RESUMEN
The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.
Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Marburgvirus/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Complejo Antígeno-Anticuerpo/química , Línea Celular , Reacciones Cruzadas , Cristalografía por Rayos X , Drosophila , Ebolavirus/química , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Enfermedad del Virus de Marburg/inmunología , Marburgvirus/genética , Marburgvirus/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Mucinas/química , Alineación de Secuencia , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.
Asunto(s)
Ebolavirus/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismo , Cristalografía por Rayos X , Dimerización , Ebolavirus/química , Ebolavirus/clasificación , Ebolavirus/genética , Modelos Moleculares , Mutagénesis , Mutación Puntual , Proteínas de la Matriz Viral/genética , Ensamble de Virus , Liberación del VirusRESUMEN
IMPORTANCE: Ebola disease (EBOD) is a public health threat with a high case fatality rate. Most EBOD outbreaks have occurred in remote locations, but the 2013-2016 Western Africa outbreak demonstrated how devastating EBOD can be when it reaches an urban population. Here, the 2022 Sudan virus disease (SVD) outbreak in Mubende District, Uganda, is summarized, and the genetic relatedness of the new variant is evaluated. The Mubende variant exhibited 96% amino acid similarity with historic SUDV sequences from the 1970s and a high degree of conservation throughout the outbreak, which was important for ongoing diagnostics and highly promising for future therapy development. Genetic differences between viruses identified during the Mubende SVD outbreak were linked with epidemiological data to better interpret viral spread and contact tracing chains. This methodology should be used to better integrate discrete epidemiological and sequence data for future viral outbreaks.
Asunto(s)
Brotes de Enfermedades , Ebolavirus , Variación Genética , Fiebre Hemorrágica Ebola , Humanos , Brotes de Enfermedades/estadística & datos numéricos , Ebolavirus/química , Ebolavirus/clasificación , Ebolavirus/genética , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/transmisión , Fiebre Hemorrágica Ebola/virología , Uganda/epidemiología , Trazado de ContactoRESUMEN
Ebola virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA1,2. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical nucleoprotein-RNA complex, VP24, VP30, VP35 and viral polymerase1,3. The nucleoprotein-RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion4,5. RNA binding and nucleoprotein oligomerization are synergistic and do not readily occur independently6. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core4,5, there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola virus nucleoprotein-RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryo-electron microscopy. Our structure reveals how the Ebola virus nucleocapsid core encapsidates its viral genome, its sequence-independent coordination with RNA by nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions that lead to the formation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion5. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development.
Asunto(s)
Microscopía por Crioelectrón , Ebolavirus/química , Ebolavirus/ultraestructura , Nucleocápside/química , ARN Viral/química , Células HEK293 , Humanos , Modelos Moleculares , Nucleocápside/ultraestructura , ARN Viral/ultraestructuraRESUMEN
Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and dephosphorylation in other viral replication processes such as nucleocapsid and virion assembly is unclear. Interestingly, VP30 is predominantly dephosphorylated by cellular phosphatases in viral inclusions, while it is phosphorylated in the released virions. Thus, uncertainties regarding how VP30 phosphorylation in nucleocapsids is achieved and whether VP30 phosphorylation provides any advantages in later steps in viral replication have arisen. In the present study, to characterize the roles of VP30 phosphorylation in nucleocapsid formation, we used electron microscopic analyses and live cell imaging systems. We identified VP30 localized to the surface of protrusions surrounding nucleoprotein (NP)-forming helical structures in the nucleocapsid, suggesting the involvement in assembly and transport of nucleocapsids. Interestingly, VP30 phosphorylation facilitated its association with nucleocapsid-like structures (NCLSs). On the contrary, VP30 phosphorylation does not influence the transport characteristics and NCLS number leaving from and coming back into viral inclusions, indicating that the phosphorylation status of VP30 is not a prerequisite for NCLS departure. Moreover, the phosphorylation status of VP30 did not cause major differences in nucleocapsid transport in authentic EBOV-infected cells. In the following budding step, the association of VP30 and its phosphorylation status did not influence the budding efficiency of virus-like particles. Taken together, it is plausible that EBOV may utilize the phosphorylation of VP30 for its selective association with nucleocapsids, without affecting nucleocapsid transport and virion budding processes. IMPORTANCE Ebola virus (EBOV) causes severe fevers with unusually high case fatality rates. The nucleocapsid provides the template for viral genome transcription and replication. Thus, understanding the regulatory mechanism behind its formation is important for the development of novel therapeutic approaches. Previously, we established a live-cell imaging system based on the ectopic expression of viral fluorescent fusion proteins, allowing the visualization and characterization of intracytoplasmic transport of nucleocapsid-like structures. EBOV VP30 is an essential transcriptional factor for viral genome synthesis, and, although its role in viral genome transcription and replication is well understood, the functional importance of VP30 phosphorylation in assembly of nucleocapsids is still unclear. Our work determines the localization of VP30 at the surface of ruffled nucleocapsids, which differs from the localization of polymerase in EBOV-infected cells. This study sheds light on the novel role of VP30 phosphorylation in nucleocapsid assembly, which is an important prerequisite for virion formation.
