干扰素诱导蛋白2/3抗狂犬病病毒作用及机制研究

Rabies, one of the oldest infectious diseases, still presents a public health threat in most parts of the world today. Its pathogen, rabies virus (RABV), is a neurotropic virus. As its unknown pathogenesis, there is no effective method to treat rabies. It has ben showed that RABV could be restricted by IFN-I, however, which kind of interferon-induced protein plays a key role remain largely unknown. The applicant has found that RABV infection significantly upregulates IFIT family gene in mice brain. The recombinant RABVs expressing IFIT2/3 developed by using the reverse genetic techniques indicated that IFIT2/3 could inhibit RABV replication in vitro. The relationship between the IFIT2/3 and RABV pathogenicity in vivo will be further analyzed in this subject. Forthermore, to understand the molecule mechanism of IFIT2/3 on restriction of RABV, apoptosis induced by IFIT2 in cells will be confirmed; the interaction between IFIT2 and RABV mRNA will be investigated; the role of IFIT3 on regulation of RIG-I signaling pathway will be analyzed. This subject will provide us new understanding on RABV pathogenesis and postexposure treatment.

狂犬病是一种古老且仍然严重威胁很多国家公共卫生安全的传染病。狂犬病病毒（RABV）是一种嗜神经病毒，因其致病机理不明，当前尚无有效治疗狂犬病的方法。现有研究表明IFN-I在体外对RABV具有限制作用，但具体由何种干扰素刺激蛋白发挥作用还不得而知。申请者前期研究发现：RABV感染可致小鼠脑内IFIT家族基因显著上调，暗示了IFIT家族蛋白可能参与限制RABV；通过构建表达IFIT2和IFIT3 (IFIT2/3)的重组狂犬病病毒证实二者在体外能够显著抑制RABV复制。本项目拟在研究IFIT2/3在体内对RABV致病力影响的同时，着重从IFIT2诱导细胞凋亡，IFIT2与RABV mRNA相互作用和IFIT3对RIG-I信号通路的调控等方面研究IFIT2/3限制RABV的分子机制，为寻找狂犬病暴露后治疗提供新的科学依据。

天然免疫应答是宿主抵抗病毒感染的第一道防线。干扰素（interferon，IFN）是重要的天然免疫分子，能促进数以百计的干扰素刺激基因（IFN stimulated genes，ISG）表达进而发挥抗病毒作用。本项目前期通过对RABV感染后的鼠脑开展RNA-seq和iTRAQ，发现Ifit1，Ifit2和Ifit3在RABV感染后均上调表达，又通过qPCR和WB在RABV感染的细胞系中证实了该现象。然后基于RABV反向遗传操作技术构建了一系列重组病毒，发现Ifit3可以显著降低RABV的致病力。于是构建了Ifit3的基因缺失小鼠， 发现rRABV-Ifit3在Ifit3-/-小鼠模型中的致死率显著低于亲本病毒rB2c。为了确定Ifit3的作用机制与核心位点，我们在N2a细胞中过表达IFITs及其突变体，然后感染rB2c-RLUC，发现Ifit2和Ifit3在抑制RABV方面具有协同作用，且作用位点不唯一。本研究证实了Ifit3在限制RABV致病力方面的关键作用，丰富了对IFIT1 家族蛋白病毒与病毒之间关系的认识。