• Abstract 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 fitness1,2,3,4,5. • 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.
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