RESUMO
Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.
Assuntos
Coronavirus Humano OC43 , Vesículas Extracelulares , Proteômica , Vírion , Vírion/metabolismo , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Coronavirus Humano OC43/fisiologia , Coronavirus Humano OC43/metabolismo , Proteômica/métodos , Proteoma/metabolismo , Proteoma/análise , Exossomos/metabolismo , Exossomos/virologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/metabolismo , Linhagem Celular , Interações Hospedeiro-PatógenoRESUMO
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.
Assuntos
Oxirredução , Espécies Reativas de Oxigênio , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo , Humanos , Estresse Oxidativo , Animais , ApoptoseAssuntos
COVID-19 , Humanos , SARS-CoV-2 , Estudos Retrospectivos , Aerossóis e Gotículas Respiratórios , Hospitais , Quartos de Pacientes , RNA ViralRESUMO
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.
Assuntos
Neutrófilos , Vírus , Humanos , Oxidases Duais/genética , Oxidases Duais/metabolismo , Células Epiteliais/metabolismo , Citocinas/metabolismo , Antivirais/farmacologia , Quimiocinas/metabolismoRESUMO
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.
Assuntos
Isomerases de Dissulfetos de Proteínas , Compostos de Sulfidrila , Isomerases de Dissulfetos de Proteínas/metabolismo , Dissulfetos/química , Tiorredoxinas/metabolismo , Proteômica , Oxirredução , Cisteína/metabolismoRESUMO
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.
Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/virologia , SARS-CoV-2 , Teste para COVID-19/instrumentação , Gerenciamento Clínico , Humanos , Nasofaringe/virologia , Reação em Cadeia da Polimerase/métodos , Impressão Tridimensional , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodosRESUMO
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.
Assuntos
COVID-19/virologia , SARS-CoV-2/genética , Genoma Viral , Humanos , Recombinação Genética , SARS-CoV-2/fisiologia , Replicação ViralRESUMO
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.
Assuntos
Vírus da Influenza A/efeitos dos fármacos , Ozônio/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Inativação de Vírus/efeitos dos fármacos , Aerossóis , Microbiologia do Ar , Infecção Hospitalar/prevenção & controle , Desinfecção/métodos , Humanos , Influenza Humana/prevenção & controle , Ozônio/administração & dosagem , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Vírus Respiratório Sincicial/prevenção & controleRESUMO
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.
Assuntos
Bancos de Espécimes Biológicos/organização & administração , COVID-19/patologia , COVID-19/epidemiologia , COVID-19/genética , COVID-19/metabolismo , Humanos , Disseminação de Informação/métodos , Pandemias , Quebeque/epidemiologia , SARS-CoV-2/isolamento & purificaçãoRESUMO
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.
Assuntos
Cisteína , Proteínas de Membrana/metabolismo , Proteômica , Cisteína/metabolismo , Humanos , Oxirredução , Processamento de Proteína Pós-Traducional , Proteoma/genética , Proteoma/metabolismoRESUMO
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.
Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Peptidil Dipeptidase A/fisiologia , Pneumonia Viral/virologia , Ligação Viral , Enzima de Conversão de Angiotensina 2 , Basigina/fisiologia , COVID-19 , Comorbidade , Infecções por Coronavirus/epidemiologia , Chaperona BiP do Retículo Endoplasmático , Regulação Enzimológica da Expressão Gênica , Heparitina Sulfato/fisiologia , Humanos , Hipertensão/epidemiologia , Hipertensão/fisiopatologia , Neuropilina-1/fisiologia , Oligopeptídeos/fisiologia , Especificidade de Órgãos , Pandemias , Pneumonia Viral/epidemiologia , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Virais , Sistema Renina-Angiotensina/fisiologia , Sistema Respiratório/enzimologia , SARS-CoV-2 , Ácidos Siálicos/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia , Internalização do VírusRESUMO
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.
