RESUMEN
Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.
Asunto(s)
Proteínas Portadoras , Proteínas de Unión al ADN , Ebolavirus/fisiología , Fiebre Hemorrágica Ebola/metabolismo , Factores de Transcripción , Proteínas Virales , Replicación Viral/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Células HeLa , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/patología , Humanos , Mapeo de Interacción de Proteínas , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.
Asunto(s)
Microscopía por Crioelectrón , Ebolavirus/fisiología , Ebolavirus/ultraestructura , Nucleocápside/ultraestructura , Nucleoproteínas/ultraestructura , Ensamble de Virus , Modelos Biológicos , Proteínas Mutantes/química , Mutación/genética , Nucleoproteínas/química , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN Viral/biosíntesis , ARN Viral/química , ARN Viral/metabolismoRESUMEN
The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Gripe Humana/inmunología , Orthomyxoviridae/fisiología , ARN Helicasas/metabolismo , ARN Polimerasa II/metabolismo , Degeneraciones Espinocerebelosas/genética , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/fisiología , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Citocinas/metabolismo , ADN Helicasas , Perros , Regulación hacia Abajo , Humanos , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Ratones Noqueados , Análisis por Micromatrices , Enzimas Multifuncionales , ARN Helicasas/genética , ARN Polimerasa II/genética , ARN Interferente Pequeño/genética , Ataxias Espinocerebelosas/congénito , Células Vero , Replicación Viral/genéticaRESUMEN
The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.
Asunto(s)
Ebolavirus/fisiología , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Prolina/metabolismo , Tirosina/metabolismo , Proteínas Portadoras , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Replicación ViralRESUMEN
Mycobacterium tuberculosis (Mtb) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving "accordion-type" amplification.
Asunto(s)
Mycobacterium tuberculosis/genética , Sistemas de Secreción Tipo VII/genética , Animales , Sistemas de Secreción Bacterianos/genética , Evolución Biológica , Evolución Molecular , Amplificación de Genes/genética , Ratones , Mycobacterium tuberculosis/metabolismo , Sistemas de Secreción Tipo VII/fisiología , Virulencia , Factores de Virulencia/genéticaRESUMEN
Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I-like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNß messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.
Asunto(s)
Proteína 58 DEAD Box , Helicasa Inducida por Interferón IFIH1 , Interferón beta , Receptores Inmunológicos , Proteínas no Estructurales Virales , Cristalografía por Rayos X , Proteína 58 DEAD Box/química , Proteína 58 DEAD Box/metabolismo , Medición de Intercambio de Deuterio , Células HEK293 , Humanos , Helicasa Inducida por Interferón IFIH1/química , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferón beta/química , Interferón beta/metabolismo , Unión Proteica , ARN Mensajero/química , ARN Mensajero/metabolismo , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
Because of its lethality, the Ebola virus often appears to be an invincible adversary. In Nature, Qiu et al. (2014) recently described the complete protection of nonhuman primates from deadly Ebola virus disease, even when treatment was begun as late as 5 days after infection.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Inmunización Pasiva , Animales , Femenino , MasculinoRESUMEN
The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5' and 3' untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3' UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5' or 3' UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3' UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3' UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3' UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3' UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3' UTR, but not the edited or deletion mutant NP 3' UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3' UTR, VP40 3' UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3' UTR edits also enhanced translation, but neither the wild-type nor the edited 3' UTRs induced IFN. These findings implicate filoviral mRNA 3' UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1. IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3' UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3' UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3' UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.
