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1.
Nature ; 619(7969): 338-347, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37380775

RESUMEN

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.


Asunto(s)
Aves , Interacciones Microbiota-Huesped , Virus de la Influenza A , Gripe Aviar , Gripe Humana , Zoonosis Virales , Animales , Humanos , Aves/virología , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/transmisión , Gripe Aviar/virología , Gripe Humana/prevención & control , Gripe Humana/transmisión , Gripe Humana/virología , Primates , Sistema Respiratorio/metabolismo , Sistema Respiratorio/virología , Medición de Riesgo , Zoonosis Virales/prevención & control , Zoonosis Virales/transmisión , Zoonosis Virales/virología , Replicación Viral
2.
PLoS Biol ; 19(9): e3001352, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34491982

RESUMEN

Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host mRNAs that encode antiviral effectors. Here, we show that highly upregulated interferon-stimulated genes (ISGs), which encode antiviral proteins, have distinctive nucleotide compositions. We propose that self-targeting by antiviral effectors has selected for ISG transcripts that occupy a less self-targeted sequence space. Following interferon (IFN) stimulation, the CpG-targeting antiviral effector zinc-finger antiviral protein (ZAP) reduces the mRNA abundance of multiple host transcripts, providing a mechanistic explanation for the repression of many (but not all) interferon-repressed genes (IRGs). Notably, IRGs tend to be relatively CpG rich. In contrast, highly upregulated ISGs tend to be strongly CpG suppressed. Thus, ZAP is an example of an effector that has not only selected compositional biases in viral genomes but also appears to have notably shaped the composition of host transcripts in the vertebrate interferome.


Asunto(s)
Fosfatos de Dinucleósidos , Factores Reguladores del Interferón/genética , ARN Viral , Proteínas de Unión al ARN/metabolismo , Células A549 , Línea Celular , Humanos , Interferón beta/farmacología , ARN Mensajero , Proteínas de Unión al ARN/genética , Fenómenos Fisiológicos de los Virus , Virus
3.
PLoS Biol ; 19(2): e3001091, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33630831

RESUMEN

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.


Asunto(s)
Vacunas contra la COVID-19 , COVID-19/diagnóstico , COVID-19/virología , Genética Inversa , SARS-CoV-2/genética , Células A549 , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Chlorocebus aethiops , Codón , Humanos , Hidrazonas/farmacología , Ratones , Morfolinas/farmacología , Sistemas de Lectura Abierta , Plásmidos/genética , Pirimidinas/farmacología , Serina Endopeptidasas/metabolismo , Células Vero , Proteínas Virales/metabolismo
4.
Biochem J ; 474(7): 1163-1174, 2017 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-28159912

RESUMEN

The double-stranded RNA mimetic poly(I:C) and lipopolysaccharide (LPS) activate Toll-like receptors 3 (TLR3) and TLR4, respectively, triggering the activation of TANK (TRAF family member-associated NF-κB activator)-binding kinase 1 (TBK1) complexes, the phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of the interferon ß (IFNß) gene. Here, we demonstrate that the TANK-TBK1 and optineurin (OPTN)-TBK1 complexes control this pathway. The poly(I:C)- or LPS-stimulated phosphorylation of IRF3 at Ser396 and production of IFNß were greatly reduced in bone marrow-derived macrophages (BMDMs) from TANK knockout (KO) mice crossed to knockin mice expressing the ubiquitin-binding-defective OPTN[D477N] mutant. In contrast, IRF3 phosphorylation and IFNß production were not reduced significantly in BMDM from OPTN[D477N] knockin mice and only reduced partially in TANK KO BMDM. The TLR3/TLR4-dependent phosphorylation of IRF3 and IFNß gene transcription were not decreased in macrophages from OPTN[D477N] crossed to mice deficient in IκB kinase ε, a TANK-binding kinase related to TBK1. In contrast with the OPTN-TBK1 complex, TBK1 associated with OPTN[D477N] did not undergo phosphorylation at Ser172 in response to poly(I:C) or LPS, indicating that the interaction of ubiquitin chains with OPTN is required to activate OPTN-TBK1 in BMDM. The phosphorylation of IRF3 and IFNß production induced by Sendai virus infection were unimpaired in BMDM from TANK KO × OPTN[D477N] mice, suggesting that other/additional TBK1 complexes control the RIG-I-like receptor-dependent production of IFNß. Finally, we present evidence that, in human HACAT cells, the poly(I:C)-dependent phosphorylation of TBK1 at Ser172 involves a novel TBK1-activating kinase(s).


