Effects Of Host Factors IGF2BP2 And SLC12A6 On Swine Influenza Virus Replication

Swine influenza caused by swine influenza virus(SIV)is an acute respiratory infectious disease.Pigs are usually infected with other bacterial and viral diseases,which seriously endanger the development of the pig industry and cause huge economic losses.In addition,because pigs act as a"mixer"during the spread of influenza viruses,they also pose a huge challenge to public health and severely threaten human life and health.This study is based on the experimental results of using CRISPR/Cas9technology to screen the swine influenza virus replication-related host genes in the early stage of the laboratory.Through individual testing and exploring some candidate genes,I finally selected IGF2BP2 and SLC12A6 genes for in-depth study Molecular mechanisms affecting influenza virus replication.1.The effect of IGF2BP2 on influenza replicationThe gene knockdown polyclonal cell line was constructed by CRISPR/Cas9technology and verified by PCR at the genomic level and Western blot at the cell protein level.After confirming that the IGF2BP2 knockdown polyclonal cell line was obtained,the selection was performed by the limiting dilution method combined with Western blot.The monoclonal cell line was further verified and tested for changes in cell viability after knocking out the IGF2BP2 gene.Finally,a completely knocked-out NPTr-KO_IGF2BP2 monoclonal cell line was screened,and the cell activity of the NPTr-KO_IGF2BP2 cell line and the wild-type NPTr cell line were not significantly different.At the same time,we successfully amplified and constructed the IGF2BP2 expression plasmid.Western blot and determination of virus titer changes confirmed that IGF2BP2 can affect the replication of influenza virus.Knockout of IGF2BP2 can inhibit influenza virus replication,and overexpression of IGF2BP2 can promote influenza virus replication.Next,we further explore the specific mechanism of how IGF2BP2 affects influenza virus replication.Through the detection of sialic acid receptors in the knockout IGF2BP2 cell line,it was found that knocking out IGF2BP2did not affect the synthesis and distribution of?-2,3 sialic acid receptors and?-2,6sialic acid receptors,and passed the virus adsorption test It is found that knocking out IGF2BP2 does not affect the process of influenza virus adsorption;through the Acid bypass test,we found that knocking out IGF2BP2 can affect the process of influenza virus endocytosis;through the Co-immunoprecipitation(Co-IP)test In the infection state,we found that IGF2BP2 can interact with the RNP complex,and is independent of RNA interaction;in the case of co-transfection,we found that IGF2BP2 can interact with influenza viruses NP and PB1,and they can interact with influenza viruses NP and PB1 respectively.There is a phenomenon of co-localization in the nucleus and cytoplasm;through the polymerase activity test,we found that in the case of overexpression of IGF2BP2,the influenza virus polymerase activity was significantly increased,but it did not affect the expression of polymerase subunits.After SIV infection of the wild-type NPTr cell line,the expression of endogenous IGF2BP2showed an increasing trend with the proliferation of the virus on the cell,and the distribution of endogenous IGF2BP2 in the cell changed significantly,from mainly distributed in the cytoplasm.It is homogeneously distributed throughout the cell.Finally,we confirmed that knocking out IGF2BP2 can broadly inhibit the proliferation of different types of influenza viruses,and inhibit the replication of human influenza H1N1/PR8,avian influenza H9N2/SH13,and swine influenza H1N1/F26 influenza strains to varying degrees.Strain specificity.2.The effect of SLC12A6 on influenza replicationWe used CRISPR/Cas9 technology to construct the SLC12A6 knockout cell line on NPTr-Cas9 cells using the same method,and obtained the gene knockout monoclonal cell line by limiting dilution.At the same time,SIV was used to infect NPTr wild-type and NPTr-KO_SLC12A6.By Western blot and the change of virus titer,it was found that knocking out SLC12A6 could significantly inhibit the proliferation of influenza virus,and after 72 hours of infection,there were still more knockout cell lines.Survive.Finally,we confirmed that knocking out SLC12A6 can broadly inhibit the proliferation of different types of influenza viruses,and inhibit the replication of human influenza H1N1/PR8,avian influenza H9N2/SH13,and swine influenza H1N1/F26 influenza strains to varying degrees.