Profiling anti-influenza activities of the pathogen recognition receptor RIG-I: Novel targets for antiviral intervention strategies
Influenza A virus (FLUAV) infections cause substantial morbidity and mortality during seasonal and pandemic outbreaks worldwide, however, currently available antiviral therapeutics are of limited efficacy. This project aims to elucidate new molecular interactions at the virus-host interface on a cellular level and within the infected lung, to be further exploited for development of putative novel antiviral compounds with improved efficacy.
RIG-I is a major pathogen recognition receptor for sensing FLUAV infections. This cytoplasmic RNA helicase binds virus-specific RNA structures and undergoes a conformational shift to expose a signaling-active domain, the 2CARD. RIG-I thereby triggers a signaling cascade that eventually results in the production of antivirally active cytokines like the type I interferons (IFNs). We have identified two novel RNA ligands of RIG-I, namely the so-called panhandle promotor of FLUAV, and short self-RNA fragments that are derived from the host cell 45s ribosomal RNA through RNase digestion. The panhandle promotor of FLUAV is a short dsRNA structure with a 5-triphosphate end that is covered by the viral polymerase on the viral nucleocapsids. We have shown that RIG-I can access the panhandle for a fraction of incoming FLUAV nucleocapsids. Importantly, decreasing polymerase-nucleocapsid affinity either by mutation to an avian-specific FLUAV adaptation (PB2-627K to PB2-627E) or by using a polymerase-disrupting peptide both result in a substantial increase in RIG-I activation. Strikingly, such hyper-activated RIG-I revealed a direct antiviral activity that was independent of IFN production and most likely due to a displacement of the viral polymerase and the panhandle-clamping of RIG-I.
Studying the different and the newly discovered antiviral activities of RIG-I may enable us to discover new strategies for anti-influenza therapy. Combining expertises in the field of virology and immunology, our project therefore aims at (i) enhancement of panhandle recognition by RIG-I (through generating RIG-I mutants with increased direct antiviral activity), (ii) the identification of additional, postulated anti-FLUAV host cell factors that directly act on the panhandle in a RIG-I-like manner, and (iii) the identification of virus-stimulated self-RNA structures with strong efficacy to activate RIG-I. Our findings will be exploited in vitro and in murine infection models in vivo, with the ultimate goal to develop therapeutic strategies combining these synergistic antiviral effects to generate antivirals with high efficacy.