The Effect Of LRRC59 Gene On Bovine Ephemeral Fever Virus Replication And The Study Of Its Mechanism

Bovine ephemeral fever virus(BEFV)can cause bovine ephemeral fever(BEF).The clinical symptoms of this disease are mainly sudden fever,lameness,temporary infertility,and decreased milk production,which cause serious economic losses to the cattle industry.Currently,the prevention and treatment of BEFV is mainly through vaccination,but vaccination has the limitations of producing low titer of virus and poor continuous protection,so it is necessary to conduct a further study on the molecular mechanism of BEFV replication.Virus infection induces host innate immune response,but viruses also develop a variety of mechanisms to antagonize innate immunity in the process of evolution.The BEFV genome encodes five structural proteins(N,P,M,G,L)and six non-structural proteins(Gns,α1,α2,α3,β,γ).This study focuses on BEFV non-structural protein α1.Previous studies have shown thatα1 protein has the structural characteristics of viroporin,which can increase the membrane permeability of bacteria and cells.At the same time,α1 protein has also been found to bind to nuclear transport receptor proteins importin β1 and importin 7.However,the role of α1 protein in host innate immunity has not been reported.Therefore,we used α1 protein as bait to screen the yeast library.LRRC59 protein was selected for further study due to its high clone number.LRRC59 protein is a member of the LRR domain family protein,which has previously been found to play an important role in mediating the entry of various proteins into the nucleus.In recent years,it has been found that the LRRC59 protein plays an important role in the regulation of host innate immunity and can inhibit the replication of vesicular stomatitis virus(VSV).However,the role of LRRC59 protein in BEFV replication is not clear.Therefore,this study investigated the effect of the host protein LRRC59 in BEFV replication and the mechanism of its interaction with the α1 protein,and the main results obtained were as follows:(1)BEFV α1 protein interacts with host protein LRRC59.The results of the yeast library screening were first validated using yeast cotransformation assay.Then,Co-Immunoprecipitation(Co-IP)and GST-Pull down expriments were performed to further confirm the interaction between α1 protein and LRRC59 protein in vivo and vitro.(2)Overexpression of LRRC59 protein inhibits BEFV replication,while knocking down LRRC59 promotes BEFV replication.The overexpression vector of LRRC59 was firstly constructed,and then the effect of overexpression of LRRC59 on BEFV replication was investigated.The experimental results showed that overexpression of LRRC59 significantly inhibited BEFV RNA level and N protein replication,and the viral titer of BEFV was also obviously reduced.Subsequently,reverse validation was performed using RNAi method,silencing LRRC59 markedly promoted the expression level of BEFV RNA and N protein,and significantly increased the viral titer.(3)LRRC59 promotes antiviral innate immunity by positively regulating the type I interferon signalling pathway.The effect of LRRC59 on BEFV-induced I-IFN signalling was explored by means of gene overexpression and silencing.The experimental results showed that overexpression of LRRC59 significantly promoted the transcription of BEFV-induced interferon-stimulated genes(ISGs)and phosphorylation of TBK1.However,silencing LRRC59 significantly reduced BEFV-induced ISGs transcription and p-TBK1 protein expression.(4)α1 protein antagonizes host innate immunity by interacting with LRRC59 protein.Furthermore,we explored the mechanism of interaction between α1 protein and LRRC59 protein.By investigating the effects of LRRC59 protein and α1 protein on RIG-I(N)-induced I-IFN signal pathway,we found that LRRC59 protein significantly increased the activity of IFN-β promoter and the activation of p-TBK1 induced by RIG-I(N).However,α1 protein significantly inhibited Poly(I:C)induced ISGs transcription and RIG-I(N)-induced TBK1 phosphorylation.Moreover,the cotransfection experiment showed that the promoting effect of LRRC59 on TBK1 phosphorylation induced by RIG-I(N)was significantly inhibited by α1 protein.Finally,the co-immunoprecipitation experiment revealed that α1 protein blocked the interaction between RIG-I(N)and LRRC59 by targeting LRRC59,and then inhibited I-IFN signal pathway.In summary,through the study of the interaction between α1 protein and LRRC59 protein,we found that LRRC59 can inhibit BEFV replication,and elucidated the molecular mechanism by which the α1 protein interacts with the host protein LRRC59,thereby inhibiting the positive regulation of RIG-I(N)by LRRC59 and ultimately antagonizing innate immunity.These findings provide a new strategy for further exploring the pathogenesis of BEFV and the development of antiviral drugs.