Inhibitory Roles Of Chicken CARD11 Protein In Newcastle Disease Virus

Newcastle disease(ND)caused by Newcastle disease virus(NDV)is an avian-specific infectious disease that poses a major threat to the poultry health.NDV can cause viral encephalitis and neurological disorders,clinical symptoms such as paralysis of the legs or wings and retractable head,pathologic changes such as congestion of blood vessel,glial cell aggregation and peripheral lymphocyte invasion(vascular cuffing)and so on.However,the mechanism between NDV and avian CNS is largely unknown.The previous study in our laboratory first employed a gene microarray to preliminarily analyze the differentially expressed genes(DEGs)in the NDV-infected chicken brains.Based on these gene microarray data,a host gene CARD11 screened from immune-related genes was found to be upregulated that was not found in other transcriptome data of NDV-infected chicken tissues and cells.Preliminary experiments indicated that CARD11 was brain-specific upregulated and could inhibit the viral replication and cytopathic effects(CPE)caused by NDV.This study aims to elucidate the mechanism of CARD11 inhibiting to NDV that provides the interaction between host and virus as well as a valuable clue of the viral neuropathogenesis.The main contents and results of this study included the following several aspects:1.Effects of CARD11 on NDV replication in chicken primary neuronal cellsThe NDV virulent strain F48E9 infected the chicken primary neuronal cells(chPNCs)caused CPE and died quickly,while chPNCs infected with avirulent strain LaSota showed no apparent CPE.All the NDV strains were replicated well in chPNCs.We analyzed statistically the numbers of up-regulated and down-regulated DEGs in gene microarray data by fold change > 2(FC>2)and P<0.05 for fourteen immune-related DEGs of F48E9 infection.The relative mRNA expression of five immune-related DEGs was verified by qPCR that was consistent with the gene microarray data.Only CARD11 gene was brain-specific upregulated expression in the F48E9-infected thirteen tissues.Due to the low expression of CARD11 protein in chPNCs,we established the immunoprecipitation enrichment(IPE)method to detect CARD11 protein and found that the F48E9 was capable of inducing high expression level of CARD11 protein in chPNCs.Using a recombinant adenovirus expression system,CARD11 was able to inhibit NDV replication in CARD11-overexpressed chPNCs.The depletion of CARD11 by specific shRNA resulted in decreasing viral replication.These results indicated that CARD11 could inhibit NDV replication in chPNCs.2.The mechanism of CARD11 in the inhibition of NDV replication in chicken primary neuronal cellsAfter CARD11 activation,Bcl10 and MALT1 are recruited to form CBM signalosome.The MALT1 activates the downstream signaling pathways including NF-?B,JNK and mTOR.To reveal the mechanism of CARD11 in inhibiting NDV replication,CBM signalosome and NF-?B pathway were blocked by an irreversible inhibitor of MALT1 and inhibitors of NF-?B in chPNCs.We found that the signaling pathways of CBM signalosome have no effect on inhibiting viral replication.Whether does CARD11 inhibit NDV replication via direct interaction with viral protein? We found that CARD11 could coimmunoprecipitated with viral P protein.The CC1 domain of CARD11 interacted with the X domain of P protein.Further studies showed that the X domain could also bind to L protein,but not bind to NP protein.There is a competitive interaction of CARD11 and L protein with P protein.Using a minigenome system,this competitive interaction led to the inhibition of viral RNA polymerase activity and further viral replication.By intracerebrally injecting with rAdV-CARD11 into the chicken brains,CARD11 could alleviate the neuropathological lesions caused by NDV and reduce viral replication in the brains.3.Effect of CARD11 on NDV replication in chicken embryo fibroblastsCARD11 could express in every chicken tissues.There is a question whether CARD11 can be induced by NDV and inhibit NDV replication in non-neural cells.Using chicken embryo fibroblast cell line(DF-1)as a non-neural cell model,both velogenic and lentogenic NDV cannot induce significant expression of CARD11 mRNA and protein.CARD11 could also co-locate with viral P protein around the nucleus in the infected DF-1 cells.The overexpression and knockdown of CARD11 stable DF-1 cell lines were established by lentivirus system.After NDV infection,CARD11 could inhibit viral replication and syncytium formation.The fusion activity induced by co-expression of the viral HN and F was also significantly suppressed.How does CARD11 inhibit NDV-induced membrane fusion activity? The expression of furin was inhibited and the cleavage efficiency of F protein was reduced in the CARD11-overexpressed cells.Using MALT1 and NF-?B inhibitors,we found the enhancement of fusion activity and increasing expression of the cellular protease furin.These results suggested that CARD11 inhibited the membrane fusion activity of NDV via the activation of CBM-NF-?B pathway.4.Other avian neurotropic viruses induce CARD11 brain-specific upregulated expression.Avian neurotropic viruses in addition to NDV include avian encephalomyelitis virus(AEV)and so on.Whether is the brain-specific upregulation of CARD11 universally induced by neurotropic viruses in chickens? AEV strain XY/Q 1410 as neurotropic viruses,and IBV strain H09 and FAdV-4 strain SXD15 as non-neurotropic viruses were used to infect chickens for observing the clinicopathological changes.We found that CARD11 mRNA expression were only upregulated in the cerebrum and cerebellum of NDV or AEV infected chickens,but not by IBV and FAdV-4 infections.CARD11 expression was not related to viral loads in the different tissues.These results preliminarily implied that CARD11 could be a potential biomarker of avian viral encephalitis.In conclusion,this study is the first to reveal that CARD11 can effectively inhibits NDV replication and CPE formation.Two mechanisms were suggested:(1)In neuronal cells,host CARD11 and viral L protein competitively bind to the viral P protein resulting in a reduction of viral polymerase activity and inhibition of viral replication;(2)In chicken embryo fibroblasts of non-neuronal cells,CARD11 inhibits membrane fusion activity of NDV by reducing the expression of host protease furin through an CBM-NF-?B pathway that reduces the cleavage efficiency of viral F protein.Meanwhile,the interaction between CARD11 and viral P protein also inhibits viral replication.This study further found that CARD11 was brain-specific upregulation in other avian neurotropic viruses,preliminarily indicating that CARD11 could serve as a potential biomarker of neurotropic virus invaded in CNS.This research provides a basis for the further exploration of the interaction between neurotropic viruses and CNS as well as a potential antiviral target.