Extended Data Fig. 7: VACV poxin degrades 2′,3′-cGAMP through hydrolysis of the 3′–5′ bond.

a, TLC analysis of poxin activity using 2′,3′-cGAMP radiolabelled at the 2′–5′-(α³²P-A) or 3′–5′-(α³²P-G) phosphodiester bonds. Radiolabelled 2′,3′-cGAMP was incubated with recombinant VACV poxin and then treated with phosphatase to remove exposed phosphates from the final product. Following hydrolysis, the guanosine phosphate is exposed for phosphatase removal, confirming the structural findings that VACV poxin specifically cleaves the 3′–5′ linkage of 2′,3′-cGAMP. The ‘−’ refers to a buffer-only control. b, Schematic of VACV poxin induced hydrolysis of 2′,3′-cGAMP. c, TLC analysis of VACV poxin 2′,3′-cGAMP-degradation activity in the presence of 5 mM EDTA metal chelation or divalent cation supplementation. Divalent cations were supplemented at the following concentrations: 5 mM Mg²⁺, 5 mM Ca²⁺, 1 mM Mn²⁺, 1 μM Co²⁺, 1 μM Ni²⁺, 1 μM Cu²⁺ or 1 μM Zn²⁺. Poxin activity is resistant to EDTA and divalent cations have no effect on the reaction, confirming the structural findings that VACV poxin activity is metal-independent. The ‘−’ refers to a buffer-only control; the ‘+’ refers to treatment with VACV poxin alone without metal addition. d, TLC analysis of mutants of the active site of VACV poxin that were incubated for 20 h with 2′,3′-cGAMP or 3′,3′-cGAMP demonstrates that all active-site mutants retain specificity for 2′,3′-cGAMP. The ‘−’ refers to a buffer-only control. All data are representative of three independent experiments.