Extended Data Fig. 10: 3′2′-cGAMP functions as a potent antiviral ligand.

a, Analysis of the effect of 3′2′-cGAMP on Drosophila C virus (DCV) viral RNA load in flies. dSTING WT and mutant flies were co-injected with DCV and 3′2′-cGAMP or buffer control. Viral RNA levels were measured at each time as indicated relative to the control gene RpL32. DCV is a picornavirus-like (+)ssRNA virus in the family Dicistroviridae. ^**P = 0.0051, ^*P = 0.0388. b, Analysis of the effect of 3′2′-cGAMP on vesicular stomatitis virus (VSV) viral RNA load in flies. dSTING WT and mutant flies were co-injected with VSV and 3′2′-cGAMP or buffer control as in a. Viral RNA levels were measured 4 days post-infection (dpi) relative to the control gene RpL32. VSV is a (-)ssRNA virus in the Rhabdoviridae family. ^*P = 0.0185. c, Analysis of DCV viral RNA load in flies injected with increasing doses of 3′2′-cGAMP, 2′3′-cGAMP or buffer control (as in a). Viral RNA levels were measured 2 dpi relative to the control gene RpL32. For 2′3′-cGAMP injection: 9E−1 ^*P = 0.0192. For 3′2′-cGAMP injection: 9E−3 ^*P = 0.0212, 9E−2 ^**P = 0.0075, 9E−1 ^**P = 0.0070. d, Survival curves after DCV infection showing the effect of injection with dose titration of 3′2′-cGAMP or 2′3′-cGAMP compared with buffer control. Both cGAMP isomers significantly delay mortality in a dose-dependent manner; 3′2′-cGAMP provides greater protection in comparison to 2′3′-cGAMP. For 2′3′-cGAMP injection: 9E−3 ^**P = 0.0047, 9E−2 ^**P = 0.0031, 9E−1 ^***P = 0.0002. For 3′2′-cGAMP injection: 9E−4 ^*P = 0.0344, 9E−3 ^***P = 0.0005, 9E−2 ^****P < 0.0001, 9E−1 ^****P < 0.0001. All data in a–d represent the mean ± s.e.m. of n = 3 independent experiments and each point represents a pool of 6 flies (a, b) or 10 flies (c, d). P value is ns unless otherwise noted; ns P > 0.05.

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