Sensing of viral and endogenous RNA by ZBP1/DAI induces necroptosis.
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.We initially wished to confirm the role of ZBP1 in cell death upon virus infection. Expression of ZBP1 was induced by IFN in Zbp1 +/− but not in Zbp1 −/− primary mouse embryonic fibroblasts (MEFs; Fig 1A). We then assessed cell viability upon virus infection by determining intracellular ATP levels using CellTiter‐Glo reagent. Infection with MCMV‐M45mutRHIM, a virus that fails to inhibit ZBP1‐RIPK3‐dependent necroptosis (Upton et al, 2012), induced cell death in Zbp1 +/− primary MEFs following IFN pre‐treatment (Fig 1B). In contrast, viability upon MCMV‐M45mutRHIM infection was much higher in IFN‐treated Zbp1 −/− primary MEFs (Fig 1B), despite comparable expression of RIPK3 and MLKL (Fig 1A). We also infected primary MEFs with MCMV‐M45wt, which did not cause much cell death in either Zbp1 +/− or Zbp1 −/− cells (Fig 1B). As a control, the induction of necroptosis by TNF stimulation in the presence of the caspase inhibitor zVAD (Vercammen et al, 1998) (hereafter simply TZ) was independent of ZBP1 (Fig 1B). Similar results were obtained in immortalised MEFs (Fig EV1A and B). These observations are consistent with an earlier report (Upton et al, 2012) and demonstrate that death of MCMV‐infected cells required ZBP1 and was antagonised by the viral M45 protein.To test if binding of Z‐form nucleic acids to ZBP1 is involved in the induction of cell death during virus infection, we mutated key conserved residues involved in Z‐RNA/DNA binding in mouse ZBP1. Based on the structures of the ZBP1 Zα1 and Zα2 domains (Schwartz et al, 2001; Ha et al, 2008; Kim et al, 2011a,b; Yang et al, 2014) and on sequence alignments (Fig 1C and D), we introduced N46A and Y50A mutations into Zα1 (Zα1mut), N122A and Y126A substitutions into Zα2 (Zα2mut), and all four mutations together (Zα1α2mut). Mutation of the corresponding residues in ADAR1's Zα ZBD completely abolishes Z‐DNA binding without altering protein stability (Schade et al, 1999); accordingly, we predicted diminished binding of our ZBP1 mutants to Z‐RNA/DNA. Wild‐type and Zα1α2mut ZBP1 proteins were equally able to induce an NF‐κB reporter upon ectopic expression together with RIPK3 in HEK293T cells, suggesting that the mutant protein folds correctly and retains signalling via its RHIMs (Figs 1E and EV1C).We reconstituted immortalised Zbp1 −/− MEFs with ZBP1 3xFLAG‐tagged ZBD mutants (Fig 1F). Cell viability following TZ treatment or MCMV‐M45wt infection was comparable between parental Zbp1 −/− and ZBP1‐reconstituted cells (Fig 1G). MCMV‐M45mutRHIM infection induced pronounced cell death in ZBP1 wild‐type reconstituted cells (Fig 1G and H). In contrast, ZBP1‐Zα1α2mut‐expressing cells displayed higher viability after infection and behaved similar to the parental Zbp1 −/− cells, while ZBP1‐Zα1mut and ZBP1‐Zα2mut cells showed intermediate phenotypes (Fig 1G and H), with a greater contribution to cell death of the Zα2 domain. Cell death induced by MCMV‐M45mutRHIM in wild‐type ZBP1‐expressing cells was also observed by live‐cell microscopy using the dye SytoxGreen, which stains cells that have lost membrane integrity, and cell death was detected from 8 h post‐infection onwards (Fig 1I). We then used mouse NIH3T3 fibroblasts, a transformed cell line. Endogenous ZBP1 expression was undetectable by Western blot, but could be induced by IFN treatment (Fig EV1D). We stably transduced NIH3T3 cells to express either 3xFLAG‐tagged wild‐type or mutant ZBP1 (Fig EV1D and F). Cell viability was comparable between these cells after MCMV‐M45wt infection (Fig EV1E) and upon TZ treatment (Fig EV1G and H). Consistent with our results in MEFs, cell viability was decreased in wild‐type ZBP1 but not in ZBP1‐Zα1α2mut‐expressing cells following infection with MCMV‐M45mutRHIM (Fig EV1E–H). Taken together, these observations suggest that induction of cell death by ZBP1 requires intact ZBDs and may imply that nucleic acids in Z‐conformation trigger this response.All mice were on the C57Bl/6 background. Tmem173 −/− (STING‐deficient) mice were a gift from J Cambier (Jin et al, 2011). The Zbp1 gene is located on chromosome 2 (GenBank NM_021394.2; Ensembl ENSMUSG00000027514). ZBP1‐Zα1α2mut transgenic mice were generated by Cyagen (see Fig EV3A) and have been designated as Zbp1 < tm1.1Jreh> (MGI:5775152). See Appendix Supplementary Methods for further details. This work was performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and institutional guidelines for animal care. This work was approved by a project license granted by the UK Home Office (PPL No. 40/3583) and was also approved by the Institutional Animal Ethics Committee Review Board at the University of Oxford.The authors thank K. Ishii for Zbp1 −/− MEFs, E. Mocarski for MCMV‐M45mutRHIM virus, A. Pichlmair for the α‐ZBP1 antibody and Q. Tang for MCMV‐IE1/3‐GFP and the α‐IE1 antibody. The authors further thank A. Jackson and members of the Rehwinkel laboratory for critical discussions and reading of the manuscript draft. This work was funded by the UK Medical Research Council (MRC core funding of the MRC Human Immunology Unit) and by the Wellcome Trust (grant number 100954). KBR and JWU were funded by the Cancer Prevention and Research Institute of Texas (CPRIT), R1202. JM was a recipient of an EMBO long‐term postdoctoral fellowship and was also supported by Marie Curie Actions (EMBOCOFUND2010, GA‐2010‐267146). Tmem173 −/− mice were provided by J. Cambier and are subject to materials transfer agreements.