Associate editor: S. Pestka
Functions of the cytoplasmic RNA sensors RIG-I and MDA-5: Key regulators of
Paola M. Barrala,e,⁎, Devanand Sarkara,b,c, Zao-zhong Sua, Glen N. Barberd, Rob DeSallef,
Vincent R. Racanielloe, Paul B. Fishera,b,c,e,⁎
aDepartment of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA
bVCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, VA
cVCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA
dDepartment of Microbiology and Immunology, UM/Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL, USA
eDepartment of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY, USA
fAmerican Museum of Natural History, Sackler Institute for Comparative Genomics, New York, NY, USA
a b s t r a c ta r t i c l ei n f o
Antiviral innate immunity
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.
© 2009 Elsevier Inc. All rights reserved.
Characterization of retinoic acid-inducible gene-I and melanoma differentiation associated gene-5 . . . . . .
rig-I and mda-5 promoter regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The retinoic acid-inducible gene-I and melanoma differentiation associated gene-5 signaling pathway . . . .
Negative regulation of the signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cell type specificity of retinoic acid-inducible gene-I and melanoma differentiation associated gene-5 . . . .
Viral countermeasures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Apoptosis and growth suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .220
Pharmacology & Therapeutics 124 (2009) 219–234
Abbreviations: Atg5, autophagy related 5 homolog (S. Cerevisiae); Atg12, autophagy related 12 homolog (S. Cerevisiae); CARD, caspase activation and recruitment domain; cDCs,
conventional dendritic cells; CTD, C-terminal domain; DISH, differentiation induction subtraction hybridization; EMCV, encephalomyocarditis virus; ER, endoplasmic reticulum;
FADD, Fas associated death domain; IFN, interferon; GMCSF, granulocyte macrophage colony stimulating factor; IKK, I kappa B kinase; IRF, interferon regulatory factor; IKKi, IkappaB-
kinase-epsilon; IPS-1, interferon β promoter stimulator 1; LGP2, laboratory of genetics and physiology 2; MDA-5, melanoma differentiation associated gene-5; MEF, mouse embryo
fibroblast; Mss4, mammalian suppressor of Sec4; NLRX1, NLR (nod-like receptor) family member X1; PAMP, pathogen-associated molecular pattern; pDCs, plasmacytoid dendritic
cells; Poly(I:C), polyinosinic:polycytidylic acid; RA, retinoic acid; RIG-I, retinoic acid-inducible gene-I; RIP1, receptor-interacting serine-threonine kinase 1; RLH, RIG-like helicases;
RNase L, ribonuclease L; RNF125, ring finger protein 125; SNP, single-nucleotide polymorphism; STING, stimulator of IFN genes; TBK1, TANK-binding kinase 1; TLR, toll-like receptor;
TRADD, TNFR1-associated death domain protein; TRAF3, TNF receptor-associated factor 3; TRAP, translocon-associated protein; VSV, vesicular stomatitis virus.
⁎ Corresponding authors. Barral is to be contacted at the Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, USA. Fisher, VCU
Institute of Molecular Medicine, Virginia Commonwealth University, Richmond, VA, USA. Tel.: +1 804 828 9632x628 3506.
E-mail addresses: email@example.com (P.M. Barral), firstname.lastname@example.org (P.B. Fisher).
0163-7258/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Pharmacology & Therapeutics
journal homepage: www.elsevier.com/locate/pharmthera
9. Role of retinoic acid-inducible gene-I in development and differentiation . . . . . . . . . . . . . . . . .
Melanoma differentiation associated gene-5 and type 1 diabetes . . . . . . . . . . . . . . . . . . . . .
Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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
responds within minutes of infection to produce type I interferons
Pestka & Baron, 1981). These antiviral proteins are produced by
infected cells and lead tothe 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,
histocompatibilityandtumorantigenexpression,gene expression and
anti-tumor effects (Fisher et al.,1983; Fisher & Grant,1984; Giacomini
et al., 1984; Grant et al., 1985; Greiner et al., 1984; Huang et al.,
1999a,1999b; Jiang et al., 2000; Moulton et al., 1992; Sen & Sarkar,
2007; Su et al., 2008; Greiner et al.,1985; Greiner el 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
presentinpathogens,suchasbacterialandfungalcell wall components,
now understand that pathogens are recognized asforeign bya 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
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, develop-
ment and differentiation, and diabetes.
2. Characterization of retinoic acid-inducible
gene-I and melanoma differentiation associated gene-5
2.1. 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 proteinwas reported. In
fact, screening an expression cDNA library obtained from IFN-β-
treated cells led to the isolation of RIG-I (Yoneyama et al., 2004). An
IRF-reporter gene was introduced in L929 cells together with the
cDNA library, and clones were selected for their ability to induce the
promoter after transfection of poly (I:C) (Yoneyama et al., 2004).
Recentlya splice variantofRIG-I hasbeen identified(Gacket al.,2008)
which carries a deletion (residues 36–80) within the first caspase
activation and recruitment domain (CARD) (Fig. 1).
MDA-5 was identified in a differentiation induction subtraction
hybridization (DISH) screen (Huang et al., 1999a,1999b) that was
designed to define genes regulated as a function of induction of
terminal differentiation in human HO-1 melanoma cells (Jiang and
Fisher, 1993). These DISH genes, many of which represented unique
sequences not reported in the 1999 gene database, were named
melanoma differentiation associated (mda) genes. mda-5 was one
such novel upregulated DISH gene in HO-1 human melanoma cells
induced to irreversibly lose growth potential and terminally differ-
entiate by treatment with IFN-β and mezerein, a protein kinase C-
activating compound (Kang et al., 2002).
2.2. Functional domains
RIG-I and MDA-5 are DExD/H RNA helicases, which possess two
(CARD) domains at their amino terminus (Fig.1). Together with LGP2,
laboratory of genetics and physiology 2, they form the RIG-I-like
receptors (RLRs) family. LGP2 possesses only the helicase domain and
lacks the CARD domain (Takeuchi & Akira, 2008; Yoneyama & Fujita,
2008). RIG-I and MDA-5 share ~25% homology within the CARD
domain regions and 40% within the helicase domain.
RIG-I and LGP2 have a similar repressor domain (RD) localized at
the carboxyl terminus (Saito et al., 2007). Evidence suggests an
interaction between the RIG-I repressor region (aa 723–925) and the
CARD and helicase domains (helicase linker region aa 420–627).
Interestingly, overexpression of the repressor domain blocks RIG-I-
Recently, a C-terminal domain (CTD) has been described, which
partly overlaps with the previously identified RD. The structure of the
CTD of RIG-I has been determined by X-ray crystallography (Cui et al.,
2008) and nuclear magnetic resonance (NMR) (Takahasi et al., 2008).
It consists of a basic concave surface and the opposite side contains
acidic residues. A positive cleft is the binding site for dsRNA and 5′-
ppp RNA and also encompasses the biological activity of signal
repression. The crystal structure of RD reveals a zinc-binding domain
coordinated by four cysteines (C810, C813, C864 and C869) conserved
also in MDA-5 and LGP2. Mutational studies have shown that the zinc
coordination site is a key structural motif and is essential for RIG-I
The CARD is the effector domain, that transduces the signal when
the molecule is activated. Overexpression of CARD induces constitu-
tivesignaling independentof viral infection(Yoneyama et al., 2004). It
has also been shown that the CARDs negatively regulate the ATPase
activity of RIG-I preventing its signaling in the absence of viral RNA
(Gee et al., 2008).
Based on structural and functional studies, a model has been
proposed for RIG-I activation. In the absence of its ligand, non-self RNA
generated during viral infection, RIG-I is in an inactivated form. Binding
of dsRNA or 5′-ppp RNA to the basic cleft in the CTD induces a
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
the CARDs, that are then able to interact with the adaptor protein IPS-1
and transduce the signal (Takahasi et al., 2008) (Fig. 2).
2.3. Subcellular distribution of retinoic acid-inducible
gene-I and melanoma differentiation associated gene-5
Recent evidence suggests that RIG-I localizes to membrane ruffles
in non-polarized epithelial cells where it associates with the F-actin
cytoskeleton (Mukherjee et al., 2009). MDA-5, however, localizes to
the cytoplasm with no appreciable co-localization with F-actin. The
specific distribution of RIG-I to the membrane ruffles is dependent on
RacGTPase activity (Mukherjee et al., 2009). Deletional analysis has
shown that association of RIG-I with the cytoskeleton was dependent
In further studies it has been shown that RIG-I is able to induce
RIG-I, unlike MDA-5, is also localized to apical junction complexes in
the interaction of RIG-I with F-actin are significant. It appears that actin
depolymerization results in RIG-I redistribution and activation of IRF3
and NF-κB and IFN-β promoter activity (Mukherjee et al., 2009).
