The Effect Of NS Protein Of Influenza Virus On Interferon-inducible Transmembrane Protein Gene Expression

The interferon family (IFNs) has been widely used in prevention and treatment of viral infections with diverse biological function. Many genes in human genome resist virus infection such as interferon-inducible transmembrane protein (IFITM) gene involved in the biological action of IFN. Biological functions of IFITM contain immune cell signal transduction, cell adhesion, tumor genesis, germ cell homing and mature, and promoting bone mineralization. Human IFITM family expresses IFITM1, IFITM2, IFITM3and IFITM5, of which genes are evolutionarily conserved and all located on chromosome11contiguously and coding polypeptide of14kDa. Since the study of IFITMs mediated cell against influenza A H1N1virus, West Nile virus and dengue virus was published on the CELL by Brass, the research on the anti-viral role of IFITM protein family is more systematic.Influenza can lead to respiratory pandemic. NS protein of Influenza virus is a non-structural protein which includes NSland NS2coded by the eighth segment of the viral genome. NS1is related to the pathogenicity of influenza viruses as interferon (IFN) antagonist. NS2protein participates in membrane transport with Matrix Protein (M1), nucleoprotein (NP).In recent years, the relationship between the influenza virus NS protein and IFITMs protein molecules has not been reported though inhibition of early replication of arbovirus by IFITM proteins was revealed.Real-time quantitative PCR (Real-time PCR) technology has been used to detect gene expression level for automatically data analyzing and precise quantitative expression levels of exogenous gene in very short time. In order to clarify the impact of NS protein of influenza virus on IFITMs protein expression, the Real-time quantitative PCR assay for accurate determination of reference gene and target gene expression was established through setting appropriate reference gene β-actin to normalized sample difference, analyzing the stability, sensitivity, specificity by adjusting PCR conditions to represent the difference of human IFITM1,2,3gene level affected by NS protein of influenza virus, protein level of IFITM1,2,3had also be detected in use of Western-blot.The results showed that:coefficient correlation of standard curve of IFITM1,2,3and β-actin was0.998,0.99,0.99,0.995respectively, the high linear correlation shows accuracy to quatitate target gene; Amplification efficiency was106.284%,101.03%,104.929%,101.962%; and interassay coefficient of variation and intraassay coefficient of variation were all below1.58%, which indicate good stability; sensitivity was2.52×100copies,1.3×100copies, and 3.7×101copies respectively shows high Specificity; no specific amplification were found with the use of cDNA in chick liver and influenza H3N2as template indicated the high sensitivity.The detection of gene expression levels of IFITM1,2,3with the established assay showed that, induction of interferon set-up the expression level of IFITM1,2,3, but expressions of IFITM1,2,3in gene level and expressions of IFITM2,3in protein level were all inhibited when NS protein exist.These results have proved unknown mechanism of interaction between IFITMs and NS protein which need lucubrate.