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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.

We isolated infectious SARS-CoV-2 from aerosol samples collected from hospital rooms of COVID19 patients. Isolated virus successfully replicated in cell cultures 14 months after collection, opening up prospects for retrospective analyses of samples stored during the previous waves of COVID-19.

In March 2018, the Canadian Association for HIV Research (CAHR) and Canadian Foundation for Infectious Diseases (CFID) collaborated to conduct a workshop targeted to mid-career virology researchers. Key objectives of the workshop included 1) sharing knowledge and expertise cutting across various viral diseases, 2) developing collaborations as we anticipate the next wave of suppressive and curative treatment for HIV, HBV, CMV, and other viral diseases, and 3) providing insights, advice, and "food for thought" as participants advance to mid- and later phases of their research careers. This article reports on the key topics contemplated including scientific misinformation within the public realm, network building, interdisciplinary collaboration, mentorship, and communicating with decision makers. Given the focus on virology, the Canadian Society for Virology was invited to highlight their efforts to build a cohesive network that is impactful in facilitating viral research in Canada including advocating for appropriate levels of peer-reviewed research funding. Many key pearls of wisdom are contained within this document which are of value to all researchers aiming for success in a continually evolving, complex, and challenging Canadian research and academic environment.En mars 2018, l’Association canadienne de recherche sur le VIH (ACRV) et la Fondation canadienne des maladies infectieuses (FCMI) ont collaboré à la tenue d’un atelier pour les chercheurs en virologie en mi-carrière. Les principaux objectifs de l’atelier s’établissaient comme suit : 1) mettre en commun les connaissances et les compétences qui recoupent diverses maladies virales, 2) établir des collaborations en prévision de la prochaine vague de traitements suppressifs et curatifs du VIH, du VHB, du CMV et d’autres maladies virales et 3) fournir des points de vue, des conseils et des éléments de réflexion aux participants qui évoluent vers le milieu et les phases suivantes de leur carrière en recherche. Le présent article rend compte des principaux sujets abordés, y compris l’information scientifique erronée qui circule dans la population, l’établissement de réseaux, les collaborations interdisciplinaires, le mentorat et la communication avec les décideurs. Puisque l’atelier portait sur la virologie, les organisateurs ont invité la Société canadienne pour la virologie à mettre en lumière leurs efforts pour mettre sur pied un réseau homogène qui contribue avec efficacité à faciliter la recherche en virologie au Canada, y compris la défense d’un financement approprié de la recherche révisée par des pairs. Le présent article contient de sages et nombreux conseils pour tous les chercheurs qui veulent réussir dans le milieu complexe, difficile et en constante évolution de la recherche et de la science universitaire au Canada.

3D-printed alternatives to standard flocked swabs were rapidly developed to provide a response to the unprecedented and sudden need for an exponentially growing amount of diagnostic tools to fight the COVID-19 pandemic. In light of the anticipated shortage, a hospital-based 3D-printing platform was implemented in our institution for the production of swabs for nasopharyngeal and oropharyngeal sampling based on the freely available, open-source design provided to the community by University of South Florida’s Health Radiology and Northwell Health System teams as a replacement for locally used commercial swabs. Validation of our 3D-printed swabs was performed with a head-to-head diagnostic accuracy study of the 3D-printed “Northwell model” with the cobas PCR Media® swab sample kit. We observed an excellent concordance (total agreement 96.8%, Kappa 0.936) in results obtained with the 3D-printed and flocked swabs, indicating that the in-house 3D-printed swab could be used reliably in the context of a shortage of flocked swabs. To our knowledge, this is the first study to report on autonomous hospital-based production and clinical validation of 3D-printed swabs.

