Mitsutoshi Yoneyama

Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.

The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.

Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity

The cellular protein retinoic acid-inducible gene I (RIG-I) senses intracellular viral infection and triggers a signal for innate antiviral responses including the production of type I IFN. RIG-I contains a domain that belongs to a DExD/H-box helicase family and exhibits an N-terminal caspase recruitment domain (CARD) homology. There are three genes encoding RIG-I-related proteins in human and mouse genomes. Melanoma differentiation associated gene 5 (MDA5), which consists of CARD and a helicase domain, functions as a positive regulator, similarly to RIG-I. Both proteins sense viral RNA with a helicase domain and transmit a signal downstream by CARD; thus, these proteins share overlapping functions. Another protein, LGP2, lacks the CARD homology and functions as a negative regulator by interfering with the recognition of viral RNA by RIG-I and MDA5. The nonstructural protein 3/4A protein of hepatitis C virus blocks the signaling by RIG-I and MDA5; however, the V protein of the Sendai virus selectively abrogates the MDA5 function. These results highlight ingenious mechanisms for initiating antiviral innate immune responses and the action of virus-encoded inhibitors.

Regulating Intracellular Antiviral Defense and Permissiveness to Hepatitis C Virus RNA Replication through a Cellular RNA Helicase, RIG-I

ABSTRACT Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.

Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF‐3 and CBP/p300

It has been hypothesized that certain viral infections directly activate a transcription factor(s) which is responsible for the activation of genes encoding type I interferons (IFNs) and interferon‐stimulated genes (ISGs) via interferon regulatory factor (IRF) motifs present in their respective promoters. These events trigger the activation of defense machinery against viruses. Here we demonstrate that IRF‐3 transmits a virus‐induced signal from the cytoplasm to the nucleus. In unstimulated cells, IRF‐3 is present in its inactive form, restricted to the cytoplasm due to a continuous nuclear export mediated by nuclear export signal, and it exhibits few DNA‐binding properties. Virus infection but not IFN treatment induces phosphorylation of IRF‐3 on specific serine residues, thereby allowing it to complex with the co‐activator CBP/p300 with simultaneous nuclear translocation and its specific DNA binding. We also show that a dominant‐negative mutant of IRF‐3 could inhibit virus‐induced activation of chromosomal type I IFN genes and ISGs. These findings suggest that IRF‐3 plays an important role in the virus‐inducible primary activation of type I IFN and IFN‐responsive genes.

LGP2 is a positive regulator of RIG-I– and MDA5-mediated antiviral responses

RNA virus infection is recognized by retinoic acid-inducible gene (RIG)-I–like receptors (RLRs), RIG-I, and melanoma differentiation–associated gene 5 (MDA5) in the cytoplasm. RLRs are comprised of N-terminal caspase-recruitment domains (CARDs) and a DExD/H-box helicase domain. The third member of the RLR family, LGP2, lacks any CARDs and was originally identified as a negative regulator of RLR signaling. In the present study, we generated mice lacking LGP2 and found that LGP2 was required for RIG-I– and MDA5-mediated antiviral responses. In particular, LGP2 was essential for type I IFN production in response to picornaviridae infection. Overexpression of the CARDs from RIG-I and MDA5 in Lgp2−/− fibroblasts activated the IFN-β promoter, suggesting that LGP2 acts upstream of RIG-I and MDA5. We further examined the role of the LGP2 helicase domain by generating mice harboring a point mutation of Lys-30 to Ala (Lgp2K30A/K30A) that abrogated the LGP2 ATPase activity. Lgp2K30A/K30A dendritic cells showed impaired IFN-β productions in response to various RNA viruses to extents similar to those of Lgp2−/− cells. Lgp2−/− and Lgp2K30A/K30A mice were highly susceptible to encephalomyocarditis virus infection. Nevertheless, LGP2 and its ATPase activity were dispensable for the responses to synthetic RNA ligands for MDA5 and RIG-I. Taken together, the present data suggest that LGP2 facilitates viral RNA recognition by RIG-I and MDA5 through its ATPase domain.

