Coxsackievirus A6 2C protein antagonizes IFN-β production through MDA5 and RIG-I depletion

As a member of the enteroviruses, coxsackievirus A6 (CV-A6) has been a major cause of hand, foot, and mouth disease (HFMD) since 2008. It can infect both pediatric and adult populations, often leading to atypical HFMD. The host innate immune system plays a vital role in the development of enteroviral infections. However, the interplay between the host antiviral response and CV-A6 has not been well investigated. In the present study, we demonstrated that the 2C protein from CV-A6 (2C(CV-A6)) suppresses interferon beta (IFN-beta) production in HEK293T cells. Further results indicated that 2C(CV-A6) interacts with both melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I) and induces the degradation of these RNA sensors through proteases in the lysosomal pathway. This function also applies to 2C proteins from enterovirus A71 (2C(EV-A71)) and coxsackievirus B3 (2C(CV-B3)) but not CV-A16 2C (2C(CV-A16)) for its incompetence in MDA5 and RIG-I recognition. Partial depletion and amino acid substitution analyses indicated that the F28A, V75A, and I96V mutations significantly compromised 2C(CV-A6)-induced MDA5/RIG-I depletion. Surprisingly, unlike V75A and I96V that interrupt the 2C(CV-A6)-MDA5/RIG-I interaction, 2C(CV-A6) F28A remained competent in MDA5/RIG-I binding, suggesting that the interaction alone is not sufficient for 2C-mediated reduction. Additional tests indicated that CV-A6 viruses containing the 2C F28A mutation were less efficient in IFN-beta suppression, which is associated with compromised viral replication and release in infected rhabdomyosarcoma (RD) cells, suggesting that 2C-mediated immune regulation plays a vital role in enteroviral replication. Taken together, our data reveal a novel mechanism by which enteroviral 2C proteins antagonize the host innate antiviral immune response.