A Dual-Functioning 5ʹ-PPP-NS1shRNA that Activates a RIG-I Antiviral Pathway and Suppresses Influenza NS1
Neetu Singh, Priya Ranjan, Weiping Cao, Jenish Patel, Shivaprakash Gangappa, Bruce A. Davidson, John M. Sullivan, Paras N. Prasad, Paul R. Knight, Suryaprakash Sambhara
Abstract
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.