Molecular mechanism of 2',3'-cGAMP degradation by monkeypox virus poxin-schlafen protein

The World Health Organization had declared the multiregional outbreak of monkeypox a global public health emergency twice since 2022. Poxin from orthopoxviruses degrades the second messenger 2',3'-cGAMP of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway to evade innate immune surveillance. Monkeypox virus (MPXV) poxin is fused to schlafen and the mechanism of how it catalyzes the degradation of 2',3'-cGAMP remains unclear. Here, we show that MPXV poxin-schlafen degrades 2',3'-cGAMP into Gp[2'-5']Ap[3'], suppressing interferon-β (IFN-β) induction mediated by the cGAS-STING pathway. The schlafen domain has no impact on the degradation of 2',3'-cGAMP mediated by the poxin domain. Crystal structures of MPXV poxin domain and its complex with Gp[2'-5']Ap[3'] reveal that 2',3'-cGAMP binds to a pocket formed by two monomers of the poxin dimer and induces closure of the pocket to trigger degradation. MPXV poxin exhibits a strict substrate specificity and does not degrade 3',3'-cGAMP, c-di-AMP, and c-di-GMP that also activate STING-mediated immune responses. These results unveil the molecular mechanism for 2',3'-cGAMP degradation by MPXV poxin-schlafen protein and provide the structural basis for the development of inhibitors against MPXV infection.