应用酵母双杂交系统筛选与流感病毒NS2蛋白相互作用的宿主蛋白

Identification of host interacting proteins of influenza virus NS2 by yeast two-hybrid system

流感病毒的NS2蛋白能够介导病毒核糖核蛋白复合体(v RNP)的核输出过程,而且可以调控病毒聚合酶的活性,参与禽流感病毒在哺乳动物宿主体内的适应过程。为鉴定与NS2相互作用的宿主蛋白,本研究利用流感病毒NS2蛋白作为"诱饵"蛋白,通过经典酵母双杂交系统筛选与其相互作用的宿主蛋白,并利用免疫共沉淀(Co-IP)和GST pull-down方法进一步验证其相互作用关系。结果表明,通过酵母双杂交系统筛选含有Calu-3、A549、THP-1和U251 4种细胞的c DNA文库,共筛选到7种蛋白,分别为HGS、EXOSC4、ZWINT、PRDX3、IRF3、NDUFB9和RMND5B,根据Gene Ontology分析结果表明,这些蛋白分别参与细胞生长发育、细胞代谢和细胞定位等过程。其中对过氧化物氧还酶3(PRDX3)进行了Co-IP和GST pull-down试验证实重组表达的NS2蛋白和PRDX3蛋白在293T细胞中存在特异性的相互作用。本研究为进一步研究两者之间相互作用如何影响NS2蛋白功能以及病毒的复制周期奠定了基础。

The NS2 protein of influenza virus mediate the nuclear export process of viral ribonucleoprotein complexes （vRNP） and also regulates the viral polymerase activity during virus replication and facilitates the adaptive processes of avian influenza viruses in the mammalian hosts. In the present study, using influenza virus NS2 as a ＂bait＂ protein, we performed the yeast two-hybrid （Y2H） screening to identify host proteins that interacted with NS2 and further valiated the interactions by Co-Immun- oprecipitation （Co-IP） and GST pull-down. As a result, we successfully identified 7 host proteins that were able to interact with the NS2 protein by screening the Y2H cDNA library constructed from Calu-3, A549, THP-1 and U251 cells. The Gene-Ontology （GO） analysis showed that these proteins were involved in cell growth and development, metabolic processes, and cellular localization, respectively. The GST pull-down and Co-IP assay demonstrated that NS2 and peroxiredoxin 3 （PRDX3） specifically interacted in the 293T cells. Our data thus provides a basis to further study the interaction between these two proteins affects the NS2 function and the virus replication cycle.