Mechanisms of nucleotide signaling in bacterial and metazoan antiviral defense

Abstract

Nucleotide-based antiviral signaling is broadly conserved between bacteria and metazoans. Mammalian cGAS and OAS1 are structurally homologous enzymes that share a pol-β-like nucleotidyltransferase fold and synthesize antiviral nucleotide signals. Mammalian cGAS synthesizes a cyclic dinucleotide using ATP and GTP with one 2′–5′ linkage and one 3′–5′ linkage, known as 2′,3′-cGAMP, that signals downstream to the effector STING, while OAS1 synthesizes a linear polyadenylate molecule with 2′–5′ linkages, known as 2′–5′ oligoadenylate, that signals downstream to the effector RNase L. In bacteria, recent work has uncovered enzymes with structural homology to cGAS, named CD-NTase enzymes, that are diverse in primary sequence and are capable of synthesizing novel cyclic oligonucleotide signals out of all four RNA nucleotides. Using biochemical and structural biology approaches, we define conserved patterns of CD-NTase product formation enabling the preliminary prediction of novel nucleotide products based on CD-NTase primary sequence. Bacterial CD-NTase enzymes are contained in operons that encode effector proteins. Effector function is dependent on recognition of the correct nucleotide signal synthesized by the cognate CD-NTase within the same operon. Effectors are comprised of several different categories of enzymes, and the diverse mechanisms of activation and regulation are open areas of study. Using X-ray crystallography, negative-stain EM, and biochemical approaches, we describe the activation and regulation of a bacterial STING homolog and its ancient effector function of cleaving NAD+. In metazoans, increasing complexity results in pathways that encode signaling functions that activate several different effector pathways, such as STING regulated activation of IRF3 and NF-κB, leading to interferon induction and inflammatory cytokine synthesis. Using enzymatically synthesized 2′–5′ oligoadenylate and sepharose bead-mediated affinity purification and mass spectrometry, we isolate and characterize a novel downstream receptor of 2′–5′ oligoadenylate, the metabolic enzyme Decr1. Altogether, functional and structural conservation of signaling pathways across bacteria and metazoans highlights broad patterns of antiviral defense.