3. 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 region of human rig-I revealed interesting findings (Su
et al., 2007). The promoter was more active in diverse normal cells
compared to their malignant counterparts that correlated with higher
rig-I mRNA expression in normal cells compared to cancer cells. In an
experimental mouse brain tumor model, rig-I expression was abun-
dantly detected in the surrounding normal brain while it was hardly
detectable in the tumor tissue. In both normal and tumor cells rig-I
promoter activity could be induced by IFN-β or by poly(I).poly(C).
factor 1 (IRF1)-binding site just proximal to the transcription start site
that conferred the basal, IFN-β- and dsRNA-mediated induction of the
rig-I promoter. As a corollary to rig-I, IRF1 expression was also higher
a similar kinetics to that of rig-I. IRF1 is a tumor suppressor and the
expression profile of rig-I together with its regulation by IRF1 indicates
that rig-I mightalso have tumor suppressor properties. Althoughmda-5
wascloned asa typeI IFN-inducible gene and mda-5induction by type I
yet no studies have been published analyzing in detail the promoter
rig-I and mda-5 promoters, to check if the mda-5 promoter also displays
normal cell specificity and whether IFN regulation of the mda-5
promoter is controlled by the classical IFN-stimulated regulatory
elements (ISRE) or by other factors, such as IRF1. More importantly,
thesepromotersmightbescreenedforidentificationof small molecules
with potential antiviral properties.
4. The retinoic acid-inducible gene-I and
melanoma differentiation associated gene-5 signaling pathway
4.1. 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). Over-
expression 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 (poly(A)) or dsDNA. Synthesis of RIG-I also
augmented the IFN response of cells infected with Newcastle disease
virus, and reduced the viral yield of vesicular stomatitis virus, a
rhabdovirus, and encephalomyocarditis virus (EMCV), a picornavirus.
Based on these observations, it was suggested that RIG-I specifically
recognizes dsRNA produced during the replication of RNA viruses
Fig. 2. Model for activation of RIG-I and MDA-5 by RNA. In the absence of RNA, the
sensor molecule is folded in an inactive state, with the CARD domain occluded by the
CTD. The latter also contains the binding sites for RNA. When RNAs are produced in
virus-infected cells (either dsRNA or ssRNA with 5′-phosphates), these bind the CTD
and cause a conformational change that exposes the CARD domain. ATP is required for
the conformational change. The CARD domain then interacts with downstream
signaling molecules, leading to IFN transcription.
Fig.1. Schematic representation of the primary structure and functional domains of MDA-5, RIG-I, RIG-I-SV (splice variant) and LGP2. CARD: caspase activating recruitment domain.
RD: repressor domain. CTD: C-terminal domain.
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
(Yoneyama et al., 2004). However, the interaction of RIG-I with its
RNA ligand was clearly not sufficient for induction of IFN. This
conclusion came from the observation that an amino acid change in
the ATP binding domain of RIG-I blocked the induction of IFN, but did
not affect the ability of the protein to bind dsRNA (Yoneyama et al.,
The importance of RIG-I in sensing RNA virus infections was
emphasized by the results of experiments with mice lacking the rig-I
gene (Kato et al., 2005). The production of IFN in response to infection
with Sendai and Newcastle disease viruses (two paramyxoviruses),
and VSV was abrogated in cDCs from rig-I−/−mice. In contrast, IFN
production by pDCs was not dependent upon RIG-I, but upon toll-like
receptors. These observations indicated that sensing of viral RNA by
RIG-I occurs in a cell-type dependent manner.
MDA-5 was originally implicated in the recognition of poly(I:C) and
thanRIG-I (Kangetal.,2002; Yoneyama etal.,2005).Overproduction of
MDA-5 inhibited the growth of EMCV and VSV, aswas alsoobserved for
5, RIG-I and TLR3 are activated during measles virus infection (Berghäll
et al., 2006) andoverexpressionofMDA-5but notRIG-IorTLR3leadsto
that MDA-5 is involved in MV-induced expression of antiviral cytokines
in human epithelial cells (Berghäll et al., 2006). It has also been
demonstrated that RIG-I and MDA-5 expression, but not TLR3, resulted
in the activation of the IFN-β promoter in influenza A virus-infected
epithelial cells (Sirén et al., 2006), suggesting that RIG-I and MDA-5
mediate influenza A virus-induced IFN-β synthesis in epithelial cells
(Sirén et al., 2006).
Gene disruptions provided evidence for differences in RNA sensing
typeorrig-I−/−miceled to similarlevels of cytokines,includingIFNs, in
sera. In contrast, mda-5−/−mice did not produce IFN or interleukins in
DCs isolated from rig-I−/−or wild type mice produced similar levels of
IFN after delivery of poly(I:C) into the cytoplasm. In contrast, after
macrophages, or fibroblasts derived from mda-5−/−mice was severely
impaired. Fibroblasts from mda-5−/−mice responded normally to
dsRNAs transcribed in vitro, while cells from rig-I−/−mice did not.
These dsRNAs were produced by in vitro transcription of the mouse
lamin A/C gene to produce (+) and (−) strand RNAs, which were
subsequently annealed. Therefore, it was concluded that MDA-5
recognizes poly(I:C) in vivo and in vitro, while RIG-I can recognize
poly(I:C) in vitro, but not in vivo; the latter protein principally
recognizes RNA transcripts.
IFN production after infection of cultured cells with paramyx-
oviruses, VSV, influenza virus, and Japanese encephalitis virus (a
flavivirus) was impaired in MEFs from rig-I−/−mice. In contrast, RIG-I
was dispensable for production of IFN in cells infected with EMCV.
GMCSF-DCs from rig-I−/−mice failed to produce IFN after transfection
with VSV RNA, but not after transfection of EMCV RNA. The authors
concluded that in mice, RIG-I recognizes dsRNA produced during RNA
virus replication, and not poly(I:C) (Kato et al., 2006).
The conclusion that the ligand for RIG-I is dsRNA was not consistent
with the fact that many negative-strand RNA viruses, such as members
of the paramyxovirus and orthomyxovirus families, do not produce
dsRNA during their replicative cycles. It was subsequently discovered
that RIG-I also recognizes RNA molecules with a 5′-ppp (Hornunget al.,
2006; Pichlmair et al., 2006; Plumet et al., 2007). The triphosphate is
found at the 5′-end of in vitro transcribed RNAs, explaining previous
results indicating that these molecules are sensed by RIG-I. In cells
5′-ppp, activates RIG-I (Hornung et al., 2006). Similarly, the influenza
ligand for RIG-I (Pichlmair et al., 2006). Removal of the 5′-phosphates
does not affect RNA binding to RIG-I, but abrogates IFN induction.
Cellular RNAs do not display a 5′-triphosphate: this structure is either
removed or replaced with a 5′-cap structure. Therefore, ‘self’ RNAs do
not activate RIG-I.
Infection of mice lacking the gene encoding MDA-5 revealed that
not flaviviruses, influenza viruses, or paramyxoviruses (Gitlin et al.,
2006; Kato et al., 2006). How this selective recognition was achieved
was explained by the finding that RIG-I, but not MDA-5, recognizes
intermediates of picornaviruses are not phosphorylated, but are
would not be recognized by RIG-I. Picornavirus infections are sensed
by MDA-5 in mice lacking RIG-I. However, in the absence of MDA-5,
picornavirus genomes are not sensed. It is not known why dsRNAs
produced during the replicative cycles of picornaviruses are not
recognizedbyRIG-I—perhaps theyaretoolong(seebelow). Although
poly(I:C) and EMCV viral RNA can activate MDA-5, the actual ligand in
virus-infected cells has not been identified. Candidates include the
the viral genome.
RNA was initially not understood. The C-terminal domain (amino acids
792–925) of recombinant RIG-I binds dsRNA (including short RNAs,
~25-bp) and 5′-ppp RNA within this RNA (Takahasi et al., 2008). The
amino acid sequence of RIG-I indicates that it is a ligand-dependent
ATP binding site at amino acid 270 is altered, RIG-I cannot induce IFN
synthesis, but it can still bind dsRNA and 5′-ppp RNA (Takahasi et al.,
2008). Although RIG-I has RNA helicase activity, it is not required
for triggering IFN synthesis — consistent with the fact that single-
stranded 5′-ppp RNA is a functional ligand for RIG-I. The precise role of
ATPin RIG-I activation is not known;it has been suggestedto playa role
in the structural change of the RNA–RIG-I complex to an active form
(Yoneyama & Fujita, 2008).
The C-terminal domain (CTD) of RIG-I contains a repressor domain
believed to act in cis by masking the CARD domain. A region that
overlaps with the repressor domain (aa 792–925) was identified by
limited protease digestion ofRIG-I bound to RNA (Takahasi et al.,2008).
