核仁蛋白LYAR调控流感病毒复制的分子机制研究

The influenza virus genome is replicated and transcribed by RNA-dependent RNA polymerase, and the polymerase function is regulated by the host proteins. However, the molecular details of the host proteins involved in the replication and transcription of influenza virus are not entirely clear. Using tandem affinity purification technique, our preliminary work has screened the host proteins interacting with influenza polymerase, and found the selected host protein LYAR played a significant role in the regulation of influenza virus replication and its function was closely correlated with its phosphorylation. We intend to take the next step on the interaction between LYAR and polymerase using Co-IP and pull-down techniques, to find the key domains and sites which are associated with the interaction between the polymerase and LYAR, and further to verify the function of these sites on polymerase with reverse genetics. Then, effects of LYAR on influenza virus polymerase activity, RNA replication and transcription, RNP complex formation and the nuclear import of polymerase will also be studied. And we aim to clarify the regulatory effect of LYAR phosphorylation on influenza virus replication and also to reveal the mechanism of LYAR phosphorylation regulated by influenza virus. Finally, using LYAR knockout mice, we will validate the regulation of LYAR on influenza virus replication in vivo. Based on the above, this research will clarify the molecular mechanism of LYAR regulation on influenza virus replication.

流感病毒基因组通过RNA依赖的RNA聚合酶来复制和转录，聚合酶的功能受宿主蛋白调控。然而，宿主蛋白参与流感病毒复制和转录的分子细节尚不完全清楚。本项目前期利用串联亲和纯化技术筛选了与流感病毒聚合酶相互作用的宿主蛋白，发现筛选出的宿主蛋白LYAR能显著影响流感病毒复制，且该蛋白功能的发挥同其磷酸化修饰密切相关。本项目首先利用免疫共沉淀及Pull-down技术研究聚合酶与LYAR的互作，寻找聚合酶与LYAR互作的关键功能区域和位点，并利用反向遗传学技术对聚合酶上互作的关键位点进行功能验证。然后，研究LYAR对流感病毒聚合酶活性、RNA复制和转录、RNP复合物形成及聚合酶入核的影响，进一步明确LYAR磷酸化对流感病毒复制的调控作用，并揭示流感病毒感染对LYAR磷酸化修饰的调控机理。最后，利用基因敲除小鼠模型对LYAR调控流感病毒复制的功能进行体内验证。最终阐明LYAR调控流感病毒复制的分子机制。

流感病毒基因组通过RNA依赖的RNA聚合酶来复制和转录，聚合酶的功能受宿主蛋白调控。然而，宿主蛋白参与流感病毒复制和转录的分子细节尚不完全清楚。本项目利用AP-MS的方法鉴定到61个新的与vRNP相互作用的宿主蛋白，发现细胞生长调控核仁蛋白LYAR在IAV复制中起重要作用。LYAR与病毒vRNP结合并促进vRNP组装，进而促进病毒RNA的合成，而LYAR在此过程中发挥作用需要它的核定位以及215和244位的丝氨酸磷酸化，并且LYAR的磷酸化受到IAV感染激活的蛋白激酶C (PKC) 的调控。发现IAV诱导的IFN-β促进宿主核仁蛋白LYAR的表达，LYAR可以与磷酸化的IRF3相互作用后阻碍其与ISRE的结合，从而抑制IFN-β和ISGs的产生，通过抑制宿主的抗病毒天然免疫反应促进病毒感染。总之，本研究鉴定到许多潜在的与vRNP相互作用的宿主因子，并进一步阐明了核仁蛋白LYAR与vRNP相互作用促进IAV复制的机制，不仅加深了对IAV转录和复制分子机制的认识，也为抗流感病毒药物的研发提供新的思路。本项目共发表SCI收录论文8篇，培养研究生5名。