MiR-155 Mediated Synergistic Replication Mechanism Of Avian Leukosis Virus Subgroup J And Reticuloendotheliosis Virus

Virus co-infections are widespread in nature which have an important impact on the disease process.In poultry breeding,avian leukosis subgroup J(J-AL)and avian reticuloendotheliosis(RE)are two common immunosuppressive diseases caused by avian leukosis virus subgroup J(ALV-J)and avian reticuloendotheliosis virus(REV),respectively.both ALV-J and REV are retroviruses with similar transmission routes,and co-infection of these two viruses is widespread in clinical.Co-infection of ALV-J and REV synergistically facilitate viral replication,leading to enhanced pathogenicity and an expanded tumor spectrum.However,studies on the synergistic mechanism of ALV-J and REV mainly focused on promoting growth inhibition and immunosuppression,and the ALV-J and REV synergistic replication mechanism has not been fully elucidated.In this study,we showed that ALV-J and REV synergistically facilitate miR-155 expression through their structural proteins,and miR-155 directly interacted with the 3′ non-coding U3 region of ALV-J and REV through the dual pathways of PRKCI-MAPK8 and TIMP3-MMP2 to promote the synergistic replication of the two viruses.To clarify the synergistic replication and pathogenicity of ALV-J and REV,an in vitro coinfection model of ALV-J and REV was established.The results showed that the replication of both viruses was accelerated when ALV-J and REV were co-infected,and their m RNA expression levels and protein expression levels were extremely significantly increased,and their viral replication efficiency reached the highest at 72-96 hpi.In order to clarify whether ALV-J and REV also co-replicate in animals,the animal model of co-infection of the two viruses was established.The results showed that the co-infected chickens had significantly lower hatching rate and body weight,higher mortality,and more severe immune organ atrophy and histopathology than the mono-infected chickens.The m RNA levels of ALV-J and REV in the liver,kidney and spleen of the co-infected chickens were significantly higher than those of the mono-infected chickens.These results suggest that ALV-J and REV have a synergistic replication effect in vitro and in vivo.The synergistic replication of viruses is mainly achieved by co-hijacking host factors and signaling pathways,such as miRNAs.To screen the crucial miRNAs that regulate the synergistic replication of ALV-J and REV,miRNAomics analysis of ALV-J and REV monoinfected and co-infected DF-1 cells were performed by using RNASeq technology.Comparative analysis of differentially expressed miRNAs in ALV-J and REV co-infected,ALV-J mono-infected,REV mono-infected and control cells showed that,compared to ALV-J mono-infected and REV mono-infected groups,17 significantly differentially expressed miRNAs were screened in ALV-J and REV co-infected groups,as 7 miRNAs were up-regulated and 10 miRNAs were down-regulated.The analysis of miRNAs that were either up-regulated or down-regulated in both co-infected and single-infected groups revealed that miR-155 has the highest relative up-regulation.miR-155 plays an important role in the replication of various viruses,and it is assumed that it also plays an important role in the co-replication of ALV-J and REV.Next,to reveal the mechanism of miR-155 in the synergistic replication of ALV-J and REV.We first investigated how ALV-J and REV regulate the expression of miR-155.Retroviruses usually regulate related miRNA expression through host cell genomic DNA integration or viral structural protein interactions.By sequencing the integration sites of genomic DNA of ALV-J and REV co-infected cells,it was found that the integration of ALVJ and REV did not regulate the expression of miR-155;moreover,to clarify whether the structural proteins of ALV-J and REV mediated the expression of miR-155,the structural proteins of ALV-J and REV were detected by overexpressing them in cultured cells.expression levels of miR-155.The results showed that co-expression of structural proteins of both ALV-J or REV synergistically promoted miR-155 expression,with the gag of ALV-J and REV most significantly synergistically promoting miR-155 expression.Subsequently,to clarify the role of miR-155 in ALV-J and REV synergistic replication,miR-155 was overexpressed or interfered in ALV-J and REV co-infected cells,and the levels of ALV-J and REV were detected.The results showed that both overexpression of miR-155 promoted ALV-J and REV synergistic replication extremely significantly,while interference with miR-155 inhibited the co-replication of ALV-J and REV extremely significantly,indicating that miR-155 played a crucial role in ALV-J and REV synergistic replication.To investigate whether miR-155 is the key factor that plays a crucial role in ALV-J and REV synergistic replication,the levels of ALV-J and REV were measured by transfecting different concentrations of miR-155 mimics into ALV-J or REV mono-infected cells to substitute the effect