The Impact On Inducing Host Cells Apoptosis By Amino Acid Mutations Of H9N2Avain Influenza Virus NS1Protein

Avian influenza (AI) is an infection or syndrome caused by avian influenza virus typeA (AIV). NS1protein is a non-structural protein of the virion, it is a multifunctionalregulatory factor and can find only in infected cells. It plays an important role in theregulation of viral pathogenicity and virulence, and also has impact on the immune responseand apoptosis of host cells. It has been confirmed that the NS1protein of H9N2WN straincould induce apoptosis in host cells. The mutations of NS1protein can change its functionusually. In this study, we mutated amino acids at the six important sites of NS1protein inorder to explore whether these mutations could affect the apoptosis induced by NS1proteinin host cells. The results were as follows:1H9N2AIV infection can induce apoptosis in DF-1cells. Microscope observed thatAIV-infected DF-1cells showed typical apoptotic features. Fluorescent microscopeobserved that AIV-infected DF-1cells which were stained by Hoechst33324, the nuclei ofcells some showed dense stained and some were fragementay shape. The chromosomalDNA fragmentation analysis showed DNA ladders could be detected as early as24h p.i,when DF-1cells were infected with AIV at0.5MOI, and the intensity of the ladder bandsincreased with time and dose of infection.2We mutated the amino acids at42,58,73,92,191and206sites of the H9N2WNstrain NS1protein, and contacted the eukaryotic expression vectors of pEGFP-NS1-42,pEGFP-NS1-58, pEGFP-NS1-73, pEGFP-NS1-92, pEGFP-NS1-191and pEGFP-NS1-206successfully. Then tansfected those vectors into DF-1cells, the subcellular localization ofNS1protein and its mutants were observed using confocal microscope. The results showedthe subcellular localization of NS1protein and its mutants were consistency, whichsuggested that the amino acids mutation at42,58,73,92,191and206sites of NS1proteinhad no influence on its subcellular localization.3The results of Flow cytometry determined the effect of the six mutants of NS1proteinon the apoptosis induced by NS1protein in host cells showed that the apoptosis rates were below the NS1protein at varying degrees, especially the191site mutation of the NS1protein. Caspase activity assay and Western blot analysis showed that the191site mutationof the NS1protein could reduce the activation and expression of Caspase-3,9induced byNS1protein significantly. These results suggested the191site mutation of the NS1proteincould reduce the apoptosis in DF-1cells induced by NS1protein.In summary, this study confirmed that the H9N2AIV could induce apoptosis in DF-1cells, and the191site mutation of the NS1protein could reduce the apoptosis in host cellsinduced by NS1protein. The results provide theoretical basis for the research on thefunction of apoptosis in cells induced by NS1protein.