This fragment is sufficient to bind dsRNA or 5′-ppp RNA. The atomic
structure of the CTD reveals similarity to the mammalian suppressor of
Sec4 (Mss4), a guanine nucleotide exchange factor that is not an RNA-
binding protein (Cui et al., 2008; Takahasi et al., 2008). A positively-
charged cleft on one side of the molecule has been suggested to be the
RNA recognition surface. Alteration of amino acids within this cleft
reduces RNA binding. Based on the observation that the CTD binds 5′-
ppp RNA as a dimer, it has been proposed that one dsRNA molecule can
bind multiple CTDs. Precise understanding of the RIG-I — ligand
interaction awaits structural determination of the complex.
Amino acid changes in the basic cleft that inactivate RNA recog-
nition do not produce constitutively active RIG-I, suggesting that the
surfaces that bind RNA and repressor functions do not overlap. This
finding, together with the observation that RNA recognition does not
require ATP binding or hydrolysis, has led to a model in which the
repressor domain maintains RIG-I in a closed, inactive conformation
(Fig. 2). Upon binding the RNA ligand on the concave surface of the
CTD, a conformational change (possibly requiring ATP) occurs,
exposing the CARD domain, and allowing the CARD domain to initiate
a signal transduction pathway leading to IFN synthesis.
The mechanism for the discrimination of RNA ligands by RIG-I
and MDA-5 is not known. Both proteins have a CTD with common
residues, although there are differences in surface charge that
may influence substrate recognition (Yoneyama & Fujita, 2008).
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
Commercially available poly(I:C), which has been frequently used to
study RNA sensing, is at least 3-kb in length. By producing poly(I:C) of
various lengths it was determined that short segments of the polymer
(~300-bp) do not activate MDA-5, but are potent ligands for RIG-I
(Kato et al., 2008). In contrast, longer segments of poly(I:C)
preferentially activate MDA-5. Consistent with these findings is the
observation that the short dsRNA segments of the reovirus genome
(1.2–1.4-kbp) selectively activate RIG-I, while the longer RNA
segments (3.4-kbp) activate MDA-5. The mechanism underlying the
discrimination between long and short dsRNA of MDA-5 and RIG-I
remains to be elucidated. One possibility is that long segments of
dsRNAbind RIG-I butinduce a conformation of theprotein thatcannot
engage in downstream signaling (Takahasi et al., 2008). This
hypothesis is supported by the observation that binding of short
dsRNA and 5′-ppp RNA to RIG-I leads to the formation of 17- and 30-
kDa protease resistant fragments; in contrast, binding of long dsRNA
causes formation of a 60-kDa protease resistant fragment (Takahasi
et al., 2008).
It is likely that there are exceptions to the simple RNA recognition
rules described above for MDA-5 and RIG-I. For example, it has been
reported that IFN production occurs during infectionwith dengue and
West Nile viruses in the absence of either RIG-I or MDA-5
(Fredericksen et al., 2008; Kato et al., 2006; Loo et al., 2008; Sumpter
et al., 2005). These results suggest that perhaps other sensors of viral
RNA remain to be discovered.
Collectively, studies to date indicate that RIG-I and MDA-5 can
recognize different types of viruses, although there is significant evi-
dence to also indicate possible redundant roles for these two helicases,
probably due to the fact that some viruses are able to inadvertently
produce both longand short typesof dsRNA species followinginfection.
Both RIG-I and MDA-5 require IPS-1 to direct innate immune defenses
and are cooperatively essential for mounting an effective response to
positive- and negative-stranded virus infection.
4.2. Induction of cytokine signaling
and the role of adaptors Fas associated death
domain/receptor-interacting serine-threonine kinase 1, interferon β
promoter stimulator 1 and a new player stimulator of interferon genes
4.2.1. Fas associated death
domain/receptor-interacting serine-threonine kinase 1
While it was apparent that RIG-I and MDA-5 were important for
intracellular recognition of viral RNAs, it was less obvious which
molecules resided downstream of these helicases and facilitated their
ability to activate IRF3, IRF7 and NF-κB, required for efficient type I IFN
transcription. At approximately the same time as RIG-I was reported to
the discovery of a TLR-independent innate immune pathway that
involved the death domain-containing proteins FADD and RIP1
(Balachandran et al., 2004). FADD (Fas associated death domain) was
previously isolated by its ability to interact with the death receptor Fas,
and to mediate caspase-8 dependent cell death in response to FasL
(Chinnaiyan et al.,1995). In contrast, RIP1 (receptor-interacting serine-
threonine kinase 1) has been reported to play a key role in facilitating
tumor necrosis factor-alpha (TNF-α) signaling and is recruited by
induce NF-κB activation (Hsu et al., 1996). Both FADD and RIP1 can
ability to produce type I IFN (Balachandran et al., 2004; Balachandran
et al., 2007). Normal MEFs lacking these death domain-containing
molecules were extremely sensitive to RNA virus infection and also
exhibited defective IFN-mediated antiviral gene activity through,
amongst other things, a lack of IRF7 production. Subsequent studies
indicated that FADD and RIP1 are essential for RIG-I- and MDA-5-
mediated signaling and likely formed an ‘innateosome’ complex with
these helicases to facilitate the activity of NF-κB and IRF3 and IRF7
is that insect homologues, referred to as IMD (for Rip1) and dFADD (for
FADD) were also shown to play a critical role in TLR-independent
signaling in Drosophila and were essential for protecting flies against
gram-negative bacteria (Lemaitre et al., 1995; Georgel et al., 2001;
Leulier et al., 2002; Naitza et al., 2002; Ferrandon et al., 2004). Insects
lacking IMD or dFADD fail to activate the Drosophila IKK complex which
is essential for the production of appropriate stress responses to anti-
be evolutionarily conserved death domain-containing molecules
important in host defense against pathogen infection.
4.2.2. Interferon β promoter stimulator 1 (IPS-1)
In addition to the studies described above related to FADD/RIP1, a
number of groups in 2005 reported the isolation of a new adaptor
molecule that also appeared to mediate RIG-I/MDA-5 function. One
group utilized an expression cloning strategy to identify molecules that
isolated the same protein by screening uncharacterized molecules
virus-induced signaling adaptor) (Xu etal., 2005).Incontrast two other
groups performed profile searches in human protein databases to
identify new CARD-domain-containing proteins, similar to domains
evident in RIG-I/MDA-5 and called the products Cardif (CARD adaptor
inducing IFN-β) and MAVS (mitochondrial antiviral signaling) (Meylan
et al., 2005; Seth et al., 2005). Herein we will term the protein IPS-1.
IPS-1 appears to be expressed in a variety of tissues and is 540
amino acids in length, predicting a protein of 65-kDa. IPS-1 was found
to activate type I IFN transcription, the NF-κB promoter as well IP-10,
RANTES, and ISRE promoters, that are also regulated by the IRF family.
IPS-1 was also found to associate with RIG-I and MDA-5, through
CARD domain interactions, as well as with FADD and RIP1, which as
mentioned earlier, are also required to facilitate RIG-I/MDA-5 activity.
RNAi knockdown of IPS-1 reduced the abilityof poly(IC) and RIG-I/
MDA-5 to induce type I IFN, but not TRIF, indicating that IPS-1 did not
play a significant role in the TLR pathway (although one group did
report that IPS-1 associated with TRIF and TRAF6 to mediate
bifurcation of the TLR3-triggered NF-κB and IRF3 activationpathways)
(Xu et al., 2005). IPS-1 was unable to activate the IFN-α and IFN-β
promoters in cells deficient in both TBK1 and IKKi, indicating that it
lies upstream of these kinases (Kawai et al., 2005; Meylan et al., 2005;
Sun et al., 2006; Xu et al., 2005). Interestingly, IPS-1 was found to
localize in the mitochondrial membrane, although little is known
about protein partners that may associate with IPS-1 in this organelle
(Sunet al., 2006). It alsoremainstobe determinedhow IPS-1is able to
associate with RIG-I/MDA-5 in the cell following virus infection.
Biochemical analysis has indicated that the CARD domain of IPS-1
is essential for signaling activity, although a transmembrane domain
was reported as being required for IPS-1 dimerization, which was also
deemed essential for activity. It was further found that NS3–4A, a
multifunctional protein of hepatitis C virus (HCV) that has serine
protease activity essential for the production of mature viral proteins,
targeted IPS-1 to subvert interferon mediated antiviral activity
(Meylan et al., 2005). Thus, HCV-mediated resistance to IFN action
may involve IPS-1 deregulation (Meylan et al., 2005). IPS-1−/−mice
appeared viable and avarietyof cells derived from these animals, such
as MEFs, macrophages and conventional dendritic cells had impaired
type I IFN responses to both RIG-I-recognized RNAviruses such as SeV,
NDV, and VSV, as well as MDA-5-recognized viruses such as EMCV
(Kumaret al., 2006; Sun et al., 2006). Howeverplasmacytoiddendritic
cells did not appear affected by the loss of IPS-1 and were able to
produce IFN in response to SeV infection.
