Tumorigenesis Mechanism Of Synergistic Infection Of Avian Leukosis Virus Subgroup J And Reticuloendotheliosis Virus

Viral synergism occurs commonly in the nature.When co-infection of two or more unrelated viruses invades the same host,the biological characteristics,such as virus replication,cell phenotype,pathological changes,host range,tissue addiction,and infection rate,are changed during synergistic.J subgroup avian leucosis virus(ALV-J)belongs to the retrovirus family,which produces the clinical symptoms characterized by immunosuppression,causing growth retardation and causing a variety of tumors.Reticuloendotheliosis(RE)are a series of symptoms pathological syndrome caused by Reticuloendotheliosis virus(REV),Spleen necrosis virus(SNV),Chicken syncytial virus(CSV)and Duck infectious anemia virus(DIA).These viruses also belong to the retrovirus family,and cause immunosuppression,dwarf syndrome and chronic tumors in lymphoid or other tissues.Over the past two decades,the epidemiological study showed co-infection of ALV-J and REV remained widespread.Co-infection of ALV-J and REV also reduces the antibodies and enhances the pathogenicity.In addition,the enhancement of tumor formation and even no-specific mortality are the key synergistic features of co-infection ALV-J with REV.However,the mechanism of tumor synergism remains unknown.As ALV-J and REV are two tumor virus models in poultry,the study on umorigenesis mechanism of these two viruses synergistic infection will provide the theoretical basis for further revealing the mechanism of tumor synergism.To verify the synergistic phenomenon of co-infection ALV-J with REV,we constructed the model of co-infection of ALV-J and REV in CEF cells,and caught the two viral particles of ALV-J and REV in the same cell by electron microscopy.Further,the CCK-8 assay showed ALV-J also synergized with REV to promote the cell survival.We also found that ALV-J synergized REV to enhance the viral replication and proteins expression by qRT-PCR and western blot analysis.To further verify the synergistic effect of co-infection ALV-J and REV in vivo,we also established the animal model of co-inoculation of ALV-J and REV in allantoic cavity of fertilized SPF eggs at 6-day of embryonic age.The synergistic effects of ALV-J and REV in chickens were the significant decline of the incubation rate,more serious immunosuppression and growth retardation.The qRT-PCR results showed both viral RNA levels of organs were higher than those in single infection group.H&E staining results showed,compared with single-infection group,more massive inflammatory cells infiltrated around the blood vessel in liver,kidney and pancreas,the demarcation between medulla and cortex in the thymus seemed to disappear,and some fibromas were observed in endocardium of co-infection group.To explore the synergistic tumorigenesis mechanism induced by ALV-J and REV,CEFs infected with ALV-J,REV or both were analyzed using iTRAQ quantitative proteomic analysis and miRNA whole-genome sequencing using the same batch of cell samples at 72 hpi.Compared with the single-infection group,there are 33 and 7 differentially expressed proteins and miRNAs in co-infected cells,respectively.Among these proteins and miRNAs,we found ALV-J might synergize with REV to activate KIAA1199 and inhibit miR-147.Further,the qRT-PCR and western blot analysis verified the results of KIAA1199 and mi R-147 expression changes were consistent with that in iTRAQ quantitative proteomic analysis and miRNA whole-genome sequencing.In vivo,the qRT-PCR results showed ALV-J also synergized with REV to increase the KIAA1199 RNA level in liver,kidney,bone marrow,bursa of Fabricius and heart.The IHC showed the protein expression levels of KIAA1199 were significantly increased in bone marrow,kidney and heart of ALV-J and REV co-infected group,while that was not significantly increased in liver and bursa of Fabricius,which meant there were also some key molecular regulators to determine the expression of KIAA1199 