Effects Of The Biological Characteristics In Influenza Virus With M2 Protein And Autophagy-related Protein LC3

Influenza A virus is one of envelope virus that need budding to release particles in its life cycle.There many studies have shown that M2 protein plays an important role in budding of Influenza A Virus.A recent study has been proved that M2 protein hijack an Autophagy-associated protein called LC3,and relocate it at the cellular membrane.Besides that,there is a LC3 interact region(LIR)in the cytoplasmic tail of M2,mutation of LIR that eliminate the interaction between the two proteins will affect the morphology and stability of the mutant virus.However,the molecular mechanism is not clear,and how the LIR region of M2 protein and LC3 protein affect the morphology and stability of the progeny virus in the process of influenza virus budding is not clear.In order to explore the role of LC3 protein in replication,morphology and stability of influenza virus,in this study,we constructed an LC3 knocked out A549 cell line(A549-LC3KO)by crispr-cas9 technology.By comparing the replication of influenza virus in A549 and A549-LC3 KO,the shape of virus particles and the tolerance to TPCK-trypsin,the results showed that influenza virus replicated in A549-LC3 KO cell was slower than that in A549,indicating that LC3 protein played a role in the replication of influenza virus.Further results showed that the virus particles propagated in A549 cells were round,uniform in size and intact in viral membrane,while the virus particles reproduced in A549-LC3 KO were uneven in shape and size.However,the tolerance of the two viruses to TPCK-trypsin treatment was similar,and there was no significant difference.The results showed that LC3 protein had some effect on the replication,morphology and size of influenza virus,but had no effect on the stability of the virus and he tolerance of exogenous pancreatinThe LIR domain of M2 protein interacting with LC3 protein is a hydrophobic domain.To further study the influence of LIR region of M2 protein on virus replication,morphology and stability,the amino acids(FVI)in LIR region of M2 protein were mutated into hydrophilic amino acids(SSS)or hydrophobic amino acids(AAA),respectively,Two mutant viruses M2-FVI/AAA and M2-FVI/SSS were rescued by reverse genetic technology,and their growth characteristics,TPCK-trypsin tolerance,virus morphology and pathogenicity were evaluated.The results showed that the replication ability of the two mutant viruses and wild virus on MDCK cells was similar to that of wild virus.The results of TPCK-trypsin tolerance test showed that M2-FVI/AAA and wild strain were tolerant to pancreatin treatment,M2-FVI/SSS was sensitive to TPCK-trypsin treatment,and the internal protein was digested and degraded by TPCK-trypsin,suggesting that the envelope structure of the virus might be incomplete.Further electron microscopy of the virus particles showed that there was a defect in the envelope of M2-FVI/SSS virus,which was the cause of the virus' s intolerance to TPCK-trypsin treatment,which also led to the decline of virus stability.The results of pathogenicity experiment also showed that the M2-FVI/SSS was significantly attenuated in mice,and the replication ability in mice lung was also significantly reduced compared with M2-FVI/AAA and wildtype virus.It is suggested that the hydrophobicity of LIR region of M2 protein is very important for the outer membrane integrity and pathogenicity of influenza virus particles.In this paper,we studied the influence of LIR domain of LC3 and M2 protein of influenza A virus on the biological characteristics of influenza A virus.It was found that the hydrophobicity of LIR region of M2 protein plays an important role in the integrity of envelope and pathogenicity of influenza virus.