3D printed alternatives to standard flocked swabs were rapidly developed to provide a response to the unprecedented and sudden need for an exponentially growing amounts of diagnostic tools to fight the pandemics of COVID-19. In light of the anticipated shortage, an hospital-based 3D printing platform was implemented in our institution for the production of swabs for nasopharyngeal and oropharyngeal sampling based on the freely available open-sourced design made available to the community by University of South Florida’s Health Radiology and Northwell Health System teams as replacement for locally used commercial swabs. Validation of our 3D printed swabs was performed by a head-to-head diagnostic accuracy study of the 3D printed “Northwell model” with the cobas PCR Media swabs sample kit. We observed an excellent concordance (total agreement 96.8%, Kappa 0.936) in results obtained with the 3D printed and flocked swabs indicating that the in-house 3D printed swab can be used reliably in a context of shortage of flocked swabs. To our knowledge, this is the first study to report on autonomous hospital-based production and clinical validation of 3D printed swabs.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution is expected, given the nature of virus replication. Selection and establishment of variants in the human population depend on viral fitness and on molecular and immunological selection pressures. Here we discuss how mechanisms of replication and recombination may contribute to the emergence of current and future variants of SARS-CoV-2.

Influenza and RSV are human viruses responsible for outbreaks in hospitals, long-term care facilities and nursing homes. The present study assessed an air treatment using ozone at two relative humidity conditions (RHs) in order to reduce the infectivity of airborne influenza. Bovine pulmonary surfactant (BPS) and synthetic tracheal mucus (STM) were used as aerosols protectants to better reflect the human aerosol composition. Residual ozone concentration inside the aerosol chamber was also measured. RSV’s sensitivity resulted in testing its resistance to aerosolization and sampling processes instead of ozone exposure. The results showed that without supplement and with STM, a reduction in influenza A infectivity of four orders of magnitude was obtained with an exposure to 1.70 ± 0.19 ppm of ozone at 76% RH for 80 min. Consequently, ozone could be considered as a virucidal disinfectant for airborne influenza A. RSV did not withstand the aerosolization and sampling processes required for the use of the experimental setup. Therefore, ozone exposure could not be performed for this virus. Nonetheless, this study provides great insight for the efficacy of ozone as an air treatment for the control of nosocomial influenza A outbreaks.

SARS-CoV-2 infection causing the novel coronavirus disease 2019 (COVID–19) has been responsible for more than 2.8 million deaths and nearly 125 million infections worldwide as of March 2021. In March 2020, the World Health Organization determined that the COVID–19 outbreak is a global pandemic. The urgency and magnitude of this pandemic demanded immediate action and coordination between local, regional, national, and international actors. In that mission, researchers require access to high-quality biological materials and data from SARS-CoV-2 infected and uninfected patients, covering the spectrum of disease manifestations. The “Biobanque québécoise de la COVID-19” (BQC19) is a pan–provincial initiative undertaken in Québec, Canada to enable the collection, storage and sharing of samples and data related to the COVID-19 crisis. As a disease-oriented biobank based on high-quality biosamples and clinical data of hospitalized and non-hospitalized SARS-CoV-2 PCR positive and negative individuals. The BQC19 follows a legal and ethical management framework approved by local health authorities. The biosamples include plasma, serum, peripheral blood mononuclear cells and DNA and RNA isolated from whole blood. In addition to the clinical variables, BQC19 will provide in-depth analytical data derived from the biosamples including whole genome and transcriptome sequencing, proteome and metabolome analyses, multiplex measurements of key circulating markers as well as anti-SARS-CoV-2 antibody responses. BQC19 will provide the scientific and medical communities access to data and samples to better understand, manage and ultimately limit, the impact of COVID-19. In this paper we present BQC19, describe the process according to which it is governed and organized, and address opportunities for future research collaborations. BQC19 aims to be a part of a global communal effort addressing the challenges of COVID–19.

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., 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-β 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.