Asunto(s)
Regiones no Traducidas 3' , Adenosina Desaminasa/metabolismo , Ebolavirus/genética , Marburgvirus/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular , Ebolavirus/metabolismo , Genes Reporteros , Humanos , Interferón Tipo I/biosíntesis , Marburgvirus/metabolismo , MicroARNs/genética , Mutación , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Polirribosomas/metabolismo , Edición de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
Infection with Zaire ebolavirus (EBOV), a member of the Filoviridae family, causes a disease characterized by high levels of viremia, aberrant inflammation, coagulopathy, and lymphopenia. EBOV initially replicates in lymphoid tissues and disseminates via dendritic cells (DCs) and monocytes to liver, spleen, adrenal gland, and other secondary organs. EBOV protein VP35 is a critical immune evasion factor that inhibits type I interferon signaling and DC maturation. Nonhuman primates (NHPs) immunized with a high dose (5 × 105 PFU) of recombinant EBOV containing a mutated VP35 (VP35m) are protected from challenge with wild-type EBOV (wtEBOV). This protection is accompanied by a transcriptional response in the peripheral blood reflecting a regulated innate immune response and a robust induction of adaptive immune genes. However, the host transcriptional response to VP35m in lymphoid tissues has not been evaluated. Therefore, we conducted a transcriptional analysis of axillary and inguinal lymph nodes and spleen tissues of NHPs infected with a low dose (2 × 104 PFU) of VP35m and then back-challenged with a lethal dose of wtEBOV. VP35m induced early transcriptional responses in lymphoid tissues that are distinct from those observed in wtEBOV challenge. Specifically, we detected robust antiviral innate and adaptive responses and fewer transcriptional changes in genes with roles in angiogenesis, apoptosis, and inflammation. Two of three macaques survived wtEBOV back-challenge, with only the nonsurvivor displaying a transcriptional response reflecting Ebola virus disease. These data suggest that VP35 is a key modulator of early host responses in lymphoid tissues, thereby regulating disease progression and severity following EBOV challenge.IMPORTANCE Zaire Ebola virus (EBOV) infection causes a severe and often fatal disease characterized by inflammation, coagulation defects, and organ failure driven by a defective host immune response. Lymphoid tissues are key sites of EBOV pathogenesis and the generation of an effective immune response to infection. A recent study demonstrated that infection with an EBOV encoding a mutant VP35, a viral protein that antagonizes host immunity, can protect nonhuman primates (NHPs) against lethal EBOV challenge. However, no studies have examined the response to this mutant EBOV in lymphoid tissues. Here, we characterize gene expression in lymphoid tissues from NHPs challenged with the mutant EBOV and subsequently with wild-type EBOV to identify signatures of a protective host response. Our findings are critical for elucidating viral pathogenesis, mechanisms of host antagonism, and the role of lymphoid organs in protective responses to EBOV to improve the development of antivirals and vaccines against EBOV.
Asunto(s)
Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/inmunología , Tejido Linfoide/inmunología , Proteínas Reguladoras y Accesorias Virales/inmunología , Inmunidad Adaptativa , Animales , Antivirales/sangre , Ebolavirus/genética , Ebolavirus/inmunología , Regulación de la Expresión Génica/inmunología , Fiebre Hemorrágica Ebola/sangre , Fiebre Hemorrágica Ebola/prevención & control , Fiebre Hemorrágica Ebola/virología , Tejido Linfoide/virología , Macaca fascicularis , Mutación , Bazo/inmunología , Transcriptoma , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus in the Paramyxoviridae family, are recently emerged, highly lethal zoonotic pathogens. The NiV and HeV nonsegmented, negative-sense RNA genomes encode nine proteins, including the W protein. Expressed from the P gene through mRNA editing, W shares a common N-terminus with P and V but has a unique C-terminus. Expressed alone, W modulates innate immune responses by several mechanisms, and elimination of W from NiV alters the course of infection in experimentally infected ferrets. However, the specific host interactions that allow W to modulate innate immunity are incompletely understood. This study demonstrates that the NiV and HeV W proteins interact with all seven isoforms of the 14-3-3 family, regulatory molecules that preferentially bind phosphorylated target proteins to regulate a wide range of cellular functions. The interaction is dependent on the penultimate amino acid residue in the W sequence, a conserved, phosphorylated serine. The cocrystal structure of the W C-terminal binding motif with 14-3-3 provides only the second structure of a complex containing a mode III interactor, which is defined as a 14-3-3 interaction with a phosphoserine/phosphothreonine at the C-termini of the target protein. Transcriptomic analysis of inducible cell lines infected with an RNA virus and expressing either wild-type W or W lacking 14-3-3 binding, identifies new functions for W. These include the regulation of cellular metabolic processes, extracellular matrix organization, and apoptosis.IMPORTANCE Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus, are recently emerged, highly lethal zoonotic pathogens that cause yearly outbreaks. NiV and HeV each encode a W protein that has roles in regulating host signaling pathways, including antagonism of the innate immune response. However, the mechanisms used by W to regulate these host responses are not clear. Here, characterization of the interaction of NiV and HeV W with 14-3-3 identifies modulation of 14-3-3-regulated host signaling pathways not previously associated with W, suggesting new avenues of research. The cocrystal structure of the NiV W:14-3-3 complex, as only the second structure of a 14-3-3 mode III interactor, provides further insight into this less-well-understood 14-3-3 binding motif.