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
IPS-1 deficient animals were severely impaired in their ability to
produce type I IFN as well as other cytokines such as IL-6 and IP-10,
following infection with EMCV. Loss of IPS-1 was not found to affect
TLR signaling, for example in response to LPS or exogenous poly(IC).
Accordingly, IPS-1 deficient animals were extremely sensitive to lethal
the importance of this molecule in both RIG-I and MDA-5-mediated
innate immune signaling (Kumar et al., 2006; Sun et al., 2006). While
one group indicated that IPS-1 was partially required to facilitate DNA
virus mediated signaling (vaccinia), other reports indicated that IPS-1
was not required for IFN induction by cytosolic DNA or Listeria
monocytogenes (Kumar et al., 2006; Sun et al., 2006).
In summary, the experimental data indicate that IPS-1 is a potent
membrane of mitochondria. IPS-1 facilitates both RIG-I and MDA-5
signaling through CARD domain association and is essential for in vivo
innate immunity to RNA, but not DNA virus infection. While the exact
signaling mechanisms remain to be determined, it has recently been
reported that TRADD may be recruited to IPS-1 to orchestrate
‘innateosome’ complex formation with the E3 ubiquitin ligase TRAF3
and TANK, and with FADD and RIP1, which culminates in the activation
et al., 2006). Further work will undoubtedly elucidate this complex
series of events.
4.2.3. Stimulator of interferon genes (STING)
components of TLR-independent innate signaling in response to RNA
viruses, less was known about innate signaling in response to DNAvirus
infection. While plasmacytoid dendritic cells wereknownto utilize TLR9
TLR-independent pathways also existed to recognize DNA in alternative
tissues, the mechanisms of action of which remain to be determined
(Takeuchi & Akira, 2007). However, in 2008, a molecule referred to as
strategy to identify molecules that activated the IFN-β promoter
(Ishikawa & Barber, 2008). This molecule was predicted to be 379-aa in
length and comprised several putative transmembrane regions in the
Confocalanalysis indicated that STING was ubiquitouslyexpressed,
resided in the endoplasmic reticulum (ER) and following over-
expression potently induced the activation of both NF-κB as well as
IRF3 to stimulate type I IFN production (Ishikawa & Barber, 2008).
STING deficient animals were generated and were viable and MEFs
lacking STING were found to be sensitive to VSV and SeV infection.
However, loss of STING did not appear to dramatically affect poly(IC)
signaling. Indeed, co-immunoprecipitation experiments indicated
that STING appeared to associate with RIG-I, rather than MDA-5,
which may explain sensitivity to VSV but not poly(IC), which are
dependent on RIG-I and MDA-5, respectively. IPS-1 was also found to
associate with STING in co-immunoprecipitation experiments but it
was not clear if this was a direct or indirect (complex) association. Of
further interest was that STING was found by two-hybrid screening to
associate with SSR2/TRAP, a member of the TRAP complex comprising
four subunits (α–D) that facilitates translocation of proteins into the
ER following translation (Hartmann et al., 1993; Ishikawa & Barber,
2008; Lipschutz et al., 2003; Menetret et al., 2005).
Fig. 3. The RIG-I and MDA-5 signaling pathway. Short dsRNAs or 5′-triphosphate ssRNAs activate RIG-I which associates with mitochondrial IPS-1 to trigger FADD/RIP1
(innateosome) controlled activation of the type I interferon genes. Longer RNAs generated from picornaviruses trigger MDA-5-mediated activation of type I IFN. The RIG-I/IPS-1
pathway also requires the assistance of STING to activate type I IFN, which resides in the endoplasmic reticulum (ER). STING may regulate the translocon composed of the TRAP
complex and Sec61, which in turn may regulate exocyst-mediated stimulation of the TBK1 pathway. STING also appears pivotal in regulating DNA-mediated triggering of type I IFN
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
The TRAP complex is known to associate with the ‘translocon’, a
protein complex that forms an aqueous pore spanning the ER
membrane, that consists of three copies of the SEC61 heterotrimer.
Thus, STING is predominantly an ER resident protein that may link
RIG-I mediated intracellular innate signaling to the ‘translocon’.
Speculatively, RIG-I may detect translating viral RNAs at the intersec-
tion of ribosome/ER ‘translocon’ association and require STING to
exert effective innate immune function. The mitochondrial network is
also known to associate with the ER and such mitochondria-
associated membranes (MAM) may provide direct physical contact
between the ER and mitochondria, thus facilitating a role for IPS-1 in
the ‘innateosome’ complex (Bozidis et al.,2007;Hartmannet al.,1993;
Ishikawa & Barber, 2008; Lipschutz et al., 2003; Menetret et al., 2005).
Alternatively, STING may participate in mediating ER stress response
pathways, but this remains to be verified.
Although it is not clear how signaling from the ‘translocon’ to IRF3/
NF-κB occurs, it has recently been established that the ‘translocon’
physically associates with the ‘exocyst’ — the octameric Sec6–Sec8
complex that also associates with the ER and tethers secretory vesicles
to membranes, and facilitates protein synthesis and secretion (Guo &
Novick, 2004; Lipschutz et al., 2003). Recently, the ‘exocyst’ complex
was found to recruit and activate TBK1 and play a role in type I IFN-β
induction (Chien et al., 2006). Preliminary analysis indicated that
STING co-immunoprecipitated with TBK1, and that RNAi ablation of
Sec5 also rendered cells defective in the STING function.
Importantly, the generation of STING deficient MEFs and macro-
phages yielded the surprising finding that STING was essential for
recognizing non CpG DNA species, such as short double-stranded DNA
oligonucleotides that are able to potently induce IFN (ISD), as well as
bacterial DNA and poly d(AdT). Thus, STING may facilitate the detection
in general, indicating convergence of these intracellular pathogen-
associated molecular pattern (PAMP) recognition pathways.
4.2.4. RNaseL and the antiviral response
The activation of signaling pathways by double-stranded RNA
molecules leads to the expression of IFNs and these, in turn, initiate
expression ofIFN-inducedgenes (ISGs).Double-strandedRNA issensed
by RIG-I and MDA-5. It had generally been considered that the RNA
molecules arose as a by-product of viral infection; however, recent
evidence suggests that cellular generated small RNAs can amplify the
antiviral response (Malathi et al., 2007). Specifically, it has been shown
that the antiviral endoribonuclease, RNaseL, is activated by 2′, 5′
oligoadenylate, and produces small RNA fragments from cellular RNA
which contribute to IFN production and this involves RIG-I, MDA-5 and
IFN stimulator protein 1 (IPS-1) and IRF3 (Malathi et al., 2007). Using
normal and RNaseL deficient mice, viral infection produces appreciably
more IFN-β if RNaseL is expressed (Malathi et al., 2007). Similarly the
introduction of 2′–5′ oligoadenylate into normal mice, but not RNaseL
null mice, caused a marked increase in levels of circulating IFN. It has
previously been demonstrated that activation of RNaseL by 2′–5′
oligoadenylate increases the expression of a number of IFN-stimulated
genes (Malathi et al., 2005). Although RNaseL is stimulated by cellular
(self) RNAs, it is interesting to note that viral dsRNAs activate 2′–5′
oligoadenylate synthetase, which causes production of 2′–5′ oligoade-
nylate, activating RNaseL. RNaseL cleaves single-stranded RNA resulting
in double-stranded (duplex) structures, which can then activate
signaling by RIG-I and MDA-5, producing IFN.
5. 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 inflamma-
tory or autoimmune diseases.
A number of mechanisms contribute to the regulation of the RIG-I
signaling pathway to limit its action under steady-state conditions. In
particular, a number of negative regulators of RIG-I signaling have
been characterized, although significantly fewer have been identified
for MDA-5. Fundamentally, some of these mechanisms contribute to a
negative feedback loop whereas others regulate steady-state levels.
