The Molecular Mechanism Of TRIM25 Restriction Against Infectious Bursal Disease Virus Replication

Infectious bursal disease(IBD)is an acute and fatal immunosuppression disease which is caused by infectious bursal disease virus(IBDV),seriously affecting the development of poultry industry.Although the current use of vaccines bring IBD under control to a certain extent,new challenges for IBD prevention and control bring with the emergence of variation of virus strains and the failure of immunity or other problems.Viruses are obligate intracellular parasite,which not only finish the replication cycle relying on the host environment,but induce antiviral activity of the host.Therefore,the researches on the interaction between virus and host can not only clarify the infection process of virus in host cells,but also contribute to the discovery of antiviral host factors.And these antiviral host factors can not only reveal the specific mechanism of regulating virus infection through interacted with virus,but also provide theoretical support for the development of new anti-virus strategies in the future.In order to screen the molecules involved in the host antiviral process,RNA-seq were used to detect the m RNA level of whole genome at 24 h after IBDV infection.3367 host factors were found to be changed upon IBDV infection,of which 1445 were upregulated and 1922 were downregulated.Furthermore,79 annotated genes related to host antiviral response were identified according to the Go function analysis,such as RSAD2(viperin),OASL,Mx1 and TRIM25.TRIM family proteins are a large protein family that significantly inhibited virus replication.For example,TRIM22 up-regulated after avian influenza virus infection can significantly inhibit virus replication,and can be used as an ideal target for developing antiviral products.In addition,RT-q PCR was used to verify the expression level of TRIM25 at different time after infection,the results showed that the expression of TRIM25 was significantly upregulated upon IBDV infection at different time point.Therefore,the study will focus on the molecular mechanism of TRIM25 regulating IBDV replication.It was found that overexpression of TRIM25 could significantly inhibit IBDV replication,and knockdown of TRIM25 could promote IBDV replication,indicating that TRIM25 can act as an antiviral factor to regulate IBDV replication.Immunoprecipitation(Co-IP)showed that TRIM25 could directly target IBDV viral protein VP3.In addition,the results further determined that TRIM25 could degrade VP3 protein through proteasome pathway.For the ubiquitin-proteasome system is the main pathway inducing protein degradation in cells,the ubiquitination assay demonstrated that TRIM25 could promote K27 ubiquitination of VP3.In addition,it was found that TRIM25 Del RING could not promote VP3 ubiquitination and could not inhibit IBDV replication,by constructing TRIM25 mutant with RING deletion(TRIM25Del RING).These results suggest that TRIM25 specifically inhibits IBDV replication by improving the ubiquitination and further degradation of structural protein VP3 depending on the ubiquitin ligase activity mediated through RING domain.In addition,to further explore which of the six lysines on VP3 as protential ubiquitination sites are the targets of TRIM25,we constructed different VP3 mutants for ubiquitination assay.The results found that lysine(K)at position 854 of VP3 was the key ubiquitination site of TRIM25 mediating.When 854 lysine was mutated to arginine(R),the ubiquitination level of VP3 catalyzed by TRIM25 was reduced.By rescuing the r Gt-VP3K854 R mutant virus and rm Gt parent virus,the animal experiment in vivo and cell infection assay in vitro demonstrated the replication ability of IBDV was improved.The results of bursal weight index and pathological section of bursa of fabricius in vivo showed that the mutation of ubiquitination site did not affect the virulence of IBDV.In conclusion,TRIM25 was upregulated upon IBDV infection,and inhibited IBDV replication by directly targeting VP3 for ubiquitination and subsequent degradation.It is also found that Lys854 of VP3 is the key ubiquitination site TRIM25 mediated.And the mutation from Lys854 to Arg854 can reduce the ubiquitination of VP3 and destroy the host's antiviral effect,thereby enhancing the replication ability of IBDV in vivo and in vitro,but not affecting its virulence.Therefore,the study not only elucidated the molecular mechanism of TRIM25 inhibiting IBDV replication,but also provided an important theoretical basis for the development of high titer IBDV live vaccine.