Molecular details of MAVS signalosomes and their regulation with camelid nanobodies

Abstract

The RIG-I like receptor (RLR) signaling pathway is crucial for the detection of viral infections and the induction of innate immune responses. However, gain-of-functions mutations in the RLR sensors can lead to type I interferonopathies, such as Aicardi-Goutière Syndrome (AGS) or Singleton Merten Syndrome (SMS), with high morbidity and mortality. Detection of viral or endogenous RNAs in the cytosol by RIG-I or MDA5 converge on the activation of mitochondrial antiviral signaling protein (MAVS). Through caspase recruitment domain (CARD) interaction, RLRs nucleate the polymerization of MAVS^CARD filaments on mitochondria and potentially other organelles. MAVS signalosomes serve as a central hub of the signaling pathway and therefore represent a potential target for therapeutic intervention. <br/> To study MAVS^CARD filaments assembly, we generated single domain antibodies (VHHs) against human MAVS^CARD and successfully characterized 11 cytoplasmic binders. We found that cytosolic expression of VHH_MAVS-8, VHH_MAVS-9 and VHH_MAVS-10 inhibit interferon and interferon-stimulated genes (ISGs) induction upon RLR stimulation. Electron-microscopy and co-immunoprecipitation studies revealed that the inhibitory VHHs are able to disrupt the formation of MAVS filaments without affecting the initial RLR:MAVS interaction, proving that cooperative assembly is critical for canonical interferon induction. This is further backed up by structure predictions, epitope mapping and functional experiments. Moreover, disruption of filament formation led to a delayed ISG response, validating the role of filament formation in speeding the kinetics of the antiviral response. <br/> Exploiting nanobodies with different binding epitopes, we identified bivalent nanobodies capable of inducing MAVS signalling, IRF3 translocation and interferon release. This agonistic nanobodies were capable of inducing MAVS^CARD oligomerization <em>in vitro</em>, and our current model suggest that they nucleate filament formation, further supporting the importance of MAVS fibrillation. <br/> Controlled doxycycline-inducible expression of Von Hippel Lindau (VHL)-VHH_MAVS-8 was able to completely degrade full-length MAVS, providing a temporally controlled and reversible knock-out system to study RLR signalling. RLR stimulation after VHL-VHH_MAVS-8 full-length MAVS degradation led to a delayed ISG induction, suggesting a novel antiviral role of the truncated MAVS isomers. <br/> In conclusion, this study validates the essential role of MAVS filament formation in amplifying and speeding the antiviral immune response. Additionally, we have developed the first MAVS-specific inhibitory, agonistic and degrader nanobodies, which serve as valuable novel molecular tools to study RLR signalling and present potential therapeutic applications for MAVS-dependent interferonopathies and for CAR-T cell therapy.