5.1. Inhibition of retinoic acid-inducible gene-I/melanoma
Of the recently characterized novel regulators of RIG-I and MDA-5
signaling, most appear to function to maintain a tight control of virus-
initiated IFN production. One such negative regulator is a member of
the RLH family, LGP2, unlike RIG-I and MDA-5, lacks the N-terminal
CARD required for activating IPS-1-dependent signaling events (see
below). It has also been observed that RIG-I-mediated production of
IFN can lead to an increase in RIG-I transcription, initiating an IFN
Under normal physiological conditions, RIG-I exists in an auto-
repressed state determined by its repressor domain (RD) located at the
end of the C-terminus (Saito et al., 2007). Besides this autoinhibition,
which occurs in the absence of stimuli and controls the basal activity of
RIG-I, several proteins have been found to have an inhibitory role in the
RIG-I-mediated signaling pathway. LGP2 was the first protein char-
acterized as an inhibitor of the RIG-I/MDA-5 signaling pathways. It
helicase, which has a high homology with RIG-I and MDA-5 in the C-
terminal region, but is completely devoid of the N-terminal region
containing the effector CARD domain. Therefore, it can bind to RNA
through its CTD, competing with RIG-I and MDA-5, but, lacking the N-
abilitytotransmitthe signal(Rothenfusseretal., 2005; Yoneyama etal.,
2004). Another way in which LGP2 exerts an inhibitory role on RIG-I is
mediated by the presence of a RD located in the C-terminal region,
domain and with a linker region between the CARD and the RNA
helicase domain and causes RIG-I dimerization that is necessary for its
activation and bindingwith the downstream protein IPS-1. It wasfound
that, through this analogous domain, LGP2 associates with RIG-I, acting
in trans preventing RIG-I dimerization necessary for its activation (Saito
transmembrane regions of IPS-1 competing for binding of the down-
2006). Therefore, LGP2 acts at two levels in the signaling pathway
attenuating the production of IFN. Interestingly LGP2 is also an IFN-
inducible gene and it is induced by viral infection playing a negative
feedback role in the antiviral signaling loop.
Interestingly LGP2−/−mice are more resistant to VSV infection
than wt animals, but are more susceptible to encephalomyocarditis
(EMCV) infection (Venkataraman et al., 2007). VSV produces RNA
intermediates during replication, which are specifically recognized by
RIG-I, whereas EMCV infection causes the activation of MDA-5. It
might therefore be postulated that LGP2 is a positive regulator of
MDA-5 mediated signaling pathways.
5.2. Inhibition by RNF125
The RIG-I and MDA-5 signaling pathway is also negatively regulated
by the RING finger E3 ubiquitin ligase RNF125 (Arimoto et al., 2007).
This protein is IFN-inducible and by conjugating ubiquitin to RIG-I and
MDA-5 causes their proteosomal degradation thereby decreasing their
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
cellular levels. By mutational analysis it was shown that the N-terminal
region of RIG-I, which contains the CARD domain, is the main sub-
strate for the RNF125 protein, with minimal ubiquitination occurring in
the C-terminal region. Overexpression of RNF125 in virus-infected or
dsRNA-stimulated cells decreases IFN-β production, whereas its reduc-
tion, mediated by siRNA knockdown, augments IFN-β expression.
RNF125 also acts downstream of RIG-I and MDA-5, by targeting, for
proteosomal degradation, the adaptor protein IPS-1. It is now clear that
ubiquitination is not only a mechanism for targeting proteins for
proteosomal degradation (usually through Lys 48-linked polyubiquiti-
nation) but can also play a role in promoting protein interactions or
catalytic activation, regulating their functions (through Lys 63-linked
which the action of ubiquitin-conjugating enzymes is counteracted by
de-ubiquitinating enzymes (DUB). In the case of RIG-I besides being
ubiquitinated by RNF125 for proteosomal degradation, it is also
ubiquitinated by TRIM25 causing activation.
5.3. Inhibition by de-ubiquitinating enzymes
Several de-ubiquitinating (DUB) enzymes have also been shown to
regulate the level of ubiquitinated RIG-I, consequentlyactingas negative
a known inhibitor of NF-κB, blocks the RIG-I-mediated signaling (Lin
et al., 2006). A20 is a peculiar protein that contains an ovarian tumor
(OTU) domain at its N-terminus that has a de-ubiquitinating function
and a C-terminal ubiquitin ligase domain. Both of these regions
are necessary for inhibition of NF-κB (Wertz et al., 2004), but only the
transfection of the constitutively active domain of RIG-I confers more
A20. In other studies, it has been shown that the de-ubiquitinating
enzyme A (DUBA) removes the lysine-63-linked polyubiquitin chain
from the TNF receptor-associated factor 3 (TRAF3), resulting in the
dissociation of TRAF3 from TANK-binding kinase 1 (TBK1) and
consequently disrupting the signal (Kayagaki et al., 2007). DUBA is
enzymes and was identified in a siRNA-based screen as an enzyme able
down DUBA by siRNA leads to more IFN production and its over-
expression has the opposite effect. The tumor suppressor CYLD
(cylindromatosis), another OTU de-ubiquitinating enzyme that removes
regulate RIG-I (Friedman et al., 2008; Zhang, Wu, et al., 2008). CYLD
negatively regulates the signaling function of RIG-I by direct interaction
enzyme of TBK1, the kinase that phosphorylates IRF3, leading to
inactivation of IRF3 signaling pathway and IFN production. RIG-I is
ubiquitinatedinits CARD domainbythe E3 ubiquitinligaseTRIM25,and
this is necessary to render RIG-I active. CYLD acts as a de-conjugating
enzyme, removing the K63 ubiquitin chain and inactivating the signal.
5.4. Inhibition by interferon-stimulated gene 15
Another protein that has an inhibitory effect on RIG-I signaling
pathway is the ubiquitin-like IFN-stimulated gene 15 (ISG15). ISG15
was found to interact with RIG-I (Zhao et al., 2005) and later it was
shown that RIG-I ISGylation negatively regulates RIG-I (Kim et al.,
2008). ISG15 is a ubiquitin-like protein, which is strongly induced by
IFNs during viral infection (Narasimhan et al., 2005).
5.5. Inhibition of melanoma differentiation
associated gene-5 by dihydroxyacetone kinase
Using a yeast two-hybrid screen the dihydroxyacetone kinase
(DAK) was shown to interact with MDA-5 and to have a specific
inhibitory effect on MDA-5-mediated IFN signaling (Diao et al., 2007).
It was observed that overexpression of DAK inhibits activation of IRF3
and the IFN-β promoter, but does not inhibit the NF-κB activation
pathway. Although MDA-5 and RIG-I are very similar in domain
structure and function, DAK does not interact with RIG-I and does not
inhibit the RIG-I-mediated signaling pathway. The interaction of DAK
with MDA-5 is disrupted during viral infection, suggesting that DAK is
a physiological inhibitor of MDA-5.
5.6. Inhibition by the autophagy complex Atg12–Atg5
TheRIG-I/MDA-5 signalingpathway is alsoinhibitedbyAtg12–Atg5,
a protein conjugate, which plays a key role in regulating autophagy
(Jounai et al., 2007). Specifically, Atg-deficient embryonic mouse
fibroblasts (MEFs) are more resistant to VSV infection and produce
complex depends on binding to the CARD of RIG-I, MDA-5 and IPS-1. In
an analogous fashion, Atg7−/−MEFs are also resistant to VSV infection,
indicating that Atg12–Atg5 complex formation, which requires Atg7, is
essential for inhibition of signaling.
5.7. Inhibition by NLRX1
NLRX1 is a member of the nucleotide-binding domain and leucine-
rich repeat containing (NLR) protein family (Moore et al., 2008).
NLRX1 is a widely expressed protein that is generally localized to the
outer mitochondrial membrane and has recently been shown to bind
to IPS-1. The CARD domain on IPS-1 determines this interaction
through a proposed nucleotide-binding domain in NLRX1 and results
in marked inhibition of RIG-I and MDA-5 mediated IFN release. Thus,
NLRX1 can be viewed as a negative regulator of RIG-I and MDA-5
activity such that depletion of NLRX1 with siRNAs results in increased
virus-induced IFN production (Moore et al., 2008). NLRX1 contains a
mitochondrial targetingsequence, andit is notablethat deletionof the
leucine-rich repeat results in loss of the IPS-1 inhibitory activity. It
appears that NLRX1 competes with RIG-I for IPS-1 possibly through
interaction between the CARDs. As the level of NLRX1 does not change
in response toIFN treatmentor MDA-5 andRIG-Iexpressionit appears
that it acts to regulate steady-state antiviral responses.
5.8. Inhibition by caspase-1
In a recent study it was demonstrated that caspase-1, which func-
tions as an inflammatory caspase and whose expression is activated
during viral infection, negatively regulates RIG-I signaling activity by
promoting RIG-I secretion, consequently controlling its intracellular
levels (Kim & Yoo, 2008). It is not clear how RIG-I is secreted, but since
RIG-I interacts with caspase-1 and it was found in the supernatant
together with caspase-1, one hypothesis is that RIG-I secretion
involves the ‘inflammasome’ complex. However, the physiological
significance of RIG-I secretion is not yet clear.
Another recent report describes a RIG-I splicing variant (RIG-I SV)
which was found to be induced during viral infection or upon IFN
stimulation and shown to suppress RIG-I signaling (Gack et al., 2008).
