RESUMO
The RNA-binding protein RIG-I is a key initiator of the antiviral innate immune response. The signaling that mediates the antiviral response downstream of RIG-I is transduced through the adaptor protein MAVS and results in the induction of type I and III interferons (IFNs). This signal transduction occurs at endoplasmic reticulum (ER)mitochondrial contact sites, to which RIG-I and other signaling proteins are recruited following their activation. RIG-I signaling is highly regulated to prevent aberrant activation of this pathway and dysregulated induction of IFN. Previously, we identified UFL1, the E3 ligase of the ubiquitin-like modifier conjugation system called ufmylation, as one of the proteins recruited to membranes at ERmitochondrial contact sites in response to RIG-I activation. Here, we show that UFL1, as well as the process of ufmylation, promote IFN induction in response to RIG-I activation. We found that following RNA virus infection, UFL1 is recruited to the membrane-targeting protein 143-3ε and that this complex is then recruited to activated RIG-I to promote downstream innate immune signaling. Importantly, we found that 143-3ε has an increase in UFM1 conjugation following RIG-I activation. Additionally, loss of cellular ufmylation prevents the interaction of 143-3ε with RIG-I, which abrogates the interaction of RIG-I with MAVS and thus the downstream signal transduction that induces IFN. Our results define ufmylation as an integral regulatory component of the RIG-I signaling pathway and as a posttranslational control for IFN induction.
Assuntos
Proteína DEAD-box 58 , Interferons , Infecções por Vírus de RNA , RNA Viral , Receptores Imunológicos , Ubiquitina-Proteína Ligases , Proteínas 14-3-3/metabolismo , Proteína DEAD-box 58/metabolismo , Humanos , Imunidade Inata , Interferons/metabolismo , Infecções por Vírus de RNA/genética , Infecções por Vírus de RNA/imunologia , RNA Viral/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Coronavirus protease nsp5 (Mpro, 3CLpro) remains a primary target for coronavirus therapeutics due to its indispensable and conserved role in the proteolytic processing of the viral replicase polyproteins. In this review, we discuss the diversity of known coronaviruses, the role of nsp5 in coronavirus biology, and the structure and function of this protease across the diversity of known coronaviruses, and evaluate past and present efforts to develop inhibitors to the nsp5 protease with a particular emphasis on new and mostly unexplored potential targets of inhibition. With the recent emergence of pandemic SARS-CoV-2, this review provides novel and potentially innovative strategies and directions to develop effective therapeutics against the coronavirus protease nsp5.
Assuntos
Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Inibidores de Protease Viral/uso terapêutico , Sequência de Aminoácidos , COVID-19/virologia , Coronavirus/enzimologia , Coronavirus/metabolismo , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/metabolismo , Humanos , Filogenia , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
Innate immune detection of viral nucleic acids during viral infection activates a signaling cascade that induces type I and type III IFNs as well as other cytokines, to generate an antiviral response. This signaling is initiated by pattern recognition receptors, such as the RNA helicase retinoic acid-inducible gene I (RIG-I), that sense viral RNA. These sensors then interact with the adaptor protein mitochondrial antiviral signaling protein (MAVS), which recruits additional signaling proteins, including TNF receptor-associated factor 3 (TRAF3) and TANK-binding kinase 1 (TBK1), to form a signaling complex that activates IFN regulatory factor 3 (IRF3) for transcriptional induction of type I IFNs. Here, using several immunological and biochemical approaches in multiple human cell types, we show that the GTPase-trafficking protein RAB1B up-regulates RIG-I pathway signaling and thereby promotes IFN-ß induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I-mediated signaling to IFN-ß and that RAB1B deletion reduces signaling of this pathway. Additionally, loss of RAB1B dampened the antiviral response, indicated by enhanced Zika virus infection of cells depleted of RAB1B. Importantly, we identified the mechanism of RAB1B action in the antiviral response, finding that it forms a protein complex with TRAF3 to facilitate the interaction of TRAF3 with mitochondrial antiviral signaling protein. We conclude that RAB1B regulates TRAF3 and promotes the formation of innate immune signaling complexes in response to nucleic acid sensing during RNA virus infection.
