Associate editor: S. PestkaFunctions of the cytoplasmic RNA sensors RIG-I and MDA-5: Key regulators of innate immunity
Introduction
The number of pathogens that we encounter daily is astronomical. Most are halted by an efficient defense system that has evolved over millions of years in the face of microbial infections. An important component of this defense arsenal is the innate immune system, which responds within minutes of infection to produce type I interferons (Isaacs and Lindenmann, 1957, Rubinstein et al., 1979, Friesen et al., 1981, Pestka and Baron, 1981). These antiviral proteins are produced by infected cells and lead to the synthesis of cell proteins, which halt viral replication, and also shape the adaptive immune response by priming T cells. Additionally, interferons also modulate a plethora of other important cellular functions including cell growth and differentiation, histocompatibility and tumor antigen expression, gene expression and anti-tumor effects (Fisher et al., 1983, Fisher and Grant, 1984, Giacomini et al., 1984, Grant et al., 1985, Greiner et al., 1984, Huang et al., 1999a, Huang et al., 1999b, Jiang et al., 2000, Moulton et al., 1992, Sen and Sarkar, 2007, Su et al., 2008, Greiner et al., 1985, Greiner et al., 1987).
Despite the discovery of interferons over 50 years ago, only recently have we begun to understand how cells sense the presence of a virus infection and initiate cytokine synthesis. First insights came from the discovery of the toll-like receptors (TLRs) during a study of genes essential for the establishment of the dorsal–ventral axis in Drosophila (Nusslein-Volhard & Wieschaus, 1980). The TLRs were subsequently shown to be transmembrane proteins that distinguish unique molecules present in pathogens, such as bacterial and fungal cell wall components, viral RNA and DNA, and lipoproteins (reviewed in Medzhitov 2007). We now understand that pathogens are recognized as foreign by a family of host pattern recognition receptors. Examination of the signaling pathways that lead from pattern recognition to cytokine induction has revealed extensive and overlapping cascades that involve protein–protein interactions and phosphorylation. These culminate in activation of transcription proteins that control the transcription of genes encoding interferons and other cytokines (reviewed in Kawai & Akira 2008).
A second family of pattern recognition receptors has recently been identified, which comprises the cytoplasmic sensors of viral nucleic acids, including MDA-5, RIG-I, and LGP2. Here we review the discovery of these proteins, how they recognize nucleic acids, the signaling pathways leading to cytokine synthesis, and viral countermeasures that have evolved to antagonize the functions of these proteins. We also consider the role of these cytoplasmic sensors in apoptosis, development and differentiation, and diabetes.
Section snippets
Identification of retinoic acid-inducible gene-I and melanoma differentiation associated gene-5
Retinoic acid-inducible gene-I (RIG-I, also known as DDX58) and melanoma differentiation associated gene-5 (MDA-5, also known as Helicard or IFIH1) are virus sensors expressed ubiquitously in the cytoplasm. RIG-I was initially identified as a gene induced in acute promyelocytic leukemia cells after treatment with retinoic acid (Sun, 1997). A few years later its role as an antiviral protein was reported. In fact, screening an expression cDNA library obtained from IFN-β-treated cells led to the
rig-I and mda-5 promoter regulation
rig-I was first cloned as a retinoic acid (RA)-inducible gene (Sun, 1997). However, the molecular mechanism by which RA regulates rig-I expression remains to be determined. The promoter region of rig-I has not been mapped for RA-responsive elements and with the accrual of information on the role of rig-I in RA signaling, efforts have not been expended in analyzing RA-regulation of rig-I. On the other hand, both rig-I and mda-5 are type I IFN-inducible genes and analysis of the ~2-kb promoter
Recognition of ribonucleic acid
A role for RIG-I as a cytoplasmic sensor of viral RNA was first discovered during a screen for molecules involved in intracellular dsRNA-induced expression of IFN (Yoneyama et al., 2004). Overexpression of RIG-I in mouse L929 cells led to increased activation of the IFN-β promoter after introduction of dsRNA into cells. Activation of this promoter was shown to be dependent upon the RIG-I helicase domain. RIG-I was found to bind poly(I:C) linked to agarose beads, but not single-stranded RNA
Negative regulation of the signaling pathway
