Preliminary Study On The Function And Mechanism Of RACK1 Gene In The Replication Of Bovine Ephemeral Fever Virus

Bovine epidemic fever(BEF)can lead to high fever,breathing difficulties,and a decrease of milk production in diseased cows.It causes damage to the development of the cattle industry.At present,the main prevention and control measures against BEF are vaccines,but they still cannot prevent or cure BEF well.There is no specific medicine for this disease.Bovine ephemeral fever virus(BEFV)is the pathogen of BEF.Studying the molecular mechanism of BEFV replication and finding feasible drug targets will provide an important theoretical basis for the prevention and control of BEF.Receptor for activated C-kinase 1(RACK1)is a conserved scaffold protein.It plays a critical role in cell proliferation,apoptosis,autophagy,and other cell activities.Although RACK1 is involved in multiple viral replications,the effect of RACK1 on BEFV replication is unclear.Therefore,this paper mainly focuses on the role of RACK1 on the BEFV replication.(1)RACK1 enhances BEFV replication.An overexpression vector for the RACK1 gene and RACK1-overexpressing stable cell lines were constructed.Then the RACK1-overexpressing stable cell lines were infected with BEFV(MOI of 0.1)for 24 h and viral titers were determined by TCID₅₀.The results suggested that overexpression of RACK1 promoted BEFV replication;At the same time,a silencing vector for the RACK1 gene and RACK1-silencing stable cell lines were constructed.The cell lines were infected with 0.1 MOI BEFV for 24 h and the viral titers were demonstrated by TCID₅₀.It showed that the knockdown of RACK1 significantly inhibited BEFV replication.All these results demonstrated that RACK1 promoted BEFV replication.(2)RACK1 targets MAVS to inhibit the type I interferon signaling pathway.The RACK1-overexpressing stable cell lines were infected with BEFV(MOI of0.1)for 24 h.It was confirmed by RT-qPCR that RACK1 inhibited the expression of IFN-?and interferon-stimulating genes,and negatively regulated the type I IFN signaling pathway;Subsequently,western blotting showed that RACK1 significantly downregulated the expression of mitochondrial antiviral signaling protein(MAVS)but not affected the expression of TANK-binding kinase 1(TBK1)and Interferon regulatory factor(IRF3);The silencing of RACK1 remarkably enhanced the expression of MAVS but not affected the expression of TBK1 and IRF3;Besides,the dual-luciferase reporter assay system experiment found that RACK1 inhibited the activation of IFN-?promoter induced by MAVS.However,IFN-?promoter activity induced by TBK1 and IRF3-5D were not affected by RACK1;Western blotting experiments suggested that RACK1 inhibited MAVS in a dose-dependent manner.These results indicated that RACK1 gene targeted MAVS to inhibit the type I interferon signaling pathway.(3)RACK1 up-regulates the expression of the E3 ubiquitin ligase STUB1,which degrades MAVS via the ubiquitin-proteasome pathway.RACK1 could degrade MAVS,and MAVS expression was reversed after treatment with the proteasome inhibitor MG132.It demonstrated that RACK1 degraded MAVS through the ubiquitin-proteasome pathway;It was predicted that STUB1(STIP1homology and U-box containing protein 1)may be the E3 ubiquitin ligase of MAVS ubiquitination by Ubi Browser.Further studies have found that overexpression of RACK1 up-regulated the expression of STUB1;Western blotting demonstrated that STUB1 inhibited the expression of MAVS in a dose-dependent manner.Treatment with MG132 reversed the expression of MAVS,indicating that STUB1 degraded MAVS through the ubiquitin-proteasome pathway;Western blotting found that the degradation and ubiquitination of MAVS could be augmented by the simultaneous overexpression of RACK1 and STUB1;The immunoprecipitation results showed that STUB1interacted with MAVS,and RACK1 enhanced the binding of STUB1 and MAVS.However,there was no interaction between RACK1 and STUB1,indicating that RACK1 degraded MAVS via promoting the interaction between MAVS and STUB1,thereby enhancing the ubiquitination and degradation of MAVS.In summary,this study firstly discovered that RACK1 may degraded MAVS by up-regulating the expression of E3 ubiquitin ligase STUB1,thereby inhibiting the expression of IFN-?and interferon-stimulating genes,and thus promoting BEFV replication.These findings revealed a new mechanism of the RACK1 gene-regulating BEFV replication through regulating the innate immune response and provided new ideas for the development of antiviral drugs.