NP磷酸化对A型流感病毒复制的调控机理

磷酸化是蛋白翻译后的一种重要修饰手段，A型流感病毒某些蛋白的磷酸化在流感病毒的复制过程中发挥着重要作用。A型流感病毒核蛋白NP（nucleoprotein）上的若干磷酸化位点已被报道，然而大多数磷酸化位点的功能仍然未知。我们的前期研究利用PAGE方法富集磷酸化的NP，通过飞行质谱鉴定得到9个新的NP磷酸化位点。本项目拟研究NP磷酸化对流感病毒复制的影响及其作用机理。首先将NP的磷酸化位点替换为模拟持续磷酸化的谷氨酸或去磷酸化状态的丙氨酸，利用反向遗传系统拯救突变病毒，检测病毒滴度及病毒多步生长曲线，筛选能够影响病毒复制的NP磷酸化位点。然后利用IFA/IP/WB等技术手段检测磷酸化是否影响NP的出核/入核，是否改变NP与病毒核糖核蛋白复合物vRNP中各组分或病毒RNA的结合从而影响病毒的聚合酶活性，进一步阐明NP磷酸化对A型流感病毒复制的调控机理。本研究将为抗流感药物的研发提供新的靶点。

Phosphorylation of viral proteins plays important roles in the influenza A virus life cycle. Many phosphorylation residues of nucleoprotein (NP) have been reported. However, the functions of these phosphorylated sites have not been elucidated systematically. In our previous study, nine phosphorylated residues on the NP of influenza virus had been identified by mass spectrometry. In the present proposal, the effect of NP phosphorylation on the replication of influenza A virus will be further investigated. We will introduce alanine or glutamic acid substitutions at these sites and examined their effects on virus growth by using the reverse genetic system and plaque assay. Then we will focus on the effect of NP phosphorylation on the cellular localization of NP, the viral polymerase activity, and its interactions with the components of viral ribonucleoprotein complex and RNA. The study will provide novel targets for the development of novel anti-influenza drugs.