Redox signaling in cell fate: Beyond damage.

This review explores the nuanced role of reactive oxygen species (ROS) in cell fate, challenging the traditional view that equates ROS with cellular damage. Through significant technological advancements in detecting localized redox states and identifying oxidized cysteines, a paradigm shift has emerged: from ROS as merely damaging agents to crucial players in redox signaling. We delve into the intricacies of redox mechanisms, which, although confined, exert profound influences on cellular physiological responses. Our analysis extends to both the positive and negative impacts of these mechanisms on cell death processes, including uncontrolled and programmed pathways. By unraveling these complex interactions, we argue against the oversimplified notion of a 'stress response', advocating for a more nuanced understanding of redox signaling. This review underscores the importance of localized redox states in determining cell fate, highlighting the sophistication and subtlety of ROS functions beyond mere damage.

DUOX2 regulates secreted factors in virus-infected respiratory epithelial cells that contribute to neutrophil attraction and activation.

The first line of defense against respiratory viruses relies on the antiviral and proinflammatory cytokine response initiated in infected respiratory epithelial cells. The cytokine response not only restricts virus replication and spreading, but also orchestrates the subsequent immune response. The epithelial Dual Oxidase 2 (DUOX2) has recently emerged as a regulator of the interferon antiviral response. Here, we investigated the role of DUOX2 in the inflammatory cytokine response using a model of A549 cells deficient in DUOX2 generated using Crispr-Cas9 and infected by Sendai virus. We found that the absence of DUOX2 selectively reduced the induction of a restricted panel of 14 cytokines and chemokines secreted in response to Sendai virus by 20 to 89%. The secreted factors produced by epithelial cells upon virus infection promoted the migration, adhesion, and degranulation of primary human neutrophils, in part through the DUOX2-dependent secretion of TNF and chemokines. In contrast, DUOX2 expression did not impact neutrophil viability or NETosis, thereby highlighting a selective impact of DUOX2 in neutrophil functions. Overall, this study unveils previously unrecognized roles of epithelial DUOX2 in the epithelial-immune cells crosstalk during respiratory virus infection.

Redox proteomics and structural analyses provide insightful implications for additional non-catalytic thiol-disulfide motifs in PDIs.

Protein disulfide isomerases (PDIs) catalyze redox reactions that reduce, oxidize, or isomerize disulfide bonds and act as chaperones of proteins as they fold. The characteristic features of PDIs are the presence of one or more catalytic thioredoxin (TRX)-like domains harboring typical CXXC catalytic motifs responsible for redox reactions, as well as non-catalytic TRX-like domain. As increasing attention is paid to oxidative post-translational modifications of cysteines (Cys ox-PTMs) with the recognition that they control cellular signaling, strategies to identify sites of Cys ox-PTM by redox proteomics have been optimized. Exploration of an available Cys redoxome dataset supported by modeled structure provided arguments for the existence of an additional non-catalytic thiol-disulfide motif, distinct from those contained in the TRX type patterns, typical of PDIAs. Further structural analysis of PDIA3 and 6 allows us to consider the possibility that this hypothesis could be extended to other members of PDI. These elements invite future studies to decipher the exact role of these non-catalytic thiol-disulfide motifs in the functions of PDIs. Strategies that would allow to validate this hypothesis are discussed.

Pinpointing cysteine oxidation sites by high-resolution proteomics reveals a mechanism of redox-dependent inhibition of human STING.

