Mechanisms Of How Viperin Promotes The Infection Of CVB3

Background and objective: The innate immune system is believed to be the first line of defense against invading viruses.Viral infection activates the cellular signaling pathway,leading to the release of cytokines,such as IFNs.IFNs plays a vital role during viral infection.The interferon family binds with specific receptor,and then activates IFNs signaling pathway,thus triggering expression of thousands of interferon stimulated genes(ISGs).These ISGs execute multiple biological functions,including inhibition of viral replication.However,large gap still be remained in understanding of the precise mechanism.Only a few ISGs have been studied in depth,including Viperin.Viperin,also known as RSAD2 and cig5,has been first identified as a HCMV-inducible gene in fibroblasts.Viperin induction has been demonstrated via stimulation with ds DNA,poly I:C,LPSs and multiple viruses,which executes broad spectrum antiviral activity.Viral myocarditis(VMC)is the most typical type of myocarditis,mainly caused by type 3 of Coxsackievirus B group virus(CVB3),which pertains to the Enterovirus of Picornaviridae.VMC is a common cardiovascular disease,and the incidence of VMC has been increasing in recent years,which is a serious threat to the health of infants and children.However,the relationship between Viperin and CVB3 replication has not been reported.Accordingly,it's quite meaningful that we focus on the function and mechanism of Viperin during CVB3 infection.Methods:(1)Cells were transfected with Viperin knockdown plasmid and then infected with CVB3.CVB3 expression was detected by Western Blot(WB)or quantitative Real-time PCR(q RT-PCR).(2)The interaction between Viperin and STAT1 has been studied by mass spectrometry(MS)or immunoprecipitation(IP)and Western Blot or immunofluorescence.(3)Mapping: Construction of STAT1 and Viperin deletion mutants;Mutantswere transfected into cell,then the interaction between Viperin deletion mutants and STAT1 deletion mutants was analyzed by IB and WB;Meanwhile,the effects of Viperin deletion mutants on STAT1-WT protein levels were evaluated by Western Blot.(4)Cells were transfected with Viperin overexpression plasmid.Then the ubiquitination of STAT1 was assessed by immunoprecipitation and Western Blot.(5)The interaction between Viperin and E3 ligase was studied by mass spectrometry or immunoprecipitation.Furthermore,the effect of E3 ligase on STAT1 protein level and ubiquitination was analyzed by IP-IB.(6)Cells were infected with CVB3,and then the correlation between STAT1 and Viperin was evaluated by Western Blot.The interaction between Viperin,STAT1 and UBE4 A was assessed by immunoprecipitation and Western Blot.Results:(1)Western Blot or quantitative Real-time PCR showed that sh Viperin limits CVB3 infection.(2)Viperin physically interacts with STAT1 and down-regulates STAT1 protein levels.(3)Mapping: SAM domain of Viperin is bound to C-terminus of STAT1;SAM domain of Viperin promotes STAT1 protein degradation.(4)Viperin induces STAT1 ubiquitination modification.(5)MS or IP-IB showed that Viperin interacts with E3 ligase UBE4 A and STAT1;Viperin facilitates the interaction between STAT1 and UBE4 A,and promotes STAT1 ubiquitination and degradation.(6)In CVB3-infected cells,there is a negative correlation between STAT1 and Viperin protein;Knockdown of Viperin inhibits the interaction between STAT1 and UBE4 A during CVB3 infection.Conclusion: Viperin increases the interaction between UBE4 A and STAT1,thus promoting STAT1 ubiquitination and degradation,and finally restricting host antiviral immunity against CVB3.