The splicing variant lacks residues 36 to 80 located in the first CARD,
therefore it cannot interact with TRIM25 or be ubiquitinated in the
second CARD, losing its ability to bind the adaptor protein IPS-1 and
consequently to carry out signal transduction. RIG-I-SV acts as a
dominant inhibitor of the RIG-I-mediated antiviral response. In fact,
RIG-I SV interacts with RIG-I WT inhibiting its multimerization and
consequent interactionwithIPS-1. This inhibitionof virus-induced IFN
signal transduction could potentially serve as a negative feedback
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
6. 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 rec-
ognizing 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 MyD88−/−TRIF−/−double
knockout cDCs), induced from bone-marrow in the presence of
GMCSF, were also defective in their ability to manufacture type I IFN
following exposure to the paramyxovirus, Newcastle disease virus
(NDV). However, plasmacytoid dendritic cells (pDCs) lacking RIG-I
were able to normally generate type I IFN following NDV infection
through activation of the TLR pathway since MyD88−/−TRIF−/−
defective pDCs had severely impaired type I IFN responses following
similar virus exposure (Kato et al., 2005, 2006). Despite this, rig-I−/−
mice remained sensitive to lethal infection by the above viruses. In
contrast, macrophages, cDCs and MEFs lacking MDA-5 were impaired
in their ability to produce type I IFN in response to the positive-
stranded virus EMCV (but not the above negative-stranded viruses) as
well as poly(IC) (Katoet al., 2006; Gitlin et al., 2006). pDCs fromMDA-
5 deficient animals exhibited normal ability to induce type I IFN
following exposure to EMCV, in contrast to pDCs lacking MyD88.
Nevertheless, mice lacking MDA-5 remained highly susceptible to
picornavirus infection. These data largely confirmed concomitant in
vitro RNA-binding analysis performed on these helicase proteins (as
described earlier) (Takeuchi & Akira, 2008; Takeuchi & Akira 2007).
For example, EMCV generates longer RNA species that are replicate
intermediates compared to negative-stranded viruses, which are
recognized by MDA-5 rather than RIG-I. However, it is worth noting
that some viruses, such as the flaviviruses, dengue virus and Japanese
encephalitis virus, were able to activate both the MDA-5 and RIG-I
pathways in MEFs, probably because these viruses generated both
long and short viral dsRNA structures following infection (Loo et al.,
2008). These studies indicate that RIG-I and MDA-5 play a pivotal role
in recognizing virus infection in most cell types, except inpDCs, where
the TLR (predominantly TLR 7) pathway appears to be the important
mechanism in manifesting antiviral host defense, for reasons that
7. 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 mayantagonize nearlyeverystepof the sensing of
RNA by RIG-I and MDA-5 (Fig. 4). Understanding such viral counter-
measures not only improves our understanding of innate sensing
pathways, but may also suggest avenues for therapeutic intervention.
7.1. Interference with ribonucleic acid sensing
As described previously, host RNAs are not detected by RIG-I or
MDA-5 because they do not contain 5′-phosphates. Many viral RNAs
are also capped, either by cellular or viral capping enzymes, or, in the
case of influenza viruses, when the viral polymerase primes the
synthesis of mRNAs with capped fragments derived from the 5′-ends
of host mRNAs (Plotch et al.,1981). A phosphatase is produced during
infection with certain negative-strand RNA viruses (Borna disease
virus, Crimean-Congo Hemorrhagic fever virus, Hantaan virus) that
converts the 5′-ppp on viral mRNAs to a monophosphate (Habjan
et al., 2008), thereby preventing detection by RIG-I. The genomic RNA
of picornaviruses is covalently linked to a viral protein, VPg (Flanegan
et al., 1977; Lee et al., 1977), which also prevents activation of RIG-I.
These modifications of viral RNAs can all be viewed as strategies to
avoid recognition by RIG-I.
Some viral genomes encode proteins that bind dsRNA and prevent
their detection by RIG-I and MDA-5 in the cytoplasm. These include
the vaccinia virus E3L protein (Smith et al., 2001; Xiang et al., 2002),
Ebola virus VP35 (Haasnoot et al., 2007), and human cytomegalovirus
proteins TRS1 and m142/m143 (Cassady, 2005; Child et al., 2004,
2006; Hakki & Geballe, 2005; Valchanova et al., 2006).
Infection of cells with members of the picornavirus family leads to
degradation of MDA-5 and RIG-I. Cleavage of MDA-5 is carried out by
cellular caspases, which are activated by virus infection (Barral et al.,
2007; Rebsamen et al., 2008). Cleavage of MDA-5 can also be
accomplished in the absence of virus infection by inducing apoptosis
pharmacologically (Kovacsovics et al., 2002; Barral et al., 2007). In
contrast, the viral 3Cproprotease cleaves RIG-I directly in cells infected
with poliovirus, rhinovirus, and other picornaviruses (Barral et al.,
submitted). It is not known if sensorcleavage plays a role in attenuating
IFN response during picornavirus infection. Cleavage of RIG-I is a
somewhat puzzling event because it is assumed that picornavirus
infections are sensed by MDA-5. Perhaps cleavage of RIG-I/MDA-5
antagonizes other functions of the sensor proteins.
Some viral proteins have been shown to bind RIG-I or MDA-5,
thereby blocking the induction of IFN. The V proteins of paramyx-
oviruses bind MDA-5 and block IFN induction (Andrejeva et al., 2004;
Childset al.,2007).TheC-terminal domain of V proteins bindsa specific
region of MDA-5, but not RIG-I; such binding prevents dsRNA binding,
significance of such antagonism is not known, because mice lacking the
gene encoding mda-5 are as susceptible to paramyxovirus infection as
wild type mice, and mount similar IFN responses (Gitlin et al., 2006;
Kato et al., 2006), and RIG-I appears to be the sensor for paramyxovirus
infections. Perhaps the conserved MDA-5 binding ability of paramyx-
ovirus V proteins targets a different activity of the protein. The NS1
protein of influenza A viruses binds RIG-I (Pichlmair et al., 2006;
Mibayashi et al., 2007) and inhibits signaling and IFN induction (Guo et
NS1 protein produce high levels of IFN in infected cells (Talon et al.,
2000). The G transmembrane glycoprotein of human metapneumo-
et al., 2008). There is some evidence that these viral proteins might not
directly interact with RIG-I, and the mechanism of interference has not
7.2. Interference with signal transduction
The downstream adaptor protein IPS-1 is cleaved in cells infected
with avarietyof viruses. As a consequence, IRF3 phosphorylation does
not occur and IFN transcription is attenuated. The 3Cproproteinase of
the picornavirus hepatitisAvirus degrades IPS-1 onlywhenpartof the
hydrophobic precursor protein 3ABC, which targets the proteinase to
the mitochondrial membrane (Yang et al., 2007). The NS3/4A protease
causing its release from the mitochondrion (Meylan et al., 2005; Lin
et al.,2006). Theseexamples demonstrated that IPS-1must be present
in the mitochondrial membrane to function in the signaling pathway
leading to IFN synthesis (Seth et al., 2005).
Members of the signaling pathway downstream of IPS-1 are also
targets of viral antagonism. The cascades that begin with ligand-
dependent activation of RIG-I and MDA-5 converge at the IKK family
of proteins (Fig. 4) whose role is to phosphorylate NF-κB, IRF3, and
IRF7. Members of the IKK protein family are targets of viral
antagonism. The G1 protein of hantavirus disrupts the interaction of
TRAF3withTBK1,therebyinterruptingsignaling(Alff etal.,2008). The
NS3/4A proteinase of hepatitis C virus and the N1L protein of vaccinia
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
virus interact with TBK1, blocking phosphorylation of downstream
transcription proteins (DiPerna et al., 2004; Otsuka et al., 2005). The
consequence of inhibiting TBK1 activity is failure to phosphorylate
IRF3, which cannot enter the nucleus to stimulate IFN transcription.
The vaccinia virus K7R protein also prevents the activation of IRF3 and
IRF7 by the TBK1–IKKe (Schroder et al., 2008). The K7R protein binds
the human DEAD box helicase DDX3, which is part of the TBK1–IKKe
complex and is required for the activation of the IFN-β promoter
(Soulat et al., 2008).