Asunto(s)
Ebolavirus , Nucleocápside , Factores de Transcripción , Proteínas Virales , Ensamble de Virus , Transporte Biológico , Ebolavirus/química , Ebolavirus/crecimiento & desarrollo , Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/virología , Humanos , Nucleocápside/biosíntesis , Nucleocápside/metabolismo , Fosforilación , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Virión/química , Virión/crecimiento & desarrollo , Virión/metabolismoRESUMEN
The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca2+ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2.
Asunto(s)
Ebolavirus/fisiología , Fusión de Membrana , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Regulación Alostérica , Calcio/metabolismo , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Niemann-Pick C1 , Polisacáridos/metabolismo , Unión Proteica , Conformación Proteica , Dominios Proteicos , Proteínas del Envoltorio Viral/genética , Internalización del VirusRESUMEN
Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of nucleocapsid or nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.
Asunto(s)
Microscopía por Crioelectrón , Ebolavirus/química , Ebolavirus/ultraestructura , Tomografía con Microscopio Electrónico , Proteínas de la Nucleocápside/ultraestructura , Nucleocápside/química , Nucleocápside/ultraestructura , Animales , Chlorocebus aethiops , Células HEK293 , Humanos , Marburgvirus/química , Modelos Moleculares , Conformación Molecular , Proteínas de la Nucleocápside/química , ARN Viral/química , ARN Viral/ultraestructura , Células VeroRESUMEN
The Ebola virus glycoprotein (GP) gene templates several mRNAs that produce either the virion-associated transmembrane protein or one of two secreted glycoproteins. Soluble glycoprotein (sGP) is the predominant product. GP1 and sGP share an amino terminal sequence of 295 amino acids but differ in quaternary structure, with GP1 being a heterohexamer with GP2 and sGP a homodimer. Two structurally different DNA aptamers were selected against sGP that also bound GP1,2. These DNA aptamers were compared with a 2'FY-RNA aptamer for their interactions with the Ebola GP gene products. The three aptamers have almost identical binding isotherms for sGP and GP1,2 in solution and on the virion. They demonstrated high affinity and selectivity for sGP and GP1,2. Furthermore, one aptamer, used as a sensing element in an electrochemical format, detected GP1,2 on pseudotyped virions and sGP with high sensitivity in the presence of serum, including from an Ebola-virus-infected monkey. Our results suggest that the aptamers interact with sGP across the interface between the monomers, which is different from the sites on the protein bound by most antibodies. The remarkable similarity in functional features of three structurally distinct aptamers suggests that aptamers, like antibodies, have preferred binding sites on proteins.
Asunto(s)
Aptámeros de Nucleótidos , Ebolavirus , Proteínas del Envoltorio Viral , Humanos , Aptámeros de Nucleótidos/química , Ebolavirus/química , Proteínas del Envoltorio Viral/química , Multimerización de ProteínaRESUMEN
Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Proteindrug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.
