RESUMEN

SARS-CoV-2, the causative agent of COVID-19, has been intensely studied in search of effective antiviral treatments. The immunosuppressant cyclosporine A (CsA) has been suggested to be a pan-coronavirus inhibitor, yet its underlying mechanism remained largely unknown. Here, we found that non-structural protein 1 (Nsp1) of SARS-CoV-2 usurped CsA-suppressed nuclear factor of activated T cells (NFAT) signaling to drive the expression of cellular DEAD-box helicase 5 (DDX5), which facilitates viral replication. Nsp1 interacted with calcineurin A (CnA) to displace the regulatory protein regulator of calcineurin 3 (RCAN3) of CnA for NFAT activation. The influence of NFAT activation on SARS-CoV-2 replication was also validated by using the Nsp1-deficient mutant virus. Calcineurin inhibitors, such as CsA and VIVIT, inhibited SARS-CoV-2 replication and exhibited synergistic antiviral effects when used in combination with nirmatrelvir. Our study delineated the molecular mechanism of CsA-mediated inhibition of SARS-CoV-2 replication and the anti-SARS-CoV-2 action of calcineurin inhibitors. IMPORTANCE: Cyclosporine A (CsA), commonly used to inhibit immune responses, is also known to have anti-SARS-CoV-2 activity, but its mode of action remains elusive. Here, we provide a model to explain how CsA antagonizes SARS-CoV-2 through three critical proteins: DDX5, NFAT1, and Nsp1. DDX5 is a cellular facilitator of SARS-CoV-2 replication, and NFAT1 controls the production of DDX5. Nsp1 is a viral protein absent from the mature viral particle and capable of activating the function of NFAT1 and DDX5. CsA and similar agents suppress Nsp1, NFAT1, and DDX5 to exert their anti-SARS-CoV-2 activity either alone or in combination with Paxlovid.

Asunto(s)

COVID-19 , SARS-CoV-2 , Transducción de Señal , Proteínas no Estructurales Virales , Humanos , Antivirales , Calcineurina/metabolismo , Inhibidores de la Calcineurina/farmacología , COVID-19/virología , Ciclosporina/farmacología , Factores de Transcripción NFATC/metabolismo , SARS-CoV-2/fisiología , Proteínas no Estructurales Virales/metabolismo

RESUMEN

Epstein-Barr virus (EBV) has developed effective strategies to evade host innate immune responses. Here we reported on mitigation of type I interferon (IFN) production by EBV deubiquitinase (DUB) BPLF1 through cGAS-STING and RIG-I-MAVS pathways. The two naturally occurring forms of BPLF1 exerted potent suppressive effect on cGAS-STING-, RIG-I- and TBK1-induced IFN production. The observed suppression was reversed when DUB domain of BPLF1 was rendered catalytically inactive. The DUB activity of BPLF1 also facilitated EBV infection by counteracting cGAS-STING- and TBK1-mediated antiviral defense. BPLF1 associated with STING to act as an effective DUB targeting its K63-, K48- and K27-linked ubiquitin moieties. BPLF1 also catalyzed removal of K63- and K48-linked ubiquitin chains on TBK1 kinase. The DUB activity of BPLF1 was required for its suppression of TBK1-induced IRF3 dimerization. Importantly, in cells stably carrying EBV genome that encodes a catalytically inactive BPLF1, the virus failed to suppress type I IFN production upon activation of cGAS and STING. This study demonstrated IFN antagonism of BPLF1 mediated through DUB-dependent deubiquitination of STING and TBK1 leading to suppression of cGAS-STING and RIG-I-MAVS signaling.

Asunto(s)

Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Humanos , Proteína 58 DEAD Box , Enzimas Desubicuitinizantes , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Inmunidad Innata , Nucleotidiltransferasas/metabolismo , Ubiquitina

RESUMEN

In February 2020, we highlighted the top nine important research questions on SARS-CoV-2 and COVID-19 concerning virus transmission, asymptomatic and presymptomatic virus shedding, diagnosis, treatment, vaccine development, origin of virus and viral pathogenesis. These and related questions are revisited at the end of 2021 to shed light on the roadmap of bringing an end to the pandemic.

