The phage prohead protease is a key determinant of Type I CBASS immune activation and evasion

Abstract

Viruses must overcome a diverse array of host immune defenses to ensure successful replication and propagation. In bacteria, CBASS (cyclic oligonucleotide-based antiphage signaling system) immunity restricts phage replication via synthesis of cyclic nucleotide-based signals which amplify antiviral signaling and halt phage propagation via abortive infection. Structural and functional analysis of CBASS operons revealed evolutionary connections with the mammalian cGAS-STING innate immune pathway, demonstrating that nucleotide-based second messenger signaling is an ancient mechanism of host antiviral defense. In mammals, cGAS-STING signaling is initiated by recognition of viral dsDNA by cGAS (Cyclic GMP-AMP Synthase) and further studies have shown that additional cGAS-like receptors (cGLRs) are also stimulated by double-stranded nucleic acid ligands. In contrast, the molecular cues which initiate antiviral signaling in bacterial CBASS immunity remain incompletely understood. We conduct a large-scale screen of 975 Type I CBASS operon-phage challenges and show that operons with distinct CD-NTases (cGAS/DncV-like NTase) and Cap (CD-NTase-associated protein) effectors exhibit marked patterns of phage restriction. We functionally characterize the CBASS-associated AGS-C immunoglobulin-like fold domain and find that it is required for defense against select phages and use X-ray crystallography to determine the 1.7 Å structure of an AGS-C domain. Escaper phages evade CBASS immunity via coding mutations in virion assembly proteins and we demonstrate that the phage Bas13 prohead protease protein interacts with the CD-NTase EcCdnD12 in cells and is sufficient to induce CBASS-dependent growth arrest in a two-plasmid system, defining phage virion assembly as a determinant of Type I CBASS immunity and demonstrating viral protein recognition as a novel putative mechanism of cGAS-like enzyme activation. To further study regulation of human cGAS–STING immunity, we determined a series of crystal structures of human-mouse chimeric TREX1 (Three prime repair exonuclease 1) proteins, a key negative regulator of cGAS, and identify human TREX1 residues critical for crystallization. We also solve the crystal structures of a >99% human TREX1 chimera and fully WT human dsDNA-bound TREX1 and perform biochemical experiments on TREX1 autoimmune disease-associated mutant proteins.