miR-155 facilitates the synergistic replication between avian leukosis virus subgroup J and reticuloendotheliosis virus by targeting a dual pathway.

Co-infection of avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) in poultry flocks induces synergistic replication, which worsens immunosuppression, growth inhibition, and increased mortality. However, the synergistic replication mechanism between ALV-J and REV is still not fully understood. We showed that microRNA-155 (miR-155) targets a dual pathway, PRKCI-MAPK8 and TIMP3-MMP2, which interacts with the U3 region of ALV-J and REV to promote synergistic replication between ALV-J and REV. Initially, we found that the RNA levels of ALV-J and REV increased significantly in ALV-J and REV co-infected cells and tissues compared with mono-infected cells and tissues. Next, miRNAomics showed that ALV-J and REV synergistically increased miR-155 levels. In co-infected cells, miR-155 promoted synergistic replication of ALV-J and REV. All structural proteins of the two viruses activated the expression of miR-155, especially gag proteins, rather than insertional activation. Proteomic study of cells co-infected with ALV-J and REV and transfected with miR-155 indicated that miR-155 directly suppressed PRKCI and TIMP3 and that PRKCI directly prevented MAPK8 expression, while TIMP3 directly interfered MMP2 expression. We also found that MAPK8 and MMP2 co-interacted with ALV-J and REV's U3 to enhance synergistic replication. In conclusion, ALV-J and REV synergistically activate miR-155, facilitating the synergistic replication of ALV-J and REV by PRKCI-MAPK8 and TIMP3-MMP2 dual-pathway interaction with the U3 region of ALV-J and REV. This discovery will assist in explaining how two retroviruses replicate synergistically. IMPORTANCE The synergy of two oncogenic retroviruses is an essential phenomenon in nature. The synergistic replication of ALV-J and REV in poultry flocks increases immunosuppression and pathogenicity, extends the tumor spectrum, and accelerates viral evolution, causing substantial economic losses to the poultry industry. However, the mechanism of synergistic replication between ALV-J and REV is still incompletely elusive. We observed that microRNA-155 targets a dual pathway, PRKCI-MAPK8 and TIMP3-MMP2, interacting with the U3 region of ALV-J and REV, enabling synergistic replication. This work gives us new targets to modulate ALV-J and REV's synergistic replication, guiding future research on the mechanism.