Protein function is regulated by post-translational modifications, among which reversible oxidation of Cys (Cys ox-PTM) emerged as a key regulatory mechanism of cellular responses. The redox regulation of virus-host interactions is well documented, but in most cases, proteins subjected to Cys ox-PTM remain unknown. The identification of Cys ox-PTM sites in vivo is essential to underpin our understanding of the mechanisms of the redox regulation. In this study, we present a proteome-wide identification of reversible Cys ox-PTM sites in vivo during stimulation by oxidants using a maleimide-based bioswitch method coupled to mass spectrometry. We identified 2720 unique Cys ox-PTM sites encompassing 1473 proteins with distinct abundance, location and functions. Label-free quantification (LFQ)-based analysis revealed the enrichment of ox-PTM in numerous pathways, many relevant to virus-host interaction. Here, we focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway induced upon detection of cytosolic DNA. We provide the first in vivo demonstration of reversible oxidation of Cys148 and Cys206 of STING. Molecular analyses led us to establish a new model in which Cys148 oxidation is constitutive, while Cys206 oxidation is inducible by oxidative stress or by the natural ligand 2’3’-cGAMP. We show that oxidation of Cys206 has an inhibitory function to prevent STING hyperactivation through the constraint of a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This provides new ground for the design of therapies targeting STING relevant to autoinflammatory disorders, immunotherapies and vaccines.Brief summary of the main resultsThe function of proteins is regulated by post-translational modifications, among which reversible oxidation of Cys recently emerged as a key component. Comprehension of redox regulation of cellular responses requires identification of specific oxidation sites in vivo. Using a bioswitch method to specifically label Cys subjected to reversible oxidation coupled to mass spectrometry, we identified thousands of novel oxidation sites. Many are relevant to virus-host interaction pathways. Here, we focused on the oxidation of STING, an adaptor critical for activating the innate immune type I interferon pathway engaged upon cytosolic DNA sensing. Molecular studies led us to establish a new model in which STING Cys148 is oxidized at basal levels, while Cys206 oxidation is induced by oxidative stress and ligand binding. We show that oxidation of Cys206 has an inhibitory function to prevent STING hyperactivation. This study provides ground for novel research avenues aimed at designing therapeutics that target this pathway.

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1(G93A) rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1(G93A) rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor–associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.

Influenza A virus (IAV) increases presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947 (H1N1) IAV significantly increased 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. We found that HLA upregulation was elicited by aberrant internally-deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs), which 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 signalling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-β and IFN-λ1, and conditioned media from these cells elicited a modest increase in HLA surface levels in naïve epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by chemical blockade of IFN receptor signal transduction. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral non-structural 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.IMPORTANCEHuman leukocyte antigens (HLA) 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 RIG-I/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 non-structural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase1(SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1G93Arat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93Arats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as with a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) as a SOD1 interactor and determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6-interacting from TRAF6 non-interacting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former, but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.

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., Highlights RNA-based agonists targeting RIG-I or MDA5 are at the center of an ongoing hunt for novel pan-antivirals, vaccine adjuvants, and antitumor strategies. Although length, structural signature, and nucleotide content determine binding to RIG-I and MDA5, features accounting for specificity between the two receptors are still unknown. Recent research has unveiled divergences in the functions mediated by RIG-I and MDA5; additional knowledge will be necessary to make an informed choice of the most appropriate ligand-based strategy for specific therapeutic or prophylactic clinical applications. RIG-I and MDA5 polymorphisms altering ligand recognition result in a spontaneous interferon signature associated with inflammatory and autoimmune diseases. Polymorphisms will certainly shape the response of patients to RLR-targeting strategies.

IFNβ typically induces an antiviral and immunoregulatory transcriptional program through the activation of ISGF3 (STAT1, STAT2 and IRF9) transcriptional complexes. The response to IFNβ is context-dependent and is prone to crosstalk with other cytokines, such as TNFα IFNβ and TNFα synergize to drive a specific delayed transcriptional program. Previous observation led to the hypothesis that an alternative STAT1-independent pathway involving STAT2 and IRF9 might be involved in gene induction by the combination of IFNβ and TNFα. Using genome wide transcriptional profiling by RNASeq, we found that the costimulation with IFNβ and TNFα induces a broad antiviral and immunoregulatory transcriptional program independently of STAT1. Additionally, STAT2 and IRF9 are involved in the regulation of only a subset of these STAT1-independent genes. Consistent with the growing literature, STAT2 and IRF9 act in concert to regulate a subgroup of these genes. Unexpectedly, STAT2 and IRF9 were also engaged in specific independent pathways to regulate distinct sets of IFNβ and TNFα-induced genes. Altogether these observations highlight the existence of distinct previously unrecognized non-canonical STAT1-independent, but STAT2 and/or IRF9-dependent pathways in the establishment of a delayed antiviral and immunoregulatory transcriptional program in conditions where elevated levels of both IFNβ and TNFα are present.

