Molecular Mechanism Of Host Protein STRAP To Inhibit PRV Replication

Pseudorabies(PR)is a severe infectious disease caused by pseudorabies virus(PRV),which is mainly characterized by reproductive disorders in breeding pigs and encephalomyelitis in piglets.Like herpes virus,PRV can form latent infection after infecting the host,establish persistent infection,and can break through the placental barrier,causing stillbirth,mummified fetus and other phenomena,resulting in extremely high mortality and seriously affecting the healthy development of the global pig industry.In recent years,PRV has produced some highly pathogenic mutant and virulent strains,which cannot be effectively protected by conventional vaccines,and there have been reports of PRV infection in humans in recent years.Pseudorabies virus(PRV)belongs to the α-herpes virus,which infects a wide range of hosts,and porcine is its natural host.The latest study found that people infected with PRV developed severe encephalitis symptoms,which aroused people’s concern about the cross-species transmission of PRV.Like other herpes viruses,PRV can establish latent infection in the body.Although there is a lot of research on PRV,there is no effective treatment and vaccine to effectively control the circulating strains of PRV.Therefore,it is particularly important to screen out host proteins that can effectively inhibit PRV replication.This study aims to screen and explore the molecular mechanism of host proteins inhibiting PRV replication.The main findings are as follows:1,Screening and identification of STRAPFirstly,the cell samples before and after PRV infection were analyzed by TMT quantitative proteomics,and 241 differentially expressed proteins were screened out,including 79 down-regulated proteins and 162 up-regulated proteins.Through RTq PCR and Western blot verification of differential proteins,four host proteins were initially screened out.Through overexpression and gene silencing methods,the host protein STRAP that inhibits PRV replication was screened out.Further through plaque assay experiments,IFA and other experiments confirmed that STRAP can significantly inhibit PRV replication.2,Molecular mechanism of STRAP inhibiting PRV replication(1)By RT-PCR and Western blot detection,it was found that the expression of STRAP was significantly up-regulated at different MOI of PRV infection and at different time of PRV infection.Nucleocytoplasmic separation detection found that STRAP was mainly distributed in the cytoplasm.(2)It was found by RT-q PCR and ELISA that STRAP could significantly promote the production of IFN-α/β.It is preliminarily shown that STRAP plays an anti-PRV role by enhancing the effect of type I IFN.Further detection by Western blot revealed that STRAP can promote the phosphorylation of TBK1 and IRF3,and the nuclear import of IRF3.Confirmed that STRAP can promote the activation of type I IFN pathway.(3)In order to further verify the molecular mechanism of STRAP promoting the type I IFN pathway,the targets of STRAP were screened by dual fluorescein reporter genes.The results showed that the overexpression of STRAP can promote the activation of IFN-β promoter activity by STING and TBK1.Co-IP further confirms that STRAP can interact with TBK1.(4)The interaction between STRAP and TBK1 significantly promoted the expression of IFN-α/β and ISGs.These results indicate that STRAP promotes the activation of type I IFN pathway by interacting with TBK1,thereby inhibiting the replication of PRV.(5)By constructing different truncations of STRAP protein,it was found that after the deletion of the WD40 domain of STRAP,the effect of STRAP on inhibiting PRV replication was significantly weakened,and the promotion of IFN-β and ISRE promoter activity by STRAP,as well as the interaction of IFN-β and various Transcription of ISGs.Co-IP experiments further verified that the WD40 domain of STRAP plays an essential role in the interaction between STRAP and TBK1.Therefore,the WD40 domain of STRAP plays an important role in the function of STRAP.In summary,the host protein STRAP screened in this study promotes the anti-PRV effect of type I IFN through the interaction with TBK1.The results of this study are expected to provide ideas for guiding STRAP as a target for inhibiting PRV.