The sensor retinoic acid-inducible gene-I (RIG-I) detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to play a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during DNA virus infection are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bind to RIG-I during herpes simplex virus 1 (HSV-1) infection. HSV-1 infection induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated RNA5SP141-interacting proteins, thereby allowing RNA5SP141 to bind RIG-I and induce type I interferon. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related Epstein-Barr virus (EBV) as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following viral depletion of their respective binding proteins