of another virus on miR-155 expression.The results showed that when the addition of miR-155 mimics resulted in no significant difference in miR-155 expression levels in monoinfected and co-infected cells of ALV-J and REV,there was also no significant difference in the replication levels of ALV-J or REV,indicating that miR-155 plays a crucial and critical role in ALV-J and REV synergistic replication.The mechanisms which miR-155 regulates viral replication are divided into two main pathways,either directly targeting the viruses or regulating viruses replication through related factors or signaling pathways.To explore the role of miR-155 in facilitating ALV-J and REV synergistic replication,initially,this study predicted the possibility of direct interaction between miR-155 and ALV-J and REV by RNA22 software.The possibility of direct interaction between miR-155 and REV is extremely low,indicating that miR-155 is not directly regulating the synergistic replication of ALV-J and REV.Therefore,to screen for miR-155-targeted hostregulated factors for ALV-J and REV synergistic replication,this study used TMT-LC/MS mass spectrometry to identify DF-1 cells overexpressed miR-155 for proteomic detection,and a total of 29 significantly differentially expressed proteins were screened by comparative analysis with the control group.By combining analysis of 29 proteins with using STRING and RNA22,two factors,PRKCI and TIMP3,were screened,which may play important roles in miR-155 promoting the synergistic replication of ALV-J and REV,but PRKCI and TIMP3 do not have the possibility of interacting with ALV-J or REV,and they may have downstream factors play their roles.Therefore,to clarify the downstream factors of PRKCI and TIMP3,further co-analysis of proteomic results with PRKCI and TIMP3 using STRING showed that PRKCI may interact with MAPK8,while TIMP3 interacts with MMP2,and both MAPK8 and MMP2 may jointly regulating miR-155 promoting ALV-J and REV synergistic replication.Subsquently,to verify the interactions between PRKCI and MAPK8,TIMP3 and MMP2,and whether miR-155 promotes ALV-J and REV synergistic replication through these two signaling pathways.The expression of PRKCI and TIMP3 were measured by overexpressing or interfering with miR-155.The results showed that overexpressed miR-155 significantly suppressed the m RNA and protein expression levels of PRKCI and TIMP3;interference with miR-155 promoted the expression of PRKCI and TIMP3 extremely significantly.It was further clarified that miR-155 inhibited the expression of PRKCI and TIMP3 by interacting with the3′UTR of PRKCI and TIMP3 through dual luciferase assay analysis.The expression of MAPK8 and MMP2 were detected in the cells overexpressed PRKCI and TIMP3 respectively.The results showed that overexpression of PRKCI extremely significantly inhibited MAPK8 expression,while overexpression of TIMP3 extremely significantly inhibited MMP2expression;Direct interaction of PRKCI with MAPK8 was determined by Co-IP assay,while interaction of TIMP3 with MMP2 has been reported.Moreover,inhibition of MAPK8 and MMP2 alone had significant effects on the synergistic replication of ALV-J and REV,whereas co-inhibition of MAPK8 and MMP2 extremely inhibited ALV-J and REV synergistic replication.The above experiments suggest that miR-155 promotes ALV-J and REV synergistic replication through regulation of the dual PRKCI-MAPK8 and TIMP3-MMP2 pathways.Moreover,to reveal the way MAPK8 and MMP2 promote the synergistic replication of ALVJ and REV,the RNA interaction possibility of MAPK8 and MMP2 with the 3′ non-coding U3 region of ALV-J and REV was predicted using RPISeq,and the results showed that the RNA interaction possibility by RF classifier and SVM classifier The results showed that the predictions by both RF classifier and SVM classifier were greater than 0.5,indicating that both MAPK8 and MMP2 may interact with the RNA of the 3′ non-coding U3 region of the virus.Using biotin-labeled probes,RNA pulldown and western blot assays showed that MAPK8 and MMP2 directly interacted with RNAs in the 3′ non-coding U3 region of ALV-J and REV.The above experiments indicate that MAPK8 and MMP2 interacted with the viral non-coding U3 region to promote the synergistic replication of ALV-J and REV.In summary,this study reveals the molecular mechanism of miR-155-mediated ALV-J and REV synergistic replication.The structural proteins of ALV-J and REV synergistically promoted the expression of miR-155.miR-155 inhibited the expression of PRKCI and TIMP3,then derepressed MAPK8 and MMP2,and the up-regulated MAPK8 and MMP2 co-interacted with the 3′ non-coding U3 region of ALV-J and REV,and facilitated the synergistic replication of ALV-J and REV.This study elucidates the molecular mechanism of miR-155-mediated synergistic replication of ALV-J and REV,which provides a reference for the study of the synergistic replication mechanism of multiple retroviruses,and provides theoretical support for the development of co-prevention and control technology for ALV-J and REV.