Detection of pathogens by the mammalian innate immune system is mediated by pattern recognition receptors. For viruses, nucleic acids are often the trigger for innate responses that culminate in antiviral gene expression, including the production of type I interferon (IFN-α/β). Foreign nucleic acids outside the cell can be recognized by transmembrane Toll-like receptors at the cell surface or in the lumen of endocytic vesicles (9
). Intracellular nucleic acids are recognized by cytoplasmic receptor proteins (24
). In both cases, receptor binding to the nucleic acid ligand triggers a signal transduction cascade that activates immediate transcriptional responses, including the induction of the antiviral cytokines in the IFN family.
An important class of receptors for the detection of cytosolic nonself RNAs is represented by the proteins encoded by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) (24
). These proteins share functional domains, including an amino-terminal protein interaction motif that functions in signal transduction and is homologous to the caspase activation and recruitment domain (CARD) (8
) and a C-terminal DECH-box family RNA helicase domain. The helicase domains share approximately 30 to 40% overall sequence identity. Experimental and structural studies have demonstrated a third functional region at the extreme C terminus of RIG-I that forms a zinc-mediated fold and functions as a regulatory domain for the recognition and discrimination of RNA 5′ ends (6
). Current evidence supports a model in which binding to RNA ligand induces a change in accessibility to the CARD, allowing it to interact with downstream signaling proteins, including the mitochondrial resident IPS-1 (also identified as MAVS, VISA, and Cardiff [12
]). This association coordinates a serine kinase-mediated cascade that activates latent transcription factors, including NF-κB and IFN regulatory factor 3, culminating in the expression of IFN-β and a number of other crucial antiviral effector genes (8
A third helicase protein, LGP2, resembles RIG-I and MDA5 in the helicase domain, also exhibiting 30 to 40% sequence identity. This overall level of similarity may underestimate the relatedness of these proteins, as they are more identical in their key helicase sequence motifs and encompassing domains (2
). Significantly, LGP2 lacks the N-terminal CARD homology region. As a result, LGP2 expression is not typically associated with the ability to directly activate downstream signaling. Ectopic expression of LGP2 interferes with double-stranded RNA (dsRNA) and virus-induced antiviral signaling, while reduction of LGP2 expression by RNA interference results in enhanced IFN-β synthesis and antiviral responses. Expression of LGP2, like that of RIG-I and MDA5, is induced by virus infection, nucleic acid transfection, and IFN stimulation, suggesting it has the properties of a negative feedback inhibitor (14
The importance of these RNA helicase proteins in antiviral responses is validated by the phenotypes of mice harboring targeted disruptions in these genes (9
). Deficiency in RIG-I leads to a widespread enhancement in replication of many RNA virus types, due to suppressed IFN biosynthesis and antiviral responses (11
). Cells deficient in RIG-I exhibit a general defect in the ability to respond to foreign dsRNAs or single-stranded RNAs (ssRNAs) bearing phosphorylated 5′ ends. MDA5 deficiency showed a more specific phenotype, resulting in increased susceptibility to picornavirus infection and insensitivity to the synthetic dsRNA analog poly(I:C) (7
LGP2 deficiency leads to a more complex phenotype (27
). LGP2-deficient mice are more sensitive to IFN induction by cytosolic poly(I:C), an MDA5 ligand, and more resistant to vesicular stomatitis virus, a virus that triggers RIG-I signaling. Negative regulation of the IFN response remains intact overall, indicating that LGP2 may not be the primary negative regulator of type I IFN production. However, LGP2 deficiency results in a suppressed IFN response to infection with encephalomyocarditis virus, a picornavirus that has been demonstrated to trigger MDA5-mediated antiviral signaling. These data suggest that LGP2 executes both negative and positive regulatory functions related to RIG-I and MDA5 signaling. The disparate effects of LGP2 deficiency are difficult to interpret without a better mechanistic understanding of LGP2 functions in cellular innate antiviral immunity.
Further affirmation of the antiviral role of the helicase proteins derives from the fact that individual viruses have evolved means to evade or disrupt their activity, either by direct interference or by antagonizing the signaling intermediates. The large Paramyxovirus
family of negative-strand RNA viruses is well known to evade IFN antiviral responses. Most viruses within this family encode a protein, called V, that is essential for IFN signaling evasion. In addition to suppressing the activity of signal transducer and activator of transcription (STAT) proteins to compromise IFN signal transduction, paramyxovirus V proteins can also limit the induction of IFN gene expression by interfering with the MDA5 protein (1
). Associations between the V protein C-terminal domain (CTD), a 60- to 70-residue zinc binding fold that is the conserved hallmark of V proteins, and the MDA5 helicase domain prevents signal transduction downstream of poly(I:C), a synthetic RNA ligand that is a known MDA5 activator (1
). This inhibition was observed for all tested V proteins of the large Paramyxovirus
). Despite the potential importance of the V protein-MDA5 interface as an antiviral target, the mechanistic basis and consequences for MDA5 interference by V proteins are poorly understood and difficult to reconcile with the lack of phenotype in MDA5-deficient mice. Recent evidence indicates that MDA5 is involved in the detection of Sendai virus-defective interfering (DI) genomes, providing plausible biological relevance to the observed V protein interference that may not have been apparent from gene disruption studies (31
Identification of the MDA5 target recognized by paramyxovirus V proteins led to the discovery that LGP2 is also a target for V protein antagonism. Results demonstrate that the helicase C domain of both proteins functions as a V protein binding region common to both MDA5 and LGP2 but absent in RIG-I. We demonstrate that interaction with the V protein interferes with the catalytic activity of both target helicases, providing a biochemical basis for V protein helicase antagonism.