Assuntos
Imunidade Inata , Fator 3 Associado a Receptor de TNF/metabolismo , Infecção por Zika virus/imunologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Animais , Chlorocebus aethiops , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Interferon beta/metabolismo , Ligação Proteica , Receptores Imunológicos , Transdução de Sinais , Células VeroRESUMO
Upon infection, both DNA and RNA viruses can be sensed by pattern recognition receptors (PRRs) in the cytoplasm or the nucleus to activate antiviral innate immunity. Sensing of viral products leads to the activation of a signaling cascade that ultimately results in transcriptional activation of type I and III interferons, as well as other antiviral genes that together mediate viral clearance and inhibit viral spread. Therefore, in order for viruses to replicate and spread efficiently, they must inhibit the host signaling pathways that induce the innate antiviral immune response. In this review, we will highlight recent advances in the understanding of the mechanisms by which viruses evade PRR detection, intermediate signaling molecule activation, transcription factor activation, and the actions of antiviral proteins.
Assuntos
Vírus de DNA/imunologia , Evasão da Resposta Imune/fisiologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Vírus de RNA/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Proteína DEAD-box 58/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Receptores Imunológicos , Transdução de Sinais/imunologiaRESUMO
PURPOSE: Sarcoidosis is a non-caseating granulomatous disease for which a role for infectious antigens continues to strengthen. Recent studies have reported molecular evidence of mycobacteria or propionibacteria. We assessed for immune responses against mycobacterial and propionibacterial antigens in sarcoidosis bronchoalveolar lavage (BAL) using flow cytometry, and localized signals consistent with microbial antigens with sarcoidosis specimens, using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS). METHODS: BAL cells from 27 sarcoidosis, 14 PPD- controls, and 9 subjects with nontuberculosis mycobacterial (NTM) infections were analyzed for production of IFN-γ after stimulation with mycobacterial ESAT-6 and Propionibacterium acnes proteins. To complement the immunological data, MALDI-IMS was performed to localize ESAT-6 and Propionibacterium acnes signals within sarcoidosis and control specimens. RESULTS: CD4+ immunologic analysis for mycobacteria was positive in 17/27 sarcoidosis subjects, compared to 2/14 PPD- subjects, and 5/9 NTM subjects (p = 0.008 and p = 0.71 respectively, Fisher's exact test). There was no significant difference for recognition of P. acnes, which occurred only in sarcoidosis subjects that also recognized ESAT-6. Similar results were also observed for the CD8+ immunologic analysis. MALDI-IMS localized signals consistent with ESAT-6 only within sites of granulomatous inflammation, whereas P. acnes signals were distributed throughout the specimen. CONCLUSIONS: MALDI-IMS localizes signals consistent with ESAT-6 to sarcoidosis granulomas, whereas no specific localization of P. acnes signals is detected. Immune responses against both mycobacterial and P. acnes are present within sarcoidosis BAL, but only mycobacterial signals are distinct from disease controls. These immunologic and molecular investigations support further investigation of the microbial community within sarcoidosis granulomas.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Mycobacterium/imunologia , Propionibacterium acnes/imunologia , Sarcoidose/imunologia , Adulto , Idoso , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/imunologia , Enterotoxinas/farmacologia , Feminino , Humanos , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Infecções por Mycobacterium/imunologia , Peptídeos/imunologia , Sarcoidose/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto JovemRESUMO
INTRODUCTION: Sarcoidosis is a multisystem granulomatous disease for which the association with mycobacteria continues to strengthen. It is hypothesized that a single, poorly degradable antigen is responsible for sarcoidosis pathogenesis. Several reports from independent groups support mycobacterial antigens having a role in sarcoidosis pathogenesis. To identify other microbial targets of the adaptive immune response, we tested the ability of CD4+ and CD8+ T cells to recognize multiple mycobacterial antigens. METHODS: Fifty-four subjects were enrolled in this study: 31 sarcoidosis patients, nine non-tuberculosis mycobacterial (NTM) infection controls, and 14 PPD- controls. Using flow cytometry, we assessed for Th1 immune responses to ESAT-6, katG, Ag85A, sodA, and HSP. RESULTS: Alveolar T-cells from twenty-two of the 31 sarcoidosis patients produced a CD4+ response to at least one of ESAT-6, katG, Ag85A, sodA, or HSP, compared to two of 14 PPD- controls (p = 0.0008) and five of nine NTM controls (p = 0.44), while eighteen of the 31 sarcoidosis subjects tested produced a CD8+ response to at least one of the mycobacterial antigens compared to two of 14 PPD- controls (p = 0.009) and three of nine NTM controls (0.26). Not only did the BAL-derived T cells respond to multiple virulence factors, but also to multiple, distinct epitopes within a given protein. The detection of proliferation upon stimulation with the mycobacterial virulence factors demonstrates that these responses are initiated by antigen specific recognition. CONCLUSIONS: Together these results reveal that antigen-specific CD4+ and CD8+ T cells responses to multiple mycobacterial epitopes are present within sites of active sarcoidosis involvement, and that these antigen-specific responses are present at the time of diagnosis.