Rescue Of Influenza A Virus And The Effect Of Interaction Between NS1 And NOLC1 Proteins On Its Infection

Influenza A virus,as the main source of disease,causes influenza outbreaks of different sizes every year.NS1 protein is the main toxic protein of influenza A virus and plays an important role in the process of viral infection.In the early stage,the laboratory has proved that there is a specific interaction between the influenza A virus NS1 protein and the host cell NOLC1 protein.And the specific sites of the two proteins have been determined,but the mechanism and effect of the interaction between NS1 and NOLC1 protein are still unclear.In this paper,an eight plasmid system of H1N1(PR8)wild-type(WT)virus was constructed using infectious cloning technology.And three sets of infectious clone transcription systems of mutant H1N1 viruses were constructed based on the sequence-specific action sites(125D and 200R)of NS1 protein and NOLC1 protein of H1N1.The effect of the interaction between NS1 protein and NOLC1 on the replication of influenza virus H1N1 was thoroughly studied.main tasks as follows:1.By constructing the PHW2000 double transcription eight plasmid system for infectious clones of H1N1 avian influenza A virus and three sets of NS1 mutation systems,four influenza virus strains were successfully rescued and obtained: H1N1(WT),H1N1-125,H1N1-200,H1N1-125 / 200;at the same time,the A549 NOLC1 protein knockdown stable transfected cell line(pLKO.1-TRC-NOLC1)was constructed and screened;the virus titer TCID50 / mL of influenza virus H1N1(PR8)amplified from chicken embryos was detected between 5.62 E + 5 and 6.40 E + 7,and the appropriate infection ratio was determined to be MOI = 0.1;2.After infecting the host cell A549 with four influenza viruses H1N1(WT),H1N1-125,H1N1-200,H1N1-125 / 200,the RNA expression level and protein level of NOLC1 in A549 cells were detected,and it was found that during the H1N1 influenza A virus infection,the amount of NOLC1 in the early stage of infection showed an upward trend compared with normal cells at the transcription level or protein level.Later in the infection,it showed a downward trend.However,after the double point mutation virus infects the cells,although the change trend of NOLC1 inhost cells is basically the same as that of wild-type infection,it has been consistently lower than that of wild-type virus-infected cells at the transcription level,and the amount of NOLC1 protein at the translation level is higher than that of wild-type virus-infected cells.3.A549 cell lines were infected with the four influenza viruses H1N1(WT),H1N1-125,H1N1-200,H1N1-125 / 200,respectively,and the stable A549 cell strain of pLKO.1-TRC-NOLC1 was infected with H1N1(WT),Samples were collected according to the infection time point,the virus titers were detected by TCID50 technology and the three RNA expression levels of viral NP gene were detected by Real-time PCR technology,and the changes of virus activity and replication level were detected.The experimental results show that the 125 D and 200 R sites on the NS1 protein have a significant effect on the replication of H1N1 influenza A virus;To sum up,this study revealed the effect of NOLC1 and NS1 interaction on influenza A virus replication during influenza virus infection,mutations in the 125 D and 200 R sites on the NS1 protein will reduce the replication of influenza A virus H1N1 in A549 cells,and provide new ideas for exploring the mechanism of action of NS1.