Host cell Z-RNAs activate ZBP1 during virus infections [HT-29]

Herpes simplex virus 1 (HSV-1) and Influenza A viruses (IAV) trigger Z-form nucleic acid Binding Protein 1 (ZBP1)-initiated cell death. ZBP1 is activated by Z-RNA, and the Z-RNAs which activate ZBP1 during HSV-1 and IAV infections were assumed to be of viral origin. However, we show here that host cell-encoded Z-RNAs are major and sufficient ZBP1 activating ligands following infection by these two human pathogens. The majority of cellular Z-RNAs mapped to intergenic endogenous retroelements (EREs) 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 which disables Cleavage and Polyadenylation Specificity Factor (CPSF)-mediated 3' processing of nascent pre-mRNAs. Mutant viruses lacking ICP27 or NS1, the virus-encoded proteins responsible for inhibiting CPSF and triggering DoTT, failed to 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. Overall design: Directional total RNA-seq of input and immunoprecipitated RNAs from FLAG-ZBP1-expressing HT-29 cells. Cells were either mock-infected or infected with HSV-1 or IAV; immunoprecipitation was performed using Z22 or anti-FLAG antibodies.