RESUMEN

Some lytic proteins encoded by Epstein-Barr virus (EBV) suppress host interferon (IFN) signaling to facilitate viral replication. In this study, we sought to identify and characterize EBV proteins antagonizing IFN signaling. The induction of IFN-stimulated genes (ISGs) by IFN-ß was effectively suppressed by EBV. A functional screen was therefore performed to identify IFN-antagonizing proteins encoded by EBV. EBV tegument protein BGLF2 was identified as a potent suppressor of JAK-STAT signaling. This activity was found to be independent of its stimulatory effect on p38 and JNK pathways. Association of BGLF2 with STAT2 resulted in more pronounced K48-linked polyubiquitination and proteasomal degradation of the latter. Mechanistically, BGLF2 promoted the recruitment of SHP1 phosphatase to STAT1 to inhibit its tyrosine phosphorylation. In addition, BGLF2 associated with cullin 1 E3 ubiquitin ligase to facilitate its recruitment to STAT2. Consequently, BGLF2 suppressed ISG induction by IFN-ß. Furthermore, BGLF2 also suppressed type II and type III IFN signaling, although the suppressive effect on type II IFN response was milder. When pretreated with IFN-ß, host cells became less susceptible to primary infection of EBV. This phenotype was reversed when expression of BGLF2 was enforced. Finally, genetic disruption of BGLF2 in EBV led to more pronounced induction of ISGs. Our study unveils the roles of BGLF2 not only in the subversion of innate IFN response but also in lytic infection and reactivation of EBV. IMPORTANCE Epstein-Barr virus (EBV) is an oncogenic virus associated with the development of lymphoid and epithelial malignancies. EBV has to subvert interferon-mediated host antiviral response to replicate and cause diseases. It is therefore of great interest to identify and characterize interferon-antagonizing proteins produced by EBV. In this study, we perform a screen to search for EBV proteins that suppress the action of interferons. We further show that BGLF2 protein of EBV is particularly strong in this suppression. This is achieved by inhibiting two key proteins STAT1 and STAT2 that mediate the antiviral activity of interferons. BGLF2 recruits a host enzyme to remove the phosphate group from STAT1 thereby inactivating its activity. BGLF2 also redirects STAT2 for degradation. A recombinant virus in which BGLF2 gene has been disrupted can activate host interferon response more robustly. Our findings reveal a novel mechanism by which EBV BGLF2 protein suppresses interferon signaling.

Asunto(s)

Infecciones por Virus de Epstein-Barr/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Virales de Fusión/metabolismo , Herpesvirus Humano 4/metabolismo , Interacciones Huésped-Patógeno , Interferones/metabolismo , Sistema de Señalización de MAP Quinasas , Fosforilación , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Transducción de Señal , Proteínas Virales de Fusión/genética , Replicación Viral

