Extended Data Fig. 7: Mechanism of 3′2′-cGAMP bond formation and resistance to degradation by viral poxin enzymes.

a, Analysis of Dm-cGLR1 reactions with pairwise combinations of α-³²P-labelled nucleotides and non-hydrolyzable nucleotides reveals reaction intermediates and identifies the order of bond formation during 3′2′-cGAMP synthesis. Left: TLC analysis demonstrates that Dm-cGLR1 forms a linear intermediate in the presence of GTP and non-hydrolyzable ATP (Apcpp), indicating that the 2′–5′ phosphodiester bond is synthesized first. Exposed γ-phosphates removed by phosphatase treatment before analysis are indicated in parentheses. Note that while a linear intermediate cannot be formed in the presence of non-hydrolyzable GTP (Gpcpp), Dm-cGLR1 will synthesize the off-product 2′3′-c-di-AMP. Mouse cGAS, which synthesizes 2′3′-cGAMP through the linear intermediate pppG[2′–5′]pA, is shown here for comparison¹⁹. Right: schematic of the reaction mechanism for each enzyme. b, Poxins are 2′3′-cGAMP-specific viral nucleases that disrupt cGAS–STING signalling. HPLC analysis of synthetic 2′3′-cGAMP or 3′2′-cGAMP treated with poxin from the insect baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) is shown^22,37. In 1 min, AcNPV poxin cleaves 2′3′-cGAMP into a mixture of intermediate and full-cleavage product; and after 1 h, turnover is complete. No cleavage of 3′2′-cGAMP is observed by AcNPV poxin under these reaction conditions. c, Using TLC as a more sensitive assay, we observed minimal cleavage of 3′2′-cGAMP following overnight incubation with AcNPV poxin. Data in a–c are representative of n = 3 independent experiments. d, Schematic highlighting how an isomeric switch in phosphodiester linkage specificity makes 3′2′-cGAMP remarkably resistant to poxin-mediated cleavage.