Asunto(s)
Antivirales/química , Antivirales/metabolismo , Ebolavirus/química , Toremifeno/química , Toremifeno/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Antivirales/farmacología , Sitios de Unión , Línea Celular , Secuencia Conservada , Ebolavirus/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ibuprofeno/química , Ibuprofeno/metabolismo , Ibuprofeno/farmacología , Ligandos , Marburgvirus/química , Fusión de Membrana/efectos de los fármacos , Modelos Moleculares , Unión Proteica , Estabilidad Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína/efectos de los fármacos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Temperatura , Toremifeno/farmacología , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Acoplamiento Viral/efectos de los fármacosRESUMEN
Cryo-electron tomography (cryo-ET) is a pivotal imaging technique for studying the structure of pleomorphic enveloped viruses and their interactions with the host at native conditions. Owing to the limited tilting range of samples with a slab geometry, electron tomograms suffer from so-called missing wedge information in Fourier space. In dual-axis cryo-ET, two tomograms reconstructed from orthogonally oriented tilt series are combined into a tomogram with improved resolution as the missing wedge information is reduced to a pyramid. Volta phase plate (VPP) allows to perform in-focus cryo-ET with high contrast transfer at low-resolution frequencies and thus its application may improve the quality of dual-axis tomograms. Here, we compare dual-axis cryo-ET with and without VPP on Ebola virus-like particles to visualize and segment viral and host cell proteins within the membrane-enveloped filamentous particles. Dual-axis VPP cryo-ET reduces the missing wedge information and ray artifacts arising from the weighted back-projection during tomogram reconstruction, thereby minimizing ambiguity in the analysis of crowded environments and facilitating 3D segmentation. We show that dual-axis VPP tomograms provide a comprehensive description of macromolecular organizations such as nucleocapsid assembly states, the distribution of glycoproteins on the viral envelope and asymmetric arrangements of the VP40 layer in non-filamentous regions of virus-like particles. Our data reveal actin filaments within virus-like particles in close proximity to the viral VP40 scaffold, suggesting a direct interaction between VP40 and actin filaments. Dual-axis VPP cryo-ET provides more complete 3D information at high contrast and allows for better interpretation of macromolecule interactions and pleomorphic organizations.
Asunto(s)
Actinas/química , Microscopía por Crioelectrón/métodos , Ebolavirus/química , Proteínas de la Matriz Viral/química , Actinas/metabolismo , Membrana Celular , Ebolavirus/metabolismo , Ebolavirus/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Células HEK293 , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/virología , Interacciones Huésped-Patógeno , Humanos , Imagenología Tridimensional , Nucleocápside/química , Proteínas de la Matriz Viral/metabolismoRESUMEN
Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.
Asunto(s)
Ebolavirus/efectos de los fármacos , Proteína Niemann-Pick C1/antagonistas & inhibidores , Receptores Virales/antagonistas & inhibidores , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas Virales de Fusión/antagonistas & inhibidores , Virión/efectos de los fármacos , Animales , Sitios de Unión , Bioensayo , Chlorocebus aethiops , Clomifeno/química , Clomifeno/farmacología , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/metabolismo , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Calor , Concentración de Iones de Hidrógeno , Simulación del Acoplamiento Molecular , Proteína Niemann-Pick C1/química , Proteína Niemann-Pick C1/genética , Proteína Niemann-Pick C1/metabolismo , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores Virales/química , Receptores Virales/genética , Receptores Virales/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/química , Tamoxifeno/farmacología , Toremifeno/química , Toremifeno/farmacología , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Virión/química , Virión/genética , Virión/metabolismoRESUMEN
Ebola virus (EBOV) RNA has the potential to form hairpin structures at the transcription start sequence (TSS) and reinitiation sites of internal genes, both on the genomic and antigenomic/mRNA level. Hairpin formation involving the TSS and the spacer sequence between promotor elements (PE) 1 and 2 was suggested to regulate viral transcription. Here, we provide evidence that such RNA structures form during RNA synthesis by the viral polymerase and affect its activity. This was analysed using monocistronic minigenomes carrying hairpin structure variants in the TSS-spacer region that differ in length and stability. Transcription and replication were measured via reporter activity and by qRT-PCR quantification of the distinct viral RNA species. We demonstrate that viral RNA synthesis is remarkably tolerant to spacer extensions of up to ~54 nt, but declines beyond this length limit (~25% residual activity for a 66-nt extension). Minor incremental stabilizations of hairpin structures in the TSS-spacer region and on the mRNA/antigenomic level were found to rapidly abolish viral polymerase activity, which may be exploited for antisense strategies to inhibit viral RNA synthesis. Finally, balanced viral transcription and replication can still occur when any RNA structure formation potential at the TSS is eliminated, provided that hexamer phasing in the promoter region is maintained. Altogether, the findings deepen and refine our insight into structure and length constraints within the EBOV transcription and replication promoter and suggest a remarkable flexibility of the viral polymerase in recognition of PE1 and PE2.