RESUMEN

BACKGROUND: miR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in carcinogenesis. Given the presence of a promotor-associated CpG island for its host gene, EVL, we hypothesized that intronic miR-342-3p is a tumor suppressor co-regulated with host gene by promoter DNA methylation in B cell lymphoma. RESULTS: By bisulfite pyrosequencing-verified methylation-specific PCR (MSP), EVL/MIR342 methylation was detected in five (50%) lymphoma cell lines but not normal peripheral blood and tonsils. EVL/MIR342 methylation correlated with repression of both miR-342-3p and EVL in cell lines. In completely methylated SU-DHL-16 cells, 5-AzadC treatment resulted in promoter demethylation and re-expression of miR-342-3p and EVL. In 132 primary lymphoma samples, EVL/MIR342 was preferentially methylated in B cell lymphomas (N = 68; 68.7%) than T cell lymphoma (N = 8; 24.2%) by MSP (P < 0.0001). Moreover, EVL/MIR342 methylation was associated with lower miR-342-3p expression in 79 primary NHL (P = 0.0443). In SU-DHL-16 cells, the tumor suppressor function of miR-342-3p was demonstrated by the inhibition of cellular proliferation and increase of cell death upon over-expression of miR-342-3p. Mechanistically, overexpression of miR-342-3p resulted in a decrease of LC3-II, a biomarker of autophagy, which was pro-survival for SU-DHL-16. Pre-treatment with 3-methyladenine, an autophagy inhibitor, abrogated tumor suppression associated with miR-342-3p overexpression. By luciferase assay, MAP1LC3B, a precursor of LC3-II, was confirmed as a direct target of miR-342-3p. Finally, in SU-DHL-16 cells, overexpression of miR-342-3p downregulated the known target DNMT1, with promoter demethylation and re-expression of tumor suppressor E-cadherin. CONCLUSIONS: Intronic miR-342-3p is co-regulated with its host gene EVL by tumor-specific promoter DNA methylation in B cell lymphoma. The tumor suppressor function of miR-342-3p was mediated via inhibition of pro-survival autophagy by targeting MAP1LC3B and downregulation of DNMT1 with demethylation and re-expression of tumor suppressor genes.

Asunto(s)

Metilación de ADN/genética , Epigénesis Genética/genética , Linfoma de Células B/genética , MicroARNs/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Islas de CpG/genética , ADN (Citosina-5-)-Metiltransferasa 1/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1/genética , Decitabina/farmacología , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Femenino , Silenciador del Gen , Genes Supresores de Tumor/efectos de los fármacos , Humanos , Linfoma de Células B/tratamiento farmacológico , Masculino , Proteínas Asociadas a Microtúbulos , Regiones Promotoras Genéticas/efectos de los fármacos

RESUMEN

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human coronavirus causing severe disease and mortality. MERS-CoV infection failed to elicit robust IFN response, suggesting that the virus might have evolved strategies to evade host innate immune surveillance. In this study, we identified and characterized type I IFN antagonism of MERS-CoV open reading frame (ORF) 8b accessory protein. ORF8b was abundantly expressed in MERS-CoV-infected Huh-7 cells. When ectopically expressed, ORF8b inhibited IRF3-mediated IFN-ß expression induced by Sendai virus and poly(I:C). ORF8b was found to act at a step upstream of IRF3 to impede the interaction between IRF3 kinase IKKε and chaperone protein HSP70, which is required for the activation of IKKε and IRF3. An infection study using recombinant wild-type and ORF8b-deficient MERS-CoV further confirmed the suppressive role of ORF8b in type I IFN induction and its disruption of the colocalization of HSP70 with IKKε. Ectopic expression of HSP70 relieved suppression of IFN-ß expression by ORF8b in an IKKε-dependent manner. Enhancement of IFN-ß induction in cells infected with ORF8b-deficient virus was erased when HSP70 was depleted. Taken together, HSP70 chaperone is important for IKKε activation, and MERS-CoV ORF8b suppresses type I IFN expression by competing with IKKε for interaction with HSP70.

Asunto(s)

Activación Enzimática/inmunología , Quinasa I-kappa B/inmunología , Interferón Tipo I/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Proteínas Virales/inmunología , Betacoronavirus , COVID-19 , Línea Celular , Infecciones por Coronavirus , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Quinasa I-kappa B/metabolismo , Interferón Tipo I/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Pandemias , Neumonía Viral , SARS-CoV-2 , Proteínas Virales/metabolismo

