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Background: Coinfection with avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) is common in chickens, and the molecular mechanism of the synergistic pathogenic effects of the coinfection is not clear. Exosomes have been identified as new players in the pathogenesis of retroviruses. The different functions of exosomes depend on their cargo components. Objectives: The aim of this study was to investigate the function of co-regulation differentially expressed proteins in exosomes on coinfection of ALV-J and REV. Methods: Here, viral replication in CEF cells infected with ALV-J, REV or both was detected by immunofluorescence microscopy. Then, we analyzed the exosomes isolated from supernatants of chicken embryo fibroblast (CEF) cells single infected and coinfected with ALV-J and REV by mass spectrometry. KEGG pathway enrichment analyzed the co-regulation differentially expressed proteins in exosomes. Next, we silenced and overexpressed tripartite motif containing 62 (TRIM62) to evaluate the effects of TRIM62 on viral replication and the expression levels of NCK-association proteins 1 (NCKAP1) and actin-related 2/3 complex subunit 5 (ARPC5) determined by quantitative reverse transcription polymerase chain reaction. Results: The results showed that coinfection of ALV-J and REV promoted the replication of each other. Thirty proteins, including TRIM62, NCK-association proteins 1 (NCKAP1, also known as Nap125), and Arp2/3-5, ARPC5, were identified. NCKAP1 and ARPC5 were involved in the actin cytoskeleton pathway. TRIM62 negatively regulated viral replication and that the inhibition of REV was more significant than that on ALV-J in CEF cells coinfected with TRIM62. In addition, TRIM62 decreased the expression of NCKAP1 and increased the expression of ARPC5 in coinfected CEF cells. Conclusions: Collectively, our results indicated that coinfection with ALV-J and REV competitively promoted each other's replication, the actin cytoskeleton played an important role in the coinfection mechanism, and TRIM62 regulated the actin cytoskeleton.

22The tumorigenesis is the result of the accumulation of multiple oncogenes and tumor 23 suppressor genes changes. Co-infection of avian leucosis virus subgroup J (ALV-J) 24 and reticuloendotheliosis virus ( REV) , as two oncogenic retroviruses, showed 25 synergistic pathogenic effects characterized by enhanced tumor initiation and 26 progression. The molecular mechanism underlying synergistic effects of ALV-J and 27 REV on the neoplasia remains unclear. Here, we found co-infection of ALV-J and 28 REV enhanced the ability of virus infection, increased viral life cycle, maintained cell 29 survival and enhanced tumor formation. We combined the high-throughput proteomic 30 readout with a large-scale miRNA screening to identify which molecules are involved 31 in the synergism. Our results revealed co-infection of ALV-J and REV activated a 32 latent oncogene of KIAA1199 and inhibited the expression of tumor suppressor miR-33 147. Further, enhanced KIAA1199, down-regulated miR-147, activated NF-κB and 34 EGFR were demonstrated in co-infected tissues and tumor. Mechanistically, we 35 showed ALV-J and REV synergistically enhanced KIAA1199 by activation of NF-κB 36 and EGFR signalling pathway, and the suppression of tumor suppressor miR-147 37 was contributed to maintain the NF-κB/KIAA1199/EGFR pathway crosstalk by 38 targeting the 3'UTR region sequences of NF-κB p50 and KIAA1199. Our results 39 contributed to the understanding of the molecular mechanisms of viral synergistic 40 tumorgenesis, which provided the evidence that suggested the synergistic actions of 41 two retroviruses could result in activation of latent pro-oncogenes. 42 Author summary 3 43The tumorigenesis is the result of the accumulation of multiple oncogenes and tumor 44 suppressor genes changes. Co-infection with ALV-J and REV showed synergistic 45 pathogenic effects characterized by enhanced tumor progression, however, the 46 molecular mechanism on the neoplasia remains unclear. Our results revealed co-47 infection of ALV-J and REV promotes tumorigenesis by both induction of a latent 48 oncogene of KIAA1199 and suppression of the expression of tumor suppressor miR-49 147. Mechanistic studies revealed that ALV-J and REV synergistically enhance 50 KIAA1199 by activation of NF-κB and EGFR signalling pathway, and the suppression 51 of tumor suppressor miR-147 was contributed to maintain the NF-52 κB/KIAA1199/EGFR pathway crosstalk by targeting the 3'UTR region sequences of 53 NF-κB p50 and KIAA1199. These results provided the evidence that suggested the 54 synergistic actions of two retroviruses could result in activation of latent pro-55 oncogenes, indicating the potential preventive target and predictive factor for ALV-J 56 and REV induced tumorigenesis. 57 Introduction 58 Viral synergism occurs commonly in the nature when co-infection of two or more 59 unrelated viruses invades the same host. As two oncogenic retroviruses, avian 60 leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) are the 61 optimal model to study the synergisti...

Exploration of the abnormal expression of exosomal molecules during the infection of avian leukosis virus subgroup J (ALV-J) is essential to provide a deeper understanding of the exosome’s role in the viral pathogenesis involved. The study aimed to investigate the differentially expressed proteins and miRNAs of the exosomes derived from DF-1 cells infected by ALV-J, their gene function and involved signal pathways. We isolated exosomes from DF-1 cells infected by ALV-J. The differentially expressed proteins and miRNAs of the exosomes were determined by proteomics and transcription detection technology. A Gene Ontology (GO) analysis and a Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway analysis identified the miRNAs target genes and the signal pathways regulated by the different proteins or/and miRNAs. A total of 116 proteins (58 upregulated and 58 downregulated) and 3 miRNAs (all upregulated) were determined. These proteins were involved in 155 signal pathways, in which the highest number of proteins involved in the cancer pathway was (up to) seven. The target genes of the miRNAs were involved in 3 signal pathways. Both the proteins and target genes of the miRNAs were involved in the Ribosome pathway and ECM-receptor interaction pathway. The results suggested that the ALV-J infection changed the proteins and miRNAs of the exosomes significantly.