Significance Cytokines interact with their receptors and activate JAK–STAT signaling pathways that lead to changes in gene expression. In mammals, there are seven STATs that have arisen due to gene duplication and genetic drift. STATs have similar DNA binding specificity, and how individual STATs have subfunctionalized to regulate very specific cytokine responses in cells is poorly understood. Here we describe X-ray structures that show how one STAT family member, STAT2, specifically pairs with a member of the IRF family of transcription factors, IRF9. Despite overall structural similarity among STAT and IRF family members, surface features in the interacting domains of IRF9 and STAT2 have diverged to enable specific interaction between these family members and to enable the antiviral response., Cytokine signaling through the JAK/STAT pathway controls multiple cellular responses including growth, survival, differentiation, and pathogen resistance. An expansion in the gene regulatory repertoire controlled by JAK/STAT signaling occurs through the interaction of STATs with IRF transcription factors to form ISGF3, a complex that contains STAT1, STAT2, and IRF9 and regulates expression of IFN-stimulated genes. ISGF3 function depends on selective interaction between IRF9, through its IRF-association domain (IAD), with the coiled-coil domain (CCD) of STAT2. Here, we report the crystal structures of the IRF9–IAD alone and in a complex with STAT2–CCD. Despite similarity in the overall structure among respective paralogs, the surface features of the IRF9–IAD and STAT2–CCD have diverged to enable specific interaction between these family members. We derive a model for the ISGF3 complex bound to an ISRE DNA element and demonstrate that the observed interface between STAT2 and IRF9 is required for ISGF3 function in cells.

The host antiviral response involves the induction of interferons and proinflammatory cytokines, but also the activation of cell death pathways, including apoptosis, to limit viral replication and spreading. This host defense is strictly regulated to eliminate the infection while limiting tissue damage that is associated with virus pathogenesis. Post-translational modifications, most notably phosphorylation, are key regulators of the antiviral defense implying an important role of protein phosphatases. Here, we investigated the role of the dual-specificity phosphatase 1 (DUSP1) in the host defense against human respiratory syncytial virus (RSV), a pathogenic virus of the Pneumoviridae family, and Sendai virus (SeV), a model virus being developed as a vector for anti-RSV vaccine. We found that DUSP1 is upregulated before being subjected to proteasomal degradation. DUSP1 does not inhibit the antiviral response, but negatively regulates virus-induced JNK/p38 MAPK phosphorylation. Interaction with the JNK-interacting protein 1 scaffold protein prevents dephosphorylation of JNK by DUSP1, likely explaining that AP-1 activation and downstream cytokine production are protected from DUSP1 inhibition. Importantly, DUSP1 promotes SeV-induced apoptosis and suppresses cell migration in RSV-infected cells. Collectively, our data unveils a previously unrecognized selective role of DUSP1 in the regulation of tissue damage and repair during infections by RSV and SeV.

