Cyclic oligonucleotide-based signaling system (CBASS) is an antiviral system that protects bacteria from phage infection and is evolutionarily related to human cGAS-STING immunity. cGAS-STING signaling is initiated by the recognition of viral DNA, but the molecular cues activating CBASS are incompletely understood. Using a screen of 975 type I CBASS operon-phage challenges, we show that operons with distinct cGAS/DncV-like nucleotidyltransferases (CD-NTases) and CD-NTase-associated protein (Cap) effectors exhibit marked patterns of phage restriction. We find that some type I CD-NTase enzymes require a C-terminal AGS-C immunoglobulin (Ig)-like fold domain for defense against select phages. Escaper phages evade CBASS via protein-coding mutations in virion assembly proteins, and acquired resistance is largely operon specific. We demonstrate that the phage Bas13 prohead protease interacts with the CD-NTase EcCdnD12 and can induce CBASS-dependent growth arrest in cells. Our results define phage virion assembly as a determinant of type I CBASS immune evasion and support viral protein recognition as a putative mechanism of cGAS-like enzyme activation.

基于环状寡核苷酸的信号系统 (CBASS) 是一种抗病毒系统，可保护细菌免受噬菌体感染，并且在进化上与人类 cGAS-STING 免疫相关。 cGAS-STING 信号传导是由病毒 DNA 识别启动的，但激活 CBASS 的分子线索尚不完全清楚。通过对 975 个 I 型 CBASS 操纵子噬菌体挑战的筛选，我们发现具有不同 cGAS/DncV 样核苷酸转移酶 (CD-NTase) 和 CD-NTase 相关蛋白 (Cap) 效应子的操纵子表现出明显的噬菌体限制模式。我们发现一些 I 型 CD-NTase 酶需要 C 端 AGS-C 免疫球蛋白 (Ig) 样折叠结构域来防御选定的噬菌体。逃逸噬菌体通过病毒粒子组装蛋白中的蛋白编码突变来逃避 CBASS，并且获得性耐药性很大程度上是操纵子特异性的。我们证明噬菌体 Bas13 前头蛋白酶与 CD-NTase EcCdnD12 相互作用，并且可以诱导细胞中 CBASS 依赖性生长停滞。我们的结果将噬菌体病毒粒子组装定义为 I 型 CBASS 免疫逃避的决定因素，并支持病毒蛋白识别作为 cGAS 样酶激活的假定机制。