Phage proteases activate CBASS antiphage immunity.

Cyclic oligonucleotide-based antiphage signaling systems (CBASS) are bacterial immune pathways evolutionarily related to cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) in humans. In CBASS, cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes sense phage infection and synthesize nucleotide signals to initiate antiviral defense. Here, we reveal phage prohead protease activity as a widespread mechanism of CD-NTase activation. We reconstitute CBASS recognition of phage T4 in vitro and identify proteolytic cleavage of a CD-NTase activation loop as a trigger of immune signal synthesis. Phage prohead proteases directly activate CBASS in vivo, explaining how immunity is initiated late during infection. Structure-based phylogenetic analyses identify activation loops in clade A, D, and G CD-NTases and define residues controlling recognition of distinct phage families. Our results establish CBASS recognition of phage protease activity as a widespread antiviral defense mechanism.