Date Published: April 25, 2013
Publisher: Public Library of Science
Author(s): Marie-Line Goulet, David Olagnier, Zhengyun Xu, Suzanne Paz, S. Mehdi Belgnaoui, Erin I. Lafferty, Valérie Janelle, Meztli Arguello, Marilene Paquet, Khader Ghneim, Stephanie Richards, Andrew Smith, Peter Wilkinson, Mark Cameron, Ulrich Kalinke, Salman Qureshi, Alain Lamarre, Elias K. Haddad, Rafick Pierre Sekaly, Suraj Peri, Siddharth Balachandran, Rongtuan Lin, John Hiscott, Ganes C. Sen.
The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5′ triphosphate (5′ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5′pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes – hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5′pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5′pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5′pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5′pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.
The innate immune system has evolved numerous molecular sensors and signaling pathways to detect, contain and clear viral infections –. Viruses are sensed by a subset of pattern recognition receptors (PRRs) that recognize evolutionarily conserved structures known as pathogen-associated molecular patterns (PAMPs). Classically, viral nucleic acids are the predominant PAMPs detected by these receptors during infection. These sensing steps contribute to the activation of signaling cascades that culminate in the early production of antiviral effector molecules, cytokines and chemokines responsible for the inhibition of viral replication and the induction of adaptive immune responses , –. In addition to the nucleic acid sensing by a subset of endosome-associated Toll-like receptors (TLR), viral RNA structures within the cytoplasm are recognized by members of the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) family, including the three DExD/H box RNA helicases RIG-I, Mda5 and LGP-2 , , , –.
RIG-I agonists are attractive potential antiviral agents, as triggering the innate cytosolic RIG-I pathway mimics the earliest steps of immune recognition and response to viral pathogens. In the present study, a short 5′pppRNA agonist of RIG-I derived from the 5′ and 3′ UTRs of the VSV genome stimulated an antiviral response that protected human lung epithelial A549 cells or human PBMCs from challenge with several viruses, including DENV, Influenza, HIV, VSV, HCV and Vaccinia virus. Intravenous administration of the 5′pppRNA agonist in mice stimulated an antiviral state in vivo that protected animals from lethal influenza virus challenge and controlled influenza virus-mediated pneumonia. Analysis of the dynamics of the host transcriptome following 5′pppRNA stimulation was characterized by antiviral and inflammation related gene expression patterns with transcriptional nodes of genes regulated by IRF, NF-κB, and STAT families. Virtually all of the genes activated by IFNα-2b were encompassed within the 5′pppRNA transcriptome; bioinformatics analysis also identified distinct gene patterns and functional processes that were uniquely induced or inhibited by 5′pppRNA. Because of its potency both in vitro and in vivo, 5′pppRNA represents a specific and powerful trigger of innate immunity and a novel approach to antiviral therapy.