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Viral infections trigger innate immune responses, including the production of type I interferons (IFN-alpha and -beta) and other proinflammatory cytokines. Novel antiviral cytokines IFN-lambda1, IFN-lambda2, and IFN-lambda3 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-lambdas is not well understood. Here, we analyzed the mechanism by which IFN-lambda 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 ifnlambda1 revealed a cluster of interferon regulatory factor-binding sites and a NF-kappaB-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.

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.

In virus-infected cells, RIG-I-like receptor (RLR) recognizes cytoplasmic viral RNA and triggers innate immune responses including production of type I and III interferon (IFN) and the subsequent expression of IFN-inducible genes. Interferon-β promoter stimulator 1 (IPS-1, also known as MAVS, VISA and Cardif) is a downstream molecule of RLR and is expressed on the outer membrane of mitochondria. While it is known that the location of IPS-1 is essential to its function, its underlying mechanism is unknown. Our aim in this study was to delineate the function of mitochondria so as to identify more precisely its role in innate immunity. In doing so we discovered that viral infection as well as transfection with 5′ppp-RNA resulted in the redistribution of IPS-1 to form speckle-like aggregates in cells. We further found that Mitofusin 1 (MFN1), a key regulator of mitochondrial fusion and a protein associated with IPS-1 on the outer membrane of mitochondria, positively regulates RLR-mediated innate antiviral responses. Conversely, specific knockdown of MFN1 abrogates both the virus-induced redistribution of IPS-1 and IFN production. Our study suggests that mitochondria participate in the segregation of IPS-1 through their fusion processes., Author Summary Virus-infections, such as influenza and chronic hepatitis C, are prominent diseases and outbreaks of newly emerging viruses are serious problems for modern society. Higher animals, including humans, are genetically equipped with mechanisms, collectively known as innate immunity, to counteract viral infections. RIG-I-like receptor (RLR), a cytoplasmic sensor, contributes to immune regulation by detecting infections by RNA viruses and triggering a series of responses which results in the activation of innate antiviral genes. Furthermore, it has been demonstrated that IPS-1, the adaptor protein of RLR, is expressed on mitochondrial outer membrane. Mitochondrion is an organelle of prokaryotic cell origin; it regulates energy production, and is involved in cell growth and cell death. Why IPS-1 is located on the mitochondrial outer membrane and how mitochondria are involved in antiviral signaling are yet to be explained clearly. In this report, we discovered that mitochondrial fusion protein MFN1 plays a novel function to mediate IPS-1 redistribution, which appears to be a critical step in RLR signaling.