Asunto(s)
Proteínas 14-3-3/metabolismo , Regulación de la Expresión Génica , Virus Hendra/metabolismo , Infecciones por Henipavirus/metabolismo , Virus Nipah/metabolismo , Proteínas Virales/metabolismo , Proteínas 14-3-3/genética , Células HEK293 , Virus Hendra/genética , Infecciones por Henipavirus/genética , Humanos , Virus Nipah/genética , Proteínas Virales/genéticaRESUMEN
Menglà virus (MLAV), identified in Rousettus bats, is a phylogenetically distinct member of the family Filoviridae Because the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) modulate host innate immunity, MLAV VP35, VP40, and VP24 proteins were compared with their EBOV and MARV homologs for innate immune pathway modulation. In human and Rousettus cells, MLAV VP35 behaved like EBOV and MARV VP35s, inhibiting virus-induced activation of the interferon beta (IFN-ß) promoter and interferon regulatory factor 3 (IRF3) phosphorylation. MLAV VP35 also interacted with PACT, a host protein engaged by EBOV VP35 to inhibit RIG-I signaling. MLAV VP35 also inhibits PKR activation. MLAV VP40 was demonstrated to inhibit type I IFN-induced gene expression in human and bat cells. It blocked STAT1 tyrosine phosphorylation induced either by type I IFN or overexpressed Jak1, paralleling MARV VP40. MLAV VP40 also inhibited virus-induced IFN-ß promoter activation, a property shared by MARV VP40 and EBOV VP24. A Jak kinase inhibitor did not recapitulate this inhibition in the absence of viral proteins. Therefore, inhibition of Jak-STAT signaling is insufficient to explain inhibition of IFN-ß promoter activation. MLAV VP24 did not inhibit IFN-induced gene expression or bind karyopherin α proteins, properties of EBOV VP24. MLAV VP24 differed from MARV VP24 in that it failed to interact with Keap1 or activate an antioxidant response element reporter gene due to the absence of a Keap1-binding motif. These functional observations support a closer relationship of MLAV to MARV than to EBOV but also are consistent with MLAV belonging to a distinct genus.IMPORTANCE EBOV and MARV, members of the family Filoviridae, are highly pathogenic zoonotic viruses that cause severe disease in humans. Both viruses use several mechanisms to modulate the host innate immune response, and these likely contribute to the severity of disease. Here, we demonstrate that MLAV, a filovirus newly discovered in a bat, suppresses antiviral type I interferon responses in both human and bat cells. Inhibitory activities are possessed by MLAV VP35 and VP40, which parallels how MARV blocks IFN responses. However, whereas MARV activates cellular antioxidant responses through an interaction between its VP24 protein and host protein Keap1, MLAV VP24 lacks a Keap1-binding motif and fails to activate this cytoprotective response. These data indicate that MLAV possesses immune-suppressing functions that could facilitate human infection. They also support the placement of MLAV in a different genus than either EBOV or MARV.
