Abstract
Introduction The host innate immune system acts as the first line of defense to prevent viral invasion. Viral products are rapidly detected through several classes of pathogen recognition receptors (PRR), like the cytosolic RIG-I like receptors (RLRs), which recognize viral RNA products in the cytoplasm of infected cells. Once activated, RIG-I binds MAVS, leading to the production of type I IFN [1] . Some viruses have developed multiple mechanisms to interfere with the RLR pathway and evade the innate immune response in infected cells. Influenza A virus (IAV) blocks the synthesis of type I IFN by targeting host effector molecules involved in activation of the RIG-I pathway [2] , [3] . Methods In order to study the steps involved in the RLR pathway, we have developed a Bimolecular Fluorescence Complementation (BiFC) assay [4] . This method allow us to visualize the subcellular localization of protein interactions by microscopy, pull down selectively BiFC complexes and isolate specific interactive complexes from transfected cells. Results We were able to detect subcellular localizations associated with interactions between RIGI, MAVS, TRIM25 and other proteins of the RLR pathway in the cell. We have analyzed the effects on these interactions of two viral proteins known to antagonize the RLR pathway. The NS3/4A of Hepatitis C Virus (HCV) changes the localization of the complexes RIGI-MAVS and MAVS-MAVS. The non structural protein 1 (NS1) from IAV prevents the formation of the complexes RIGI-MAVS and TRIM25-TRIM25, but does not have any effect on the complex RIGI-TRIM25. These complexes are localized in different compartments in the host cells. Conclusion The use of the BiFC technique has provided us with a powerful tool to analyze known and novel protein interactions of the RLR pathway. We have been able to isolate and track directly protein interactions, analyze their localization in the host cell and the effects of viral IFN antagonist proteins on these complexes.
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