Associate editor: S. Pestka
Functions of the cytoplasmic RNA sensors RIG-I and MDA-5: Key regulators of innate immunity

https://doi.org/10.1016/j.pharmthera.2009.06.012Get rights and content

Abstract

The innate immune system responds within minutes of infection to produce type I interferons and pro-inflammatory cytokines. Interferons induce the synthesis of cell proteins with antiviral activity, and also shape the adaptive immune response by priming T cells. 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. Two families of pattern recognition receptors have been shown to distinguish unique molecules present in pathogens, such as bacterial and fungal cell wall components, viral RNA and DNA, and lipoproteins. The first family includes the membrane-bound toll-like receptors (TLRs). Studies of the signaling pathways that lead from pattern recognition to cytokine induction have revealed extensive and overlapping cascades that involve protein–protein interactions and phosphorylation, and culminate in activation of transcription proteins that control the transcription of genes encoding interferons and other cytokines. 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. In this review we summarize the discovery of these cytoplasmic sensors, 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 function of these cytoplasmic sensors in apoptosis, development and differentiation, and diabetes.

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

References (165)

  • GeeP. et al.

    Essential role of the N-terminal domain in the regulation of RIG-I ATPase activity

    J Biol Chem

    (2008)
  • GeorgelP. et al.

    Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis

    Dev Cell

    (2001)
  • GrantS. et al.

    Recombinant human interferon sensitizes resistant myeloid leukemic cells to induction of terminal differentiation

    Biochem Biophys Res Commun

    (1985)
  • GreinerJ.W. et al.

    Modulation of tumor associated antigen expression and shedding by recombinant human leukocyte and fibroblast interferons

    Pharmacol & Therapeut

    (1985)
  • GuoW. et al.

    The exocyst meets the translocon: a regulatory circuit for secretion and protein synthesis?

    Trends Cell Biol

    (2004)
  • HsuH. et al.

    TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex

    Immunity

    (1996)
  • HuangF. et al.

    Differentiation induction subtraction hybridization (DISH): an approach for cloning genes differentially expressed during growth arrest and terminal differentiation in human melanoma cells

    Gene

    (1999)
  • KatoH. et al.

    Cell type-specific involvement of RIG-I in antiviral response

    Immunity

    (2005)
  • KelliherM.A. et al.

    The death domain kinase RIP mediates the TNF-induced NF-kappaB signal

    Immunity

    (1998)
  • KovacsovicsM. et al.

    Overexpression of Helicard, a CARD-containing helicase cleaved during apoptosis, accelerates DNA degradation

    Curr Biol

    (2002)
  • LeulierF. et al.

    Inducible expression of double-stranded RNA reveals a role for dFADD in the regulation of the antibacterial response in Drosophila adults

    Curr Biol

    (2002)
  • LinR. et al.

    Negative regulation of the retinoic acid-inducible gene I-induced antiviral state by the ubiquitin-editing protein A20

    J Biol Chem

    (2006)
  • LipschutzJ.H. et al.

    The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum

    J Biol Chem

    (2003)
  • LuL.L. et al.

    Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1

    J Biol Chem

    (2008)
  • MattaH. et al.

    Kaposi's sarcoma-associated herpesvirus (KSHV) oncoprotein K13 bypasses TRAFs and directly interacts with the IkappaB kinase complex to selectively activate NF-kappaB without JNK activation

    J Biol Chem

    (2007)
  • MelroeG.T. et al.

    Recruitment of activated IRF-3 and CBP/p300 to herpes simplex virus ICP0 nuclear foci: potential role in blocking IFN-beta induction

    Virology

    (2007)
  • AggarwalK. et al.

    Positive and negative regulation of the Drosophila immune response

    BMB Rep

    (2008)
  • AlffP.J. et al.

    The NY-1 hantavirus Gn cytoplasmic tail coprecipitates TRAF3 and inhibits cellular interferon responses by disrupting TBK1–TRAF3 complex formation

    J Virol

    (2008)
  • AndrejevaJ. et al.

    The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFN-beta promoter

    Proc Natl Acad Sci U S A

    (2004)
  • ArimotoK. et al.

    Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125

    Proc Natl Acad Sci U S A

    (2007)
  • AwomoyiA.A. et al.

    Association of TLR4 polymorphisms with symptomatic respiratory syncytial virus infection in high-risk infants and young children

    J Immunol

    (2007)
  • BalachandranS. et al.

    A FADD-dependent innate immune mechanism in mammalian cells

    Nature

    (2004)
  • BalachandranS. et al.

    Fas-associated death domain-containing protein-mediated antiviral innate immune signaling involves the regulation of Irf7

    J Immunol

    (2007)
  • BaoX. et al.

    Human metapneumovirus glycoprotein G inhibits innate immune responses

    PLoS Pathog

    (2008)
  • BarralP.M. et al.

    MDA-5 is cleaved in poliovirus-infected cells

    J Virol

    (2007)
  • BarroM. et al.

    Rotavirus NSP1 inhibits expression of type I interferon by antagonizing the function of interferon regulatory factors IRF3, IRF5, and IRF7

    J Virol

    (2007)
  • BauhoferO. et al.

    Classical swine fever virus Npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation

    J Virol

    (2007)
  • BozidisP. et al.

    Isolation of endoplasmic reticulum, mitochondria, and mitochondria-associated membrane fractions from transfected cells and from human cytomegalovirus-infected primary fibroblasts

    Curr Protoc Cell Biol

    (2007)
  • BurckstummerT. et al.

    An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome

    Nat Immunol

    (2009)
  • CassadyK.A.

    Human cytomegalovirus TRS1 and IRS1 gene products block the double-stranded-RNA-activated host protein shutoff response induced by herpes simplex virus type 1 infection

    J Virol

    (2005)
  • ChenR.A. et al.

    Inhibition of IkappaB kinase by vaccinia virus virulence factor B14

    PLoS Pathog

    (2008)
  • ChildS.J. et al.

    Evasion of cellular antiviral responses by human cytomegalovirus TRS1 and IRS1

    J Virol

    (2004)
  • ChildS.J. et al.

    Double-stranded RNA binding by a heterodimeric complex of murine cytomegalovirus m142 and m143 proteins

    J Virol

    (2006)
  • ChildsK.S. et al.

    Mechanism of mda-5 inhibition by paramyxovirus V proteins

    J Virol

    (2008)
  • CocudeC. et al.

    A novel cellular RNA helicase, RH116, differentially regulates cell growth, programmed cell death and human immunodeficiency virus type 1 replication

    J Gen Virol

    (2003)
  • DiaoF. et al.

    Negative regulation of MDA5- but not RIG-I-mediated innate antiviral signaling by the dihydroxyacetone kinase

    Proc Natl Acad Sci U S A

    (2007)
  • FisherP.B. et al.

    Effects of interferon on differentiation in normal and tumor cells

    Pharmacol & Therapeut

    (1984)
  • FisherP.B. et al.

    Opposing effects of interferon produced in bacteria and of tumor promoters on myogenesis in human myoblast cultures

    Proc Natl Acad Sci USA

    (1983)
  • FlaneganJ.B. et al.

    Covalent linkage of a protein to a defined nucleotide sequence at the 5′-terminus of virion and replicative intermediate RNAs of poliovirus

    Proc. Natl. Acad. Sci. USA

    (1977)
  • FredericksenB.L. et al.

    Establishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1

    J Virol

    (2008)
  • Cited by (0)

    View full text