Gut-associated lymphoid tissues are enriched in CCR6+ Th17-polarized CD4+ T cells that contribute to HIV-1 persistence during antiretroviral therapy (ART). This raises the need for Th17-targeted immunotherapies. In an effort to identify mechanisms governing HIV-1 permissiveness/persistence in gut-homing Th17 cells, we analyzed the transcriptome of CCR6+ versus CCR6– T cells exposed to the gut-homing inducer retinoic acid (RA) and performed functional validations in colon biopsies of HIV-infected individuals receiving ART (HIV+ART). Although both CCR6+ and CCR6– T cells acquired gut-homing markers upon RA exposure, the modulation of unique sets of genes coincided with preferential HIV-1 replication in RA-treated CCR6+ T cells. This molecular signature included the upregulation of HIV-dependency factors acting at entry/postentry levels, such as the CCR5 and PI3K/Akt/mTORC1 signaling pathways. Of note, mTOR expression/phosphorylation was distinctively induced by RA in CCR6+ T cells. Consistently, mTOR inhibitors counteracted the effect of RA on HIV replication in vitro and viral reactivation in CD4+ T cells from HIV+ART individuals via postentry mechanisms independent of CCR5. Finally, CCR6+ versus CCR6– T cells infiltrating the colons of HIV+ART individuals expressed unique molecular signatures, including higher levels of CCR5, integrin β7, and mTOR phosphorylation. Together, our results identify mTOR as a druggable key regulator of HIV permissiveness in gut-homing CCR6+ T cells.

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.

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 IFNβ 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 IFNβ 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.

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.

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.

STAT1 plays a central role in the immune system, in which it mediates signaling induced by different cytokines, including type I (α/β) and type II Interferon (IFN), and regulates expression of genes involved in many physiological processes, including antiviral and antibacterial defense, immune tolerance, suppression of cell proliferation, and induction of apoptosis. Dysregulation or mutation of STAT1 causes life threatening conditions resulting from inefficient defense against pathogens, autoimmunity or cancer development. Single nucleotide polymorphisms of STAT1 Cys residues are associated with chronic mucocutaneous candidiasis disease (CMCD), suggesting a role of Cys in the capacity of STAT1 to drive an appropriate anti-pathogen response. Regulation of STAT1 activity through different post-translational modifications is well-characterized. However, the role of Reactive Oxygen species (ROS) and Cys reversible oxidative post-translational modifications (ox-PTMs) on STAT1 function remained to be investigated. Using maleimide-derivative switch methods to label Cys ox-PTMs, we found that Diamide, a thiol-oxidizing agent, and the myeloperoxidase-generated HOCl induce ox-PTMs of STAT1 Cys in epithelial cells (A549), monocytes (THP-1) and ectopically expressed STAT1 in STAT1 deficient fibroblasts (U3A). Analysis of Cys/Ala (C/A) mutants of STAT1 in response to IFNγ and IFNβ revealed gain of function phenotype of mutations in two different functional domains of STAT1. Altogether, our data supports a model in which previously unrecognized ox-PTMs of STAT1 Cys residues are essential to dampen STAT1 activity. Current studies are aimed at further characterizing the specific Cys ox-PTMs and their consequences on STAT1-dependent transcriptional program induction and antiviral immune response.

Chronic infection and inflammation have been associated with progressive airway epithelial damage in patients with cystic fibrosis (CF). However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Our objective was to study the regulation of repair mechanisms by tumor necrosis factor-α (TNF-α), a major component of inflammation in CF, in a model of mechanical wounding, in two bronchial cell lines, non-CF NuLi and CF CuFi. We observed that TNF-α enhanced the NuLi and CuFi repair rates. Chronic exposure (24–48 h) to TNF-α augmented this stimulation as well as the migration rate during repair. The cellular mechanisms involved in this stimulation were then evaluated. First, we discerned that TNF-α induced metalloproteinase-9 release, epidermal growth factor (EGF) shedding, and subsequent EGF receptor transactivation. Second, TNF-α-induced stimulation of the NuLi and CuFi wound-closure rates was prevented by GM6001 (metalloproteinase inhibitor), EGF antibody (to titrate secreted EGF), and EGF receptor tyrosine kinase inhibitors. Furthermore, we recently reported a relationship between the EGF response and K+channel function, both controlling bronchial repair. We now show that TNF-α enhances KvLQT1 and KATPcurrents, while their inhibition abolishes TNF-α-induced repair stimulation. These results indicate that the effect of TNF-α is mediated, at least in part, through EGF receptor transactivation and K+channel stimulation. In contrast, cell proliferation during repair was slowed by TNF-α, suggesting that TNF-α could exert contrasting actions on repair mechanisms of CF airway epithelia. Finally, the stimulatory effect of TNF-α on airway wound repair was confirmed on primary airway epithelial cells, from non-CF and CF patients.