RESUMEN

Mutation and adaptation have driven the co-evolution of coronaviruses (CoVs) and their hosts, including human beings, for thousands of years. Before 2003, two human CoVs (HCoVs) were known to cause mild illness, such as common cold. The outbreaks of severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) have flipped the coin to reveal how devastating and life-threatening an HCoV infection could be. The emergence of SARS-CoV-2 in central China at the end of 2019 has thrusted CoVs into the spotlight again and surprised us with its high transmissibility but reduced pathogenicity compared to its sister SARS-CoV. HCoV infection is a zoonosis and understanding the zoonotic origins of HCoVs would serve us well. Most HCoVs originated from bats where they are non-pathogenic. The intermediate reservoir hosts of some HCoVs are also known. Identifying the animal hosts has direct implications in the prevention of human diseases. Investigating CoV-host interactions in animals might also derive important insight on CoV pathogenesis in humans. In this review, we present an overview of the existing knowledge about the seven HCoVs, with a focus on the history of their discovery as well as their zoonotic origins and interspecies transmission. Importantly, we compare and contrast the different HCoVs from a perspective of virus evolution and genome recombination. The current CoV disease 2019 (COVID-19) epidemic is discussed in this context. In addition, the requirements for successful host switches and the implications of virus evolution on disease severity are also highlighted.

Asunto(s)

Betacoronavirus/aislamiento & purificación , Quirópteros/virología , Coronavirus/clasificación , Evolución Molecular , Zoonosis/transmisión , Zoonosis/virología , Animales , COVID-19 , China , Coronavirus/aislamiento & purificación , Infecciones por Coronavirus , Reservorios de Enfermedades/veterinaria , Reservorios de Enfermedades/virología , Interacciones Huésped-Patógeno , Humanos , Pandemias , Neumonía Viral , Roedores/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , SARS-CoV-2 , Síndrome Respiratorio Agudo Grave

RESUMEN

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an ongoing global health emergency. Here we highlight nine most important research questions concerning virus transmission, asymptomatic and presymptomatic virus shedding, diagnosis, treatment, vaccine development, origin of virus and viral pathogenesis.

RESUMEN

World Health Organization has declared the ongoing outbreak of coronavirus disease 2019 (COVID-19) a Public Health Emergency of International Concern. The virus was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses. Human infection with SARS-CoV-2 leads to a wide range of clinical manifestations ranging from asymptomatic, mild, moderate to severe. The severe cases present with pneumonia, which can progress to acute respiratory distress syndrome. The outbreak provides an opportunity for real-time tracking of an animal coronavirus that has just crossed species barrier to infect humans. The outcome of SARS-CoV-2 infection is largely determined by virus-host interaction. Here, we review the discovery, zoonotic origin, animal hosts, transmissibility and pathogenicity of SARS-CoV-2 in relation to its interplay with host antiviral defense. A comparison with SARS-CoV, Middle East respiratory syndrome coronavirus, community-acquired human coronaviruses and other pathogenic viruses including human immunodeficiency viruses is made. We summarize current understanding of the induction of a proinflammatory cytokine storm by other highly pathogenic human coronaviruses, their adaptation to humans and their usurpation of the cell death programmes. Important questions concerning the interaction between SARS-CoV-2 and host antiviral defence, including asymptomatic and presymptomatic virus shedding, are also discussed.

Asunto(s)

Betacoronavirus/fisiología , Infecciones por Coronavirus/virología , Interacciones Huésped-Patógeno , Neumonía Viral/virología , Animales , Betacoronavirus/aislamiento & purificación , COVID-19 , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/transmisión , Vectores de Enfermedades , Humanos , Neumonía Viral/epidemiología , Neumonía Viral/prevención & control , Neumonía Viral/transmisión , SARS-CoV-2

