A Preliminary Study On The Effects Of NS1-NOLC1 Interaction On CK2 Expression Level,Cell Proliferation And Virus Replication In A549-NS1 Stable Strains

The single-stranded RNA influenza A virus belongs to the genus orthomyxoviridae,causes all world-wide of the flu epidemic outbreaks.The new flu virus has been easier to spread between people,so the infectivity of the avian influenza virus should arouse wide attention.Non-structural protein 1(NS1)of influenza A virus exists only in infected cells,plays a central role in antagonising host innate immune response,ensuring efficient virus replication through the NS1 protein only or interacting with host factor.Human nucleolar and coiled-body phosphoprotein 1(NOLC1)is a host protein which interacts with NS1,had been screening and verified by our previous works,plays important role in cell apoptosis and development.Human nucleolar and coiled-body phosphoprotein 1(NOLC1)is a highly phosphorylated mammalian protein that participates in the development of nucleolus and rDNA transcription,and is involved in the pathogenesis of inflammation,cell growth,apoptosis,and tumorigenesis.In this study,we mainly studied the interaction between NS1 protein and NOLC1 protein on the proliferation,apoptosis and viral replication of host cells and its related signaling pathways.In this study,stably transfected A549-NS1 cell lines were constructed by lentivirus.The expression of NS1 protein was detected by immunofluorescence and Western Blot.The results showed that the stable cell lines were successfully constructed.In order to study the effect of NS1 and NOLC1 on the apoptosis,the expression level of CK2 in A549,A549-FLAG and A549-FLAG-NS1 cells was determined by ELISA.The mRNA and protein levels of NOLC1 and NF-?B in A549,A549-FLAG and A549-FLAG-NS1 cells were detected by Real-time PCR and western blot.The results showed that the interaction between NS1 and NOLC1 reduced the expression level of CK2,up regulated the mRNA level of NOLC1 and NF-?B,and inhibited the protein expression of NOLC1.CK2,NOLC1 and NF-?B are the key factors to regulate the apoptosis of cells.Combined with the results of this study,we speculate that the expression level of CK2,NOLC1 and NF-?B are the key factors in the regulation of apoptosis,but the specific mechanism remains to be further studied.In order to detect the effect of interaction of NS1 and NOLC1 on the ability of virus replication.A549,A549-FLAG and A549-FLAG-NS1 cells were infected with H3N2 avian influenza virus.The virus NP was detected by Real-time PCR to reflect viral replication.The results showed that the interaction between NS1 and NOLC1 promoted the replication level of the virus.In order to detect the effect of NS1 and NOLC1 on the proliferation of host cells,the proliferation ability of A549,A549-FLAG and A549-FLAG-NS1 cells was detected by MTT assay.The results showed that NS1 and NOLC1 enhanced the proliferation ability of host cells.In this study,we investigated the effect of NS1 and NOLC1 on the proliferation and apoptosis of host cells.In order to further study the related signaling pathways,we used Real-time PCR to study the genes involved in proliferation and apoptosis of A549,A549-FLAG,A549-FLAG-NS1.The results showed that the ?-catenin and cyclinD genes in the Wnt signaling pathway,Bcl2 and CDK4 genes were related to the interaction between the two proteins,and the interaction between NS1 and NOLC1 up regulated ?-catenin,cyclinD and Bcl2 mRNA levels,down-regulated CDK4 mRNA levels.NS1 may regulate these genes and their signaling pathway to regulate cell proliferation and apoptosis.To study the effect of NS1 and NOLC1 on the proliferation,apoptosis and viral replication of host cells,is helpful for us to further study the mechanism of interaction between NS1 and NOLC1,which provides a new idea for the study of pathogenesis of avian influenza.