Study On The Mechanism Of Activating Fcγ Rs Inhibiting The Antiviral Innate Immunity During PRRSV-ADE Infection

Since it was introduced into China from the United States in the 1990 s,porcine reproductive and respiratory syndrome virus(PRRSV)has spread rapidly in pig herds all over the country.Meanwhile,because it is difficult to prevent PRRSV infection,the Chinese swine industry has suffered incalculable economic losses over the years.Therefore,PRRSV has been one of the most important animal viruses in the field of veterinary medicine.Although the most effective way of preventing PRRSV infection still was the immunization with commercial vaccines,the clinical effect of immunization was unsatisfactory so far.Except for the genetic diversity of PRRSV itself,ADE effect may be another major factor reducing the clinical immune effect of commercial PRRSV vaccines.The ADE effect is that virus-antibody immune complex could enhance the infection and replication of PRRSV at sub-neutralizing antibody levels or in the presence of non-neutralizing antibodies.In addition,several studies also showed that the ADE effect might also aggravate clinical symptoms of PRRSV infected pigs.Obviously,the ADE effect could be a huge obstacle for the development of clinical immunization schedules and the high-efficiency PRRSV vaccines.Therefore,it is particularly important to comprehensively and deeply explore the mechanism of ADE of PRRSV infection.Then,based on proteomic characterization of PAMs in PRRSV-ADE infection,the new mechanism of PRRSV-ADE infection inhibiting the antiviral innate immune response in PAMs was comprehensively explored in this study.In this study,we firstly performed the analysis of proteomic characteristics of the PAMs in the early period of PRRSV-ADE infection by the tandem mass tag(TMT)labeled liquid chromatographytandem mass spectrometry(LC-MS/MS).The results showed that 6813 proteins were identified and quantified by the unique peptide.Based on t-test(P<0.05)and the fold change of PRRSV-ICs/PRRSV-NI ratio(greater than 1.2 or less than 0.87),a total of 3935 differentially expressed proteins(DEPs)including 2004up-regulated expressed proteins and 1931 down-regulated expressed proteins were obtained.Subsequently,the nine selected nine DEPs associated with the antiviral innate immune response were verified by RT-q PCR and/or Western Blot,and the results were consistent with those selected from the proteomic data.Therefore,it suggested that the proteomic data were accurate and reliable in this study.Based on GO function annotation,KEEG pathway annotation and PPI analysis,we found that the expression of ribosome large/small subunits and proteasome complex subunits were significantly decreased,but the expression of mitochondrial respiratory chain complex subunits were significantly increased in PAMs with PRRSV-ADE infection.Furthermore,it was worth noting that the expression of multiple molecules associated with the antiviral innate immune response also were significantly decreased in PAMs with PRRSV-ADE infection in this study.It suggested that PRRSV-ADE infection could strengthen the function of mitochondrial,and inhibit function of the ribosome,the ubiquitin proteasome pathway and the antiviral innate immune response.In order to investigate the inhibition mechanism of antiviral innate immunity during PRRSV-ADE infection,we explored the expression changes of multiple molecules associated with TLR3,RIG-I and MDA-5 mediated innate immune signals and antiviral proteins in PAMs with PRRSV-ADE infection in this study.The results showed that the expression of RIG-I and MDA-5 in PAMs of the PRRSV-ICs group was significantly decreased,compared with the PRRSV-NI group.Significantly,there was no obvious difference in the expression level of MDA-5 between the PRRSV group and the PRRSV-ICs group.Therefore,the increased expression of MDA-5 in PAMs of the PRRSV-NI group may be attributed to the stimulation of pig negative Ig G.Furthermore,there was no obvious difference for the expression of TLR3 between the PRRSV-ICs group and the PRRSV-NI group.Simultaneously,compared with the PRRSV-NI group,the expression levels and phosphorylation levels of TBK-1,IRF-3 and p65 were also significantly decreased in PAMs of the PRRSV-ICs group.Then,the expression levels of IFN-α and TNF-α were also significantly down-regulated during PRRSV-ADE infection.In subsequent experiments,we found that the expression levels of STAT-1,STAT-2,ISG15,ISG56,OAS1,OAS2,Mx1 and RSAD2 in PAMs of the PRRSV-ICs group were also significantly decreased,compared with the PRRSV-NI group.To sum up,it suggested that PRRSV-ADE infection could inhibit the RIG-I mediated the innate immune response.To investigate the mechanism of Fcγ RI and Fcγ RIII inhibiting RIG-I mediated the antiviral natural immune response.We analyzed the effects of Fcγ RI、Fcγ RIII and γchain on RIG-I mediated the antiviral natural immune response in PAMs during PRRSV-ADE infection by the RNA interference technique.The results showed that the expression level of RIG-I and its downstream signaling molecules were significantly up-regulated,and the expression of STAT signals and antiviral proteins were also significantly increased in PAMs after PRRSV-ADE infection when the expression of FcγRI、Fcγ RIII or γ chain was inhibited.Subsequently,we found that the expression level of FGR was significantly up-regulated during PRRSV-ADE infection,while the expression level of FGR was also down regulated when Fcγ RI、Fcγ RIII or γ chain were knocked down in this study.Moreover,Co-IP analysis indicated that there was an interaction between FGR and Fcγ RI,which indicated that the activated Fcγ RI may transmit negative regulatory signals by recruiting FGR during PRRSV-ADE infection.In addition,we also found that the expression of RIG-I and its downstream key molecules were significantly increased,and the expression of antiviral proteins were also significantly increased in PAM with PRRSV-ADE infection when the expression of FGR was inhibited in this study.When FGR and RIG-I co-expressed in HEK-293 T cells,FGR significantly inhibited the expression of RIG-I in a dose-dependent manner.The Co-IP analysis also showed that FGR could interact with RIG-I and TBK-1.It indicated that FGR could inhibit RIG-I mediated antiviral innate immune response in PAMs with PRRSV-ADE infection.In conclusion,the above results indicated that the activating Fcγ RI could inhibit RIG-I mediated antiviral innate immune response in PAMs with PRRSV-ADE infection by up-regulating and recruiting FGR.This study revealed a new mechanism of PRRSV-ADE infection inhibiting antiviral innate immunity,and provided important information for the development of clinical immunization procedures and the novel and high-efficiency PRRSV vaccines.