Protein function is regulated by posttranslational modifications (PTMs), among which reversible oxidation of cysteine residues has emerged as a key regulatory mechanism of cellular responses. Given the redox regulation of virus-host interactions, the identification of oxidized cysteine sites in cells is essential to understand the underlying mechanisms involved. Here, we present a proteome-wide identification of reversibly oxidized cysteine sites in oxidant-treated cells using a maleimide-based bioswitch method coupled to mass spectrometry analysis. We identified 2720 unique oxidized cysteine sites within 1473 proteins with distinct abundances, locations, and functions. Oxidized cysteine sites were found in numerous signaling pathways, many relevant to virus-host interactions. We focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway, which is stimulated in response to the detection of cytosolic DNA by cGAS. We demonstrated the reversible oxidation of Cys148 and Cys206 of STING in cells. Molecular analyses led us to establish a model in which Cys148 oxidation is constitutive, whereas Cys206 oxidation is inducible by oxidative stress or by the natural ligand of STING, 2'3'-cGAMP. Our data suggest that the oxidation of Cys206 prevented hyperactivation of STING by causing a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This finding should aid the design of therapies targeting STING that are relevant to autoinflammatory disorders, immunotherapies, and vaccines.

ACE2: Evidence of role as entry receptor for SARS-CoV-2 and implications in comorbidities.

Pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus 19 disease (COVID-19) which presents a large spectrum of manifestations with fatal outcomes in vulnerable people over 70-years-old and with hypertension, diabetes, obesity, cardiovascular disease, COPD, and smoking status. Knowledge of the entry receptor is key to understand SARS-CoV-2 tropism, transmission and pathogenesis. Early evidence pointed to angiotensin-converting enzyme 2 (ACE2) as SARS-CoV-2 entry receptor. Here, we provide a critical summary of the current knowledge highlighting the limitations and remaining gaps that need to be addressed to fully characterize ACE2 function in SARS-CoV-2 infection and associated pathogenesis. We also discuss ACE2 expression and potential role in the context of comorbidities associated with poor COVID-19 outcomes. Finally, we discuss the potential co-receptors/attachment factors such as neuropilins, heparan sulfate and sialic acids and the putative alternative receptors, such as CD147 and GRP78.

Defective Influenza A Virus RNA Products Mediate MAVS-Dependent Upregulation of Human Leukocyte Antigen Class I Proteins.

Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-ß and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity.IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.

The Combination of IFN ß and TNF Induces an Antiviral and Immunoregulatory Program via Non-Canonical Pathways Involving STAT2 and IRF9.

Interferon (IFN) ß and Tumor Necrosis Factor (TNF) are key players in immunity against viruses. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFNß is reprogrammed by crosstalk with TNF. IFNß mainly induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2, and IRF9. The signaling pathways engaged downstream of the combination of IFNß and TNF remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFNß and TNF costimulation. Additional stratification of this transcriptional response revealed that STAT2 and IRF9 mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of antiviral and immunoregulatory genes in conditions with elevated levels of both IFNß and TNF.

Therapeutic Targeting of RIG-I and MDA5 Might Not Lead to the Same Rome.

RIG-I and MDA5 receptors are key sensors of pathogen-associated molecular pattern (PAMP)-containing viral RNA and transduce downstream signals to activate an antiviral and immunomodulatory response. Fifteen years of research have put them at the center of an ongoing hunt for novel pharmacological pan-antivirals, vaccine adjuvants, and antitumor strategies. Current knowledge testifies to the redundant, but also distinct, functions mediated by RIG-I and MDA5, opening opportunities for the use of specific and potent nucleic acid agonists. We critically discuss the evidence and remaining knowledge gaps that have an impact on the choice and design of optimal RNA ligands to achieve an appropriate immunostimulatory response, with limited adverse effects, for prophylactic and therapeutic interventions against viruses and cancer in humans.

Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes.

Interleukin (IL)-30, the IL-27p28 subunit of the heterodimeric cytokine IL-27, acts as an antagonist of IL-27 and IL-6 signaling in murine cells via glycoprotein 130 (gp130) receptor and additional binding partners. Thus far, functions of IL-30 have not been fully elucidated in human cells. We demonstrate that like IL-27, IL-30 upregulated TLR4 expression to enhance lipopolysaccharide-induced TNF-α production in human monocytes; however, these IL-30-mediated activities did not reach the same levels of cytokine induction compared to IL-27. Interestingly, IL-30- and IL-27-mediated interferon-γ-induced protein 10 (IP-10) production required WSX-1 engagement and signal transducer and activator of transcription (STAT) 3 phosphorylation; furthermore, IL-30 induced STAT phosphorylation after 16 h, whereas IL-27 induced STAT phosphorylation within 30 min. This prompted us to examine if a secondary mediator was required for IL-30-induced pro-inflammatory functions, and hence we examined IL-6-related molecules. Combined with inhibition of soluble IL-6 receptor α (sIL-6Rα) and data showing that IL-6 inhibited IL-30/IL-27-induced IP-10 expression, we demonstrate a role for sIL-6Rα and gp130 in IL-30-mediated activity in human cells.

