Study On STUB1 Suppressing SVCV Replication In Zebrafish By Promoting The Expression Of RIG-I

Spring viraemia of carp virus(SVCV)is a single-stranded negative RNA virus belonging to the genus Spirivivirus of Rhabdoviridae family.SVCV is a fierce pathogen of spring viraemia of carp(SVC)causing high mortality in the common carp(Cyprinus carpio).At present,there is no effective treatment for SVC.Innate immunity is the host’s first line of defense against microbial pathogens.RIG-I activation plays an essential role in the antiviral immune response.Virus infection can activate the RIG-I signaling and induce the production of interferon(IFN)and the expression of IFN-stimulated genes(ISGs).STUB1(STIP1 homology and U-box containing protein 1)is a highly conserved cytoplasmic protein.This protein is known to exist widely in many biological systems and plays an important role in the process of immune regulation,but little is known in fish.In addition,the previous study of our research group found that Hsc70 protein has antiviral function and can inhibit the proliferation of SVCV.STUB1 is a chaperone of Hsc70,the role of STUB1 in SVCV infection remains to be explored.Zebrafish STUB1 gene was cloned and analyzed in this study and its function was verified at the cellular level,to explore the influence of STUB1 of zebrafish on SVCV and the regulation of SVCV on zebrafish STUB1.The main research results are as follows:1.Zebrafish STUB1 up-regulates interferon and downstream ISG moleculesIn this study,a eukaryotic expression plasmid of zebrafish STUB1 was successfully constructed.Subcellular localization by indirect immunofluorescence showed that STUB1 was located in the cytoplasm.Overexpression experiments were performed in EPC cells,and the up-regulation of interferon and downstream ISG molecules was detected by q RT-PCR,which proved that zebrafish STUB1 could activate the interferon signaling pathway.2.Zebrafish STUB1 activates RIG-I signaling pathway and promotes RIG-I expressionSTUB1 was overexpressed in EPC cells,stimulated with mock stimuli poly(I:C)and poly(d A:d T),and STUB1 was co-transfected with RIG-I,and downstream interferon and ISG15 were found to be upregulated,demonstrating STUB1 activation RIG-I signaling pathway.By co-transfection of STUB1 with RIG-I,MAVS,TBK1 and IRF7,zebrafish STUB1 could promote RIG-I expression by q RT-PCR and Western blot detection.3.Zebrafish STUB1 inhibits SVCV proliferation through the N-terminal TPR domainIn this experiment,STUB1 truncations STUB1-N,STUB1-C and mutant STUB1-N110 S were successfully constructed and overexpressed in EPC cells.The overexpression of STUB1 and STUB1-N was detected by q RT-PCR,Western blot and plaque experiments.Afterwards,the proliferation of SVCV was significantly inhibited,and the antiviral ability of STUB1 was weakened after overexpression of STUB1-N110 S.It is proved that zebrafish STUB1 can exert antiviral function through the N-terminal TPR domain,and asparagine 110 is a key antiviral site.4.SVCV inhibits the expression of STUB1 in zebrafishIn the preliminary study of SVCV immune evasion,the expression of STUB1 could be inhibited by both treatment with simulated stimuli poly(I:C)and poly(d A:d T).and SVCV infection of cellsIn summary,this study proved that zebrafish STUB1 can activate the RIG-I signaling pathway,promote the expression of RIG-I,and induce the production and expression of IFN and downstream ISG molecules,thereby inhibiting the proliferation of viruses.When the host protein STUB1 resisting SVCV invasion,SVCV also inhibits the expression of host protein STUB1 through its own corresponding escape strategy.These findings may provide a basis for the development of host antiviral targets and antiviral drugs in the future,and also lay the foundation for the study of virus escape strategies.