Fig. 5

Screening and identification of viral proteins interacting with vDPOL (ADRV-47L). A Schematic diagram of the NanoLuc complementation assay in aquatic animal cells. The coding sequence of ADRV-47L was fused with N-terminus of NanoLuciferase (NlucN) in both orientation and Flag tag. Other genes under testing (X) were fused with the C-terminus of NanoLuciferase (NlucC) in both orientation and HA tag. Plasmids containing ADRV-47 and genes under testing were cotransfected into EPC cells. The luciferase activity was determined 48 h post transfection. The plasmid expressing NlucN was cotransfected with the genes under testing and used as a control. B NanoLuc complementation assay showed that twenty-three viral proteins (*p < 0.01) showed significantly higher luciferase activity in cells cotransfected with plasmids as described above. C, D Co-IP analysis of the protein interactions revealed by NanoLuc complementation assay. HEK293T cells were cotransfected with pcDNA-47L-3Flag and the plasmid expressing viral protein-NlucC-HA. The cells were then coimmunoprecipitated with anti-HA beads (C) or anti-Flag beads (D). The bands corresponding to the theoretical molecular weight of viral proteins are marked with #