The Molecular Mechanism Of Resveratrol Against Pseudorabies Virus Based On IE180 Protein And Its Protective Effect For Brain Tissue Damage Of PRV-infected Mice

Pseudorabies virus(PRV)belongs to Alphaherpesvirinae subfamily,which transcription was regulated in a cascade-like process including three temporally regulated phases: immediate-early(IE),early(E)and late(L).IE180 gene is the only immediate early gene of PRV.When the genome of PRV enters the nucleus,the IE180 gene was immediately transcribed and translated,while the IE180 protein was essential for the transcription of downstream genes.Therefore,the IE180 protein is essential for its own replication,which is considered as an important target for anti-PRV drugs.Resveratrol(Res)is the most well-known polyphenolic stilbenoid,which possessed antiviral,anti-inflammatory and Neuroprotective properties.Previous studies have provided evidence that Res mainly inhibited the early replication stage of PRV in vitro.Based on this,on the one hand,this study investigated the anti-PRV effect of Res on transcription level,protein level and protein transcriptional activation function of PRV IE180 gene.At the same time,the molecular docking was used to simulate the binding of Res and PRV IE180 protein,to obtain and verify the binding amino acid sites,which aimed to reveal the antiviral mechanism of Res against PRV.On the other hand,PRV is a kind of neurotropic virus,its infection can cause inflammation of the nervous system.Moreover,this study evaluated the efficacy of Res on PRV-infected mice and the protected effect of Res on brain tissue damage,which revealed the indirect antiviral effect and mechanism of Res from the perspective of the body,providing a theoretical basis for the clinical application of Res.The main research contents and results are as follows:1.The effect of Res on mRNA expression level of PRV genesThe transcription of PRV genomes follows a strict cascade of regulation whereby immediate early gene IE180 is transcribed first,followed by early and late genes.Therefore,the IE180 protein is required for PRV replication.In this study,the effect of Res on the mRNA expression level of PRV immediate early gene(IE180),early gene(EP0,US1 and UL54)and genes necessary for viral DNA synthesis(UL5,UL8,UL9,UL29,UL30,UL42 and UL52)was determined by RT-PCR.The results showed that Res has no significant effect on the mRNA expression level of immediate early gene IE180 after PRV infection.However,Res can inhibit the mRNA expression level of early gene.These results revealed that Res may affect the expression of PRV IE180 protein,thereby blocking the transcription of downstream genes and inhibiting the replication of PRV.2.The effect of Res on expression of PRV IE180 proteinIn this part,the p IE180 recombinant plasmid was constructed to test the effect of Res on expression of PRV IE180 protein.Firstly,the effect of Res on the expression of PRV IE180 protein in PRV infection and p IE180 recombinant plasmid overexpression were detected by Western blot.Additionally,we examined the effect of Res on the localization of PRV IE180 protein in cells after PRV infection.The results showed that after PRV infection and p IE180 recombinant plasmid overexpression,Res has no significant effect on PRV IE180 protein expression at 4 h,6 h and 8 h,respectively.However,Res can inhibit the distribution of IE180 protein in the nucleus.It suggested that Res can inhibit PRV replication by affecting the transcriptional activation of IE180 protein.3.The effect of Res on transcriptional activation of PRV IE180 proteinPRV IE180 gene can be rapidly transcribed and translated into proteins after infection,and it possesses the function of activating downstream early gene and late gene transcription.Therefore,in this study,the p GL3-TK recombinant plasmid was constructed to detect the influence of Res on this function.The effect of Res on the transcriptional activation of PRV IE180 protein was performed using dual-luciferase reporter assay and RT-PCR.The results showed that the recombinant plasmid p IE180 and p GL3-TK was co-transfected,and p RL-TK was used as the internal reference.It was found that PRV IE180 protein could activate the early gene promoter.However,the ability of PRV IE180 protein to activate the early gene promoter decreased in a dose-dependent manner after Res treatment.Furthermore,after the p IE180 recombinant plasmid was transfected,the mRNA expression of early genes was promoted,while Res treatment inhibited the mRNA expression of early genes.Collectively,it indicated that Res could inhibit the transcriptional activation function of PRV IE180 protein and reduce the activity of its activation of early gene promoters,resulting in the failure of early gene transcription and ultimately blocking the replication of PRV.4.Molecular docking and verification of PRV IE180 protein and ResIn order to excavate the anti-PRV target of Res,binding sites were obtained by simulated docking,and the binding sites were confirmed by site-specific mutation,dual-luciferase reporter assay and RT-PCR.The results showed that the IE180 protein was similarly to ICP4 of herpes simplex virus.Additionally,Thr601,Ser603 and Pro606 are the binding sites of IE180 protein and Res by molecular docking.p IE180Thr601Ala/p IE180Ser603Ala/p IE180Pro606 Ala recombinant plasmid activated the early gene promoter,and Res activated early gene promoter after