A widespread family of viral sponge proteins reveals specific inhibition of nucleotide signals in anti-phage defense.

Cyclic oligonucleotide-based anti-phage signaling systems (CBASSs) are bacterial anti-phage defense operons that use nucleotide signals to control immune activation. Here, we biochemically screen 57 diverse E. coli and Bacillus phages for the ability to disrupt CBASS immunity and discover anti-CBASS 4 (Acb4) from the Bacillus phage SPO1 as the founding member of a large family of >1,300 immune evasion proteins. A 2.1 Ã crystal structure of Acb4 in complex with 3'3'-cyclic guanosine monophosphate (GMP)-AMP (3'3'-cGAMP) reveals a tetrameric assembly that functions as a sponge to sequester CBASS signals and inhibit immune activation. We demonstrate that Acb4 alone is sufficient to disrupt CBASS activation in vitro and enable immune evasion in vivo. Analyzing phages that infect diverse bacteria, we explain how Acb4 selectively targets nucleotide signals in host defense and avoids disruption of cellular homeostasis. Together, our results reveal principles of immune evasion protein evolution and explain a major mechanism phages use to inhibit host immunity.