Asunto(s)
Ebolavirus/genética , Estabilidad del ARN/genética , ARN Viral/química , Replicación Viral/genética , Ebolavirus/química , Ebolavirus/fisiología , Genoma Viral/fisiología , Células HEK293 , Fiebre Hemorrágica Ebola/virología , Humanos , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN Viral/genética , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Ebola virus (EBOV) belongs to the Filoviridae family, which can cause severe hemorrhagic fever in humans and nonprimates. The neutralization of EBOV by monoclonal antibody (mAb) ADI-15946 was reported recently. In the present study, the molecular interactions between the receptor GPcl of EBOV and ADI-15946 were studied by molecular dynamics (MD) simulation and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis. Hydrophobic interaction was identified as the main driving force for the binding of ADI-15946 on EBOV. Moreover, the contribution of each amino acid residue for the binding was evaluated. Then, an affinity binding model (ABM) was constructed using the residues favorable for the binding, including Y107, F108, D109, W110, and R113. The biomimetic design of neutralizer against EBOV according to the ABM of ADI-15946 was then performed, followed by screening using docking, structural similarity. Two neutralizers YFDWHMR and YFDWRYR were obtained, which were proven to be capable of strong binding on GPcl and then neutralizing GPcl. These results would be helpful for the development of neutralizers for Ebola virus.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Antivirales/metabolismo , Ebolavirus/química , Péptidos/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Anticuerpos Monoclonales/química , Antivirales/química , Biomimética , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Biblioteca de Péptidos , Péptidos/química , Unión Proteica , Termodinámica , Proteínas del Envoltorio Viral/químicaAsunto(s)
Pruebas Diagnósticas de Rutina/economía , Pruebas Diagnósticas de Rutina/estadística & datos numéricos , Brotes de Enfermedades/estadística & datos numéricos , Ebolavirus/aislamiento & purificación , Fiebre Hemorrágica Ebola/diagnóstico , Fiebre Hemorrágica Ebola/epidemiología , República Democrática del Congo/epidemiología , Brotes de Enfermedades/prevención & control , Industria Farmacéutica/economía , Ebolavirus/química , Ebolavirus/genética , Medicina de Emergencia/economía , Medicina de Emergencia/métodos , Medicina de Emergencia/tendencias , Fiebre Hemorrágica Ebola/virología , Humanos , Reacción en Cadena de la Polimerasa , Factores de TiempoRESUMEN
Ebola virus (EBOV) is a human pathogen with the ability to cause hemorrhagic fever and bleeding diathesis in hosts. The life cycle of EBOV depends on its nucleocapsid. The Ebola nucleocapsid consists of a helical assembly of nucleoproteins (NPs) encapsidating single-stranded viral RNA (ssRNA). Knowledge of the molecular determinants of Ebola nucleocapsid stability is essential for the development of therapeutics against EBOV. However, large degrees of freedom associated with the Ebola nucleocapsid helical assembly pose a computational challenge, thereby limiting the previous simulation studies to the level of monomers. In the present work, we have performed all atom molecular dynamics (MD) simulations of the helical assembly of EBOV nucleoproteins in the absence and presence of ssRNA. We found that ssRNA is essential for maintaining structural integrity of the nucleocapsid. Other molecular determinants observed to stabilize the nucleocapsid include NP-RNA and NP-NP interactions and ion distributions. Additionally, the structural and dynamical behavior of the nucleocapsid monomer depends on its position in the helical assembly. NP monomers present on the longitudinal edges of the helical tube are more exposed, flexible, and have weaker NP-NP interactions than those residing in the center. This work provides key structural features stabilizing the nucleocapsid that may serve as therapeutic targets.
Asunto(s)
Ebolavirus/química , Simulación de Dinámica Molecular , Nucleocápside/análisis , HumanosRESUMEN
Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.
Asunto(s)
Ebolavirus/química , Proteínas del Envoltorio Viral/química , Proteínas Virales de Fusión/química , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/virología , Ebolavirus/fisiología , Dominios Proteicos , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/metabolismo , Internalización del VirusRESUMEN
Members of Mononegavirales, the order that includes nonsegmented negative sense RNA viruses (NNSVs), encode a small number of multifunctional proteins. In members of the Filoviridae family, virus protein 35 (VP35) facilitates immune evasion and functions as an obligatory cofactor for viral RNA synthesis. VP35 functions in a manner orthologous to that of phosphoproteins from other NNSVs. Although the critical roles of Ebola viral VP35 (eVP35) in immune evasion and RNA synthesis are well-appreciated, a complete understanding of its organization and its role in carrying out its many functions has yet to be fully realized. In particular, we currently lack information about the role of the oligomerization domain within eVP35. To address this limitation, we report here an investigation of the oligomer structure of eVP35 using hybrid methods that include multiangle light scattering, small-angle X-ray scattering, and cross-linking coupled with mass spectrometry to determine the shape and orientation of the eVP35 oligomer. Our integrative results are consistent with a parallel tetramer in which the N-terminal regions that are required for RNA synthesis are all oriented in the same direction. Furthermore, these results define a framework for targeting the symmetric tetramer for structure-based antiviral discovery.