Limitation of IFN production is an essential physiological requisite necessary for the overall well-being of the organism. Following initiation of antiviral and antiproliferative responses or the activation of innate immunity, restriction of production of excess IFN must occur. The fact that there exist multiple mechanisms to control IFN levels confirms the importance of counteracting deleterious effects of IFN, that include chronic cellular toxicity and the initiation of inflammatory or
Cell type specificity of retinoic acid-inducible gene-I and melanoma differentiation associated gene-5
In vivo evaluation of the importance of RIG-I and MDA-5 in recognizing virus infection came through the generation of viable knockout mice that lacked these genes. rig-I−/− MEFs did not significantly produce type I IFN in response to a variety of negative-stranded RNA viruses, such as paramyxoviruses, rhabdoviruses and orthomyxoviruses, since the major mechanism of 5′-triphosphate viral RNA recognition was absent (Kato et al., 2005). Conventional dendritic cells (cDCs) lacking rig-I (but not
Viral countermeasures
The various defense mechanisms that operate in healthy hosts have evolved for millions of years, yet are imperfect because viral genomes encode gene products that block every step of host defense. The RNA sensing pathways described in this review are no exception. Viral gene products may antagonize nearly every step of the sensing of RNA by RIG-I and MDA-5 (Fig. 4). Understanding such viral countermeasures not only improves our understanding of innate sensing pathways, but may also suggest
Apoptosis and growth suppression
mda-5 was cloned as a transcript induced during induction of terminal differentiation of human melanoma cells and initiates an irreversible growth arrest program in these cancer cells (Kang et al., 2002). Indeed, the first report on cloning and characterization of mda-5 revealed its growth suppressor properties (Kang et al., 2002). Follow-up studies demonstrated that adenovirus-mediated delivery of mda-5 induced apoptosis that could be inhibited by active Ras/Raf pathway (Kang et al., 2004; Lin
Role of retinoic acid-inducible gene-I in development and differentiation
As has been discussed above, the best-characterized role of RIG-I is in viral RNA-induced type 1 IFN generation. However, recent evidence has suggested that it can act, in the absence of viral infection, to regulate myeloid cell differentiation (Zhang, Shen, et al., 2008). Initial experiments suggested that treatment of retinoic acid-sensitive myeloid leukemia cell lines with all-trans-retinoic acid (ATRA) resulted in increased expression of RIG-I. As this is considered partially to mimic
Melanoma differentiation associated gene-5 and type 1 diabetes
In a large screen of single-nucleotide polymorphisms (SNPs) associated with type 1 diabetes, a number of mutations were observed in the IFN induced with helicase C domain 1 (IFIH1/MDA-5) linkage disequilibrium (LD) block on chromosome 2q. Indeed there was a strong correlation between SNPs in the MDA-5 gene itself and in the 3′ intergenic region and susceptibility to type 1 diabetes (Liu et al., 2009, Smyth et al., 2006, Todd et al., 2007). Furthermore, after screening a large panel of controls
Evolution
The origin of the MDA-5 and RIG-I proteins has been examined from a phylogenetic perspective (Sarkar et al., 2008). What at first might seem a simple task is complicated by the presence of four protein domains in MDA-5 and RIG-I (Fig. 5A). Because the proteins are made up of these four domains that are themselves members of their own large domain families, it is possible that the MDA-5 and RIG-I proteins have a diverse and chimeric evolutionary history. In addition, the proteins that are
Perspectives
In just 6 years we have progressed from the discovery of the cytoplasmic RNA sensors of the innate immune response, to developing a detailed understanding of RNA recognition and the signaling pathways that lead to interferon induction. The molecular mechanisms by which MDA-5 and RIG-I recognize specific RNA ligands remain to be determined; we expect that additional structural work will be required to answer this question. Specifically, it will be necessary to solve the atomic structures of
Acknowledgments
The present studies were supported in part by the National Institutes of Health grants GM068448 (PBF), AI079336 (GNB) and AI50754 and AI068017 (VRR). Support was also provided by the Samuel Waxman Cancer Research Foundation (SWCRF) (PBF). RD thanks the Sackler Institute for Comparative Genomics, the Lewis and Dorothy B Cullman Program in Molecular Systematics and the Korein Family Foundation all at the American Museum of Natural History for their continued support. Support from the VCU
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