Viral infection or stimulation of TLR3 triggers signaling cascades, leading to activation of the transcription factors IRF-3 and NF-kappaB, which collaborate to induce transcription of type I interferon (IFN) genes. In this study, we identified a protein termed VISA (for virus-induced signaling adaptor) as a critical component in the IFN-beta signaling pathways. VISA recruits IRF-3 to the cytoplasmic viral dsRNA sensor RIG-I. Depletion of VISA inhibits virus-triggered and RIG-I-mediated activation of IRF-3, NF-kappaB, and the IFN-beta promoter, suggesting that VISA plays a central role in virus-triggered TLR3-independent IFN-beta signaling. Our data also indicate that VISA interacts with TRIF and TRAF6 and mediates bifurcation of the TLR3-triggered NF-kappaB and IRF-3 activation pathways. These findings suggest that VISA is critically involved in both virus-triggered TLR3-independent and TLR3-mediated antiviral IFN signaling.

We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non-self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/ retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.innate immunity ͉ type I IFN ͉ functional cDNA library screening ͉ cytosolic RNA and dsDNA ͉ ER retention signal M icrobial infection-induced host immune responses are initiated by the germline-encoded pattern recognition receptors, which recognize components specific to microorganisms. There are 3 major classes of such receptors: Toll-like receptors (TLRs), RIG-I-like helicases (RLHs) and NOD-like receptors (1). During infection, nucleic acids derived from microbes are recognized by TLRs and RLHs, which then trigger a series of signaling events leading to the production of type I IFNs and proinflammatory cytokines.RLHs have recently been identified to sense the invading viruses in the cytoplasm. Unlike TLRs, which are expressed in specific cells like macrophages and dendritic cells, RLHs are found in most cell types (2). They contain caspase recruitment domain (CARD) and DExD/H helicase domain. RLHs interact with microbial nucleotides through their helicase domain. The N-terminal CARDs are responsible for activating downstream signaling pathways that mediate type I IFN production. Genetic analyses demonstrate that RIG-I and MDA5 sense distinct types of viruses (3-5). RIG-I and MDA5 use a common adaptor molecule, IPS-1 (also known as Cardif, MAVS, or VISA) (6-9). IPS-1 is found to reside on the mitochondrial membrane by its C-terminal transmembrane (TM) domain. It also contains a CARD-like domain at its N-terminus, which mediates the interaction with MDA5 or RIG-I. IPS-1 transmits the signal to TANK-binding kinase-1 (TBK1)/I B kinase i (IKKi; also known as IKK ) and the IKK complex to activate interferon regulatory factor (IRF)-3/IRF-7 and NF-B, respectively, collectively eliciting innate antiviral immune responses, including type I IFN production.On the other hand, dsDNA in the cytosol, for example, genomic DNA from intracellular bacteria (e.g., Listeria, Legionella), also causes a strong host immune response independent of TLRs, leading to the induction of type I IFN. A recent report has indicated that the molecule DAI (also known as ZBP1) might serve as a cytosolic dsDNA sensor (10). However, ZBP1 Ϫ/Ϫ cells showed normal type I IFN production in response to dsDNA stimulation (11). Meanwhile, reports showed that IPS-1/Cardif/MAVS/VISA was not required for dsDNA-caused innate immune activation (12).The signaling induced by cytoplasmic dsDNA leading ...

MAVS is critical in innate antiviral immunity as the sole adaptor for RIG-I-like helicases. MAVS regulation is essential for the prevention of excessive harmful immune responses. Here we identify PCBP2 as a negative regulator in MAVS-mediated signaling. Overexpression of PCBP2 abrogated cellular responses to viral infection, whereas knockdown of PCBP2 exerted the opposite effect. PCBP2 was induced after viral infection, and its interaction with MAVS led to proteasomal degradation of MAVS. PCBP2 recruited the HECT domain-containing E3 ligase AIP4 to polyubiquitinate and degrade MAVS. MAVS was degraded after viral infection in wild-type mouse embryonic fibroblasts but remained stable in AIP4-deficient (Itch(-/-)) mouse embryonic fibroblasts, coupled with greatly exaggerated and prolonged antiviral responses. The PCBP2-AIP4 axis defines a new signaling cascade for MAVS degradation and 'fine tuning' of antiviral innate immunity.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that triggers caspase-independent apoptosis. We describe here the cloning and characterization of a novel AIF-homologous molecule designated AMID (AIFhomologous mitochondrion-associated inducer of death). AMID lacks a mitochondrial localization sequence but shares significant homology with AIF and NADH oxidoreductases from bacteria to mammalian species. Immunofluorescent staining and biochemical experiments indicated that AMID was co-localized with mitochondria. Overexpression of AMID induced cell death with characteristic apoptotic morphology. Furthermore, AMID-induced apoptosis was independent of caspase activation and p53 and was not inhibited by Bcl-2. These findings suggest that AMID induces a novel caspaseindependent apoptotic pathway.

Viral infection or TLR3 engagement causes activation of the transcription factors IRF-3 and NF-jB, which collaborate to induce transcription of type I IFN genes. IKKe and TBK1 are two IKK-related kinases critically involved in virus-and TLR3-triggered activation of IRF-3. We identified a protein termed SIKE (for Suppressor of IKKe) that interacts with IKKe and TBK1. SIKE is associated with TBK1 under physiological condition and dissociated from TBK1 upon viral infection or TLR3 stimulation. Overexpression of SIKE disrupted the interactions of IKKe or TBK1 with TRIF, RIG-I and IRF-3, components in virus-and TLR3-triggered IRF-3 activation pathways, but did not disrupt the interactions of TRIF with TRAF6 and RIP, components in TLR3-triggered NF-jB activation pathway. Consistently, overexpression of SIKE inhibited virus-and TLR3-triggered interferon-stimulated response elements (ISRE) but not NF-jB activation. Knockdown of SIKE potentiated virus-and TLR3-triggered ISRE but not NF-jB activation. Moreover, overexpression of SIKE inhibited IKKe-and TBK1-mediated antiviral response. These findings suggest that SIKE is a physiological suppressor of IKKe and TBK1 and plays an inhibitory role in virus-and TLR3-triggered IRF-3 but not NF-jB activation pathways.