Nathalie Grandvaux

The interferon antiviral response: from viral invasion to evasion.

One of the initial responses of an organism to infection by pathogenic viruses is the synthesis of antiviral cytokines such as the type I interferons (interferon-α/β), interleukins, and other proinflammatory cytokines and chemokines. Interferons provide a first line of defence against virus infections by generating an intracellular environment that restricts virus replication and signals the presence of a viral pathogen to the adaptive arm of the immune response. Interferons stimulate cells in the local environment to activate a network of interferon-stimulated genes, which encode proteins that have antiviral, antiproliferative and immunomodulatory activities. The present review focuses on recent reports that describe the activation of multiple signalling pathways following virus infection, new candidate genes that are implicated in the establishment of the antiviral state, and the strategies used by viruses and their specific viral products to antagonize and evade the host antiviral response.

Inhibition of RIG-I-Dependent Signaling to the Interferon Pathway during Hepatitis C Virus Expression and Restoration of Signaling by IKKε

ABSTRACT Interferon (IFN) is one important effector of the innate immune response, induced by different viral or bacterial components through Toll-like receptor (TLR)-dependent and -independent mechanisms. As part of its pathogenic strategy, hepatitis C virus (HCV) interferes with the innate immune response and induction of IFN-β via the HCV NS3/4A protease activity which inhibits phosphorylation of IRF-3, a key transcriptional regulator of the IFN response. In the present study, we demonstrate that inhibition by the protease occurs upstream of the noncanonical IKK-related kinases IKKε and TBK-1, which phosphorylate IRF-3, through partial inhibition of the TLR adapter protein TRIF/TICAM1-dependent pathway. Use of TRIF−/− mouse embryo fibroblasts however revealed the presence of a TRIF-independent pathway involved in IFN induction that was also inhibited by NS3/4A. Importantly, we show that NS3/4A can strongly inhibit the ability of the recently described RIG-I protein to activate IFN, suggesting that RIG-I is a key factor in the TRIF-independent, NS3/4A-sensitive pathway. Expression of IFN signaling components including IKKε, TBK-1, TRIF, and wild type or constitutively active forms of RIG-I in the HCV replicon cells resulted in IFN-β promoter transactivation, with IKKε displaying the highest efficiency. Subsequently, overexpression of IKKε resulted in 80% inhibition of both the positive and negative replicative strands of the HCV replicon. The partial restoration of the capacity of the host cell to transcribe IFN-β indicates that IKKε expression is able to bypass the HCV-mediated inhibition and restore the innate antiviral response.

Recognition of the Measles Virus Nucleocapsid as a Mechanism of IRF-3 Activation

ABSTRACT The mechanisms of cellular recognition for virus infection remain poorly understood despite the wealth of information regarding the signaling events and transcriptional responses that ensue. Host cells respond to viral infection through the activation of multiple signaling cascades, including the activation of NF-κB, c-Jun/ATF-2 (AP-1), and the interferon regulatory factors (IRFs). Although viral products such as double-stranded RNA (dsRNA) and the processes of viral binding and fusion have been implicated in the activation of NF-κB and AP-1, the mechanism(s) of IRF-1, IRF-3, and IRF-7 activation has yet to be fully elucidated. Using recombinant measles virus (MeV) constructs, we now demonstrate that phosphorylation-dependent IRF-3 activation represents a novel cellular detection system that recognizes the MeV nucleocapsid structure. At low multiplicities of infection, IRF-3 activation is dependent on viral transcription, since UV cross-linking and a deficient MeV containing a truncated polymerase L gene failed to induce IRF-3 phosphorylation. Expression of the MeV nucleocapsid (N) protein, without the requirement for any additional viral proteins or the generation of dsRNA, was sufficient for IRF-3 activation. In addition, the nucleocapsid protein was found to associate with both IRF-3 and the IRF-3 virus-activated kinase, suggesting that it may aid in the colocalization of the kinase and the substrate. Altogether, this study suggests that IRF-3 recognizes nucleocapsid structures during the course of an MeV infection and triggers the induction of interferon production.