RESUMEN

Epstein-Barr virus (EBV) successfully persists in the vast majority of adults but causes lymphoid and epithelial malignancies in a small fraction of latently infected individuals. Innate immunity is the first-line antiviral defense, which EBV has to evade in favor of its own replication and infection. EBV uses multiple strategies to perturb innate immune signaling pathways activated by Toll-like, RIG-I-like, NOD-like, and AIM2-like receptors as well as cyclic GMP-AMP synthase. EBV also counteracts interferon production and signaling, including TBK1-IRF3 and JAK-STAT pathways. However, activation of innate immunity also triggers pro-inflammatory response and proteolytic cleavage of caspases, both of which exhibit proviral activity under some circumstances. Pathogenic inflammation also contributes to EBV oncogenesis. EBV activates NFκB signaling and induces pro-inflammatory cytokines. Through differential modulation of the proviral and antiviral roles of caspases and other host factors at different stages of infection, EBV usurps cellular programs for death and inflammation to its own benefits. The outcome of EBV infection is governed by a delicate interplay between innate immunity and EBV. A better understanding of this interplay will instruct prevention and intervention of EBV-associated cancers.

RESUMEN

Severe acute respiratory syndrome coronavirus (SARS-CoV) is capable of inducing a storm of proinflammatory cytokines. In this study, we show that the SARS-CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). SARS-CoV and its ORF3a protein were found to be potent activators of pro-IL-1ß gene transcription and protein maturation, the 2 signals required for activation of the NLRP3 inflammasome. ORF3a induced pro-IL-1ß transcription through activation of NF-κB, which was mediated by TRAF3-dependent ubiquitination and processing of p105. ORF3a-induced elevation of IL-1ß secretion was independent of its ion channel activity or absent in melanoma 2 but required NLRP3, ASC, and TRAF3. ORF3a interacted with TRAF3 and ASC, colocalized with them in discrete punctate structures in the cytoplasm, and facilitated ASC speck formation. TRAF3-dependent K63-linked ubiquitination of ASC was more pronounced in SARS-CoV-infected cells or when ORF3a was expressed. Taken together, our findings reveal a new mechanism by which SARS-CoV ORF3a protein activates NF-κB and the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of p105 and ASC.-Siu, K.-L., Yuen, K.-S., Castaño-Rodriguez, C., Ye, Z.-W., Yeung, M.-L., Fung, S.-Y., Yuan, S., Chan, C.-P., Yuen, K.-Y., Enjuanes, L., Jin, D.-Y. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.

Asunto(s)

Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ubiquitinación , Proteínas Estructurales Virales/metabolismo , Células A549 , Animales , Chlorocebus aethiops , Células HEK293 , Humanos , Inflamasomas/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Factor 3 Asociado a Receptor de TNF/metabolismo , Células Vero

RESUMEN

Mouse p202 is a disease locus for lupus and a dominant-negative inhibitor of AIM2 inflammasome activation. A human homolog of p202 has not been identified so far. Here, we report a novel transcript isoform of human IFI16-designated IFI16-ß, which has a domain architecture similar to that of mouse p202. Like p202, IFI16-ß contains two HIN domains, but lacks the pyrin domain. IFI16-ß is ubiquitously expressed in various human tissues and cells. Its mRNA levels are also elevated in leukocytes of patients with lupus, virus-infected cells, and cells treated with interferon-ß or phorbol ester. IFI16-ß co-localizes with AIM2 in the cytoplasm, whereas IFI16-α is predominantly found in the nucleus. IFI16-ß interacts with AIM2 to impede the formation of a functional AIM2-ASC complex. In addition, IFI16-ß sequesters cytoplasmic dsDNA and renders it unavailable for AIM2 sensing. Enforced expression of IFI16-ß inhibits the activation of AIM2 inflammasome, whereas knockdown of IFI16-ß augments interleukin-1ß secretion triggered by dsDNA but not dsRNA Thus, cytoplasm-localized IFI16-ß is functionally equivalent to mouse p202 that exerts an inhibitory effect on AIM2 inflammasome.

