Aims: The RIG-I like receptor (RLR) signaling is an essential pathway for the initiation of host IFN-mediated antiviral responses. The activation of this pathway is complex and well characterized, but most of the spatio-temporal events, and the subcellular localization where the essential proteins interact, are still under interrogation.

Methods: The interaction of key partners in the RLR pathway was examined by Bimolecular Fluorescence Complementation (BiFC). This technique allowed us to isolate, visualize and analyze protein-protein interactions in living cells.

Results: We have defined spatially in the cell different complexes formed between RIG-I, TRIM25 and MAVS, in the presence and absence of viral IFN antagonistic proteins. Dimers and/or multimers of RIG-I, TRIM25 and MAVS localize into different compartments in the cytoplasm of the cell. The impact of several viral proteins in the RLR pathway was investigated through BiFC:  HCV NS3/4A, IAV NS1, NiV V and SFTSV NSs proteins. In order to inhibit the IFN response, these viral proteins target specific complexes and interact in defined areas in the host cell.

Conclusion: For the first time, the interactions among proteins formed along the RLR pathway have been tracked and visualized in living cells. We found different complexes localized in concrete subcellular compartments not known before. This method has provided us new information to further understand the mechanisms that regulate the specific distributions of these complexes in the activation of the RLR pathway.