蛋白甲基化转移酶Smyd2在病毒感染诱发的天然免疫反应中的调控作用

Identifying the key molecules responsible for the efficient initiation of antiviral innate response and how the viruses can feedback inhibit the innate response for their immune escape is the key issue to be addressed in the field of molecule immunology. Transcriptional factor IRF3 plays critical role in induction of type interferons (IFNs) transcription to eliminate virus infection. There are reports about the activation and deactivation of IRF3, however, negative regulation of IRF3 activation process is not fully clarified. In this project, we screened the differentially expressed genes in mouse peritoneal macrophages with or without VSV infection by cDNA microarray. We found that, Smyd2, one member of SMYD (SET and MYND domain) family, was significantly downregulated in macrophages upon RNA virus infection. Also, knock-down of Smyd2 significantly increased typeⅠinterferons in macrophages. We further prepared Smyd2-/- mice, and confirmed typeⅠinterferons substantially increased in the serum and organs, which led to decreased virus load and increased survival ratio. We further found that phosphorylation of IRF3 significantly increased in Smyd2-deficient macrophages infected with VSV. Therefore, our results indicate that Smyd2 negatively regulate innate immune response by reduction of phosphorylation of IRF3. Therefore, the primary aim of this application is to investigate the molecular mechanisms involved this process from a view of epigenetic enzyme, trying to figure out the epigenetic regulation of IRF3-initiated antiviral innate immune response. This project will shed new light on the regulations of IRF3 activation, which might provide theoretical support and new target for control of virus infection.

病毒感染触发天然免疫反应以及病毒如何通过负向调控天然免疫反应而逃逸免疫的机制是分子免疫领域的前沿热点。转录因子IRF3促进Ⅰ类干扰素转录进而在防御病毒感染过程中起关键作用，因此其活化受到严格调控，大量研究揭示了其活化及失活过程，但其详细的调控过程仍未完全揭示。我们通过cDNA芯片筛选VSV病毒感染前后小鼠腹腔巨噬细胞的差异基因，发现蛋白甲基化转移酶Smyd2 mRNA在病毒刺激后表达显著降低，RNA干扰巨噬细胞的Smyd2表达，发现Ⅰ类干扰素产生显著升高.通过制备Smyd2-/-小鼠，并且体内感染VSV，证实Smyd2抑制Ⅰ类干扰素产生,从而抑制病毒的清除。进一步Western blot检测发现IRF3的磷酸化显著升高。上述结果表明Smyd2通过降低IRF3的磷酸化水平从而负向调控病毒诱发的天然免疫反应过程而达到逃逸免疫得作用，其详细的调控机制需要进一步阐明，该课题将从负向调控以及蛋白甲基

病毒感染性疾病根据其发病过程又分为急性病毒感染性疾病和慢性病毒感染性疾病。目前病毒感染性疾病仍然严重威胁人类的健康，抗病毒药的应用对治疗病毒性感染有了很大进展，但是鉴于病毒的高突变率产生，使得治疗病毒感染性疾病具有严峻的挑战性。Ⅰ型干扰素在抵抗及清除病毒干扰过程中具有关键性的作用，因此其产生过程受到严格的调控。干扰素调节因子3(Interferon regulatory Factor 3, IRF3)是调控细胞在病毒感染过程中Ⅰ型干扰素产生的关键转录因子， 磷酸化的IRF3促进其形成同源二聚体，进而进入细胞核，促进Ⅰ型干扰素的转录过程。我们发现病毒感染后，巨噬细胞中Smyd2基因mRNA的表达下调，进而我们发现干扰或者敲除Smyd2基因表达后，病毒感染的巨噬细胞的Ⅰ型干扰素的mRNA及蛋白产生显著上调。Smyd2敲除小鼠（Smyd2 -/-）体内实验证实，Smyd2 -/-小鼠血清中Ⅰ型干扰素的量显著升高，Smyd2 -/-小鼠清除病毒的能力显著升高，表现为敲除小鼠脏器病毒滴度显著低于野生对照组，因此病毒感染后Smyd2 -/-小鼠的生存率显著高于野生对照组。机制研究发现，Smyd2基因能够抑制IRF3的磷酸化，因此负向调控Ⅰ型干扰素的产生。继而研究发现此负向调控过程并不依赖于其甲基化酶活性，而是通过SMYD2蛋白与磷酸化酶PP1相互作用，从而促进PP1对IRF3的去磷酸化过程。我们的此项研究发现了负向调控Ⅰ型干扰素产生的Smyd2基因，从而为治疗病毒感染性疾病找到了潜在的治疗靶点。