MDA5

Abstract: RIG-I recognizes molecular patterns in viral RNA to regulate the induction of type I interferons. The C-terminal domain (CTD) of RIG-I exhibits high affinity for 5' triphosphate (ppp) dsRNA as well as blunt-ended dsRNA. Structures of RIG-I CTD bound to 5'-ppp dsRNA showed that RIG-I recognizes the termini of dsRNA and interacts with the ppp through electrostatic interactions. However, the structural basis for the recognition of non-phosphorylated dsRNA by RIG-I is not fully understood. Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5' ppp dsRNA and interacts with both strands of the dsRNA. Overlapping sets of residues are involved in the recognition of blunt-ended dsRNA and 5' ppp dsRNA. Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I. These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.

Abstract: Oxidative stress is linked to the inflammation observed in asthma and COPD and might be involved in exacerbations. Both COPD and asthma exacerbations often involve rhinovirus (RV) infection. Impaired anti-viral signaling and attenuated viral clearance in the bronchial epithelium has been hypothesized for these diseases. However, it is not known whether oxidative stress affects the anti-viral signaling capacity. Bronchial epithelial cells from asthma and COPD donors were infected with RV or treated with the oxidative stressor H2O2 followed by exposure to the synthetic RV replication intermediate poly(I:C). Gene and protein levels of antioxidants and anti-viral responses were measured 3h and 24h post poly(I:C) exposure. RV infection and poly(I:C) stimulation induced protein levels of the antioxidants SOD1 and SOD2. poly(I:C) was used to ascertain a constant infection-like burden in the interaction experiments. Upon pre-treatment with increasing doses of H2O2 the antioxidant response to dsRNA was dose-dependently decreased. This data suggests exaggerated H2O2-induced oxidative stress. Further, poly(I:C)-induced IFNs gene expression was reduced after pre-treatment with H2O2. This was associated with a reduced expression of the pattern recognition receptors RIG-I, MDA5 and TLR3 both on gene and protein level. Knockdown of TLR3 but not RIG-I/MDA5 abrogated impairment of poly(I:C)-induced IFNs gene expression by H2O2. Oxidative stress impairs anti-viral signaling in asthma and COPD bronchial epithelium, likely by reducing RV-responsiveness of TLR3. These findings shed light on molecular mechanisms potentially causing reduced IFNs at exacerbation.

Abstract: The early detection of invading viruses by the host depends on their identification by pathogen sensors. These include Toll-like receptors (TLRs) as well as cytoplasmic RNA helicases such as retinoic acid inducible protein I (RIG-I) and melanoma differentiation associated gene 5 (MDA-5). These pathogen sensors recognize specific molecular patterns found in viruses and trigger inflammatory and antiviral responses that result in the eradication of invading pathogens. In this study we investigated the specific recognition of Human rhinovirus 6 (HRV6) the common cold pathogen by the innate immune response in lung epithelial cells. Our experiments established that in the first stages on infection the TLRs play a crucial role in HRV recognition and that different constituents of HRV6 are recognized by different TLRs, while upon viral replication and generation of dsRNA the type I IFN inflammatory response is mediated by MDA-5. The HRV6 capsid is recognized via TLR2, whereas upon HRV6 ssRNA internalization the virus genome is recognized by TLR7 and TLR8. Upon generation of dsRNA the type I IFN response is mediated by MDA-5. The combined recognition by different TLRs and MDA5 and their upregulation concurs with the huge inflammatory response seen in the common cold caused by human rhinoviruses.