Activation of TBK1 and IKKε Kinases by Vesicular Stomatitis Virus Infection and the Role of Viral Ribonucleoprotein in the Development of Interferon Antiviral Immunity

ABSTRACT Mounting an immune response to a viral pathogen involves the initial recognition of viral antigens through Toll-like receptor-dependent and -independent pathways and the subsequent triggering of signal transduction cascades. Among the many cellular kinases stimulated in response to virus infection, the noncanonical IKK-related kinases TBK1 and IKKε have been shown to phosphorylate and activate interferon regulatory factor 3 (IRF-3) and IRF-7, leading to the production of alpha/beta interferons and the development of a cellular antiviral state. In the present study, we examine the activation of TBK1 and IKKε kinases by vesicular stomatitis virus (VSV) infection in human lung epithelial A549 cells. We demonstrate that replication-competent VSV is required to induce activation of the IKK-related kinases and provide evidence that ribonucleoprotein (RNP) complex of VSV generated intracellularly during virus replication can activate TBK1 and IKKε activity. In TBK1-deficient cells, IRF-3 and IRF-7 activation is significantly reduced, although transcriptional upregulation of IKKε following treatment with VSV, double-stranded RNA, or RNP partially compensates for the loss of TBK1. Biochemical analyses with purified TBK1 and IKKε kinases in vitro demonstrate that the two kinases exhibit similar specificities with respect to IRF-3 and IRF-7 substrates and both kinases target serine residues that are important for full transcriptional activation of IRF-3 and IRF-7. These data suggest that intracellular RNP formation contributes to the early recognition of VSV infection, activates the catalytic activity of TBK1, and induces transcriptional upregulation of IKKε in epithelial cells. Induction of IKKε potentially functions as a component of the amplification mechanism involved in the establishment of the antiviral state.

Disruption of NF-κB Signaling Reveals a Novel Role for NF-κB in the Regulation of TNF-Related Apoptosis-Inducing Ligand Expression

The NF-κB family of transcription factors functions broadly in the host control of immunoregulatory gene expression, inflammation, and apoptosis. Using Jurkat T cells engineered to inducibly express a transdominant repressor of IκBα, we examined the role of NF-κB in the regulation of cytokine and apoptotic gene expression. In this T cell model, as well as in primary T lymphocytes, expression of TNF-related apoptosis-inducing ligand (TRAIL) apoptotic signaling protein was dramatically down-regulated by inhibition of NF-κB binding activity. TRAIL acts through membrane death receptors to induce apoptosis of activated T lymphocytes and can be up-regulated by a variety of physiological and pharmacological inducers. However, regulation of TRAIL gene expression has not been defined. Treatment with TCR mimetics (PMA/ionomycin, PHA, and anti-CD3/CD28 Abs) resulted in a rapid increase in the expression of TRAIL mRNA and cell surface TRAIL protein. Induction of the transdominant repressor of IκBα dramatically down-regulated surface expression of TRAIL, indicating an essential role for NF-κB in the regulation of TRAIL. The induced expression of TRAIL was linked to a c-Rel binding site in the proximal TRAIL promoter at position −256 to −265; mutation of this site or an adjacent κB site resulted in a complete loss of the inducibility of the TRAIL promoter. The regulation of TRAIL expression by NF-κB may represent a general mechanism that contributes to the control of TRAIL-mediated apoptosis in T lymphocytes.

Requirement of NOX2 and Reactive Oxygen Species for Efficient RIG-I-Mediated Antiviral Response through Regulation of MAVS Expression

