A Dual-Functioning 5ʹ-PPP-NS1shRNA that Activates a RIG-I Antiviral Pathway and Suppresses Influenza NS1

Retinoic acid-inducible gene-I (RIG-I) is a cytosolic pathogen sensor that is crucial against a number of viral infections. Many viruses have evolved to inhibit pathogen sensors to suppress host innate immune responses. In the case of influenza, nonstructural protein 1 (NS1) suppresses RIG-I function, leading to viral replication, morbidity, and mortality. We show that silencing NS1 with in-vitro-transcribed 5′-triphosphate containing NS1 short hairpin RNA (shRNA) (5′-PPP-NS1shRNA), designed using the conserved region of a number of influenza viruses, not only prevented NS1 expression but also induced RIG-I activation and type I interferon (IFN) expression, resulting in an antiviral state leading to inhibition of influenza virus replication in vitro. In addition, administration of 5′-PPP-NS1shRNA in prophylactic and therapeutic settings resulted in significant inhibition of viral replication following viral challenge in vivo in mice with corresponding increases of RIG-I, IFN-β, and IFN-λ, as well as a decrease in NS1 expression.