All viruses of the genus Mammarenavirus possess an exoribonuclease (ExoN) domain in their nucleoproteins (NP). Through this domain, the NP efficiently prevents the activation of the interferon (IFN) response, presumably by degrading double-stranded RNA (dsRNA) produced during the viral replication. While the importance of this ExoN activity regarding Mammarenavirus virulence is well established, little is known about the dsRNA molecules that are targeted by NP for degradation, and the contribution of cellular sensors activated by these molecules has yet to be described. Here, we addressed these questions using recombinant viruses with abrogated ExoN domains that are no longer able to control the IFN response. We infected RIG-I, MDA5, and MAVS deficient cells and demonstrated that ExoN mutants activate the interferon response through RIG-I, but not MDA5. We then purified RIG-I-associated RNA from infected cells and confirmed its immunostimulatory activity in transfected cells. We sequenced the RIG-I-associated RNA and identified different enriched sequences in the Mopeia virus (MOPV) or Lassa virus (LASV) RNA. RIG-I activating sequences corresponded to the extremities of the 5' ends and intergenic regions of the MOPV genome and to a glycoprotein precursor complex (GPC) sequence in the LASV genome. We produced corresponding synthetic RNA molecules and confirmed their RIG-I-dependent activation of the IFN response. These results underline the central role of the ExoN domain in MOPV and LASV NP for immune escape and identify new virus-derived RNA molecules with high immunostimulatory properties.IMPORTANCEArenaviruses prevent the activation of the interferon response due to the exonuclease activity of their nucleoprotein, suggesting that infection leads to the production of immunostimulatory RNA molecules. However, neither the exact nature of the immune RNA sensors nor the identity of the RNA activating these sensors is clearly identified. By taking advantage of recombinant MOPV and LASV deficient for their exonuclease activity and that are strong activators of the interferon response, we have identified RIG-I as the major sensor of arenaviruses in infected cells. We also identified the RNA molecules recognized by RIG-I upon infection, and we highlighted differences between MOPV and LASV viruses. Our results represent critical information regarding the factors of immunogenicity and pathogenicity of Old-World arenaviruses and can help us explain key differences between pathogenic and non-pathogenic arenaviruses.
Keywords: RIG-I-like receptors; arenaviruses; interferon response; viral RNA.