Virus Infection Triggers MAVS Polymers of Distinct Molecular Weight.

The mitochondrial antiviral signaling (MAVS) adaptor protein is a central signaling hub required for cells to mount an antiviral response following virus sensing by retinoic acid-inducible gene I (RIG-I)-like receptors. MAVS localizes in the membrane of mitochondria and peroxisomes and in mitochondrial-associated endoplasmic reticulum membranes. Structural and functional studies have revealed that MAVS activity relies on the formation of functional high molecular weight prion-like aggregates. The formation of protein aggregates typically relies on a dynamic transition between oligomerization and aggregation states. The existence of intermediate state(s) of MAVS polymers, other than aggregates, has not yet been documented. Here, we used a combination of non-reducing SDS-PAGE and semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) to resolve whole cell extract preparations to distinguish MAVS polymerization states. While SDD-AGE analysis of whole cell extracts revealed the formation of previously described high molecular weight prion-like aggregates upon constitutively active RIG-I ectopic expression and virus infection, non-reducing SDS-PAGE allowed us to demonstrate the induction of lower molecular weight oligomers. Cleavage of MAVS using the NS3/4A protease revealed that anchoring to intracellular membranes is required for the appropriate polymerization into active high molecular weight aggregates. Altogether, our data suggest that RIG-I-dependent MAVS activation involves the coexistence of MAVS polymers with distinct molecular weights.

Detection of IKKε by immunohistochemistry in primary breast cancer: association with EGFR expression and absence of lymph node metastasis.

BACKGROUND: IKKε is an oncogenic kinase that was found amplified and overexpressed in a substantial percentage of human breast cancer cell lines and primary tumors using genomic and gene expression analyses. Molecular studies have provided the rational for a key implication of IKKε in breast cancer cells proliferation and invasiveness through the phosphorylation of several substrates. METHODS: Here, we performed immunohistochemical detection of IKKε expression on tissue microarrays constituted of 154 characterized human breast cancer tumors. We further determined the association with multiple clinicopathological parameters and 5-years overall, disease-free and distant disease free survival. RESULTS: We observed expression of IKKε in 60.4% of the breast cancer tumors. IKKε expression status showed no association with a panel of markers used for molecular classification of the tumors, including ER/PR/HER2 status, or with the molecular subtypes. However, IKKε expression was inversely associated with lymph node metastasis status (p = 0.0032). Additionally, we identified a novel association between IKKε and EGFR expression (p = 0.0011). CONCLUSIONS: The unexpected observation of an inverse association between IKKε and lymph node metastasis advocates for larger scale immunohistochemical profiling of primary breast tumors to clarify the role of IKKε in metastasis. This study suggests that breast cancer tumors expressing EGFR and IKKε may be potential targets for drugs aiming at inhibiting IKKε activity or expression.

1st Workshop of the Canadian Society for Virology.

The 1st Workshop of the Canadian Society for Virology (CSV2016) was a Special Workshop of the 35th Annual Meeting for the American Society for Virology, held on 18 June 2016 on the beautiful Virginia Tech campus in Blacksburg, Virginia. The workshop provided a forum for discussion of recent advances in the field, in an informal setting conducive to interaction with colleagues. CSV2016 featured two internationally-renowned Canadian keynote speakers who discussed translational virology research; American Society for Virology President Grant McFadden (then from University of Florida, now relocated to Arizona State University) who presented his studies of oncolytic poxviruses, while Matthew Miller (McMaster University) reviewed the prospects for a universal influenza vaccine. The workshop also featured a variety of trainee oral and poster presentations, and a panel discussion on the topic of the future of the CSV and virus research in Canada.