Asunto(s)
Factor 3 Regulador del Interferón/genética , Interferón beta/genética , Macrófagos/inmunología , Proteínas Serina-Treonina Quinasas/genética , Receptor Toll-Like 3/genética , Receptor Toll-Like 4/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Animales , Proteínas de Ciclo Celular , Línea Celular Transformada , Proteínas del Ojo/genética , Proteínas del Ojo/inmunología , Técnicas de Sustitución del Gen , Humanos , Quinasa I-kappa B/deficiencia , Quinasa I-kappa B/genética , Factor 3 Regulador del Interferón/inmunología , Interferón beta/inmunología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Lipopolisacáridos/farmacología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/inmunología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Proteínas de Transporte de Membrana , Ratones , Ratones Noqueados , Fosforilación , Poli I-C/farmacología , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 4/inmunología
5.
Biochem Biophys Res Commun ; 474(3): 452-461, 2016 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-27133719

RESUMEN

We have reported previously that activation of the MyD88-signaling network rapidly induces the formation of hybrid ubiquitin chains containing both Lys63-linked and Met1-linked ubiquitin (Ub) oligomers, some of which are attached covalently to Interleukin Receptor Associated kinase 1. Here we show that Lys63/Met1-Ub hybrids are also formed rapidly when the TNFR1/TRADD, TLR3/TRIF- and NOD1/RIP2-signaling networks are activated, some of which are attached covalently to Receptor-Interacting Protein 1 (TNFR1 pathway) or Receptor-Interacting Protein 2 (NOD1 pathway). These observations suggest that the formation of Lys63/Met1-Ub hybrids are of general significance for the regulation of innate immune signaling systems, and their potential roles in vivo are discussed. We also report that TNFα induces the attachment of Met1-linked Ub chains directly to TNF receptor 1, which do not seem to be attached covalently to Lys63-linked or other types of ubiquitin chain.


Asunto(s)
Inmunidad Innata/inmunología , Factores Inmunológicos/inmunología , Lisina/inmunología , Metionina/inmunología , Monocitos/inmunología , Ubiquitina/inmunología , Animales , Células Cultivadas , Humanos , Ratones , Unión Proteica/inmunología , Transducción de Señal/inmunología , Ubiquitinación/inmunología
6.
mBio ; 14(3): e0010123, 2023 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-37097030

RESUMEN

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.


Asunto(s)
Lengua Azul , Interferón Tipo I , Femenino , Ovinos , Animales , Bovinos , Interferón Tipo I/genética , Lengua Azul/metabolismo , Viremia , Antivirales
8.
PLoS One ; 6(12): e28594, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22163042

RESUMEN

The Nairoviruses are an important group of tick-borne viruses that includes pathogens of man (Crimean Congo hemorrhagic fever virus) and livestock animals (Dugbe virus, Nairobi sheep disease virus (NSDV)). NSDV is found in large parts of East Africa and the Indian subcontinent (where it is known as Ganjam virus). We have investigated the ability of NSDV to antagonise the induction and actions of interferon. Both pathogenic and apathogenic isolates could actively inhibit the induction of type 1 interferon, and also blocked the signalling pathways of both type 1 and type 2 interferons. Using transient expression of viral proteins or sections of viral proteins, these activities all mapped to the ovarian tumour-like protease domain (OTU) found in the viral RNA polymerase. Virus infection, or expression of this OTU domain in transfected cells, led to a great reduction in the incorporation of ubiquitin or ISG15 protein into host cell proteins. Point mutations in the OTU that inhibited the protease activity also prevented it from antagonising interferon induction and action. Interestingly, a mutation at a peripheral site, which had little apparent effect on the ability of the OTU to inhibit ubiquitination and ISG15ylation, removed the ability of the OTU to block the induction of type 1 and the action of type 2 interferons, but had a lesser effect on the ability to block type 1 interferon action, suggesting that targets other than ubiquitin and ISG15 may be involved in the actions of the viral OTU.


Asunto(s)
Interferones/metabolismo , Virus de la Enfermedad de los Ovinos de Nairobi/genética , Animales , Chlorocebus aethiops , ARN Polimerasas Dirigidas por ADN/química , Genes Reporteros , Cabras , Células HEK293 , Humanos , Inmunidad Innata , Interferón beta/metabolismo , Interferón gamma/metabolismo , Virus de la Enfermedad de los Ovinos de Nairobi/metabolismo , Fosforilación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Ovinos , Transcripción Genética , Ubiquitina/química , Células Vero
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