Background <p>MAVS is a mitochondrial and peroxisomal protein that adapts innate RNA sensing by RIG-I-like receptors to downstream signalling. MAVS aggregation is required for the propagation of the activation signal, however whether this requirement varies with respect to subcellular localization remains unclear.</p> Methods <p>MAVS mutants were expressed in transfected, lentivirus-transduced and Sendai virus-infected cells. They were then analysed for subcellular localization by confocal immunofluorescence microscopy, for aggregation by semi-denaturing gel electrophoresis, and for activation of type I interferon production by Western blotting of phosphorylated IRF3 and ELISA of interferon β.</p> Results <p>We identified that MAVS aggregation is required for innate immune signalling triggered by mitochondrial but not peroxisomal MAVS. An aggregation-deficient mutant of peroxisomal MAVS retained its ability to induce type I interferon production. We further demonstrated that the magnitude of the activation signal and the distribution of MAVS to mitochondria and peroxisomes are governed by the tail charge at the C-terminus of MAVS. An increase in tail charge directs MAVS to peroxisomes predominantly, leading to a more modest activation of innate immune signalling.</p> Conclusion <p>These results suggest that MAVS displays compartment-specific properties that are mechanistically and functionally distinct in RNA sensing.</p>