RNA recognition and signal transduction by RIG-I-like receptors.

Summary:  Viral infection is detected by cellular sensor molecules as foreign nucleic acids and initiates innate antiviral responses, including the activation of proinflammatory cytokines and type I interferon (IFN). Recent identification of cytoplasmic viral sensors, such as retinoic acid‐inducible gene‐I‐like receptors (RLRs), highlights their significance in the induction of antiviral innate immunity. Moreover, it is intriguing to understand how they can discriminate endogenous RNA from foreign viral RNA and initiate signaling cascades leading to the induction of type I IFNs. This review focuses on the current understanding of the molecular machinery underlying RNA recognition and subsequent signal transduction by RLRs.

Viral infections activate types I and III interferon genes through a common mechanism

Viral infections trigger innate immune responses, including the production of type I interferons (IFN-α and -β) and other proinflammatory cytokines. Novel antiviral cytokines IFN-λ1, IFN-λ2, and IFN-λ3 are classified as type III IFNs and have evolved independently of type I IFNs. Type III IFN genes are regulated at the level of transcription and induced by viral infection. Although the regulatory mechanism of type I IFNs is well elucidated, the expression mechanism of IFN-λs is not well understood. Here, we analyzed the mechanism by which IFN-λ gene expression is induced by viral infections. Loss- and gain-of-function experiments revealed the involvement of RIG-I (retinoic acid-inducible gene I), IPS-1, TBK1, and interferon regulatory factor-3, key regulators of the virus-induced activation of type I IFN genes. Consistent with this, a search for the cis-regulatory element of the human ifnλ1 revealed a cluster of interferon regulatory factor-binding sites and a NF-κB-binding site. Functional analysis demonstrated that all of these sites are essential for gene activation by the virus. These results strongly suggest that types I and III IFN genes are regulated by a common mechanism.

Induction of IRF-3/-7 kinase and NF-kappaB in response to double-stranded RNA and virus infection: common and unique pathways.

Infection by virus or treatment with double‐stranded RNA (dsRNA) results in the activation of transcription factors including IRF‐3, IRF‐7 and a pleiotropic regulator NF‐κB by specific phosphorylation. These factors are important in triggering a cascade of antiviral responses. A protein kinase that is yet to be identified is responsible for the activation of these factors and plays a key role in the responses.

Function of RIG-I-like Receptors in Antiviral Innate Immunity

Various cells in the body are capable of sensing infectious viruses and initiating reactions collectively known as antiviral innate responses. These responses include the production of antiviral cytokines such as type I interferon (IFN)2 and subsequent synthesis of antiviral enzymes, which are responsible for the impairment of viral replication and promoting adaptive immune responses (1). In this minireview, we focus on a subset of molecules known as RIG-I-like receptors, which sense viral RNA molecules that trigger a danger signal.

Recognition of viral nucleic acids in innate immunity.

Viral infections are detected by sensor molecules, which initiate innate antiviral responses, including the activation of type I interferons (IFNs) and proinflammatory cytokines. These cytokines are responsible for not only inhibiting viral replication in infected cells but also regulating the induction of adaptive immunity, leading to the swift eradication of viruses. Recent advances in the identification of pathogen receptors in the innate immune system have revealed that distinct types of sensors play a role in the detection of viral nucleic acids in different ways; Toll‐like receptors (TLRs), which detect viral DNA or RNA in endosomal compartments in immune cells, retinoic acid inducible gene‐I (RIG‐I)‐like receptors (RLRs), which recognise viral RNA in the cytoplasm, and DNA sensors, which detect cytoplasmic viral DNA. Since these sensors have to exclusively recognise viral infections, it is intriguing to understand how they distinguish self nucleic acids from foreign viral ones. Here, we review the current knowledge of the recognition of viral nucleic acids by these sensor molecules and the signal transduction machinery. Copyright