狂犬病毒通过犬唾液中泛素蛋白酶体系统逃避免疫识别机制的研究

狂犬病在我国是一种危害严重的人畜共患传染病，致死率几乎100%。狂犬病毒（RV）通过患病犬、猫等动物咬伤宿主传播，病毒在外周短暂复制顺神经轴突逆向进入中枢神经系统。病毒如何逃避外周免疫系统识别是感染的关键，其免疫与分子机制仍待阐明。先前研究报道泛素蛋白酶体与宿主抗病毒免疫密切相关，申请人研究发现：犬唾液处理后的RV对小鼠的致病力显著上升；犬唾液和RV粒子均含有大量蛋白酶体；泛素化结合位点突变的RV泛素化降低，导致RV糖蛋白含量上升。以上结果暗示犬唾液中蛋白酶体可能通过影响RV糖蛋白泛素化从而改变RV糖蛋白的结构和含量，导致RV躲避免疫识别并增强病毒致病力。本项目以生物信息学、病毒反向遗传学和CRISPR-Cas9等方法，深入研究犬唾液成份如何扮演病毒增强因子的角色。该研究不仅从翻译后修饰的角度揭示RV利用犬唾液实现免疫逃避的分子机制，还为RV疫苗株的研制与疫苗生产工艺的提高提供科学依据。

Rabies remains a serious zoonotic infectious disease with an almost 100% fatality rate. China is one of the most seriously affected countries by rabies. Rabies virus (RV) is transmitted through the bite of infected dogs, cats and other animals, and RV briefly replicates in axons and reverse-enters the central nervous system. However, the immune and molecular mechanisms of RV evade recognition by the peripheral immune system remains obscure. Previous studies have reported that the ubiquitin proteasome system is important to the antiviral immune response of the host. The previous results of our lab show that the virulence on mice of RV treated with dog saliva is significantly increased. A large number of proteasomes were found in dog saliva and RV particles. The level of ubiquitination on RV was decreased after ubiquitination sites on RV mutated, and the expression of glycoproteins in RV was increased. The above results suggest that the proteasome in dog saliva may affect the ubiquitination modification of RV glycoprotein, thereby changing the structure and expression of RV glycoprotein, leading to RV evading immune cell recognition and enhancing the infection of virus to animals. On this basis, this project intends to explain how components in dog saliva work as virus enhancer with recombinant viruses, primary cells, mice and dogs as models, using protein spectrum, immunoprecipitation, bioinformatics analysis, viral reverse genetics and crispr-cas9 methods. This study will not only reveal the molecular mechanism of RV hijacking dog saliva to adjust post-translational modification of proteins to evade the immune systerm, but also provide scientific basis for the development of RV vaccine strain and the improvement of vaccine production process.