Antiviral immunity against a pathogen is mounted upon recognition by the host of virally associated structures. One of these viral ‘signatures’, double-stranded (ds) RNA, is a replication product of most viruses within infected cells and is sensed by Toll-like receptor 3 (TLR3) and the recently identified cytosolic RNA helicases RIG-I (retinoic acid inducible gene I, also known as Ddx58) and Mda5 (melanoma differentiation-associated gene 5, also known as Ifih1 or Helicard)¹. Both helicases detect dsRNA, and through their protein-interacting CARD domains, relay an undefined signal resulting in the activation of the transcription factors interferon regulatory factor 3 (IRF3) and NF-κB. Here we describe Cardif, a new CARD-containing adaptor protein that interacts with RIG-I and recruits IKKα, IKKβ and IKKɛ kinases by means of its C-terminal region, leading to the activation of NF-κB and IRF3. Overexpression of Cardif results in interferon-β and NF-κB promoter activation, and knockdown of Cardif by short interfering RNA inhibits RIG-I-dependent antiviral responses. Cardif is targeted and inactivated by NS3-4A, a serine protease from hepatitis C virus known to block interferon-β production. Cardif thus functions as an adaptor, linking the cytoplasmic dsRNA receptor RIG-I to the initiation of antiviral programmes.

Sensing viruses by pattern recognition receptors (PRR) triggers the innate immune system of the host cell and activates immune signaling cascades such as the RIG-I/IRF3 pathway. Mitochondrial antiviral-signaling protein (MAVS, also known as IPS-1, Cardif, and VISA) is the crucial adaptor protein of this pathway localized on mitochondria, peroxisomes and mitochondria-associated membranes of the endoplasmic reticulum. Activation of MAVS leads to the production of type I and type III interferons (IFN) as well as IFN stimulated genes (ISGs). To refine the role of MAVS subcellular localization for the induction of type I and III IFN responses in hepatocytes and its counteraction by the hepatitis C virus (HCV), we generated various functional and genetic knock-out cell systems that were reconstituted to express mitochondrial (mito) or peroxisomal (pex) MAVS, exclusively. Upon infection with diverse RNA viruses we found that cells exclusively expressing pexMAVS mounted sustained expression of type I and III IFNs to levels comparable to cells exclusively expressing mitoMAVS. To determine whether viral counteraction of MAVS is affected by its subcellular localization we employed infection of cells with HCV, a major causative agent of chronic liver disease with a high propensity to establish persistence. This virus efficiently cleaves MAVS via a viral protease residing in its nonstructural protein 3 (NS3) and this strategy is thought to contribute to the high persistence of this virus. We found that both mito- and pexMAVS were efficiently cleaved by NS3 and this cleavage was required to suppress activation of the IFN response. Taken together, our findings indicate comparable activation of the IFN response by pex- and mitoMAVS in hepatocytes and efficient counteraction of both MAVS species by the HCV NS3 protease. Mammalian cells developed several defense mechanisms against viral infection. One major strategy involves pattern recognition receptors (PRRs) recognizing non-self motifs in viral RNA and triggering the production of type I and III interferon (IFN) that induce an antiviral state. One central signaling molecule in this cascade is MAVS (Mitochondrial Antiviral Signaling protein), residing on mitochondria, mitochondria-associated membranes of the endoplasmic reticulum, and peroxisomes. Here we characterized the role of mitochondrial and peroxisomal MAVS for the activation of the IFN response and their counteraction by the hepatitis C virus (HCV), a major causative agent of chronic liver disease with a high propensity to establish persistence. By using various functional and genetic knock-out cell systems reconstituted to express exclusively mitochondrial or peroxisomal MAVS, we observed comparable activation of type I and III IFN response by either MAVS species. In addition, we found that the HCV protease residing in nonstructural protein 3 (NS3) efficiently cleaves MAVS independent from its subcellular localization. This cleavage is required for suppression of the IFN response and might contribute to HCV persistence. Our results indicate a largely localization-independent activation of the IFN response by MAVS in hepatocytes and its efficient counteraction by the HCV NS3 protease.