RESUMEN
The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52-p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence.
Asunto(s)
Ciclo Celular/genética , Regulación de la Expresión Génica , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cristalización , Humanos , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteína p107 Similar a la del Retinoblastoma/química , Proteína p107 Similar a la del Retinoblastoma/metabolismo , Proteína p130 Similar a la del Retinoblastoma/química , Proteína p130 Similar a la del Retinoblastoma/metabolismo , Alineación de Secuencia , Transactivadores/metabolismoRESUMEN
Filoviruses, marburgvirus (MARV) and ebolavirus (EBOV), are causative agents of highly lethal hemorrhagic fever in humans. MARV and EBOV share a common genome organization but show important differences in replication complex formation, cell entry, host tropism, transcriptional regulation, and immune evasion. Multifunctional filoviral viral protein (VP) 35 proteins inhibit innate immune responses. Recent studies suggest double-stranded (ds)RNA sequestration is a potential mechanism that allows EBOV VP35 to antagonize retinoic-acid inducible gene-I (RIG-I) like receptors (RLRs) that are activated by viral pathogen-associated molecular patterns (PAMPs), such as double-strandedness and dsRNA blunt ends. Here, we show that MARV VP35 can inhibit IFN production at multiple steps in the signaling pathways downstream of RLRs. The crystal structure of MARV VP35 IID in complex with 18-bp dsRNA reveals that despite the similar protein fold as EBOV VP35 IID, MARV VP35 IID interacts with the dsRNA backbone and not with blunt ends. Functional studies show that MARV VP35 can inhibit dsRNA-dependent RLR activation and interferon (IFN) regulatory factor 3 (IRF3) phosphorylation by IFN kinases TRAF family member-associated NFkb activator (TANK) binding kinase-1 (TBK-1) and IFN kB kinase e (IKKe) in cell-based studies. We also show that MARV VP35 can only inhibit RIG-I and melanoma differentiation associated gene 5 (MDA5) activation by double strandedness of RNA PAMPs (coating backbone) but is unable to inhibit activation of RLRs by dsRNA blunt ends (end capping). In contrast, EBOV VP35 can inhibit activation by both PAMPs. Insights on differential PAMP recognition and inhibition of IFN induction by a similar filoviral VP35 fold, as shown here, reveal the structural and functional plasticity of a highly conserved virulence factor.
Asunto(s)
Marburgvirus/inmunología , Marburgvirus/patogenicidad , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cristalografía por Rayos X , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Inmunidad Innata , Interferón Tipo I/antagonistas & inhibidores , Enfermedad del Virus de Marburg/etiología , Enfermedad del Virus de Marburg/inmunología , Enfermedad del Virus de Marburg/virología , Marburgvirus/química , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Estructura Terciaria de Proteína , ARN/química , ARN/genética , ARN/metabolismo , Homología de Secuencia de Aminoácido , Virulencia/inmunologíaRESUMEN
The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression.
Asunto(s)
Genes cdc , Nucleosomas/metabolismo , Unión Proteica , Sitio de Iniciación de la Transcripción , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Cromatina , ADN/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-alpha/beta responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-alpha/beta production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.
Asunto(s)
Ebolavirus/genética , Ebolavirus/patogenicidad , Cobayas/virología , Mutación Puntual , ARN Bicatenario/metabolismo , Proteínas Reguladoras y Accesorias Virales , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Ebolavirus/inmunología , Femenino , Humanos , Factores Inmunológicos/genética , Factores Inmunológicos/inmunología , Interferón-alfa/genética , Interferón-alfa/inmunología , Interferón beta/genética , Interferón beta/inmunología , Interferones/antagonistas & inhibidores , Interferones/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Bicatenario/genética , Alineación de Secuencia , Células Vero , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6(1)22, P2(1)2(1)2(1) and P2(1), respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source.
Asunto(s)
Ebolavirus/química , Interferones/antagonistas & inhibidores , Mutación , Proteínas Reguladoras y Accesorias Virales/química , Secuencia de Aminoácidos , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Ebolavirus/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.
Asunto(s)
Antivirales/química , Coenzimas/antagonistas & inhibidores , Ebolavirus/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Antivirales/farmacología , Coenzimas/química , Simulación por Computador , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/metabolismo , Ebolavirus/enzimología , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/fisiología , Pirroles/química , Pirroles/metabolismo , Pirroles/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Reguladoras y Accesorias Virales/químicaRESUMEN
The cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencias de Aminoácidos , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/genética , Ebolavirus/química , Ebolavirus/genética , Fiebre Hemorrágica Ebola/enzimología , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/virología , Humanos , Unión Proteica , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Receptores Inmunológicos , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
The Filoviridae family of viruses, which includes the genera Ebolavirus (EBOV) and Marburgvirus (MARV), causes severe and often times lethal hemorrhagic fever in humans. Filoviral infections are associated with ineffective innate antiviral responses as a result of virally encoded immune antagonists, which render the host incapable of mounting effective innate or adaptive immune responses. The Type I interferon (IFN) response is critical for establishing an antiviral state in the host cell and subsequent activation of the adaptive immune responses. Several filoviral encoded components target Type I IFN responses, and this innate immune suppression is important for viral replication and pathogenesis. For example, EBOV VP35 inhibits the phosphorylation of IRF-3/7 by the TBK-1/IKKε kinases in addition to sequestering viral RNA from detection by RIG-I like receptors. MARV VP40 inhibits STAT1/2 phosphorylation by inhibiting the JAK family kinases. EBOV VP24 inhibits nuclear translocation of activated STAT1 by karyopherin-α. The examples also represent distinct mechanisms utilized by filoviral proteins in order to counter immune responses, which results in limited IFN-α/ß production and downstream signaling.
Asunto(s)
Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Evasión Inmune/inmunología , Enfermedad del Virus de Marburg/inmunología , Marburgvirus/inmunología , Proteínas Virales/metabolismo , Inmunidad Adaptativa , Animales , Ebolavirus/metabolismo , Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/virología , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Enfermedad del Virus de Marburg/virología , Marburgvirus/metabolismo , Marburgvirus/patogenicidad , Fosforilación , Transducción de Señal , Replicación ViralRESUMEN
Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I-like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I-like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also important for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.