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

Cyclic oligonucleotide-based antiviral signaling systems (CBASS) are bacterial operons that defend against phage infection by using nucleotide signals to trigger immune responses. In this study, we performed a biochemical screen of 57 diverse E. coli and Bacillus phages and identified anti-CBASS 4 (Acb4) from Bacillus phage SPO1 as the first characterized member of a widespread family comprising over 1,300 immune evasion proteins. A 2.1 Å crystal structure of Acb4 bound to 3’3′-cGAMP reveals a 3′,3′-cGAMP tetrameric assembly that acts as a molecular sponge, sequestering CBASS signaling molecules and blocking immune activation. We show that Acb4 alone is sufficient to inhibit CBASS both in vitro and to promote phage immune evasion in vivo. By analyzing phages infecting a variety of bacterial hosts, we demonstrate how Acb4 specifically targets host defense nucleotide signals while sparing cellular processes. Collectively, these findings uncover key principles underlying immune evasion protein evolution and define a major strategy used by phages to suppress bacterial immunity.