The Effects Of Alternatively Spliced Porcine FcγRI On Antibody-dependent Enhancement Of PRRSV Infection

Porcine reproductive and respiratory syndrome(PRRS),also known as “blue ear” disease,is caused by highly contagious PRRS virus(PRRSV),resulting in great economic losses in the swine industry worldwide.Antibody dependent enhancement(ADE)of PRRSV is an important cause of the persistent viral infections,immune failure and poor vaccine effectiveness.Fcγ receptors(FcγRs)are membrane receptors highly expressed on porcine alveolar macrophages(PAMs),which are target cells of PRRSV.FcγRs mediate ADE of virus through binding to the virus-antibody immune complex(IC)and participate in the regulation of intracellular signal and inflammatory response.FcγRs have several different isoforms,including FcγRI,FcγRII,FcγRIII and FcγRIV,and previous studies have identified the alternative splicing(AS)of FcγRs in human and mouse.However,little is known about the AS of porcine FcγRI,and the involvements of which in pathogenesis of PRRSV ADE infection remain unclear.To uncover the effects of alternatively spliced FcγRI on PRRSV ADE infection,we analyzed the different mechanism of membrane or soluble FcγRI in endocytosis,antigen presentation and pro-inflammatory effects through a virus-effected cell model in vitro.Together the characteristics of cell responds in ADE of PRRSV infection were also explored.These results provided evidences for understanding of the mechanism of PRRSV ADE,the characteristics of immune response,and the unique pathogenic and immunological mechanisms of PRRSV infection.The main results were listed as follows:(1)There are many kinds of alternatively spliced transcriptions of porcine FcγRI: We identified the mechanism of alternative splicing of porcine FcγRI by sequencing and GenomeScan assay,and reported six different isoforms including two membrane FcγRI and four soluble FcγRI lacking the transmembrane region,which were generated by 3’ alternative splicing mechanism.The alternative splicing of FcγRI was commonly found in primary PAMs according to the 125 clones from three different porcine individuals and 3D4/21 cell line by single-genome amplification(SGA)and highresolution melting(HRM)analysis.Here we showed that the membrane FcγRI was dominantly expressed on PAM cells,which accounts for 74%~91% of the total FcγRI.(2)Porcine membrane FcγRI participate in the internalization of PRRSV-IC and mediate PRRSV ADE: The membrane-and soluble-FcγRI eukaryotic expression vectors were respectively transfected into PRRSV susceptible 3D4/21 cells or notsusceptible 293 T cells.By using blocking of FcγRI antibody,virus load detection and confocal assay,the cellular immune response and different function of membrane or soluble FcγRI in ADE of PRRSV infection were studied.Polyclonal antibody against porcine FcγRI was obtained in rabbit after the immunization with FcγRI-GST fusion protein.After blocking of FcγRI antibody,we found FcγRI was involved in ADE of PRRSV.In this present work,qRT-PCR,immunoblotting and confocal analysis showed overexpression of membrane FcγRI but not soluble FcγRI could enhance infection of PRRSV-IC in PAM and 293 T cells.Results of confocal assay illustrated that the membrane FcγRI were synergistically facilitated entry of PRRSV-antibody complexes into PAMs through stimulating the expression and recruitment of F-actin,and the formation and transportation of mature lysosomes.Instead of enhancement of ADE,soluble FcγRI up-regulated IFN-β,TNF-α and IL-1β,and promoted processing and presentation of antigen by MHC I and II,resulting in promotion of inflammatory reaction and negative regulation of the PRRSV ADE.However,membrane FcγRI inhibited the activation of TLR7 and down-regulated the production of interferons.(3)The expression of soluble FcγRI on PAMs was enhanced by PRRSV,and ADE of PRRSV infection not only up regulated FcγR-mediated phagocytosis and altered immune response,but also induced proliferation of specific TCRVβ cells.The expression of FcγR on 3D4/21 cells were measured by qRT-PCR after PRRSV infection with or without PRRSV specific antibodies.FcγRI but not FcγRIIB was up-regulated in PRRSV infected 3D4/21 cells whereas in the PRRSV specific antibodies treated cells,FcγRIIB were up-regulated four-fold after PRRSV infection,along with inhibition of FcγRI.Alternative splicing of genes was changed in PRRSV infected cells,and the proportion of soluble FcγRI was increased from 19.5%(before PRRSV infection)to 33.3%(after PRRSV infection)by using Single-genome Amplification and Highresolution melting(SGA-HRM)method.According to the RNA-seq assay,we discovered that ADE of PRRSV infection enhanced FcγR-mediated phagocytosis,broken the balance of FcγR on PAMs by up-regulated FcγRIIB gene expression,resulting in suppression of antiviral immune responds.Verified by RT-PCR method,the activation of TLR7,RIG-I/ MDA5 and transcription of IL-1β、TNF-α、IFN-β and MHC involved in antiviral responds were all repressed by PRRSV ADE in vitro,but the Th2 cytokine IL-4 was up-regulated.By construction of the TCRVβ gene library(90 clones),we further confirmed a significant increase of PRRSV-specific T cell types,pTVB11,by SGA assay.Taken together,we identified six alternatively spliced porcine FcγRIs,including two membrane and four soluble FcγRIs;ADE of PRRSV infection facilitated the attachment and internalization of the virus onto PAMs through membrane FcγRImediated endocytosis;the characteristics of immune response against PRRSV were studied,presenting as up-regulation of soluble FcγRI,activation of Th2 cells,but decrease of the inflammatory factor secretion and antigen presentation induced by ADE;furthermore,we confirmed a significant increase of PRRSV-specific T cell types,pTVB11.