We recently provided evidence that the ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 (HSV-1 and -2) protect cells against tumor necrosis factor alpha- and Fas ligand-induced apoptosis by interacting with caspase 8. Double-stranded RNA (dsRNA) is a viral intermediate known to initiate innate antiviral responses. Poly(I · C), a synthetic analogue of viral dsRNA, rapidly triggers caspase 8 activation and apoptosis in HeLa cells. Here, we report that HeLa cells after HSV-1 and HSV-2 infection were quickly protected from apoptosis caused by either extracellular poly(I · C) combined with cycloheximide or transfected poly(I · C). Cells infected with the HSV-1 R1 deletion mutant ICP6Δ were killed by poly(I · C), indicating that HSV-1 R1 plays a key role in antiapoptotic responses to poly(I · C). Individually expressed HSV R1s counteracted caspase 8 activation by poly(I · C). In addition to their binding to caspase 8, HSV R1s also interacted constitutively with receptor-interacting protein 1 (RIP1) when expressed either individually or with other viral proteins during HSV infection. R1(1-834)-green fluorescent protein (GFP), an HSV-2 R1 deletion mutant protein devoid of antiapoptotic activity, did not interact with caspase 8 and RIP1, suggesting that these interactions are required for protection against poly(I · C). HSV-2 R1 inhibited the interaction between the Toll/interleukin-1 receptor domain-containing adaptor-inducing beta interferon (IFN-β) (TRIF) and RIP1, an interaction that is essential for apoptosis triggered by extracellular poly(I · C) plus cycloheximide or TRIF overexpression. TRIF silencing reduced poly(I · C)-triggered caspase 8 activation in mock- and ICP6Δ-infected cells, confirming that TRIF is involved in poly(I · C)-induced apoptosis. Thus, by interacting with caspase 8 and RIP1, HSV R1s impair the apoptotic host defense mechanism prompted by dsRNA.

La kinase IKKe, membre atypique de la famille des kinases IKK, a recemment ete reconnue comme etant un oncogene majeur surexprime dans une large proportion de cancers du sein. Outre son role dans la regulation du facteur de transcription NF-κB, dont l’action dans le developpement du cancer du sein est largement etablie, IKKe participe a la phosphorylation de divers substrats. L’identification de certains de ces substrats a permis de trouver des voies oncogeniques qui pourraient constituer des cibles therapeutiques. De plus, IKKe semble jouer un role dans la resistance au tamoxifene. Ces nouvelles donnees ouvrent des perspectives prometteuses en vue de la mise au point de therapies ciblant IKKe dans les cancers du sein.

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.

Detection of oxidative post-translational modifications (ox-PTMs) of Cys residues in mammalian signaling proteins in vivo is one of the current challenges in redox biology. Ox-PTM residues labeling using maleimide switch methods coupled to mass spectrometry (M/S) or immunoblot have rendered possible the identification of signaling proteins subjected to ox-PTM in vivo. However, the low sensitivity remains a major hurdle to the detection of the modified residue(s) for the low abundant less reactive proteins. In most cases, identification of the Cys that undergo oxidative modification is only achieved using recombinant proteins oxidized in vitro. Here, we report a modified method of ox-PTM protein labeling coupled to M/S that highly improved the enrichment of oxidized peptides and the identification of specific ox-PTM Cys residues in vivo. In cell lines treated with diamide, a thiol-oxidizing agent, or HOCl, we identified 2699 peptides, covering 1473 proteins, containing at least 1 ox-PTM Cys residue. Previously reported ox-PTM residues were confirmed in vivo, validating our approach. Signaling proteins, including kinases, phosphatases and transcription and translation factors are largely represented in proteins containing ox-PTM Cys. Altogether, the use of an improved method with higher sensitivity for the detection of ox-PTM Cys residues in vivo allowed us to deepen our understanding of redox signaling. Network analyses allowed us to highlight signaling pathways enriched in oxidized proteins related to innate immunity, antiviral response and inflammation.