The RLR-MAVS-IRF3 axis activates the IFN pathway to restrict Tonate virus (TONV) infection.

Tonate virus (TONV) is a neglected mosquito-borne alphavirus of the Venezuelan equine encephalitis complex associated with febrile illness, encephalitis, and fetal central nervous system abnormalities. Yet, host pathways that sense TONV infection and restrict its replication remain poorly defined. Here, we investigated the interaction between TONV and the type I interferon (IFN-I) system in human T98G and A549 cells. We show that TONV infection led to the accumulation of cytosolic double-stranded RNA and a robust IFN response. RIG-I and MDA5 depletion as well as that of MAVS and IRF3 strongly reduced TONV-induced IFN response. Disruption of RIG-I, MDA5, MAVS, or IRF3 resulted in an increase of dsRNA-positive cells and a higher viral RNA accumulation. Finally, we found that treatment with exogenous IFN-I strongly inhibits TONV replication reducing both viral RNA loads and infectious particle production. Thus, together, our results identify the RIG-I/MDA5-MAVS-IRF3 axis as a major pathway sensing TONV infection and establishing an IFN-I-dependent antiviral state, providing the first molecular characterization of TONV innate immune sensing in human cells.