Functional characterizations of RIG-I to GCRV and viral/bacterial PAMPs in Grass Carp Ctenopharyngodon idella

Abstract

Background: RIG-I (retinoic acid inducible gene-I) is one of the key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) and mediating the induction of type I interferon and inflammatory cytokines in innate immune response. Though the mechanism is well characterized in mammals, the study of the accurate function of RIG-I in teleosts is still in its infancy. Methodology/Principal Findings: To clarify the functional characterizations of RIG-I in grass carp Ctenopharyngodon idella (CiRIG-I), six representative overexpression plasmids were constructed and transfected into C. idella kidney (CIK) cell lines to obtain stably expressing recombinant proteins, respectively. A virus titer test and 96-well plate staining assay showed that all constructs exhibited the antiviral activity somewhat. The quantitative real-time RT-PCR (qRT-PCR) demonstrated that mRNA expressions of CiIPS-1, CiIFN-I and CiMx2 were regulated by not only virus (GCRV) or viral PAMP (poly(IC) challenge but also bacterial PAMPs (LPS and PGN) stimulation in the steadily transfected cells. The results showed that the full-length CiRIG-I played a key role in RLR pathway. The repressor domain (RD) exerted an inhibitory function of the signaling channel under all utilized challenges. Caspase activation and recruitment domains (CARDs) showed a positive role in GCRV and poly(I:C) challenge. Helicase motifs were crucial for the signaling pathway upon LPS and PGN stimulation. Interestingly, ΔCARDs (CARDs deleted) showed postive modulation in RIG-I signal transduction. Conclusions/Significance: The results provided some novel insights into RIG-I sensing with a strikingly broad regulation in teleosts, responding not only to the dsRNA virus or synthetic dsRNA but also bacterial PAMPs. © 2012 Chen et al.