treatment.Furthermore,transfection p IE180Thr601Ala/p IE180Ser603Ala/p IE180Pro606 Ala recombinant plasmid,Res had no effect on mRNA expression of early genes.These results demonstrated that the target of Res on PRV is Thr601,Ser603 and Pro606 of PRV IE180 protein.After being bond with these three amino acids,the transcriptional activation function of PRV IE180 protein was inhibited,so as to block the activation of downstream early gene transcription and finally achieve the antiviral effect.5.The effect of Res on early PRV infection in miceIn vitro studies revealed that Res could inhibit the transcriptional activation of PRV IE180 protein,thus blocking the downstream early and late gene transcription.To examinate the effect of Res in vivo,the mice model of PRV infection was established.The effect of Res on PRV infection was evaluated by clinical observation,body weight and organ coefficient,viral copy number,viral reactivation analysis and immunohistochemistry.The results showed that the mice infected with PRV at 80 μL1×106 TCID50 for 24 h had no significant effects on body weight and organ coefficient.However,Res decreased the number of viral copies in the brain and spinal cord of mice infected with PRV.Additionally,after PRV infection,viruses retained in brain tissue,spinal cord and trigeminal nerve were able to re-infect PK-15 cells,while the ability of viruses in Res treated mice to infect PK-15 cells in vitro was reduced.Further study showed that Res had no effect on IE180 mRNA expression after PRV infection,but the mRNA expression of early genes was inhibited.What’s more,Res could decrease the distribution of PRV in brain tissue.In summary,these results suggested that Res plays an anti-PRV role by reducing viral load in brain tissue and spinal cord,blocking the reactivation ability of virus,inhibiting mRNA expression level of early genes,and reducing the distribution of PRV in brain tissue.6.The Protective effect of Res on brain tissue injury induced by PRV infection in micePRV is a neurotropic swine alpha herpesvirus causing inflammation of brain tissue after infection.The immunohistochemistry,HE/Nissl/TUNEL staining,RT-PCR and ELISA were performed to detect the effects of Res on brain tissue damage in PRV-infected mice.The results showed that after PRV infection,it could decrease the content of Evans blue in brain tissue,inhibit the expression of MMP-2/MMP-9,increase the expression of ZO-1 by treating with Res.The pathological were alleviated by Res treatment,the Nissl body returned to normal level,and the neuronal apoptosis was reduced.Furthermore,after PRV infection,it can significantly inhibit the expression of pro-inflammatory factors(IL-6,TNF-α)and chemokines(MCP-1,CCL3,CXCL10)by treated with Res.Contrastingly,anti-inflammatory factors(IL-4 and IL-10)and neurotrophic factors(TGF-β and GNDF)the expression levels were augmented.These results converged to suggest that Res administration can ameliorate the permeability of blood-brain barrier.In addition,Res can improve the pathological morphological changes characteristic of brain tissue injury and inhibit the pro-inflammatory response to alleviate the brain tissue injury caused by PRV infection.7.The effect of Res on primary microglia and neurons infected with PRVMicroglia are resident macrophages of the central nervous system that can regulate brain development,injury repair and maintenance of neuronal networks.The effect of Res on primary microglia and neurons infection with PRV were performed by immunofluorescence,flow cytometry and ELISA.These results showed that it has no significant effect on the survival rate of primary microglia when Res concentration< 30 μg/m L.After PRV infecting primary microglia,Res can promote the transformation of primary microglia from M1 to M2 and secret anti-inflammatory factors.Additionally,the Transwell was used to establish primary microglia-neuron co-culture system.The results indicated that Res treatment could inhibit neuronal apoptosis,reduce pro-inflammatory factors expression after PRV infection.Contrastingly,the expression level of anti-inflammatory factors was increased treating with Res.These results suggested that Res can mainly regulate the polarization of primary microglia from M1 to M2 after PRV infection.The anti-inflammatory factors were released to decrease the damage in neurons by M2-type microglia,thus alleviating the inflammation of brain tissue caused by PRV infection.In summary,the anti-PRV mechanism of Res is mainly reflected in two aspects:on the one hand,Res does not affect the transcription level and protein expression level of PRV IE180,but blocks its transcriptional activation function,making inhibition the transcription of downstream early genes.Furthermore,Res could combine with amino acids(Thr601,Ser603 and Pro606)of PRV IE180 protein,resulting in the failure of viral replication.On the other hand,Res treatment can significantly decrease viral load in brain and spinal cord after PRV-infected mice.Additionally,it can improve the permeability of the blood-brain barrier,regulate inflammation and alleviate the pathological injury of brain tissue by treating with Res.What’s more,Res also could influence the polarization process by promoting the inflammatory M2 phenotype on the basis of M1 phenotype,so as to alleviate the neuroinflammatory response caused by PRV infection and achieve the role of antiviral protection of brain tissue.