Nucleotide signals coordinate activation and inhibition of bacterial immunity.

The cellular nucleotide pool is a major focal point of the host immune response to viral infection. Immune effector proteins that disrupt the nucleotide pool enable animal and bacterial cells to broadly restrict diverse viruses, but reduced nucleotide availability induces cellular toxicity and can limit host fitness1-5. Here we identify Clover, a bacterial anti-phage defence system that overcomes this trade-off by encoding a deoxynucleoside triphosphohydrolase enzyme (CloA) that dynamically responds to both an activating phage cue and an inhibitory nucleotide immune signal produced by a partnering regulatory enzyme (CloB). Analysis of phage restriction by Clover in cells and reconstitution of enzymatic function in vitro demonstrate that CloA is a dGTPase that responds to viral enzymes that increase cellular levels of dTTP. To restrain CloA activation in the absence of infection, we show that CloB synthesizes a dTTP-related inhibitory nucleotide signal, p3diT (5'-triphosphothymidyl-3'5'-thymidine), that binds to CloA and suppresses activation. Cryo-electron microscopy structures of CloA in activated and suppressed states reveal how dTTP and p3diT control distinct allosteric sites and regulate effector function. Our results define how nucleotide signals coordinate both activation and inhibition of antiviral immunity and explain how cells balance defence and immune-mediated toxicity.