RIG-I and MDA5 cooperatively recognize the DENV replicative-form dsRNA to activate innate immunity

RIG-I like receptors (RLRs) are a family of cytosolic RNA sensors that sense RNA virus infection to activate innate immune response. It is generally believed that different RNA viruses are recognized by either RIG-I or MDA5, two important RLR members, depending on the nature of pathogen-associated molecular patterns (PAMPs) that are generated by RNA virus replication. Dengue virus (DENV) is an important RNA virus causing serious human diseases. Despite extensive investigations, the molecular basis of the DENV PAMP recognized by the host RLR has been poorly defined, and which RLR is involved in sensing the DENV PAMP remains controversial. Here, we demonstrated that the DENV infection-induced interferon response is dependent upon both RIG-I and MDA5, with RIG-I playing a predominant role. Next we purified the DENV PAMP RNA from the DENV-infected cells, and demonstrated that the purified DENV PAMP is viral full-length double-stranded RNA bearing 5ppp modifications, likely representing the viral replicative-form RNA. Finally, we confirmed the nature of the DENV PAMP by reconstituting the viral replicative-form RNA from in vitro synthesized DENV genomic RNA. In conclusion, our work not only defined the molecular basis of the RLR-PAMP interaction during DENV infection, but also revealed the previously underappreciated recognition of the distinct moiety of same PAMP by different RLRs in innate immunity against RNA viruses. ImportanceThe molecular interaction between PAMPs and RLRs plays a crucial role in innate immune response against the RNA virus infection. To our knowledge, the exact molecular basis of the DENV PAMPs and which RLR member (RIG-I or MDA5) is involved in this recognition remain controversial. In this study, we demonstrated that the DENV PAMP is likely DENV replicative-form RNA, a double-stranded RNA. RIG-I and MDA5 can sense different moieties of this DENV PAMP to active innate immune response. Our work not only clarified which RLR and what viral PAMP are involved in innate immune sensing of DENV infection, but also revealed previously underappreciated recognition of the same PAMPs by different RLRs in innate immunity against RNA viruses.