Asunto(s)

Proteínas de Unión al ADN/genética , Inflamasomas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Animales , Núcleo Celular/genética , ADN/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-1beta/genética , Ratones , Isoformas de Proteínas/genética , ARN Bicatenario/genética , ARN Mensajero/genética

RESUMEN

STING is a core adaptor in innate nucleic acid sensing in mammalian cells, on which different sensing pathways converge to induce type I interferon (IFN) production. Particularly, STING is activated by 2'3'-cGAMP, a cyclic dinucleotide containing mixed phosphodiester linkages and produced by cytoplasmic DNA sensor cGAS. Here, we reported on a novel transcript isoform of STING designated STING-ß that dominantly inhibits innate nucleic acid sensing. STING-ß without transmembrane domains was widely expressed at low levels in various human tissues and viral induction of STING-ß correlated inversely with IFN-ß production. The expression of STING-ß declined in patients with lupus, in which type I IFNs are commonly overproduced. STING-ß suppressed the induction of IFNs, IFN-stimulated genes and other cytokines by various immunostimulatory agents including cyclic dinucleotides, DNA, RNA and viruses, whereas depletion of STING-ß showed the opposite effect. STING-ß interacted with STING-α and antagonized its antiviral function. STING-ß also interacted with TBK1 and prevented it from binding with STING-α, TRIF or other transducers. In addition, STING-ß bound to 2'3'-cGAMP and impeded its binding with and activation of STING-α, leading to suppression of IFN-ß production. Taken together, STING-ß sequesters 2'3'-cGAMP second messenger and other transducer molecules to inhibit innate nucleic acid sensing dominantly.

Asunto(s)

Proteínas de la Membrana/metabolismo , Nucleótidos Cíclicos/metabolismo , Animales , Línea Celular , ADN/fisiología , Humanos , Factor 3 Regulador del Interferón/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , FN-kappa B/metabolismo , Fosforilación , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fenómenos Fisiológicos de los Virus

RESUMEN

Epstein-Barr virus (EBV) infects more than 90% of the world's adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%. The suppressive effect on EBV DNA load lasted for weeks but could not be further enhanced by re-transfection of gRNA. Suppression of EBV by CRISPR/Cas9 did not affect survival of C666-1 cells but sensitized them to chemotherapeutic killing by cisplatin and 5-fluorouracil. Our work provides the proof-of-principle for suppressing EBV DNA load with CRISPR/Cas9 and a potential new strategy to sensitize EBV-infected NPC cells to chemotherapy.

Asunto(s)

Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , ADN Viral/genética , Endonucleasas/genética , Edición Génica/métodos , Genoma Viral , Herpesvirus Humano 4/genética , ARN Guía de Kinetoplastida/genética , Antineoplásicos/farmacología , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Viral/metabolismo , Endonucleasas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Epiteliales/virología , Fluorouracilo/farmacología , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/crecimiento & desarrollo , Herpesvirus Humano 4/metabolismo , Humanos , Nasofaringe/efectos de los fármacos , Nasofaringe/patología , Nasofaringe/virología , Plásmidos/química , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Carga Viral/efectos de los fármacos , Latencia del Virus/genética , Replicación Viral

RESUMEN

Severe complications of Zika virus (ZIKV) infection might be caused by inflammation, but how ZIKV induces proinflammatory cytokines is not understood. In this study, we show opposite regulatory effects of the ZIKV NS5 protein on interferon (IFN) signaling. Whereas ZIKV and its NS5 protein were potent suppressors of type I and type III IFN signaling, they were found to activate type II IFN signaling. Inversely, IFN-γ augmented ZIKV replication. NS5 interacted with STAT2 and targeted it for ubiquitination and degradation, but it had no influence on STAT1 stability or nuclear translocation. The recruitment of STAT1-STAT2-IRF9 to IFN-ß-stimulated genes was compromised when NS5 was expressed. Concurrently, the formation of STAT1-STAT1 homodimers and their recruitment to IFN-γ-stimulated genes, such as the gene encoding the proinflammatory cytokine CXCL10, were augmented. Silencing the expression of an IFN-γ receptor subunit or treatment of ZIKV-infected cells with a JAK2 inhibitor suppressed viral replication and viral induction of IFN-γ-stimulated genes. Taken together, our findings provide a new mechanism by which the ZIKV NS5 protein differentially regulates IFN signaling to facilitate viral replication and cause diseases. This activity might be shared by a group of viral IFN modulators.IMPORTANCE Mammalian cells produce three types of interferons to combat viral infection and to control host immune responses. To replicate and cause diseases, pathogenic viruses have developed different strategies to defeat the action of host interferons. Many viral proteins, including the Zika virus (ZIKV) NS5 protein, are known to be able to suppress the antiviral property of type I and type III interferons. Here we further show that the ZIKV NS5 protein can also boost the activity of type II interferon to induce cellular proteins that promote inflammation. This is mediated by the differential effect of the ZIKV NS5 protein on a pair of cellular transcription factors, STAT1 and STAT2. NS5 induces the degradation of STAT2 but promotes the formation of STAT1-STAT1 protein complexes, which activate genes controlled by type II interferon. A drug that specifically inhibits the IFN-γ receptor or STAT1 shows an anti-ZIKV effect and might also have anti-inflammatory activity.

