Extended Data Fig. 4: Construction and validation of poxin-expressing cells and poxin knockout virus.
From: Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS–STING signalling
a, TLC analysis of lysates from HEK293T cells after transduction with poxin(WT) or the poxin(H17A) catalytically inactive construct, and selection with puromycin. HEK293T poxin(WT) cells but not control cells show degradation of 2′,3′-cGAMP after a 1-h reaction. Data are representative of two independent experiments. The ‘−’ refers to a buffer-only control. b, Western blot analysis of poxin-transduced cell lines demonstrating expression of both VACV poxin(WT) and poxin(H17A) proteins. Data are representative of two independent experiments. Gel source data are available in Supplementary Fig. 1. c, Schematic demonstrating strategy for poxin (B2R) knockout by homologous recombination and replacement with sfGFP. Coloured arrows depict primers used for PCR and sequencing validation of selected viral clones. d, PCR analysis of parental VACV and VACV ΔPoxin confirming removal of B2R and replacement with the sfGFP gene. Data are representative of two independent experiments. e, Sequencing trace confirming replacement of B2R with sfGFP in the genome of VACV ΔPoxin. f, Bright-field and fluorescence microscopy showing Vero cells infected with wild-type VACV poxin or VACV ΔPoxin after 20 h at MOI = 1. VACV ΔPoxin-infected cells express sfGFP. Data are representative of three independent experiments. g, TLC analysis of 2′,3′-cGAMP after incubation with lysates of cells infected with wild-type or ΔPoxin viruses. VACV ΔPoxin-infected cells lack detectable 2′,3′-cGAMP degradation activity. The ‘−’ refers to a buffer-only control; M refers to a mock-infection control. Data are representative of three independent experiments. h, Multiple cycle growth curve (MOI = 0.01) of wild-type and ΔPoxin VACV strains in Vero cells, demonstrating that poxin knockout has no effect on viral growth kinetics in interferon-deficient cells in cell culture (n = 2). Data are mean ± s.e.m. i, qRT–PCR analysis of the transcriptional induction of IFNβ and an interferon-stimulated gene (CXCL10) following infection of A549 cells with wild-type or ΔPoxin VACV after 5 h at MOI = 5 (n = 2). Poxin deletion does not increase IFNβ-dependent signalling in cell culture under these conditions. As a positive control, STING-dependent signalling in A549 cells was stimulated with 2′,3′-cGAMP and digitonin permeabilization.