The innate immune response is essential to the host defense against viruses, through restriction of virus replication and coordination of the adaptive immune response. Induction of antiviral genes is a tightly regulated process initiated mainly through sensing of invading virus nucleic acids in the cytoplasm by RIG-I like helicases, RIG-I or Mda5, which transmit the signal through a common mitochondria-associated adaptor, MAVS. Although major breakthroughs have recently been made, much remains unknown about the mechanisms that translate virus recognition into antiviral genes expression. Beside the reputed detrimental role, reactive oxygen species (ROS) act as modulators of cellular signaling and gene regulation. NADPH oxidase (NOX) enzymes are a main source of deliberate cellular ROS production. Here, we found that NOX2 and ROS are required for the host cell to trigger an efficient RIG-I-mediated IRF-3 activation and downstream antiviral IFNβ and IFIT1 gene expression. Additionally, we provide evidence that NOX2 is critical for the expression of the central mitochondria-associated adaptor MAVS. Taken together these data reveal a new facet to the regulation of the innate host defense against viruses through the identification of an unrecognized role of NOX2 and ROS.

Memory CCR6+CD4+ T Cells Are Preferential Targets for Productive HIV Type 1 Infection Regardless of Their Expression of Integrin β7

HIV type 1 infection is associated with a rapid depletion of Th17 cells from the GALT. The chemokine receptor CCR6 is a marker for Th17 lineage polarization and HIV permissiveness in memory CD4+ T cells. CCR6+ T cells have the potential to migrate into the GALT via the gut-homing integrin α4β7, a newly identified HIV-gp120 binding receptor. In this study, we investigated whether memory T cells coexpressing CCR6 and integrin β7 are selective HIV targets and whether retinoic acid (RA)-induced imprinting for gut-homing selectively increases CCR6+ T cell permissiveness to infection. We demonstrated that β7−R6+ and β7+R6+ compared with β7−R6− and β7+R6− T cells were highly permissive to HIV, produced Th17 cytokines, and their frequency was decreased in the peripheral blood of HIV-infected subjects. RA upregulated integrin α4 and β7 coexpression in both CCR6+ and CCR6− T cells, but increased HIV permissiveness selectively in CCR6+ T cells via entry (CCR5 upregulation) and postentry mechanisms. In conclusion, these results demonstrate that CCR6, but not the integrin β7, is a discriminative marker for memory T cells imprinted with a transcriptional program favorable to HIV replication. Nevertheless, given the ability of integrin β7 to regulate cell migration into the GALT and bind HIV-gp120, CCR6+ T cells coexpressing integrin β7 and CCR5 might have an extraordinary ability to disseminate HIV from the portal sites of entry. Understanding the molecular mechanisms of memory CCR6+ T cell differentiation is critical for the design of new therapeutic strategies that should interfere with viral permissiveness but not Th17 lineage commitment and gut-homing potential in CCR6+ T cells.

Nuclear Accumulation of cRel following C-Terminal phosphorylation by TBK1/IKKε

The NF-κB transcription factors are key regulators of immunomodulatory, cell cycle, and developmental gene regulation. NF-κB activity is mainly regulated through the phosphorylation of IκB by the IκB kinase (IKK) complex IKKαβγ, leading to proteasome-mediated degradation of IκB, nuclear translocation of NF-κB dimers, DNA binding, and gene induction. Additionally, direct posttranslational modifications of NF-κB p65 and cRel subunits involving C-terminal phosphorylation has been demonstrated. The noncanonical IKK-related homologs, TNFR-associated factor family member-associated NF-κB activator (TANK)-binding kinase (TBK)1 and IKKε, are also thought to play a role in NF-κB regulation, but their functions remain unclear. TBK1 and IKKε were recently described as essential regulators of IFN gene activation through direct phosphorylation of the IFN regulatory factor-3 and -7 transcription factors. In the present study, we sought to determine whether IKKε and TBK1 could modulate cRel activity via phosphorylation. TBK1 and IKKε directly phosphorylate the C-terminal domain of cRel in vitro and in vivo and regulate nuclear accumulation of cRel, independently of the classical IκB/IKK pathway. IκBα degradation is not affected, but rather IKKε-mediated phosphorylation of cRel leads to dissociation of the IκBα-cRel complex. These results illustrate a previously unrecognized aspect of cRel regulation, controlled by direct IKKε/TBK1 phosphorylation.