Asunto(s)

Interferón gamma/metabolismo , Proteínas no Estructurales Virales/inmunología , Virus Zika/inmunología , Línea Celular , Humanos , Unión Proteica , Factor de Transcripción STAT2/metabolismo , Transducción de Señal

RESUMEN

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein-Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.

Asunto(s)

Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Infecciones por Virus de Epstein-Barr/virología , Genoma Humano/genética , Herpesvirus Humano 4/genética , Mutagénesis/genética , Edición de ARN/genética , Línea Celular , Cromosomas Artificiales Bacterianos/genética , Virus ADN/genética , Ingeniería Genética/métodos , Células HEK293 , Humanos , Plásmidos/genética

RESUMEN

Middle East respiratory syndrome coronavirus (MERS-CoV) infection has claimed hundreds of lives and has become a global threat since its emergence in Saudi Arabia in 2012. The ability of MERS-CoV to evade the host innate antiviral response may contribute to its severe pathogenesis. Many MERS-CoV-encoded proteins were identified to have interferon (IFN)-antagonizing properties, which correlates well with the reduced IFN levels observed in infected patients and ex vivo models. In this study, we fully characterized the IFN-antagonizing property of the MERS-CoV M protein. Expression of MERS-CoV M protein suppressed type I IFN expression in response to Sendai virus infection or poly(I:C) induction. This suppressive effect was found to be specific for the activation of IFN regulatory factor 3 (IRF3) but not nuclear factor-κB. MERS-CoV M protein interacted with TRAF3 and disrupted TRAF3-TBK1 association leading to reduced IRF3 activation. M proteins from MERS-CoV and SARS-CoV have three highly similar conserved N-terminal transmembrane domains and a C-terminal region. Using chimeric and truncation mutants, the N-terminal transmembrane domains of the MERS-CoV M protein were found to be sufficient for its inhibitory effect on IFN expression, whereas the C-terminal domain was unable to induce this suppression. Collectively, our findings suggest a common and conserved mechanism through which highly pathogenic MERS-CoV and SARS-CoV harness their M proteins to suppress type I IFN expression at the level of TBK1-dependent phosphorylation and activation of IRF3 resulting in evasion of the host innate antiviral response.

Asunto(s)

Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/biosíntesis , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Fosfotransferasas , Proteínas Serina-Treonina Quinasas/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Proteínas de la Matriz Viral/fisiología , Proteínas M de Coronavirus , Proteína 58 DEAD Box/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Evasión Inmune , Inmunidad Innata , Factor 3 Regulador del Interferón/inmunología , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Arabia Saudita , Virus Sendai/genética , Virus Sendai/inmunología , Alineación de Secuencia , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/inmunología , Proteínas de la Matriz Viral/genética

