Screening And Preliminary Study Of Crucial RNA-binding Proteins In The Life Cycle Of Influenza Virus Infection

Influenza virus is a highly contagious pathogen of zoonotic diseases,with high morbidity and mortality worldwide,and poses a serious threat to global public health security.At present,the only way to control seasonal influenza outbreaks around the world is to use vaccines.However,due to the high mutation rate of the virus strains,the coverage and effectiveness of vaccines have been greatly reduced,making influenza infection a continuing public health problem.Influenza virus is a negative-stranded RNA virus with envelope and gene component segments,and each of its genome segments and encoded proteins has a certain role in the replication process.When influenza virus invades and infects host cells through receptor-mediated endocytosis,it usually induces a series of complex cellular responses in the host cell.The replication of influenza virus requires the use of the host's transcription and translation systems.Therefore,the host proteins play an extremely important role in the life cycle and pathogenicity of influenza virus.Therefore,this study carried out relevant screening and verification of key host proteins that interact with viral RNA during the life cycle of influenza viruses.Through siRNA silencing technology,q-PCR,Western blot,and half-cell infection experiments,the effect of RNA binding protein(RBP)on influenza virus infection and replication was verified.This research includes three parts:1.Influenza A virus was used to infect human lung cancer cell lines(A549)and human mononuclear macrophages(THP-1)cultured in vitro.Through in vitro vRNA-pull down combined with mass spectrometry test methods,the key factors in the life cycle of influenza virus RNA binding protein(RBP).The experimental results show that there are 69 RBPs that can interact with 3 or more viral RNA(vRNA)fragments of influenza virus in the life cycle of influenza virus.2.Through siRNA silencing technology,q-PCR,Western blot and half of the cell infection experiment,the key RNA binding protein(RBP)screened out was further explored.The results showed that the four proteins of DHX9,IFI27,RBBP6 and SRSF5 in the 69 RBPs screened had obvious effects on influenza virus.3.The half-cell infection test and Western blot were used to further verify the effect of SRSF5 on influenza virus replication and infection.The verification results show that knocking out SRSF5 gene can significantly inhibit influenza virus infection and replication.In summary,this study used relevant experiments to screen and verify the host proteins that interact with vRNA during the influenza virus life cycle,in order to further provide a research basis on the impact of these RBPs on the host or viral gene expression after influenza virus infection and their binding mechanism in regulating influenza influenza virus infection.