(A) SDS-PAGE analysis of purified amarillovirus poxin constructs. All proteins are expected to migrate as a SUMO-poxin fusion ~40–50 kDa, except Gamboa mosquito virus and Gentian Kobu-sho-associated virus which encode two poxin sequences in tandem (SUMO-poxin-poxin). Fragments corresponding in size to SUMO which migrates ~17 kDa appear for most constructs which did not yield a substantial soluble poxin fragment, potentially indicating N-terminal cleavage and self-excision from the N-terminal 6 × His-SUMO2 tag used for purification. (B) TLC analysis of poxin specificity for 2′3′-cGAMP compared to 3′3′-cGAMP. Most poxins, including the amarillovirus XCV poxin, exclusively degrade 2′3′-cGAMP. However, Dendrolimus punctatus cypovirus 22 (DpCV 22) exhibits relaxed specificity and catalyzes some degradation of both 2′3′-cGAMP and 3′3′-cGAMP. Proteins are abbreviated as in Figures 1B and 5C. (C) SDS-PAGE analysis of XCV poxin mutant self-cleavage. Left: Removal of the XCV poxin cleavage site (C-term Δ5) completely abrogates proteolysis. Right: Alanine scanning of the XCV poxin cleavage site motif shows that H1007 at the P1 position, adjacent to the scissile peptide bond, is critical for efficient autoproteolysis. (D) Schematic of the XCV polyprotein showing the two loci at which the cleavage site motif NxQH occurs as dotted vertical lines. The solid black lines indicate a magnified region from amino acid 700–1,100, depicting in red the cloned construct used in this study. This construct included the C-terminal cleavage site but excluded a putative N-terminal cleavage motif. (E) Alignment of the XCV putative N-terminal cleavage motif with the experimentally identified C-terminal cleavage site. (F) Putative model explaining how amarillovirus enzymes like XCV poxin might self-excise from the polyprotein and assemble into functional 2′3′-cGAMP nucleases for immune antagonism.