7.3. Inhibition of interferon regulatory factors
The transcription proteins IRF3 and IRF7 are prime targets for
inhibition by a variety of viral proteins. The papain-like protease of
SARS virus binds IRF3 and prevents dimerization, thereby inhibiting
nuclear translocation (Devaraj et al., 2007). Other viral proteins act as
IRF mimics. For example, the V proteins of paramyxoviruses bind
TBK1–IKKe and compete with IRF3, thereby inhibiting its phosphor-
ylation (Lu et al., 2008). The genome of Kaposi's sarcoma-associated
herpesvirus (KSHV) forms dimers with IRF7, blocking its ability to
bind DNA (Joo et al., 2007). Several human herpesvirus 8 proteins are
dominant-negative inhibitors of IRF3, preventing its association with
the co-activators CBP and p300 (Lin et al., 2001). The transcription
protein K-bZIP of KSHV blocks the activation of the IFN-β promoter by
competing with IRF3 for binding sites in the promoter (Lefort et al.,
2007), while ICP0 of HSV binds IRF3 and localizes the protein in
nuclear bodies where it cannot activate the IFN-β promoter (Melroe
et al., 2007).
is proteolytic degradation. The ICP0 protein from bovine herpesvirus
degrades IRF3 insteadof sequestering it like the HSVorthologue (Saira
et al. 2007). Degradation of IRF3 is also brought about by the Vpr and
Vif proteins of HIV-1, the N protease of flaviviruses, and the NSP1
protein of rotaviruses; the latter also induces degradation of IRF7
(Barro & Patton, 2007; Bauhofer et al., 2007; Okumura et al., 2008). Of
great interest is the observation that the vaccinia virus E3L protein
disablesthe cellular ISG15 protein(Guerra et al., 2008). ISG15 is an ISG
that is believed to counter virus-induced proteolysis of IRF3; for
example, it can prevent degradation of IRF3 by paramyxovirus V
protein (Lu et al., 2006). The vaccinia virus E3L protein therefore
disables the cell's attempt to block a viral antagonism of the innate
NF-κB function is also modulated during virus infection. This
but also inhibits apoptosis and causes cell proliferation, which may
benefit viral replication. Consequently, in some viral infections, NF-κB
Fig. 4. Viralcountermeasures inthe RNA sensing and signaling pathway. The steps leading fromdetectionof RNAbyRIG-I and MDA-5through signaling and transcription of IFN genes
are depicted. Viral gene products that interfere at different steps of this pathway are listed. An arrow indicates stimulation, and a bar indicates inhibition of the pathways.
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
clearingof infection.The A238Lprotein of African swine fever virus acts
as a homolog of IκBα; it binds NF-κB and prevents its entry into the
nucleus (Tait et al., 2000). Later in infection, the viral A224L protein
activates NF-κB, causing inhibition of apoptosis and cellular prolifera-
tion. Why cleavage of the p65-RelA subunit of NF-κB by poliovirus
proteinase 3Cproat a late stage in infection would be beneficial to virus
et al., 2005).
Other viral proteins regulate NF-κB by disrupting IKK. The B14R
protein of vaccinia virus binds IKKβ and prevents its phosphorylation
and therefore activation (Chen et al., 2008), while the K13 protein of
KSHV binds the IKKα–IKKβ complex in a way that causes activation of
NF-κB (Matta et al., 2007). Curiously, cleavage of IκBα by the 3Cpro
proteinase of the picornavirus coxsackvirus B3 limits NF-κB mediated
NF-κB, which enters the nucleus but cannot transactivate; therefore
apoptosis increases, limiting viral replication (Zaragoza et al., 2006).
The authors suggest that IκBα acts as a sensor for viral replication.
8. 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 et al., 2006). In genetically modified
rodent fibroblasts as well as in human pancreatic and colorectal
cancer cell lines, constitutively active Ras/Raf/MEK/ERK pathway
precluded mda-5-induced apoptosis that could be reversed by
antisense inhibition of Ras or chemical inhibition of the ERK pathway
(Lin, Lacoste, et al., 2006; Lin, Su, et al., 2006; Lin, Yang, et al., 2006).
Deletion analysis revealed that compared to the full-length mda-5,
deletion of either the CARD or the helicase domain significantly
abrogated the growth suppressor properties of mda-5 (Kang et al.,
2004). This loss-of-function was more pronounced when the CARD
domain was deleted compared to the deletion of the helicase domain
indicating that CARD domain is the active domain of the molecule, an
observation later confirmed for RIG-I.
of the murine mda-5 gene, named Helicard (Kovacsovics et al., 2002).
Apoptoticstimuli, suchasFasL,resulted incleavageofMDA-5/Helicard
by caspases with subsequent nuclear translocation of the helicase
domain, the consequences of which remain to be determined. MDA-5/
Helicard augmented FasL-mediated DNA degradation while a mutant,
resistant to caspase-cleavage, and a second mutant, with loss of
helicase activity, lost this activity. However, MDA-5/Helicard alone did
not induce apoptosis in 293T cells. Caspase-mediated cleavage of
human MDA-5 has also been observed in poliovirus-infected cells
(Barral et al., 2007).
RH116, having 99.5% identity to mda-5, was cloned as a gene
downregulated by Murabutide, an HIV-suppressive immunomodulator
not induce apoptosis and the mechanism of this growth inhibition was
not analyzed. Interestingly, counterintuitive to the antiviral function of
MDA-5, RH116 actually increased HIV-1 replication. HIV-1 treatment
itself induced RH116 expression, which was localized predominantly in
the nucleus. Thus, although it is accepted that MDA-5 inhibits growth,
there is debate about induction of apoptosis by MDA-5 and possible
differentialeffects of thehumanvs.the murineversion of this gene. The
lack of follow-up studies on Helicard or RH116 precludes resolution of
this issue at the present time.
Similar to MDA-5, RIG-I also displays apoptosis-inducing proper-
ties. Cytosolic poly(I:C) or influenza A virus infection activates
caspases 1 and 3 that results in pro-IL-18 processing (Rintahaka
et al., 2008). Overexpression of RIG-I or its active form (only the CARD
domains) or IPS-1 activated caspases, processed pro-IL-18 and
induced apoptosis in HaCaT keratinocytes cells. The reverse experi-
ment, i.e., inhibition of rig-I to block these phenomena, was not
carried out to strengthen the direct link of RIG-I in virus-induced
apoptosis. The apoptosis induction by a synthetic RA (CD437) was
shown to be inhibited by the hepatitis C virus (HCV) non-structural
(NS)3/4A protease, which is know to cleave RIG-I, thereby indirectly
showing an involvement of RIG-I in apoptosis induction (Pan et al.,
2008). Infection of cells with Sendai virus activates RIG-I and that
leads to the activation of IRF3. IRF3 induces the expression of virus
stress-inducible genes and causes host cell apoptosis by activating
caspase-8 (Peters et al., 2008). A dominant-negative inhibitor of RIG-I,
containing only the helicase domain, inhibits IRF3 activation, prevents
apoptosis and allows persistent virus infection. A novel strategy was
used to capitalize on specific properties of RIG-I as an anti-cancer
therapy (Poeck et al., 2008). RIG-I, not MDA-5, is activated by 5′-
triphosphates. An siRNA, with 5′-triphosphate ends, was generated
against Bcl-2. The recognition of 5′-triphosphate by RIG-I activated
innate immune cells, such as dendritic cells and directly induced
expression of IFNs and apoptosis in tumor cells. These RIG-I-mediated
effects synergized with Bcl-2 siRNA to induce apoptosis of tumor cells
in lung metastasis in animal models. The observation that systemic
administration of this bifunctional RNA molecule resulted in tumor-
specific apoptosis offers promise of potential therapeutic use of this
9. Role of retinoic acid-inducible
gene-I in development and differentiation
viral RNA-induced type 1 IFN generation. However, recentevidence has
suggested that it can act, in the absence of viral infection, to regulate
myeloid cell differentiation (Zhang, Shen, et al., 2008). Initial experi-
ments 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
normal myelopoiesis it has been concluded that RIG-I is developmen-
A potential role for MDA-5 in differentiationwas not investigated and is
an area worth further exploration.
It had been shown that most rig-I−/−mice die before birth (Kato
et al., 2005). However, in a later study, inwhich a different region of the
granulocytosis, indicative of defects in myeloid development. In the
same study it was noted that the granulocytosis in the rig-I−/−mice is
attributable to reduction in expression of IFN consensus sequence-
binding protein (Icsbp) which plays a central role in regulating myeloid
differentiation (Zhang, Shen, et al., 2008).
10. Melanoma differentiation
associated gene-5 and type 1 diabetes
In a large screen of single-nucleotide polymorphisms (SNPs)
associated with type 1 diabetes, a numberof mutations wereobserved
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 geneitself andin the 3′
intergenic region and susceptibility totype 1 diabetes (Liu et al., 2009;
Smyth et al., 2006; Todd et al., 2007). Furthermore, after screening a
large panel of controls and type 1 diabetes patients for MDA-5
expression it was suggested that SNPs leading to increased expression
of MDA-5 may be associated with increased risk of developing the
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
disease and that there might be an increase in MDA-5 expression
overall in diabetes although this latter observation did not appear to
be statisticallysignificant in thiscohort(Liuet al.,2009). Thesestudies
suggest an intriguing possibility that blocking MDA-5 expression
could provide a protective mechanism in the context of diabetes.
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
theirownlarge domainfamilies,it is possiblethatthe MDA-5 andRIG-
I proteins have a diverse and chimeric evolutionary history. In
addition, the proteins that are closely related to MDA-5 and RIG-I
can be determined using phylogenetic approaches.
Three steps were used to unravel the evolutionary history of these
proteins. The first step was to examine if any of the domains had
ancient linkage to each other or to other domains in other proteins.
