Effect Of Interaction Between NS1 And NOLC1 On Influenza Virus(H1N1) Replication

Influenza A virus is the main cause of the outbreak of a large-scale influenza epidemic.The NS1 protein is an important viral factor encoded by influenza A virus and plays an important role in infected cells.The nucleolar and coiled-body phosphoprotein 1?NOLC1?is a highly phosphorylated mammalian protein.In the early stage of the laboratory,it has been proved by various experiments that the H5N1subtype NS1 protein interacts with the host cell NOLC1 protein,however,the effect of NS1 interaction with NOLC1 protein on influenza virus replication remains unclear.In this thesis,we first verified the interaction between H1N1 subtype influenza virus NS1 protein and host cell NOLC1 protein by co-immunoprecipitation?Co-IP?technology,the effects of NS1 interaction with NOLC1 on the replication of influenza virus H1N1?PR8?were studied in depth.The results are as follows:1.Construction and screening of NOLC1 protein knockdown stable transgenic strain?pLKO.1-TRC-NOLC1?and overexpression stable transgenic strain?pBABE-puro-NOLC1?.The virus titer of the influenza virus H1N1?PR8?amplified by chicken embryo was determined to be 1.0×10⁸,the trypsin concentration was2?g.mL^-1,and MOI=1.0,which was the optimal condition for the proliferation of influenza virus H1N1?PR8?in host cells.2.Infected pBABE-puro-NOLC1,pLKO.1-TRC-NOLC1 and A549 cell lines with influenza virus H1N1?PR8?,and collected samples at different infection times.On the one hand,the virus titer was detected by TCID50 technology;on the other hand,the expression level of three RNAs of the viral NP gene was detected by Real-time PCR.The results showed that the virus titer of influenza virus H1N1?PR8?in the three cell lines reached the highest at 24h,followed by a downward trend.Compared with normal cells,the titer of virus in NOLC1 over-expressing stable strain increased,the level of viral NP RNA increased,while the titer of virus in NOLC1knockdown stable strain decreased,and the level of viral NP RNA decreased.From the above test results,it was concluded that there was a positive correlation between the level of viral replication and the expression level of NOLC1 in the host.3.In order to explore the effect of NS1 interaction with NOLC1 on the replication of influenza virus H1N1?PR8?,the expression level of NOLC1 mRNA was detected at different time after host infection with H1N1?PR8?.The expression level of NOLC1 mRNA showed a decreasing trend within 48 hours after virus infection of host cells.Combined with the above experimental results,we can know that during the viral infection process,the host interacts with the NS1 protein of the influenza virus through the NOLC1 protein,which reduces the level of virus replication and thus affects the infection process of the influenza virus.In summary,this study reveals the effect of NOLC1 and NS1 protein interaction on the process of influenza virus infection,and provides new ideas for the development of drugs for the treatment of diseases caused by IAV.