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3.

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon ß and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Redox-modulating agents target NOX2-dependent IKKε oncogenic kinase expression and proliferation in human breast cancer cell lines.

Oxidative stress is considered a causative factor in carcinogenesis, but also in the development of resistance to current chemotherapies. The appropriate usage of redox-modulating compounds is limited by the lack of knowledge of their impact on specific molecular pathways. Increased levels of the IKKε kinase, as a result of gene amplification or aberrant expression, are observed in a substantial number of breast carcinomas. IKKε not only plays a key role in cell transformation and invasiveness, but also in the development of resistance to tamoxifen. Here, we studied the effect of in vitro treatment with the redox-modulating triphenylmethane dyes, Gentian Violet and Brilliant Green, and nitroxide Tempol on IKKε expression and cell proliferation in the human breast cancer epithelial cell lines exhibiting amplification of IKKε, MCF-7 and ZR75.1. We show that Gentian Violet, Brilliant Green and Tempol significantly decrease intracellular superoxide anion levels and inhibit IKKε expression and cell viability. Treatment with Gentian Violet and Brilliant Green was associated with a reduced cyclin D1 expression and activation of caspase 3 and/or 7. Tempol decreased cyclin D1 expression in both cell lines, while activation of caspase 7 was only observed in MCF-7 cells. Silencing of the superoxide-generating NOX2 NADPH oxidase expressed in breast cancer cells resulted in the significant reduction of IKKε expression. Taken together, our results suggest that redox-modulating compounds targeting NOX2 could present a particular therapeutic interest in combination therapy against breast carcinomas exhibiting IKKε amplification.

Lung epithelial NOX/DUOX and respiratory virus infections.

Determining the role of NADPH oxidases in the context of virus infection is an emerging area of research and our knowledge is still sparse. The expression of various isoforms of NOX/DUOX (NADPH oxidase/dual oxidase) in the epithelial cells (ECs) lining the respiratory tract renders them primary sites from which to orchestrate the host defence against respiratory viruses. Accumulating evidence reveals distinct facets of the involvement of NOX/DUOX in host antiviral and pro-inflammatory responses and in the control of the epithelial barrier integrity, with individual isoforms mediating co-operative, but surprisingly also opposing, functions. Although in vivo studies in mice are in line with some of these observations, a complete understanding of the specific functions of epithelial NOX/DUOX awaits lung epithelial-specific conditional knockout mice. The goal of the present review is to summarize our current knowledge of the role of individual NOX/DUOX isoforms expressed in the lung epithelium in the context of respiratory virus infections so as to highlight potential opportunities for therapeutic intervention.

Fludarabine downregulates indoleamine 2,3-dioxygenase in tumors via a proteasome-mediated degradation mechanism.

Indoleamine 2,3-dioxygenase (IDO) is found in multiple malignancies and exerts immunosuppressive effects that are central in protecting tumors from host T lymphocyte rejection. IDO is an enzyme involved in the catabolism of tryptophan resulting in inhibition of T lymphocyte function. While inhibition of IDO enzymatic activity results in tumor rejection, it is still unknown how we can directly target IDO expression within tumors using drugs. We have chosen to interfere with IDO expression by targeting the key-signaling event signal transducer and activator of transcription 1 (STAT1). We evaluated the efficacy of fludarabine, previously described to inhibit STAT1 phosphorylation. Interestingly, fludarabine was efficient in suppressing protein expression and consequently IDO activity in two different cell lines derived from breast cancer and melanoma when IDO was activated with interferon-gamma (IFN-γ) or supernatants prepared from activated T lymphocytes. However, fludarabine had no inhibitory effect on STAT1 phosphorylation. Other IFN-γ-responsive genes were only marginally inhibited by fludarabine. The level of IDO transcript was unaffected by this inhibitor, suggesting the involvement of post-transcriptional control. Strikingly, we have found that the inhibition of proteasome partially protected IDO from fludarabine-induced degradation, indicating that fludarabine induces IDO degradation through a proteasome-dependent pathway. Currently used in the clinic to treat some malignancies, fludarabine has the potential for use in the treatment of human tumors through induction of IDO degradation and consequently, for the promotion of T cell-mediated anti-tumor response.