protein.The expressions of miR-147 were decreased in kidney,bone marrow and heart,and increased in liver and bursa of Fabricius,which were correlated with the oncogene KIAA1199 activation by co-infection of ALV-J and REV,implying that there might be some association between miR-147 and KIAA1199 in the co-infection of ALV-J and REV.To explore the relationship between miR-147 and KIAA1199,one putative miR-147 binding site in the 3’UTR of KIAA1199 was identified by RNA22,NCBI,ensemble and mi Randa bioinformatics analysis.We constructed the mi R-147 mimics and inhibitors,andthen transferred into the CEF cells co-infected ALV-J and REV.The western blot analysis results showed miR-147 inhibited expressions of KIAA1199 in a dose-dependent manner,and inhibition of miR-147 promoted expression of KIAA1199.KIAA1199 3’UTR luciferase reporter assay showed that mi R-147 significantly inhibited the activity of KIAA1199 3’UTR reporter but not that of the control reporter,while mutation of this site abolished the miR-147 inhibitory effect on KIAA1199 3’UTR reporter activity.These data suggested that miR-147 directly targeted KIAA1199.Previous study showed HPV promote the KIAA1199 expression by the activation of NF-κB and EGFR.In order to explore the relationship among NF-κB,KIAA1199 and EGFR in co-infection of ALV-J and REV,the in vitro expressions of NF-κB p50 、 NF-κB p65,pho-NF-κB p65 and EGFR were detected by qRT-PCR、western blot and ELISA analysis.Compared with single-infection group,these analysis results showed the expressions of NF-κB p50 、 NF-κB p65,pho-NF-κB p65 and EGFR were increased significantly in co-infection group.In vivo,we also tested the expressions of p-IκBα and EGFR in organ tissues by ELISA and qRT-PCR analysis.The P-IκBα ELISA results showed that co-infection of ALV-J and REV increased the levels of phosphorylated IκBα in liver,kidney,bone marrow,bursa of Fabricius and heart.Furthermore,ALV-J synergized with REV to enhance the EGFR levels in kidney,bone marrow,bursa of Fabricius and heart,while that decreased in liver.To further determine whether KIAA1199 is required for induction of NF-κB and EGFR,we constructed and transfected the NF-κB p65 shRNAs into the CEF cells.Indeed,when NF-κB p65 was knockdown,the RNA levels of KIAA1199 and EGFR were decreased at 48 hpi.Identitical results were observed in KIAA1199 inhibition.Together these data suggest that ALV-J and REV synergistically activated the NF-κB/KIAA1199/EGFR pathway crosstalk.Interestingly,previous study showed miR-147 is a member of suppressors for targeting EGFR signaling proteins.As the close cooperation of NF-κB,KIAA1199 and EGFR,mi R-147 might also have some connection to NF-κB in mediating the synergistic effect of ALV-J and REV.As expected,bioinformatics analysis identified one putative miR-147 binding site in the 3’UTR of NF-κB p50.Further,NF-κB p50 3’UTR luciferase reporter assay showed that miR-147 significantly inhibited the activity of NF-κB p50 3’UTR reporter but notthat of the control reporter.Mi R-147 inhibited the activity of the NF-κB 3’UTR reporter and the NF-κB p50 expression in CEF in a dose-dependent fashion.A miR-147 inhibitor up-regulated the NF-κB p50 expression level in a dose-dependent manner.Mutation of this site abolished the miR-147 inhibitory effect on NF-κB p50 3’UTR reporter activity.These data suggested that miR-147 also directly targeted NF-κB p50 besides KIAA1199.In this study,we investigated the synergistic phenomena of ALV-J and REV in vivo and in vitro.We showed that ALV-J and REV synergistically activated a latent oncogene KIAA1199 and inhibited a tumor suppressor miR-147.Mechanistically,ALV-J and REV synergistically enhanced KIAA1199 by activation of NF-κB and EGFR signalling pathway,and the suppression of tumor suppressor mi R-147 was contributed to maintain the NF-κB/KIAA1199/EGFR pathway crosstalk by targeting the 3’UTR region sequences of NF-κB p50 and KIAA1199,providing the theoretical basis for further revealing the mechanism of tumor synergism.