RESUMEN

UNLABELLED: Infection with human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and tropical spastic paraparesis. Type I interferons (IFNs) are key effectors of the innate antiviral response, and IFN-α combined with the nucleoside reverse transcriptase inhibitor zidovudine is considered the standard first-line therapy for ATL. HTLV-1 oncoprotein Tax is known to suppress innate IFN production and response but the underlying mechanisms remain to be fully established. In this study, we report on the suppression of type I IFN production by HTLV-1 Tax through interaction with and inhibition of TBK1 kinase that phosphorylates IRF3. Induced transcription of IFN-ß was severely impaired in HTLV-1-transformed ATL cells and freshly infected T lymphocytes. The ability to suppress IRF3 activation was ascribed to Tax. The expression of Tax alone sufficiently repressed the induction of IFN production by RIG-I plus PACT, cGAMP synthase plus STING, TBK1, IKKε, IRF3, and IRF7, but not by IRF3-5D, a dominant-active phosphomimetic mutant. This suggests that Tax perturbs IFN production at the step of IRF3 phosphorylation. Tax mutants deficient for CREB or NF-κB activation were fully competent in the suppression of IFN production. Coimmunoprecipitation experiments confirmed the association of Tax with TBK1, IKKε, STING, and IRF3.In vitrokinase assay indicated an inhibitory effect of Tax on TBK1-mediated phosphorylation of IRF3. Taken together, our findings suggested a new mechanism by which HTLV-1 oncoprotein Tax circumvents the production of type I IFNs in infected cells. Our findings have implications in therapeutic intervention of ATL. IMPORTANCE: Human T-cell leukemia virus type 1 (HTLV-1) is the cause of adult T-cell leukemia (ATL), an aggressive and fatal blood cancer, as well as another chronic disabling disease of the spinal cord. Treatments are unsatisfactory, and options are limited. A combination of antiviral cellular protein alpha interferon and zidovudine, which is an inhibitor of a viral enzyme called reverse transcriptase, has been recommended as the standard first-line therapy for ATL. Exactly how HTLV-1 interacts with the cellular machinery for interferon production and action is not well understood. Our work sheds light on the mechanism of action for the inhibition of interferon production by an HTLV-1 oncogenic protein called Tax. Our findings might help to improve interferon-based anti-HTLV-1 and anti-ATL therapy.

Asunto(s)

Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Interferón beta/antagonistas & inhibidores , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Productos del Gen tax/genética , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/biosíntesis , Células Jurkat , Leucemia-Linfoma de Células T del Adulto/virología , FN-kappa B/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/metabolismo , Linfocitos T/virología

RESUMEN

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen causing significant morbidity and mortality in Asia. NSs protein of SFTSV is known to perturb type I IFN induction and signalling, but the mechanism remains to be fully understood. Here, we showed the suppression of both type I and type III IFN signalling by SFTSV NSs protein is mediated through inhibition of STAT1 phosphorylation and activation. Infection with live SFTSV or expression of NSs potently suppressed IFN-stimulated genes but not NFkB activation. NSs was capable of counteracting the activity of IFN-α1, IFN-ß, IFN-λ1 and IFN-λ2. Mechanistically, NSs associated with STAT1 and STAT2, mitigated IFN-ß-induced phosphorylation of STAT1 at S727, and reduced the expression and activity of STAT1 protein in IFN-ß-treated cells, resulting in the inhibition of STAT1 and STAT2 recruitment to IFNstimulated promoters. Taken together, SFTSV NSs protein is an IFN antagonist that suppresses phosphorylation and activation of STAT1.

Asunto(s)

Interferón-alfa/genética , Interferón beta/genética , Interleucinas/genética , Fiebre por Flebótomos/genética , Phlebovirus/metabolismo , Factor de Transcripción STAT1/metabolismo , Proteínas no Estructurales Virales/metabolismo , Humanos , Interferón-alfa/metabolismo , Interferón beta/metabolismo , Interferones , Interleucinas/metabolismo , Fiebre por Flebótomos/metabolismo , Fiebre por Flebótomos/virología , Phlebovirus/genética , Fosforilación , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismo , Transducción de Señal , Proteínas no Estructurales Virales/genética