Identification Of Interacting Protein Of Influenza Virus NS2 By Yeast Two-hybrid System

The NS2 protein of influenza virus mediates the nuclear export process of viral ribonucleoprotein complexes(v RNP)and also regulates the viral polymerase activity during virus replication and facilitates the adaptive processes of avian influenza viruses in the mammalian hosts. Other influenza virus proteins but relatively speaking,NS2 studies rarely reported. After the capture of this study to a new influenza virus NS2 interaction with host proteins, not only to expand the interaction network of influenza virus and host factors, and to further explore the replication cycle of influenza virus pathogenicity and immune mechanisms of the foundation.Yeast two-hybrid system as the classical techniques of protein interactions, has been host protein interaction studies with influenza A virus protein is applied. This test uses the technology as the basis for H5N1 influenza virus NS2 protein as "bait" protein, from containing Calu-3, A549, THP-1 and U251 hybrid c DNA library screening of four cells and their interacting proteins, researchers mutual for biological function. The main contents are as follows:(1) With avian influenza virus A/Anhui/02/2005(H5N1)NS2 protein as bait, from containing Calu-3, A549, THP-1 and U251 hybrid c DNA library screening of its four cells interacting proteinH5NS2 was cloned into yeast two-hybrid bait vector(p GBKT7 vector) and transformed yeast Y2 HGold, bait plasmid detect non-cytotoxic and non-existence of the phenomenon of self-activated after the p GBKT7-H5NS2/Y2 HGold Y187 c DNA library with four kinds of cell lines were hybridized, after screening the final total capture seven kinds of proteins, respectively HGS, EXOSC4, ZWINT, PRDX3, IRF3,NDUFB9 and RMND5 B. The gene-ontology(GO) analysis showed that these proteins were involved in cell growth and development, metabolic processes, and cellular localization, respectively.(2) co-immunoprecipitation(Co-IP) to verify its interactionThe test selected seven kinds of proteins peroxiredoxin 3(PRDX3) conducted a more in-depth research. The H5NS2, PRDX3 were cloned into p EGFP-C1, p CAGGS,in 293 T cells transfected with p EGFP-C1 and p CAGGS-myc-PRDX3 or p EGFP-C1-H5NS2 and p CAGGS-myc-PRDX3 plasmids were Co-IP and Western blot testing.Results are shown in 293 T cells, exogenous expression of H5NS2 and PRDX3 has a specific interaction.(3) GST pull-down to further validate the interactionThe H5NS2 cloned in p GEX-6p-1 and GST-H5NS2 prepare recombinant proteins and GST-tagged proteins expressed in E. coli. Cell lysates were incubated with GST-H5NS2 fusion protein as bait for the control GST protein or transfected with p CAGGS-myc-PRDX3 plasmid 293 T cells were GST pull-down. By GST pull-down experiments showed, H5NS2 and PRDX3 presence of specific proteins,direct interaction. Our data thus provides a basis to further study the interaction between these two proteins affects the NS2 function and the virus replication cycle.