Monkeypox virus protein OPG188 antagonizes cGAS-STING antiviral signaling pathway to mediate immune evasion.

Innate immune evasion is critical for productive viral replication. Activation of the cGAS-STING antiviral signaling pathway and its downstream effector genes plays a pivotal role in restricting viral replication during early DNA virus infection. Through a comprehensive genomic screen of monkeypox virus (MPXV), we identified three viral genes, OPG147, OPG188, and OPG200, whose expression potently suppresses cGAS-STING pathway activation. Notably, the N-terminal domain of the Poxin protein encoded by OPG188 exhibits nuclease activity and cleaves the cyclic dinucleotide second messenger 2'3'-cGAMP. Using site-directed mutagenesis, we further delineated nine conserved regions and four key amino acid residues-H15, K140, R182, and I79-within Poxin that are essential for antagonism of cGAS-STING signaling. Moreover, via molecular docking and high-throughput screening of 7,155 small molecules targeting the catalytic pocket of Poxin, we identified two compounds that competitively bind to Poxin, inhibiting its cGAMP-degrading activity and consequently restoring cGAS-STING-mediated antiviral signaling upon MPXV infection. Collectively, these findings underscore the pivotal role of OPG188 in modulating antiviral immune responses and highlight NAD+ and Theaflavin-3'-gallate as promising candidates for the development of anti-MPXV therapeutics.