Anti-influenza Virus Functional Comparison Of RIG-Is Derived From Waterfowl And Mammal

Retinoic acid-induced gene I (RIG-I), is the most crucial pattern recognition receptor (PRR) of innate immunity, which senses the single strand RNA (ssRNA) and double strand RNA (dsRNA) of RNA viruses and triggers the production of IFN-β.Bioinformatics analysis showed that avian RIG-I is close to reptilian RIG-I, not RIG-Ⅰ of mammal. The number of phosphorylation sites and ubiquitination sites in RIG-I increased on the fish-reptiles-mammal axis. However, ubiquitination sites of RIG-Ⅰ increased first and then decreased on the fish-reptiles-birds axis. These results suggest that regulatory mechanism of RIG-Ⅰ became complexification from reptiles to mammal and simplification from reptiles to birds.Mammal-derived and waterfowl-derived RIG-I and CARDs expression levels and celluar localization analysis demonstrated that waterfowl RIG-Is/CARDs expression levels significantly higher than mammal counterparts in both DF-1and A549cells. Codon usage bias analysis revealed that waterfowl-derived RIG-I contain less rare codon and have higher adaptability in human expression system than mammal counterparts.The comparison in anti-influenza virus activity between mammal-derived and waterfowl-derived RIG-I and CARDs show that duck and goose RIG-I (dRIG-I and gRIG-Ⅰ) significantly increase antiviral genes levels, and decrease the viral titers in DF-1cells upon LAV infection, whereas human and mouse RIG-I (hRIG-I and mRIG-I) are not functional. On the contrary, in human A549cells, hRIG-I had the most pronounced ability to induce antiviral genes and decrease viral titers, followed by mRIG-I and dRIG-I. gRIG-I even blocked the antiviral genes expression. Taken together, these data suggest that RIG-I can only activate the IFN signaling pathway effectively in its own or very similar cellular systems (dRIG-I and gRIG-I in DF-1, and hRIG-I and mRIG-I in A549,293T, and MDCK cells).To assess the differences in IFN-inducing activity between dRIG-I and gRIG-I, we replaced gCARD1with dCARD1, gCARD2with dCARD2. The replacement of gCARDl with dCARDl facilitated the IFN-inducing activity of gCARDs, which implying that some of the amino acid mutant in CARD1might lead to the different IFN-inducing activity of dRIG-I and gRIG-I. The dCARDs-S8G and gCARDs-G8S mutant further confirmed that ser8negatively regulated dCARDs.To determine whether dRIG-I is responsible for the recognition of influenza virus in duck embyro fibroblast (DEF), we used RNAi to inhibit endogenous dRIG-I. We observed that dRIG-I knockdown up-regulated influenza virus M gene expression and reduced IFN-β mRNA level, which suggests that dRIG-I is essential to sense influenza virus and activation of interferon response in DEF. Furthermore, the apoptosis and cytopathic effect analysis demonstrated that dRIG-I protect DF-1cells from influenza virus-induced apoptosis.Taken together, our study revealed the RIG-I evolution, functional differences between waterfowl RIG-Ⅰ and mammalian RIG-I, and the requirement of dRIG-I for sensing influenza virus in DEF, Which lay a foundation for the functional research of avian RIG-I.