Enterovirus A71 3AB protein facilitates immune evasion by blocking cGAS recognition of mtDNA

Mitochondrial DNA (mtDNA) is a powerful stimulator of the innate immune system and has been shown to trigger cytosolic DNA-sensing signaling during picornavirus infection. In this study, we observe that EV-A71 infection induces mitochondrial damage and leads to the release of mtDNA into the cytoplasm, which was mediated by the viral 2B protein. Despite this release, EV-A71 effectively suppresses the cGAS-STING-mediated type I interferon (IFN-I) response. We identify the nonstructural protein 3AB as a key viral antagonist of mtDNA sensing. Mechanistically, 3AB directly binds cytosolic mtDNA and disrupts cGAS-DNA phase separation, thereby suppressing cGAS-STING-dependent antiviral signaling. The immunosuppressive function of 3AB depends on the "3B + 7" region, with mutations impairing its mtDNA binding and IFN-I suppression. Moreover, the 3AB proteins from coxsackievirus A9 (CVA9) and A16 (CVA16) also exhibit mtDNA-binding ability. This study reveals a novel immune evasion strategy by blocking mtDNA-triggered immune signaling, providing new insights into the interplay between viral infection and mitochondrial immune defense.