The Role Of Pseudorabies Virus Tegument Protein UL13 In Regulation Of RLR-Mediated Signaling Pathway

Pseudorabies virus(Pseudorabies virus,PRV)is susceptible to a variety of animals,including livestock,poultry and wild animals.Among them,adult pigs are prone to respiratory diseases after infection,causing huge economic losses to the pig industry.Studies have shown that PRV can establish latent infection in pigs.In addition,it is reported that It is reported that multiple proteins of herpes viruses(such as some proteins of HSV-1 and HSV-2)can participate in the regulation of the RLR signaling pathways,whose recepetors in this signaling pathways recognize RNA viruses,thereby achieving immune escape.Since RLR-mediated type ? IFN production plays a pivotal role in elevating host immunity for viral clearance and cancer immune surveillance.However,it is unclear whether PRV is involved in the regulations for the RLR signaling pathway and which PRV proteins are involved in this process.Our preliminary research found that the PRV tegument protein UL13 may be an important immune escape protein of the virus,but the specific mechanisms of its effect on the host cell is still unclear.At present,there are few reports on the mechanism of PRV acting on RLR signaling pathway for immune escape.The tegument protein of PRV is a highly complex structural layer between the nucleocapsid and the virion envelope,which plays a connecting role between the viral nucleocapsid and the viral envelope.These proteins also have the functions of both structural and non-structural proteins.Among them,UL13 is a conserved protein in the herpesvirus family and has threonine/serine kinase activity.Preliminary studies have shown that overexpression of UL13 protein can inhibit the production of type ? interferon.In order to investigate the effect of UL13 on the innate immune RLR signaling pathway and its mechanisms,we used Lentivirus expression vectors to construct HEK 293 T and PK-15 monoclonal cell lines that stably expressing PRV UL13,and verified the expression of UL13 in monoclonal cell lines.Subsequently,using Poly(I: C)transfection and Sendai virus(Se V)infection on control cells and UL13 expressing cells simultaneously,we found that UL13 can significantly inhibit the type ? interferon production and downstream ISG expression which were induced by the RLR signaling pathway activation.Further research found that UL13 expression can reduce the mRNA and protein levels of RLR receptors such as RIG-I and MDA5,and UL13 inhibitory effect on the mRNA level of downstream antiviral molecules have weakened after supplementing RIG-I.Consistent with this,UL13 expression also significantly inhibits the activation of TBK1 and NF-?B,which are molecules of the RLR signaling pathway.This indicates that UL13 may inhibit the innate immune response by targeting the expression of intracellular RNA recognition receptors such as RIG-I and MDA5.The dual-luciferase reporter gene system was used to detect the effect of UL13 on the activity of the RIG-I promoter region.It was found that UL13 can significantly inhibit the activity of the RIG-I promoter region,further illustrating that UL13 can inhibit the gene expression of RIG-I.In order to further investigate the molecular mechanisms of UL13 inhibiting the activation of the RLR signaling pathway,we used a variety of signaling pathway inhibitors to pretreat cells.It was found that UL13 could not inhibit IFN production after U0126 treatment,indicating that the inhibitory effect of UL13 on IFN is related to the ERK signaling pathway.In summary,this study shows that the PRV interstitial protein UL13 can inhibit the activity of the RIG-I promoter region,thereby inhibiting the expression of RIG-I,affecting the protein levels of downstream MAVS and TBK1,and inhibiting the activation of NF-?B,resulting in the expression of downstream IFN and ISG reduced.And the inhibition of UL13 on interferon is related to the activation of ERK signaling pathway.Therefore,this study perliminarily explored the inhibitory effect of the PRV tegument protein UL13 on the activation of the RLR signaling pathway,providing new ideas for the further study of PRV opposes innate immune response.