黄热病毒顺式作用RNA元件cHP2调控基因组复制的机制研究

黄热病毒为黄病毒科黄病毒属成员，经蚊虫叮咬感染人类后可导致高致死率的黄热病。目前对黄热病毒复制机制的认识尚不深入。申请人在前期研究中发现，黄热病毒基因组5′末端存在可直接参与基因组环化的独特“开关”元件cHP2，提示与其它重要蚊媒黄病毒相比，黄热病毒具备特有的复制策略。基于上述发现，本项目以黄热17D疫苗株为模型，首先在复制子水平分析两类宿主中cHP2对病毒基因组复制的作用，然后基于反向遗传学操作，在完整病毒中对该元件的功能加以鉴定。进一步利用RNA pull-down、质谱分析等手段，筛选与cHP2互作的哺乳动物及蚊虫蛋白。在此基础上，结合EMSA、SHAPE、病毒酶活性试验等技术，对cHP2调节病毒基因组环化与RNA合成的机制展开研究。本项目将阐明不同宿主条件下cHP2在黄热病毒复制周期中的功能与相关分子机制，为深入认识黄热病毒复制规律奠定基础，并为抗病毒药物设计提供新思路。

Yellow fever virus (YFV) belongs to the Flavivirus genus of the Flaviviridae family. YFV is mainly transmitted by the biting of mosquitoes, and human infections of YFV can lead to the high fatality yellow fever disease, posing a great threat to the public health. Currently, the mechanistic details of yellow fever virus replication are poorly understood. In the previous research, the applicant has found that a special “RNA switching” element in the genomic 5′ end, designated as the cHP2, may directly participate in the genome cyclization process of YFV. The above finding suggests that YFV may utilize a specialized replication strategy, different from that used by other medically important mosquito-borne flaviviruses. Based on these work, in this project, the live attenuated 17D vaccine strain will be utilized as a model to study the role of cHP2 in YFV replication. Firstly, the mutations targeting the cHP2’s structure will be introduced into YFV-17D replicons, and the effect on viral RNA replication will be investigated in both mammalian and mosquito cell lines. Then, virus mutants will be generated to characterize the cHP2’s function under the condition of complete viral propagation by using reverse genetics approaches. Furthermore, the cHP2-interacting proteins from both mammalian and mosquito cells will be screened by RNA pull-down assay followed by LC-MS/MS analysis. Based on the above results, the molecular mechanisms how cHP2 regulates genome cyclization and viral RNA synthesis will be characterized by a combination of the electrophoretic mobility shift assay (EMSA), selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) as well as in vitro RNA-dependent RNA polymerase (RdRp) and helicase activity assays. By unveiling the function and the corresponding mechanisms of cHP2 in YFV replication in different hosts, this project will not only deepen the understandings about the regulation of YFV replication, but also provide novel concepts for the design of antiviral drugs.