Asunto(s)
Proteínas Reguladoras y Accesorias Virales/química , Secuencia de Aminoácidos , Ebolavirus/química , Escherichia coli/genética , Dominios Proteicos , Multimerización de ProteínaRESUMEN
Ebola virus (EBOV) VP40 is a major driving force of nascent virion production and a negative regulator of genome replication/transcription. Here, we showed that the YIGL sequence at the C-terminus of EBOV VP40 is important for virus-like particle (VLP) production and the regulation of genome replication/transcription. Accordingly, a mutation in the YIGL sequence caused defects in VLP production and genome replication/transcription. The residues I293 and L295 in the YIGL sequence were particularly critical for VLP production. Furthermore, an in silico analysis indicated that the amino acids surrounding the YIGL sequence contribute to intramolecular interactions within VP40. Among those surrounding residues, F209 was shown to be critical for VLP production. These results suggested that the VP40 YIGL sequence regulates two different viral replication steps, VLP production and genome replication/transcription, and the nearby residue F209 influences VLP production.
Asunto(s)
Ebolavirus/fisiología , Fiebre Hemorrágica Ebola/virología , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismo , Virión/fisiología , Replicación Viral , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Ebolavirus/química , Ebolavirus/genética , Genoma Viral , Humanos , Alineación de Secuencia , Proteínas de la Matriz Viral/genética , Virión/química , Virión/genética , Liberación del VirusRESUMEN
Although natural herbs have been a rich source of compounds for drug discovery, identification of bioactive components from natural herbs suffers from low efficiency and prohibitive cost of the conventional bioassay-based screening platforms. Here we develop a new strategy that integrates virtual screening, affinity mass spectrometry (MS) and targeted metabolomics for efficient discovery of herb-derived ligands towards a specific protein target site. Herb-based virtual screening conveniently selects herbs of potential bioactivity whereas affinity MS combined with targeted metabolomics readily screens candidate compounds in a high-throughput manner. This new integrated approach was benchmarked on screening chemical ligands that target the hydrophobic pocket of the nucleoprotein (NP) of Ebola viruses for which no small molecule ligands have been reported. Seven compounds identified by this approach from the crude extracts of three natural herbs were all validated to bind to the NP target in pure ligand binding assays. Among them, three compounds isolated from Piper nigrum (HJ-1, HJ-4 and HJ-6) strongly promoted the formation of large NP oligomers and reduced the protein thermal stability. In addition, cooperative binding between these chemical ligands and an endogenous peptide ligand was observed, and molecular docking was employed to propose a possible mechanism. Taken together, we established a platform integrating in silico and experimental screening approaches for efficient discovery of herb-derived bioactive ligands especially towards non-enzyme protein targets.
Asunto(s)
Productos Biológicos/metabolismo , Espectrometría de Masas/métodos , Metabolómica/métodos , Nucleoproteínas/metabolismo , Extractos Vegetales/metabolismo , Proteínas del Núcleo Viral/metabolismo , Sitios de Unión , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Descubrimiento de Drogas/métodos , Ebolavirus/química , Ligandos , Simulación del Acoplamiento Molecular , Proteínas de la Nucleocápside , Nucleoproteínas/química , Ophiopogon/química , Piper nigrum/química , Componentes Aéreos de las Plantas/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Unión Proteica , Salvia miltiorrhiza/química , Semillas/química , Proteínas del Núcleo Viral/químicaRESUMEN
The Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes severe hemorrhagic fever with a high fatality rate in humans. The EBOV encodes a glycoprotein that when cleaved, produces the delta peptide. Experimental evidence suggests that the delta peptide functions as a viroporin that enhances virus particle release through the host cell membrane. However, the viroporin forming mechanism of the delta peptide is still not well understood. Guided by experimental information, we have computationally investigated the pore formation by different oligomers of the delta peptide. We have performed all-atom molecular dynamics (MD) simulations in an explicit membrane environment to investigate the pore-forming mechanism and stability of the pores. Our results suggest that the delta peptide forms stable pentameric pores. In addition, the pore is selective with respect to chloride ions, and the disulfide bond formed between Cys-29 and Cys-38 in the C-terminal of the peptide is essential for the pore stabilization and ion permeation. Our study provides helpful information on the pore-forming mechanism of filovirus delta peptides and such structural information can be important in designing and developing molecular modulators that target the delta peptide pore and disrupt the pathology of the Ebola virus.