Sustained activation of interferon regulatory factor 3 during infection by paramyxoviruses requires MDA5.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the main cytosolic sensors of single-stranded RNA viruses, including paramyxoviruses, and are required to initiate a quick and robust innate antiviral response. Despite different ligand-binding properties, the consensus view is that RIG-I and MDA5 trigger common signal(s) to activate interferon regulatory factor 3 (IRF-3) and NF-κB, and downstream antiviral and proinflammatory cytokine expression. Here, we performed a thorough analysis of the temporal involvement of RIG-I and MDA5 in the regulation of IRF-3 during respiratory syncytial virus (RSV) infection. Based on specific RNA interference-mediated knockdown of RIG-I and MDA5 in A549 cells, we confirmed that RIG-I is critical for the initiation of IRF-3 phosphorylation, dimerization and downstream gene expression. On the other hand, our experiments yielded the first evidence that knockdown of MDA5 leads to early ubiquitination and proteasomal degradation of active IRF-3. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Altogether, we provide novel mechanistic insight into the temporal involvement of RIG-I and MDA5 in the innate antiviral response. While RIG-I is essential for initial IRF-3 activation, engagement of induced MDA5 is essential to prevent early degradation of IRF-3, thereby sustaining IRF-3-dependent antiviral gene expression. MDA5 plays a similar role during Sendai virus infection suggesting that this model is not restricted to RSV amongst paramyxoviruses.

Genome-wide RNAi screen reveals a new role of a WNT/CTNNB1 signaling pathway as negative regulator of virus-induced innate immune responses.

To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-ß (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of ß-catenin (CTNNB1) upon virus infection negatively regulate expression of representative inducible genes IFNB1, IFIT1 and TNF in a CTNNB1-dependent effector mechanism. The antiviral response is drastically reduced by glycogen synthase kinase 3 (GSK3) inhibitors but restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of antiviral innate immunity by a canonical-like WNT/CTNNB1 signaling pathway. The study identifies novel avenues for broad-spectrum antiviral targets and preventing immune-mediated diseases upon viral infection.

IFNß/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH oxidase-mediated airway antiviral response.

Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNß secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNß and TNFα, and signals through a non-canonical STAT2- and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H2O2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction.

Individual interferon regulatory factor-3 thiol residues are not critical for its activation following virus infection.

The interferon regulatory factor (IRF)-3 transcription factor plays a central role in the capacity of the host to mount an efficient innate antiviral immune defense, mainly through the regulation of type I Interferon genes. A tight regulation of IRF-3 is crucial for an adapted intensity and duration of the response. Redox-dependent processes are now well known to regulate signaling cascades. Recent reports have revealed that signaling molecules upstream of IRF-3, including the mitochondrial antiviral-signalling protein (MAVS) and the TNF receptor associated factors (TRAFs) adaptors, are sensitive to redox regulation. In the present study, we assessed whether redox regulation of thiol residues contained in IRF-3, which are priviledged redox sensors, play a role in its regulation following Sendai virus infection, using a combination of mutation of Cysteine (Cys) residues into Alanine and thiols alkylation using N-ethyl maleimide. Alkylation of IRF-3 on Cys289 appears to destabilize IRF-3 dimer in vitro. However, a detailed analysis of IRF-3 phosphorylation, dimerization, nuclear accumulation, and induction of target gene promoter in vivo led us to conclude that IRF-3 specific, individual Cys residues redox status does not play an essential role in its activation in vivo.