Abstract
Nucleic acids are potent triggers for innate immunity. Double‐stranded DNA and RNA adopt different helical conformations, including the unusual Z‐conformation. Z‐DNA/RNA is recognised by Z‐binding domains (ZBDs), which are present in proteins implicated in antiviral immunity. These include ZBP1 (also known as DAI or DLM‐1), which induces necroptosis, an inflammatory form of cell death. Using reconstitution and knock‐in models, we report that mutation of key amino acids involved in Z‐DNA/RNA binding in ZBP1's ZBDs prevented necroptosis upon infection with mouse cytomegalovirus. Induction of cell death was cell autonomous and required RNA synthesis but not viral DNA replication. Accordingly, ZBP1 directly bound to RNA via its ZBDs. Intact ZBP1‐ZBDs were also required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross‐linked to endogenous RNA. These observations show that Z‐RNA may constitute a molecular pattern that induces inflammatory cell death upon sensing by ZBP1.
Highlights
Using reconstitution and knock-in models, we report that mutation of key amino acids involved in Z-DNA/RNA binding in ZBP1’s Z-binding domains (ZBDs) prevented necroptosis upon infection with mouse cytomegalovirus
Intact ZBP1-ZBDs were required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross-linked to endogenous RNA
Viability upon mouse cytomegalovirus (MCMV)-M45mutRHIM infection was much higher in IFN-treated Zbp1À/À primary mouse embryonic fibroblasts (MEFs) (Fig 1B), despite comparable expression of RIPK3 and MLKL (Fig 1A)
Summary
Double-stranded (ds) DNA typically adopts the so-called B-conformation, while dsRNA is usually in the A-conformation. Both DNA and RNA can adopt a Z-form double helix that is characterised by a left-handed helical arrangement, a zigzag pattern of the phosphodiester backbone and the absence of major grooves (Wang et al, 1979; Hall et al, 1984; Rich & Zhang, 2003). Antibodies raised against Z-form RNA or DNA stain mammalian cells and chromosomes, respectively, suggesting that nucleic acids in this unusual conformation occur naturally in cells (Viegas-Pequignot et al, 1983; Zarling et al, 1990). Biological functions of the Z-conformation are only partially understood (Rich & Zhang, 2003; Wang & Vasquez, 2007).
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