Asunto(s)
Infecciones por Filoviridae/fisiopatología , Filoviridae/genética , Animales , Quirópteros/inmunología , Quirópteros/virología , Ebolavirus , Filoviridae/metabolismo , Filoviridae/patogenicidad , Células HEK293 , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/inmunología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Marburgvirus , Factor 2 Relacionado con NF-E2/metabolismo , Factor de Transcripción STAT1 , Proteínas Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
The question as to whether RNA viruses produce bona fide microRNAs (miRNAs) during infection has been the focus of intense research and debate. Recently, several groups using computational prediction methods have independently reported possible miRNA candidates produced by Ebola virus (EBOV). Additionally, efforts to detect these predicted RNA products in samples from infected animals and humans have produced positive results. However, these studies and their conclusions are predicated on the assumption that these RNA products are actually processed through, and function within, the miRNA pathway. In the present study, we performed the first rigorous assessment of the ability of filoviruses to produce miRNA products during infection of both human and bat cells. Using next-generation sequencing, we detected several candidate miRNAs from both EBOV and the closely related Marburg virus (MARV). Focusing our validation efforts on EBOV, we found evidence contrary to the idea that these small RNA products function as miRNAs. The results of our study are important because they highlight the potential pitfalls of relying on computational methods alone for virus miRNA discovery.IMPORTANCE Here, we report the discovery, via deep sequencing, of numerous noncoding RNAs (ncRNAs) derived from both EBOV and MARV during infection of both bat and human cell lines. In addition to identifying several novel ncRNAs from both viruses, we identified two EBOV ncRNAs in our sequencing data that were near-matches to computationally predicted viral miRNAs reported in the literature. Using molecular and immunological techniques, we assessed the potential of EBOV ncRNAs to function as viral miRNAs. Importantly, we found little evidence supporting this hypothesis. Our work is significant because it represents the first rigorous assessment of the potential for EBOV to encode viral miRNAs and provides evidence contrary to the existing paradigm regarding the biological role of computationally predicted EBOV ncRNAs. Moreover, our work highlights further avenues of research regarding the nature and function of EBOV ncRNAs.
Asunto(s)
Ebolavirus/metabolismo , MicroARNs/metabolismo , Interferencia de ARN , ARN Viral/metabolismo , Animales , Línea Celular , Quirópteros , Ebolavirus/genética , Humanos , Marburgvirus/genética , Marburgvirus/metabolismo , MicroARNs/genética , ARN Viral/genéticaRESUMEN
In March 2021, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.
Asunto(s)
Mononegavirales , Virus , HumanosRESUMEN
In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.
Asunto(s)
Mononegavirales/clasificación , Terminología como AsuntoRESUMEN
Members of the family Filoviridae produce variously shaped, often filamentous, enveloped virions containing linear non-segmented, negative-sense RNA genomes of 15-19 kb. Several filoviruses (e.g., Ebola virus) are pathogenic for humans and are highly virulent. Several filoviruses infect bats (e.g., Marburg virus), whereas the hosts of most other filoviruses are unknown. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on Filoviridae, which is available at www.ictv.global/report/filoviridae.
Asunto(s)
Filoviridae/clasificación , Animales , Filoviridae/genética , Genoma Viral/genética , Humanos , ARN Viral/genéticaRESUMEN
In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).
Asunto(s)
Mononegavirales/clasificación , Mononegavirales/genética , Mononegavirales/aislamiento & purificación , Filogenia , Virología/organización & administraciónRESUMEN
In February 2019, following the annual taxon ratification vote, the order Mononegavirales was amended by the addition of four new subfamilies and 12 new genera and the creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).
