion of these responses by viruses represent major selective forces for both types of organisms. Among the hallmarks of this battle were the acquisition by eukaryotic organisms of sensors of viral RNA products that trigger secretion of antiviral cytokines, such as type I interferon, and the subsequent acquisition by viruses of mechanisms to evade recognition by these sensors. The 5' cap structures of higher eukaryote mRNAs have guanine-N-7 and ribose 2'-O-methylation. Likewise, Vesicular stomatitis virus (VSV) which replicates in the cytoplasm of eukaryotes have evolved guanine-N-7 and ribose 2'-O methyltransferases(MTase) to autonomously modify its mRNAs.The role(s) of guanine-N-7 and ribose 2'-O methylation have proved more elusive in innate immunity. Previous research we found that VSV mutants that lack MTase activity was attenuated in wild type primary cells and mice. This link between innate immunity sensing of viral RNA and methylation of mRNA suggests that RNA modifications such as G-N-7-methylation provide a molecular signature for the discrimination of self and non-self mRNA. Since replication was inhibited in type I interferon treated fibroblast, methylation of viral mRNA may be critically involved in subverting the induction of type I interferon..Here we aimed to address the mechanism by which guanine-N-7 and ribose 2'-O methylations in discrimination of self and non-self mRNA by host innate immunity.We hypothesize that, specifically, the induction of type I interferon by viruses defective in MTase may dependent on the cytoplasmic RNA sensor PRRs or methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFN-stimulated genes (ISGs)-mediated suppression. The gene-knockout mice and MTase defective VSV(G-N-7 MTase defective VSV, 2'-O MTase defective VSV and G-N-7/2'-O MTase defective VSV)will be used to screen the specific PRRsand ISGs, which may highly sensitive to the MTase-defective VSV. The successful outcomes of our proposal will not only contribute to our understanding of mRNA cap methylation in viral pathogenesis, but also fill in a major gap in understanding how host innate immunity controls viral infection.

病毒的侵染与宿主的抗病毒作用一直交织推进着双方的进化。水疱性口炎病毒(Vesicular Stomatitis Virus, VSV)在与宿主细胞的协同进化过程中，进化出了与宿主mRNA相一致的甲基化修饰。甲基转移酶致弱（Methyltransferase defective, MTase-defective）的VSV在毒力和复制能力方面严重致弱。VSV mRNA G-N-7和2′-O位点甲基化作为区分自身与非自身的分子标签，在抵御宿主先天免疫识别与抗病毒功能方面可能起到相当积极的作用。在本项目研究中，我们将以基因敲除小鼠为实验动物，结合分子生物学和免疫学实验方法，来筛选能特异性识别不同MTase-defective VSV的模式识别受体和干扰素刺激因子。本研究的预期成果不仅能让我们进一步了解甲基转移酶在病毒致病性方面的作用，更能填补负链RNA病毒侵染与机体先天抗病毒免疫之间的空白。

在前期研究中，本课题组利用成熟的VSV反向遗传系统和定点技术，已成功拯救获得一系列甲基转移酶致弱的水疱性口炎病毒(MTase-defective VSV)，包括guanine-N-7和ribose 2'-O 两个位点甲基化严重致弱的rVSV-Y1650A, rVSV-F1691A和rVSV-E1833Q以及单独G-N-7位点甲基化严重致弱的rVSV-G1670A，. 在本次研究中，我们通过动物实验分析MTase-defective VSV对小鼠的毒力，初步探索病毒mRNA G-N-7和2'-O位点甲基化在机体免疫反应中扮演的角色。在动物实验中，接种rVSV-Y1650A, rVSV-F1691A和 rVSV-E1833Q组小鼠体内严重致弱，没有引起显著的病变。而接种rVSV-G1670A组小鼠，则出现了轻度的病变，G-N-7位点的甲基化致弱仍然使病毒保留有部分毒力，突破机体的免疫防线。. 为了进一步深入研究guanine-N-7和ribose 2'-O位点甲基化在机体先天免疫中所起的作用，我们使用了基因缺陷小鼠胚胎成纤维细胞系MEF-MDA5和MEF-IFIT2（分别敲除了MDA5和IFIT2基因），以及对照组MEF-WT作为研究平台。通过一步法生长曲线和噬斑实验，研究MTase-defective VSV在3个细胞系的复制情况。实验结果显示，相比较于接种MEF-WT细胞系，rVSV-E1833Q接种MEF-MDA5和MEF-IFIT2两个细胞系后，在不同的时间点都体现出极高的病毒滴度，而rVSV-G1670A，在基因敲除细胞系的滴度与MEF-WT组相比，远远没有像rVSV-E1833Q那样差别显著。. 我们的初步实验结果显示，rVSV-E1833Q虽然在MEF-WT细胞系毒力致弱，但是在病原相关模式识别受体MDA5基因缺失的细胞系，表现出较强的毒力。由于MDA5具有识别病原RNA的功能，所以在VSV，2'-O位点的甲基化为病毒躲避机体先天免疫系统的识别提供了分子标签。我们的实验结果还表明，2'-O位点的甲基化能够调整IFIT的抗病毒作用，起到躲避先天免疫系统干扰素的抗病毒作用。而G-N-7位点的甲基化在病毒躲避机体的先天免疫机能中所起的作用不显著。. 我们的研究使我们进一步了解了单股负链RNA病毒mRNA甲基化与病毒毒力的关系，也对先天免疫