Proteomics Analysis Of IBV-infected Chicken PBMCs-M? And The Effects Of STING On ISGs Gene Expressions And Virus Replication In Infected Cells

Avian infectious bronchitis(IB)is a highly contagious respiratory disease with worldwide distribution,caused by infectious bronchitis virus(IBV).IBV belongs to the Coronavirus family,which is a single-stranded,positive-sense enveloped RNA virus.IBV was first discovered and discribed in the 1930s in the United States.The available commercial IB vaccines may not provide powerful cross-immunity and protection because of different serotypes of IBV infections,and lead to clinical outbreak and huge economic losses.Therefore,it is very necessary to study the host immune response mechanisms in IBV infection.Macrophages are important innate immune cells which can resist microbial infections and participate in innate and adaptive immune responses.Macrophages recognize pathogens and subsequently trigger various signaling pathways to produce immune regulatory molecules.Currently,there is no study on the proteomic analysis of IBV-infected macrophages.Interferon stimulating genes(ISGs)have the anti-microbial biological activities.Among them,ISGs genes myxovirus-resistant(Mx),interferon-inducible protein 5(IFIT5),2',5'-oligoadenylate synthase-like protein(OASL)can directly control pathogen infection and multiplication.Stimulator of interferon genes(STING)is an immunomodulatory molecule.STING can regulate the immune response and protect the host in the process of virus infection.This study aimed to establish the IBV-infected chicken peripheral blood mononuclear cells-derived macrophages(PBMCs-M?)model,use proteomics to analyze the differentially expressed proteins post IBV infection and quantify the target proteins,and clarify the effects of STING on ISGs expressions and virus replication in IBV-infected PBMCs-M?.In this study,PBMCs-M?were firstly cultured.Flow cytometry was used to identify the purity of the cultured cells.Results showed that the percentages of KUL01+cells in PBMCs-M?were 91.3±1.2%,indicating the extracted cells were satisfied the needs of the followed experiments.After adaption culture of PBMCs-M?with IBV 41 strain infection,PBMCs-M?were infected IBV M41 strain(10 MOI),real-time fluorescent quantitative PCR(qRT-PCR),indirect immunofluorescence(IFA),TCID₅₀ method were used to detect virus content post infection for clarifing virus growth in PBMCs-M?.Results showed that PBMCs-M?exhibit typical CPE at 36 h post IBV M41 strain infection,and the virus could replicate on them.In the previous study,we detected the dynamic changes of immune-related gene expressions in PBMCs-M?post IBV infection by qRT-PCR.Results showed that the expressions of immune-related genes in PBMCs-M?were inhibited at the early stage of IBV infection,and subsequently activated,and the dynamic changes of gene expressions had a closely relationship with virus replication.According to the immune-related gene expressions and the growth of IBV in PBMCs-M?,we chose 24 hours post infection to extract and analyze the differentially expressed proteins in both mock cells(Mock group)and IBV-infected cells(IBV group).Results showed that a total of 5662 proteins were identified in this study,of which 4921 proteins contained quantitative information.Furthermore,114 quantified proteins were up-regulated in the IBV group compared with Mock group,and 53 quantified proteins were down-regulated.We subsequently performed protein classification,functional enrichment and cluster analysis.There showed that differential expressed proteins were widely distributed in the processing of cell life.The analysis of protein-protein interaction indicated that anti-viral related ISGs proteins Mx,IFIT5 and OASL which have interaction relationship were significantly up-regulated.At the same time,the expression of STING,a key protein in the interferons(IFNs)pathways,was also significantly up-regulated.The above results indicate that the cellular immune responses were activated in PBMCs-M?post IBV infection.And Mx,IFIT5 and OASL may play key roles in anti-IBV infection.Finally,we quantified ISGs and STING proteins using the PRM method.The PRM results were consistent with the proteome sequencing results,indicating that the proteome analyses in this experiment were reliable.In order to study whether STING can regulate ISGs expression and affect virus replication in IBV-infected PBMCs-M?,we constructed the STING recombinant eukaryotic expression plasmid(p3×Flag-cmv-10-STING),optimize the transfection conditions of the above plasmid and STING-si RNA.The cells were divided into Mock group,IBV group,NC-si RNA+IBV group,STING-si RNA+IBV group,p3×Flag-cmv-10+IBV group,and p3×Flag-cmv-10-STING+IBV group.The effects of STING slience and overexpression on the expressions of ISGs and virus replication of IBV-infected PBMCs-M?were tested using qRT-PCR,Western Blot,TCID₅₀method.Results showed that Mx,IFIT5 and OASL m RNA expressions of PBMCs-M?were inhibited at the early stage of infection,and then activated.STING activated Mx,IFIT5,OASL m RNA expressions in IBV-infected PBMCs-M?and inhibited IBV replication.Meanwhile,STING could reduce virus titer and alleviate the pathological lesion of IBV on IBV-infected cells.The above results indicated that STING could exert its antiviral role via regulating the expressions of ISGs genes,and may become an antiviral target in IBV infection.The results of this experiment proved that the IBV M41 strain could replicate on PBMCs-M?,and CPE were showed at 36 hours post infection.At the same time,proteins which have antiviral effects in differentially expressed proteins were significantly up-regulated post IBV infection,such as ISGs and STING.By silence and overexpression of STING in PBMCs-M?,it was found that STING promoted ISGs expressions and inhibited virus replication in IBV-infected PBMCs-M?.The results of this study provided new ideas for the studies of IBV pathogenesis,prevention and treatment,and antiviral drugs.