Assuntos
Genes MHC Classe I/genética , Antígenos HLA/metabolismo , Vírus da Influenza A Subtipo H1N1/genética , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína DEAD-box 58/genética , Bases de Dados Genéticas , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata , Vírus da Influenza A/genética , Influenza Humana/genética , Células Matadoras Naturais/metabolismo , Pulmão/virologia , RNA Viral/genética , Transdução de Sinais , Ativação Transcricional , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genéticaRESUMO
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 SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93A 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.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Superóxido Dismutase-1/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Anticorpos/imunologia , Linhagem Celular , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , NF-kappa B/metabolismo , Agregados Proteicos , Dobramento de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Transgênicos , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/imunologia , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/genética , UbiquitinaçãoRESUMO
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.
Assuntos
Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Interferon beta/fisiologia , Fator de Transcrição STAT2/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Células A549 , HumanosRESUMO
The 2nd Symposium of the Canadian Society for Virology (CSV2018) was held in June 2018 in Halifax, Nova Scotia, Canada, as a featured event marking the 200th anniversary of Dalhousie University. CSV2018 attracted 175 attendees from across Canada and around the world, more than double the number that attended the first CSV symposium two years earlier. CSV2018 provided a forum to discuss a wide range of topics in virology including human, veterinary, plant, and microbial pathogens. Invited keynote speakers included David Kelvin (Dalhousie University and Shantou University Medical College) who provided a historical perspective on influenza on the 100th anniversary of the 1918 pandemic; Sylvain Moineau (Université Laval) who described CRISPR-Cas systems and anti-CRISPR proteins in warfare between bacteriophages and their host microbes; and Kate O'Brien (then from Johns Hopkins University, now relocated to the World Health Organization where she is Director of Immunization, Vaccines and Biologicals), who discussed the underlying viral etiology for pneumonia in the developing world, and the evidence for respiratory syncytial virus (RSV) as a primary cause. Reflecting a strong commitment of Canadian virologists to science communication, CSV2018 featured the launch of Halifax's first annual Soapbox Science event to enable public engagement with female scientists, and the live-taping of the 499th episode of the This Week in Virology (TWIV) podcast, hosted by Vincent Racaniello (Columbia University) and science writer Alan Dove. TWIV featured interviews of CSV co-founders Nathalie Grandvaux (Université de Montréal) and Craig McCormick (Dalhousie University), who discussed the origins and objectives of the new society; Ryan Noyce (University of Alberta), who discussed technical and ethical considerations of synthetic virology; and Kate O'Brien, who discussed vaccines and global health. Finally, because CSV seeks to provide a better future for the next generation of Canadian virologists, the symposium featured a large number of oral and poster presentations from trainees and closed with the awarding of presentation prizes to trainees, followed by a tour of the Halifax Citadel National Historic Site and an evening of entertainment at the historic Alexander Keith's Brewery.
Assuntos
Congressos como Assunto , Virologia , Distinções e Prêmios , Canadá , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Vacinas contra Influenza , Vírus Sinciciais RespiratóriosRESUMO
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.
Assuntos
Proteína DEAD-box 58/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Antivirais/farmacologia , Doenças Autoimunes/tratamento farmacológico , Proteína DEAD-box 58/química , Proteína DEAD-box 58/imunologia , Humanos , Helicase IFIH1 Induzida por Interferon/química , Helicase IFIH1 Induzida por Interferon/imunologia , Ligantes , Terapia de Alvo Molecular , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismoRESUMO
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.
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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.
Assuntos
Inflamação/imunologia , Interleucina-6/imunologia , Interleucinas/imunologia , Monócitos/imunologia , Humanos , Células THP-1RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Polímeros/metabolismo , Infecções por Respirovirus/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Eletroforese em Gel de Ágar/métodos , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Peso Molecular , Agregados Proteicos , Receptores Imunológicos , Vírus Sendai , Serina Proteases/genética , Serina Proteases/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
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.