Myriam Vilasco

A functional C-terminal TRAF3-binding site in MAVS participates in positive and negative regulation of the IFN antiviral response

Recognition of viral RNA structures by the cytosolic sensor retinoic acid-inducible gene-I (RIG-I) results in the activation of signaling cascades that culminate with the generation of the type I interferon (IFN) antiviral response. Onset of antiviral and inflammatory responses to viral pathogens necessitates the regulated spatiotemporal recruitment of signaling adapters, kinases and transcriptional proteins to the mitochondrial antiviral signaling protein (MAVS). We previously demonstrated that the serine/threonine kinase IKKε is recruited to the C-terminal region of MAVS following Sendai or vesicular stomatitis virus (VSV) infection, mediated by Lys63-linked polyubiquitination of MAVS at Lys500, resulting in inhibition of downstream IFN signaling (Paz et al, Mol Cell Biol, 2009). In this study, we demonstrate that C-terminus of MAVS harbors a novel TRAF3-binding site in the aa450-468 region of MAVS. A consensus TRAF-interacting motif (TIM), 455-PEENEY-460, within this site is required for TRAF3 binding and activation of IFN antiviral response genes, whereas mutation of the TIM eliminates TRAF3 binding and the downstream IFN response. Reconstitution of MAVS−/− mouse embryo fibroblasts with a construct expressing a TIM-mutated version of MAVS failed to restore the antiviral response or block VSV replication, whereas wild-type MAVS reconstituted antiviral inhibition of VSV replication. Furthermore, recruitment of IKKε to an adjacent C-terminal site (aa 468–540) in MAVS via Lys500 ubiquitination decreased TRAF3 binding and protein stability, thus contributing to IKKε-mediated shutdown of the IFN response. This study demonstrates that MAVS harbors a functional C-terminal TRAF3-binding site that participates in positive and negative regulation of the IFN antiviral response.

Ubiquitin-regulated recruitment of IkappaB kinase epsilon to the MAVS interferon signaling adapter.

ABSTRACT Induction of the antiviral interferon response is initiated upon recognition of viral RNA structures by the RIG-I or Mda-5 DEX(D/H) helicases. A complex signaling cascade then converges at the mitochondrial adapter MAVS, culminating in the activation of the IRF and NF-κB transcription factors and the induction of interferon gene expression. We have previously shown that MAVS recruits IκB kinase ε (IKKε) but not TBK-1 to the mitochondria following viral infection. Here we map the interaction of MAVS and IKKε to the C-terminal region of MAVS and demonstrate that this interaction is ubiquitin dependent. MAVS is ubiquitinated following Sendai virus infection, and K63-linked ubiquitination of lysine 500 (K500) of MAVS mediates recruitment of IKKε to the mitochondria. Real-time PCR analysis reveals that a K500R mutant of MAVS increases the mRNA level of several interferon-stimulated genes and correlates with increased NF-κB activation. Thus, recruitment of IKKε to the mitochondria upon MAVS K500 ubiquitination plays a modulatory role in the cascade leading to NF-κB activation and expression of inflammatory and antiviral genes. These results provide further support for the differential role of IKKε and TBK-1 in the RIG-I/Mda5 pathway.

Polo-like kinase 1 (PLK1) regulates interferon (IFN) induction by MAVS.

The mitochondria-bound adapter MAVS participates in IFN induction by recruitment of downstream partners such as members of the TRAF family, leading to activation of NF-κB, and the IRF3 pathways. A yeast two-hybrid search for MAVS-interacting proteins yielded the Polo-box domain (PBD) of the mitotic Polo-like kinase PLK1. We showed that PBD associates with two different domains of MAVS in both dependent and independent phosphorylation events. The phosphodependent association requires the phosphopeptide binding ability of PBD. It takes place downstream of the proline-rich domain of MAVS, within an STP motif, characteristic of the binding of PLK1 to its targets, where the central Thr234 residue is phosphorylated. Its phosphoindependent association takes place at the C terminus of MAVS. PLK1 strongly inhibits the ability of MAVS to activate the IRF3 and NF-κB pathways and to induce IFN. Reciprocally, depletion of PLK1 can increase IFN induction in response to RIG-I/SeV or RIG-I/poly(I)-poly(C) treatments. This inhibition is dependent on the phosphoindependent association of PBD at the C terminus of MAVS where it disrupts the association of MAVS with its downstream partner TRAF3. IFN induction was strongly inhibited in cells arrested in G2/M by nocodazole, which provokes increased expression of endogenous PLK1. Interestingly, depletion of PLK1 from these nocodazole-treated cells could restore, at least partially, IFN induction. Altogether, these data demonstrate a new function for PLK1 as a regulator of IFN induction and provide the basis for the development of inhibitors preventing the PLK1/MAVS association to sustain innate immunity.

The protein kinase IKKε can inhibit HCV expression independently of IFN and its own expression is downregulated in HCV-infected livers†

During a viral infection, binding of viral double‐stranded RNAs (dsRNAs) to the cytosolic RNA helicase RIG‐1 leads to recruitment of the mitochondria‐associated Cardif protein, involved in activation of the IRF3‐phosphorylating IKKε/TBK1 kinases, interferon (IFN) induction, and development of the innate immune response. The hepatitis C virus (HCV) NS3/4A protease cleaves Cardif and abrogates both IKKε/TBK1 activation and IFN induction. By using an HCV replicon model, we previously showed that ectopic overexpression of IKKε can inhibit HCV expression. Here, analysis of the IKKε transcriptome profile in these HCV replicon cells showed induction of several genes associated with the antiviral action of IFN. Interestingly, IKKε still inhibits HCV expression in the presence of neutralizing antibodies to IFN receptors or in the presence of a dominant negative STAT1α mutant. This suggests that good IKKε expression levels are important for rapid activation of the cellular antiviral response in HCV‐infected cells, in addition to provoking IFN induction. To determine the physiological importance of IKKε in HCV infection, we then analyzed its expression levels in liver biopsy specimens from HCV‐infected patients. This analysis also included genes of the IFN induction pathway (RIG‐I, MDA5, LGP2, Cardif, TBK1), and three IKKε‐induced genes (IFN‐β, CCL3, and ISG15). The results show significant inhibition of expression of IKKε and of the RNA helicases RIG‐I/MDA5/LGP2 in the HCV‐infected patients, whereas expression of TBK1 and Cardif was not significantly altered. In conclusion, given the antiviral potential of IKKε and of the RNA helicases, these in vivo data strongly support an important role for these genes in the control of HCV infection.(HEPATOLOGY 2006;44:1635–1647.)