The second step was to identify whether any of the four domains had
similar evolutionary histories, thus supporting the hypothesis that
fusion events of previously independent domains occurred. The third
step was to examine the evolutionary patterns of these singular and/
or linked domains.
It is well known that helicase and DEAH domains are found linked
in the genomes of species from the organismal superdomain Archaea
as well as throughout the genomes of Eukaryotes (Story et al., 2001;
Abdelhaleem et al., 2003). There are no known linked helicase DEAH
domains in the organismal superdomain Bacteria. Given the well
accepted topology of the tree of life with respect to the three
organismal superdomains (Bacteria, Eukarya, Archaea), these obser-
vations suggest that the helicase DEAH linkage occurred in the
common ancestor of Archaea and Eukarya. However, the helicase
DEAH domain linkage could be a more recent phenomenon due to
secondaryfusion of these domains.If thelatterscenariois correct then
the evolutionary histories of the two domains will be divergent and if
the former scenario is correct the evolutionary histories of the two
domains will be congruent. When we examined the congruence of
helicase and DEAH domains with respect to their evolutionary
histories using the Incongruence Length Difference (ILD; 47–49) test,
we found that both have statistically significant congruent histories
(pb0.05; (Sarkar et al., 2008) allowing us to reject the hypothesis that
these two domains have fused after the divergence of Archaea. This
result strongly implies that the helicase DEAH domain linkage in RIG-I
and MDA-5 is an ancient relationship that arose in the common
ancestor of the Archaea and Eukarya.
Fig. 5. A. Schematic diagramshowingthedomainstructureofRIG-IandMDA-5.Thenumbersabovethecartoonindicatethesizeof thedomains.Abbreviations:C1=CARDdomain1;
C2 = CARD domain 2; hel = helicase and DEAH = DEAH boxdomain. B. Phylogenetic tree showing the overall topologyof the helicase/DEAH domains. C. Stepwise diagram showing
the duplication events involved in the evolution of the helicase/DEAH linked domains leading to RIG-I and MDA-5. Each double-headed arrow represents a gene duplication event.
D. Phylogenetic tree showing the topology for the CARD domains.
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
Understanding the ancient linkage of the CARD domains is more
in a wide variety of proteins from a broad array of vertebrates (Inohara
number of protein families and some of these protein families are not
related, we suggest that the CARD domains may have experienced
different evolutionary histories to the helicase DEAH domains. Most
caspases (cysteine-dependent aspartate-specific proteases) have a
single CARD domain, with the exceptions being MDA-5, RIG-I, and
Nachtproteins. Because the NachtCARD domains have diverged greatly
from the MDA-5 and RIG-I CARD domains (see below), we can rule out
an ancient linkage involving Nacht CARD domains. Furthermore,
because the linked CARD1/CARD2 state in MDA-5 is found in all
vertebrates, we conclude that the linkage of the two CARD domains in
this protein occurred in the common ancestor of vertebrates.
The second step involved understanding the CARD1/CARD2 linked
arrangement in RIG-I and MDA-5. The linkage of the CARD1/CARD2
domains could have arisen by two very different processes in the
vertebrates. First, a single CARD domain could have simply duplicated
to produce two linked domains. Second, two independently derived
CARD domains could have fused to produce the linked state. Again the
former scenario would result in the two CARD domains having
congruent evolutionary history and in the latter the two domains
and CARD2 revealed strong incongruence of CARD1 and CARD2 using
the ILD test (p=0.68). In addition, when the congruence of CARD1 is
tested relative to the helicase and DEAH domains there is also strong
incongruence (p=0.45 and p=0.91 respectively). On the other hand,
the CARD2 domain shows strong congruence with both the helicase
and DEAH domains (both pb0.01). The ILD tests indicate that the
evolutionary history of the CARD1 domain is divergent from the other
three domains and suggests that the CARD1 domain was grafted onto
an existing CARD2-helicase-DEAD/DEAH structure more than likely in
the common ancestor of vertebrates.
The congruence analysis discussed above using the ILD test
suggests that there are two independently evolving domain linkages
that make up these proteins. Consequently, we constructed phylo-
genies for the domains in these proteins to examine the evolutionary
history of each. We first examined the linked helicase/DEAD-DEAH
domains and second, the genealogical relationships of the two CARD
Fig. 5B shows a phylogenetic analysis of a large representation of
helicase, DEAH domains proteins in the database. This phylogenetic
tree can be directly translated into the diagram shown in Fig. 5C. This
scenario suggests that the helicase DEAH proteins have undergone at
least seven duplication events to produce new families of helicase
proteins in the evolution of eukaryotes. The final two duplications
produced the LGP2 helicases and then the MDA-5-RIG-I proteins.
Because LGP2, MDA-5, and RIG-I are found only in vertebrates and
DICER proteins are broadly distributed phylogenetically, we propose
that the duplication event of MDA-5 and RIG-I occurred in the
common ancestor of vertebrates.
The CARD domain phylogenetic analysis is consistent with our
observations based on the ILD tests (Fig. 5D). The phylogenetic and
incongruence results indicate that the CARD2 boxes were the first N-
terminal elements to be grafted to the anciently duplicated helicase/
DEAD domains. Next, the MDA-5 CARD1 domain was grafted to the
CARD2-helicase/DEAD domains. Because RIG-I and MDA-5 genes are
only found in vertebrates we propose that the CARD1 fusion occurred
in the common ancestor of vertebrates.
The evolutionary history of the MDA-5 and RIG-I proteins that we
propose here, demonstrates an intriguing and circuitous pathway that
is consistent with intense selection pressure for the existence and
maintenance of these genes. Gene duplication is generally considered
the easiest way to construct proteins with similar domain structure. In
other words, the most parsimonious route to RIG-I and MDA-5
proteins would have been to make one and then duplicate the entire
assemblage. However, according to the phylogenetic analyses, there
was independent fusion of CARD2 to the RIG-I or MDA-5 helicase/
DEAD domains and then another independent fusion of CARD1 to the
CARD2/helicase/ DEAD domain. This pathway rules out simple
duplications as the only route to the eventual structure of the RIG-I
and MDA-5 proteins. We suggest, from these data, that the MDA-5 and
RIG-I domain structures are a case where the most parsimonious
evolutionary path has not been taken, because the scenario the trees
support has nearly twice as many steps in them than required to
perform simple duplication events (see Sarkar et al., 2008).
In just 6 years we have progressed from the discovery of the
cytoplasmic RNA sensors of the innate immune response, to developing
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 full-length RIG-I and MDA-5 with and without an
RNA ligand to provide a detailed understanding of the recognition
process. The requirement for theRNA helicase activity is also enigmatic.
Similarly, RNA unwinding is not needed for RNA recognition, although
this activity may be important for the conformational change that leads
to exposure of the CARD domains.
A relatively uncharted area of innate immunity is the recognition
of viral DNA. There have been several reports of cytoplasmic DNA
sensors, but precisely how these recognize nucleic acid and induce
cytokine synthesis remains to be determined (Stetson & Medzhitov,
2006; Ishii et al., 2006; Burckstummer et al., 2009). STING is essential
for interferon production in response to cytoplasmic dsDNA, but it is
not a nucleic acid sensor (Ishikawa & Barber, 2008). It will be of
interest to determine whether DNA sensing pathways overlap with
those described here for detection of RNA.
Viral genomes encode proteins that block every step of the RNA
antagonize nearlyevery step of the sensingof RNA by RIG-I and MDA-5,
including RNA recognition, signaling, and induction of transcription.
Understanding such viral countermeasures not only improves our
understanding of innate sensing pathways, but may also suggest
avenues for therapeutic intervention.
Animportantquestion is whetherwecanuse our understandingof
RNA sensing pathways for health benefit. We know that polymorph-
isms in genes encoding TLRs may influence the outcome of virus
infection (Awomoyi et al., 2007). Therefore it would be useful to
identify changes in MDA-5 and RIG-I that might alter susceptibility to
infection. Other approaches include induction of MDA-5 or RIG-I
transcription with specific promoter activators, or restoring defective
innate responses thatoccurduringchronic diseasessuch as hepatitis C
virus infection. It might also be possible to suppress the inappropriate
activation of innate responses that take place during autoimmune and
inflammatory disease. One approach to such therapy would be to
design inhibitors of key components that are targeted by viral
antagonism. Such approaches could bring unexpected health benefits
beyond the prevention of infectious diseases.
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
P.M. Barral et al. / Pharmacology & Therapeutics 124 (2009) 219–234
Support from the VCU Institute of Molecular Medicine to DS and PBF is
acknowledged. DS is a Harrison Scholar in Cancer Research at the VCU
Massey Cancer Center, VCU School of Medicine. PBF holds the Thelma
Newmeyer Corman Chair in Cancer Research at the VCU Massey Cancer
Center, VCU School of Medicine and is a SWCRF Investigator.
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