The Effect Of Deletion Of NS1 Protein C-terminus Of H5 Subtype Avian Influenza Viruses On Viral Replication

The 8^th segment NS gene of influenza A virus encodes NS1 protein which is a critical virulence factor of influenza A virus.NS1 protein is multifunctional and strain-specific.NS1 protein from the different subtypes has different capacity in regulating viral virulence on diverse stage and the domains of NS1 exerting the various functions are different as well.It has been revealed that NS1 protein enhances viral replication by antagonizing the interferon responses and inhibiting apoptosis.The purpose of this study is to understand the effect of deletion of 113 amino acids at the C-terminus of H5 subtype NS1 protein on the virus replication in vitro and in vivo,and further explore the mechanism of this effect.These results would enrich the basic research of influenza A virus,also provide the supportive data for the development of NS1-truncated attenuated vaccines against H5 influenza.A strain of H3N8 equine influenza virus A/equine/Kyonggi/SA01/2011（KG11）was reported to be isolated in South Korea.Sequence analysis indicated that NS1 of KG11 strain lacks 23 nucleotides at positions 327-329by a frameshift mutation,resulting in the deletion of C-terminal 113 amino acids.According to the mutation of the KG11 NS1 sequence,using reverse genetic system,we rescued two pairs of recombinant strains,namely HN109,HN109 NS1/112 and SX06,SX06 NS1/112.All four strains possess the same genes except NS.HN109and HN109 NS1/112 NS genes are derived from H5N6 subtype strain（A/Goose/Hunan/109/2014,HN109）,whereas SX06 and SX06 NS1/112 NS genes are from H5N1（A/Chicken/Shanxi/2/2006,SX06）.The NS1protein of HN109 NS1/112 and SX06 NS1/112 only remain the N-terminal 112 amino acids compared with those of HN109 and SX06.The results of the replication of full-length NS1 and NS1/112 strains in MDCK,A549,CEF,SPF chickens and BALB/c mice showed that the replication of HN109 NS1/112 in cells and SPF chickens was significantly reduced compared to that of HN109,but no significant difference was observed between SX06 and SX06NS1/112 in MDCK,CEF and BALB/c mice.To preliminarily investigate the mechanism of replication capacity change of the NS1/112 strains,we examined their ability to induce interferon and apoptosis.The result indicated that the ability of NS1/112 protein to inhibit typeⅠinterferon promoter activity was attenuated compared with NS1.And HN109 NS1/112 induced stronger interferon and apoptosis respond than HN109.However,SX06NS1/112 induced the same level of interferon transcription as SX06.The discrepancy may relate to the specificity of NS1 derived from different strains.The C-terminal deletion of NS1 protein of HN109reduced its ability of inhibiting interferon and apoptosis responses,and hence attenuated the viral replication.Nevertheless,the C-terminal deletion of NS1 protein of SX06 did not alter the ability to induce interferon transcription and the viral replication.