Research On The Mechanism Of SVCV Escaping Host Innate Immunity Through Negatively Regulating The Expression Of Fish Viperin＿sv1

Spring Viremia of Carp Virus(SVCV)is an aquatic virus that causes serious damage to fish species of the Cyprinus carpio family.The break of SVC would cause an acute systemic hemorrhagic syndrome and extremely high mortality rates,ultimately lead to serious economic losses.Severe abdominal inflammation is the main cause of mortality in SVCV-infected fish.However,the research on its pathogenic mechanism and its relationship with host immunity is still unclear.Increasing studies have shown that SVCV can regulate the expression or enzyme activity of various host immune proteins to escape the monitoring and clearance of the host antiviral immune system.On the other hand,SVCV infection induces a host antiviral immune response,which activates the expression of antiviral proteins to inhibit viral replication.As one of the classical antiviral proteins,viperin exists in a wide range of species,and there are many studies on the function of mammalian viperin,but the antiviral function of fish viperin and its relationship with viral infection need to be further explored.A novel splicing variant of viperin＿sv1 was identified in the SVCV-infected FHM cell and named it viperin＿sv1,which plays a much stronger antiviral effect than viperin.More interestingly,SVCV significantly downregulated the protein level of viperin＿sv1 but not viperin.This thesis focuses on the specific mechanism of negative regulation on viperin＿sv1 protein induced by SVCV infection,the identification of host E3 ubiquitin ligases related to viperin＿sv1 protein and the mechanism of innate immunity signaling regulation by viperin＿sv1.These studies aim to investigate and reveal the characteristics of fish viperin＿sv1 in the natural immunity against SVCV and provide the scientific basis for the pathogenesis of SVCV and the products development of SVCV prevention.1.Ubiquitination degradation of viperin＿sv1 protein induced by SVCVThe treatment of different pathway inhibitors and in vivo ubiquitination assay were carried to verify the effect of SVCV infection on viperin＿sv1 protein.The results demonstrated that viperins＿sv1 protein was degraded through the ubiquitin-proteasome pathway during SVCV infection.The material basis of SVCV which induced viperin＿sv1 degradation was investigated by exogenous expressing different viral structural proteins of SVCV and viperin＿sv1,and the SVCV N protein was identified as the key protein that plays the effect on the degradation of viperin＿sv1.The interaction relationship between SVCV N protein and viperin＿sv1 was confirmed by Co-Immunoprecipitation(Co-IP)and indirect immune-fluorescence(IFA).By constructing and co-transfecting different regions truncated mutants and point mutants of viperin＿sv1 followed SVCV infection,the Lys201 was the target site of viperin＿sv1to be ubiquitinated and degraded.In the meanwhile,comparing the antiviral effect of the viperin＿sv1 and its Lysine201 mutant,it was found that the protein level of the Lys201 mutant was no longer degraded during SVCV infection and could exert a stronger and durable antiviral effect than viperin＿sv1.This study reveals a novel defense mechanism for SVCV to inhibit host antiviral response by ubiquitinating and degrading viperin＿sv1.2.TRIM33 negatively regulates viperin＿sv1 to promote SVCV proliferationIn the process of exploring the degradation mechanism of viperin＿sv1,a combination of immunoprecipitation(Co-IP)and mass spectrometry analysis(LCMS/MS)was used to screen host proteins that may interact with viperin＿sv1 in FHM cells.There are three possible E3 ubiquitin ligases that were screened from the results which may participate in the ubiquitination and degradation of viperin＿v1.The cotransfection of E3 ligases and viperin＿sv1 demonstrated that only TRIM33 negatively regulated the protein level of viperin＿sv1,then the Co-IP assay and indirect immunofluorescence assay demonstrated that TRIM33 and viperin＿sv1 were interacted and co-localized to induce its degradation.Besides,truncated mutation of TRIM33 was contrasted and overexpressed to detect the effect of a different region of TRIM33 on the viperin＿sv1 protein expression,the results of immunoblotting experiments confirmed that the N-terminal region of TRIM33 was critical for interacting with viperin＿sv1 protein and inducing its degradation.In addition,SVCV infection significantly enhanced TRIM33 expression,which was found to reduce interferon(IFN)expression and promote viral replication.This study showed that TRIM33 enhanced SVCV replication by inhibiting the expression of the antiviral protein viperin＿sv1 and followed the production of IFN.These findings may provide the foundation for the development of new therapeutic strategies to suppress SVCV infection.3.Viperin＿sv1 regulates the innate immunity signaling pathway through RIG-Ⅰ proteinSVCV infection activates host innate immune responses,including the production of IFN and interferon-stimulated genes(ISGs).The expression of viperin＿sv1 is upregulated after SVCV infection and has been shown to have a positive effect on the regulation of IFN production.However,the underlying mechanism remains unresolved.In this study,the P protein of SVCV was identified to play a critical role in the induction of viperin＿sv1 during viral infection.In addition,viperin＿sv1 was able to trigger the RLR signaling cascade to activate the IFN expression and production.Additional analysis revealed that viperin＿sv1 promotes the stability and function of RIG-Ⅰ,which leads to more production of IFN and ISGs.Moreover,the central SAM domain of viperin＿sv1 was shown to be critical in regulating RIG-Ⅰ protein expression and inducing IFN production.Furthermore,the SVCV replication was confirmed to be inhibited by the SAM structural domain of viperin＿sv1.This study shows that viperin＿sv1 reduces SVCV replication by promoting the stability of RIG-Ⅰ protein.Our results identify the antiviral function performed by the SAM structural domain of viperin＿sv1 and suggest an antiviral mechanism conserved across different species.