Asunto(s)
Mononegavirales/clasificación , Mononegavirales/genética , Genoma Viral/genética , ARN Viral/genéticaRESUMEN
BST2 is a host protein with dual functions in response to viral infections: it traps newly assembled enveloped virions at the plasma membrane in infected cells, and it induces NF-κB activity, especially in the context of retroviral assembly. In this study, we examined whether Ebola virus proteins affect BST2-mediated induction of NF-κB. We found that the Ebola virus matrix protein, VP40, and envelope glycoprotein, GP, each cooperate with BST2 to induce NF-κB activity, with maximal activity when all three proteins are expressed. Unlike human immunodeficiency virus type 1 Vpu protein, which antagonizes both virion entrapment and the activation of NF-κB by BST2, Ebola virus GP does not inhibit NF-κB signaling even while it antagonizes the entrapment of virus-like particles. GP from Reston ebolavirus, a nonpathogenic species in humans, showed a phenotype similar to that of GP from Zaire ebolavirus, a highly pathogenic species, in terms of both the activation of NF-κB and the antagonism of virion entrapment. Although Ebola virus VP40 and GP both activate NF-κB independently of BST2, VP40 is the more potent activator. Activation of NF-κB by the Ebola virus proteins either alone or together with BST2 requires the canonical NF-κB signaling pathway. Mechanistically, the maximal NF-κB activation by GP, VP40, and BST2 together requires the ectodomain cysteines needed for BST2 dimerization, the putative BST2 tetramerization residue L70, and Y6 of a potential hemi-ITAM motif in BST2's cytoplasmic domain. BST2 with a glycosylphosphatidylinositol (GPI) anchor signal deletion, which is not expressed at the plasma membrane and is unable to entrap virions, activated NF-κB in concert with the Ebola virus proteins at least as effectively as wild-type BST2. Signaling by the GPI anchor mutant also depended on Y6 of BST2. Overall, our data show that activation of NF-κB by BST2 is independent of virion entrapment in the case of Ebola virus. Nonetheless, BST2 may induce or amplify proinflammatory signaling during Ebola virus infection, potentially contributing to the dysregulated cytokine response that is a hallmark of Ebola virus disease.IMPORTANCE Understanding how the host responds to viral infections informs the development of therapeutics and vaccines. We asked how proinflammatory signaling by the host protein BST2/tetherin, which is mediated by the transcription factor NF-κB, responds to Ebola virus proteins. Although the Ebola virus envelope glycoprotein (GP1,2) antagonizes the trapping of newly formed virions at the plasma membrane by BST2, we found that it does not inhibit BST2's ability to induce NF-κB activity. This distinguishes GP1,2 from the HIV-1 protein Vpu, the prototype BST2 antagonist, which inhibits both virion entrapment and the induction of NF-κB activity. Ebola virus GP1,2, the Ebola virus matrix protein VP40, and BST2 are at least additive with respect to the induction of NF-κB activity. The effects of these proteins converge on an intracellular signaling pathway that depends on a protein modification termed neddylation. Better mechanistic understanding of these phenomena could provide targets for therapies that modulate the inflammatory response during Ebola virus disease.
Asunto(s)
Antígenos CD/metabolismo , Ebolavirus/metabolismo , FN-kappa B/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas de la Matriz Viral/metabolismo , Virión/metabolismo , Secuencias de Aminoácidos , Antígenos CD/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/virología , Ebolavirus/genética , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Humanos , FN-kappa B/genética , Dominios Proteicos , Multimerización de Proteína , Proteínas del Envoltorio Viral/genética , Proteínas de la Matriz Viral/genética , Virión/genéticaRESUMEN
Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. IMPORTANCE: The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis.
Asunto(s)
Ebolavirus/fisiología , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Interferones/metabolismo , Proteínas Virales/metabolismo , alfa Carioferinas/metabolismo , Secuencia de Aminoácidos , Línea Celular , Fiebre Hemorrágica Ebola/genética , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Estabilidad Proteica , Proteínas Recombinantes de Fusión , Relación Estructura-Actividad , Proteínas Virales/química , alfa Carioferinas/química , alfa Carioferinas/genéticaRESUMEN
Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection.IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.