Herpes simplex virus 1 (HSV-1) and influenza A viruses (IAV) induce Z-form-nucleic-acid-binding protein 1 (ZBP1)-initiated cell death1–8. ZBP1 is activated by Z-RNA1,7,9, and the Z-RNAs that trigger ZBP1 during HSV-1 and IAV infections were assumed to be of viral origin1. Here, however, we show that host cell-encoded Z-RNAs are major and sufficient ZBP1-activating ligands after infection by these two human pathogens. The majority of cellular Z-RNAs mapped to intergenic endogenous retroelements embedded within abnormally long 3′ extensions of host cell mRNAs. These aberrant host cell transcripts arose as a consequence of disruption of transcription termination (DoTT)—a virus-driven phenomenon that disables cleavage and polyadenylation specificity factor (CPSF)-mediated 3′ processing of nascent pre-mRNAs10–15. Mutant viruses lacking ICP27 or NS1—the virus-encoded proteins responsible for inhibiting CPSF and triggering DoTT13,15—did not induce host cell Z-RNA accrual and were attenuated in their ability to stimulate ZBP1. Ectopic expression of HSV-1 ICP27 or IAV NS1 or pharmacological blockade of CPSF activity induced accumulation of host cell Z-RNAs and activated ZBP1. These results demonstrate that DoTT-generated cellular Z-RNAs are bona fide ZBP1 ligands, and position ZBP1-activated cell death as a host response to counter viral disruption of the cellular transcriptional machinery.
