The cyclic-oligonucleotide-based anti-phage signalling system (CBASS) is a type of innate prokaryotic immune system. Composed of a cyclic GMP–AMP synthase (cGAS) and CBASS-associated proteins, CBASS uses cyclic oligonucleotides to activate antiviral immunity. One major class of CBASS contains a homologue of eukaryotic ubiquitin-conjugating enzymes, which is either an E1–E2 fusion or a single E2. However, the functions of single E2s in CBASS remain elusive. Here, using biochemical, genetic, cryo-electron microscopy and mass spectrometry investigations, we discover that the E2 enzyme from Serratia marcescens regulates cGAS by imitating the ubiquitination cascade. This includes the processing of the cGAS C terminus, conjugation of cGAS to a cysteine residue, ligation of cGAS to a lysine residue, cleavage of the isopeptide bond and poly-cGASylation. The poly-cGASylation activates cGAS to produce cGAMP, which acts as an antiviral signal and leads to cell death. Thus, our findings reveal a unique regulatory role of E2 in CBASS.

基于环状寡核苷酸的抗噬菌体信号系统（CBASS）是一种先天原核免疫系统。 CBASS 由环状 GMP-AMP 合酶 (cGAS) 和 CBASS 相关蛋白组成，利用环状寡核苷酸激活抗病毒免疫。 CBASS 的一个主要类别包含真核泛素结合酶的同源物，它可以是 E1-E2 融合酶，也可以是单个 E2。然而，CBASS 中单个 E2 的功能仍然难以捉摸。在这里，通过生化、遗传学、冷冻电子显微镜和质谱研究，我们发现粘质沙雷氏菌的 E2 酶通过模仿泛素化级联来调节 cGAS。这包括 cGAS C 末端的加工、cGAS 与半胱氨酸残基的缀合、cGAS 与赖氨酸残基的连接、异肽键的裂解和聚 cGAS 化。多聚 cGAS 化激活 cGAS 产生 cGAMP，作为抗病毒信号并导致细胞死亡。因此，我们的研究结果揭示了 E2 在 CBASS 中的独特调节作用。