Regulation Of Ubiquitination Modification And Protein Levels Of Viperin

Objective: Viperin plays an important role in host defense against DNA or RNA viral infection.This study firstly found that it is very limited to induce the expression of Viperin protein during viral infection.Interestingly,viral infection significantly promotes ubiquitiation and degradation of Viperin.To promote Viperin protein expression during viral infection,we plan to identify the Ubiquitin E3 Ligase and the deubiquitinase(DUB)which regulate Viperin protein levels,and further investigate the regulatory mechanism by which the E3 ligase and deubiquitinase affect host antiviral function.Our study aims to uncover new mechanisms of host antiviral defense and to provide potential targets for clinical antiviral therapies.Methods:(1)Lung cancer cells were transfected with Viperin overexpression plasmids or Viperin knockdown plasmids,and then were infected with VSV,or SeV,or H1N1 virus.The viral RNA and protein levels were analyzed by quantitative RT-PCR or Western Blot;Mouse lung tissues or cells infected with viruses were collected and then the protein levels of Viperin were tested by Western Blot;Meanwhile,the protein levels of Viperin and the ubiquitination of Viperin regulated by viruses were investigated by immunoprecipitation and Western Blot.(2)Cells were transfected with Flag-Viperin overexpression plasmids.Then the proteins interacting with Viperin were identified by Mass Spectrum(MS)to screen the related E3 ligase.The protein levels of Viperin and the ubiquitination of Viperin regulated by UBE4 A were evaluated by immunoprecipitation and Western Blot.Cells were infected with viruses,and the interaction between UBE4 A and Viperin protein was explored by Western Blot.(3)We designed peptides interfering UBE4A-Viperin interaction.Cells were treated by peptides and the interaction between UBE4 A and Viperin was analyzed.The protein levels of Viperin and the ubiquitination of Viperin were detected.Furthermore,the mice were injected intraperitoneally with VSV-GFP virus.Then the survival curve of mice was observed,and the VSV viral load was measured by qRT-PCR.(4)The DUB library was used to transfect cells,and the DUB interacting with Viperin was screened by MS and Western Blot.The protein stability of Viperin and the ubiquitination of Viperin regulated by USP33 was identified by MS and Western Blot.Cells transfected with USP33 or shUSP33 were infected with viruses and the antiviral defense mediated by interferon signaling pathway was analyzed.(5)Screening from common ionic compounds by Western Blot,we identified NaCl as a factor which could regulate the protein levels of USP33.Cells were treated with NaCl and then collected to detect the protein levels of USP33,Viperin,and the ubiquitination of Viperin by immunoprecipitation and Western Blot.In vivo mice experiments: we investigated the effect of High Salt Diet on host antiviral abilities.Results:(1)Realtime qPCR and Western Blot results showed that viruses could induce the degradation of viperin via UPS pathway.(2)The MS result showed that UBE4 A could interact with Viperin.UBE4 A increased the ubiquitination level of Viperin and induced Viperin degradation.Viruses increased protein levels of UBE4 A which inhibited the expression of Viperin protein.In addition,UBE4 A promoted Lys6-linkages of Viperin on lysine 206 residue.(3)Peptides were designed to interfere with UBE4A-Viperin interaction.We found that obviously peptide 3# could stabilize Viperin by inhibiting Viperin ubquitination.In vivo experiments showed that peptide 3# could promote innate antiviral immune responses.(4)Deubiquitinase USP33 could stabilize Viperin by inhibiting the ubiquitination level of Viperin.USP33 promoted the IFN-mediated antiviral defense.(5)Screening results indicated that NaCl could decrease the protein levels ofUSP33 and inhibit Viperin protein expression by promoting its ubquitination.In vivo experiments of mice showed that NaCl inhibited antiviral activity by regulating USP33.Conclusion: The protein expression of Viperin could be seriously restricted by UPS pathway.Viruses can increase UBE4 A protein levels which results in Viperin ubquitination and degradation,and inhibits host antiviral defense.USP33 stabilizes Viperin protein levels by deubiquitinating effects,and therefore promotes host ativiral defense.NaCl could decrease USP33 protein to inhibit host antiviral activity.