Respiratory Syncytial Virus-Mediated NF-κB p65 Phosphorylation at Serine 536 Is Dependent on RIG-I, TRAF6, and IKKβ

ABSTRACT Respiratory syncytial virus (RSV) is the etiological agent of acute respiratory diseases, such as bronchiolitis and pneumonia. The exacerbated production of proinflammatory cytokines and chemokines in the airways in response to RSV is an important pillar in the development of these pathologies. As such, a keen understanding of the mechanisms that modulate the inflammatory response during RSV infection is of pivotal importance to developing effective treatment. The NF-κB transcription factor is a major regulator of proinflammatory cytokine and chemokine genes. However, RSV-mediated activation of NF-κB is far from characterized. We recently demonstrated that aside from the well-characterized IκBα phosphorylation and degradation, the phosphorylation of p65 at Ser536 is an essential event regulating the RSV-mediated NF-κB-dependent promoter transactivation. In the present study, using small interfering RNA and pharmacological inhibitors, we now demonstrate that RSV sensing by the RIG-I cytoplasmic receptor triggers a signaling cascade involving the MAVS and TRAF6 adaptors that ultimately leads to p65ser536 phosphorylation by the IKKβ kinase. In a previous study, we highlighted a critical role of the NOX2-containing NADPH oxidase enzyme as an upstream regulator of both the IκBαSer32 and p65Ser536 in human airway epithelial cells. Here, we demonstrate that inhibition of NOX2 significantly decreases IKKβ activation. Taken together, our data identify a new RIG-I/MAVS/TRAF6/IKKβ/p65Ser536 pathway placed under the control of NOX2, thus characterizing a novel regulatory pathway involved in NF-κB-driven proinflammatory response in the context of RSV infection.

Binding of kaposi's sarcoma-associated herpesvirus K-bZIP to interferon-responsive factor 3 elements modulates antiviral gene expression

ABSTRACT Kaposis sarcoma-associated herpesvirus encodes numerous regulatory proteins capable of modulating viral and cellular gene expression and affecting host cell functions. K-bZIP, a leucine zipper-containing transcription factor encoded by ORFK8, is one such protein. During infection, transcription of the ORFK8 early gene is turned on by the immediate-early replication and transcription factor activator (RTA). One described function of the K-bZIP nuclear protein is to interact with and repress RTA-mediated transactivation of viral promoters, including that of the K8 gene. In the present work, we provide evidence that the expression of K-bZIP results in the activation of the ifn-β gene. Of interest, ifn-β gene activation by K-bZIP is independent of interferon (IFN)-responsive factor 3 (IRF-3) and nuclear factor κB (NF-κB) activation. Using a DNA binding affinity assay and electromobility shift assay, we report that K-bZIP binds efficiently to the PRDIII-I region of the beta IFN (IFN-β) promoter, and, in doing so, it prevents the attachment of activated IRF-3 but not that of NF-κB or ATF2/c-Jun to the IFN-β promoter sequence. As a consequence, ifn-β gene activation in response to IFN inducers such as Sendai virus infection or expression of retinoic acid-inducible gene I, mitochondrial antiviral signaling protein, or TANK-binding kinase 1 (TBK-1) is severely impaired (>90%) by the presence of K-bZIP. K-bZIP also prevents the activation of RANTES and CXCL11, whose promoters are also regulated by IRF-3. Lysine 158 (target for SUMO conjugation), threonine 111, and serine 167 (targets for phosphorylation) mutants of K-bZIP were equally effective as wild-type K-bZIP in mediating the repression of TBK-1-activated ifn-β gene expression. Lastly, the overexpression of CREB binding protein could not reverse the K-bZIP repression of TBK-1-activated ifn-β gene expression. In all, our results indicate that K-bZIP binds directly to the PRDIII-I region of the IFN-β promoter and, as a consequence, causes a low level of ifn-β gene transcription. In doing so, K-bZIP prevents IRF-3 from binding to the IFN-β promoter and precludes the formation of the enhanceosome, which is required for maximal ifn-β gene transcription. A new role for K-bZIP as a protein involved in immune evasion is therefore uncovered.