RESUMEN
The human monoclonal antibody (HmAb) C10 potently cross-neutralizes Zika virus (ZIKV) and dengue virus. Analysis of antibody fragment (Fab) C10 interactions with ZIKV and dengue virus serotype 2 (DENV2) particles by cryoelectron microscopy (cryo-EM) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS) shows that Fab C10 binding decreases overall ZIKV particle dynamics, whereas with DENV2, the same Fab causes increased dynamics. Testing of different Fab C10:DENV2 E protein molar ratios revealed that, at higher Fab ratios, especially at saturated concentrations, the Fab enhanced viral dynamics (detected by HDXMS), and observation under cryo-EM showed increased numbers of distorted particles. Our results suggest that Fab C10 stabilizes ZIKV but that with DENV2 particles, high Fab C10 occupancy promotes E protein dimer conformational changes leading to overall increased particle dynamics and distortion of the viral surface. This is the first instance of a broadly neutralizing antibody eliciting virus-specific increases in whole virus particle dynamics.
Asunto(s)
Anticuerpos Neutralizantes , Virus del Dengue , Dengue , Proteínas del Envoltorio Viral , Infección por el Virus Zika , Virus Zika , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Dengue/inmunología , Dengue/virología , Virus del Dengue/inmunología , Virus del Dengue/fisiología , Humanos , Unión Proteica , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Virus Zika/inmunología , Virus Zika/fisiología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Zika virus (ZIKV), a mosquito-borne flavivirus, causes devastating congenital birth defects. We isolated a human monoclonal antibody (mAb), ZKA190, that potently cross-neutralizes multi-lineage ZIKV strains. ZKA190 is highly effective in vivo in preventing morbidity and mortality of ZIKV-infected mice. NMR and cryo-electron microscopy show its binding to an exposed epitope on DIII of the E protein. ZKA190 Fab binds all 180 E protein copies, altering the virus quaternary arrangement and surface curvature. However, ZIKV escape mutants emerged in vitro and in vivo in the presence of ZKA190, as well as of other neutralizing mAbs. To counter this problem, we developed a bispecific antibody (FIT-1) comprising ZKA190 and a second mAb specific for DII of E protein. In addition to retaining high in vitro and in vivo potencies, FIT-1 robustly prevented viral escape, warranting its development as a ZIKV immunotherapy.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Infección por el Virus Zika/terapia , Virus Zika/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/administración & dosificación , Anticuerpos Antivirales/química , Microscopía por Crioelectrón , Epítopos , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Alineación de Secuencia , Proteínas del Envoltorio Viral/química , Virus Zika/inmunologíaRESUMEN
We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/genética , Epítopos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Subunidades de Proteína , Microscopía por Crioelectrón , Proteínas del Envoltorio Viral/genética , Anticuerpos MonoclonalesRESUMEN
Different strains within a dengue serotype (DENV1-4) can have smooth, or "bumpy" surface morphologies with different antigenic characteristics at average body temperature (37°C). We determined the neutralizing properties of a serotype cross-reactive human monoclonal antibody (HMAb) 1C19 for strains with differing morphologies within the DENV1 and DENV2 serotypes. We mapped the 1C19 epitope to E protein domain II by hydrogen deuterium exchange mass spectrometry, cryoEM and molecular dynamics simulations, revealing that this epitope is likely partially hidden on the virus surface. We showed the antibody has high affinity for binding to recombinant DENV1 E proteins compared to those of DENV2, consistent with its strong neutralizing activities for all DENV1 strains tested regardless of their morphologies. This finding suggests that the antibody could out-compete E-to-E interaction for binding to its epitope. In contrast, for DENV2, HMAb 1C19 can only neutralize when the epitope becomes exposed on the bumpy-surfaced particle. Although HMAb 1C19 is not a suitable therapeutic candidate, this study with HMAb 1C19 shows the importance of choosing a high-affinity antibody that could neutralize diverse dengue virus morphologies for therapeutic purposes.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Epítopos/inmunología , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Dengue/virología , Virus del Dengue/química , Virus del Dengue/metabolismo , Epítopos/metabolismo , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , SerogrupoRESUMEN
Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Aedes , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/ultraestructura , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/uso terapéutico , Anticuerpos Antivirales/ultraestructura , Sitios de Unión/efectos de los fármacos , Fiebre Chikungunya/inmunología , Fiebre Chikungunya/virología , Virus Chikungunya/inmunología , Chlorocebus aethiops , Microscopía por Crioelectrón , Modelos Animales de Enfermedad , Humanos , Inmunoglobulinas/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Células Vero , Proteínas Virales de Fusión/antagonistas & inhibidores , Proteínas Virales de Fusión/inmunología , Acoplamiento Viral/efectos de los fármacosRESUMEN
Zika virus (ZIKV), formerly a neglected pathogen, has recently been associated with microcephaly in fetuses, and with Guillian-Barré syndrome in adults. Here we present the 3.7 Å resolution cryo-electron microscopy structure of ZIKV, and show that the overall architecture of the virus is similar to that of other flaviviruses. Sequence and structural comparisons of the ZIKV envelope (E) protein with other flaviviruses show that parts of the E protein closely resemble the neurovirulent West Nile and Japanese encephalitis viruses, while others are similar to dengue virus (DENV). However, the contribution of the E protein to flavivirus pathobiology is currently not understood. The virus particle was observed to be structurally stable even when incubated at 40 °C, in sharp contrast to the less thermally stable DENV. This is also reflected in the infectivity of ZIKV compared to DENV serotypes 2 and 4 (DENV2 and DENV4) at different temperatures. The cryo-electron microscopy structure shows a virus with a more compact surface. This structural stability of the virus may help it to survive in the harsh conditions of semen, saliva and urine. Antibodies or drugs that destabilize the structure may help to reduce the disease outcome or limit the spread of the virus.
Asunto(s)
Temperatura , Virión/química , Virión/ultraestructura , Virus Zika/química , Virus Zika/ultraestructura , Microscopía por Crioelectrón , Virus del Dengue/química , Virus del Dengue/clasificación , Virus del Dengue/patogenicidad , Virus de la Encefalitis Japonesa (Especie)/química , Humanos , Modelos Moleculares , Estabilidad Proteica , Saliva/virología , Semen/virología , Orina/virología , Proteínas del Envoltorio Viral/química , Virión/patogenicidad , Virus del Nilo Occidental/química , Virus Zika/patogenicidadRESUMEN
The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to "bumpy" surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become "bumpy". These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus.
Asunto(s)
Virus del Dengue/clasificación , Virus del Dengue/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos Virales/química , Antígenos Virales/genética , Línea Celular , Microscopía por Crioelectrón , Virus del Dengue/ultraestructura , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Dominios y Motivos de Interacción de Proteínas , Homología de Secuencia de Aminoácido , Serogrupo , Temperatura , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
With severe disease manifestations including microcephaly, congenital malformation, and Guillain-Barré syndrome, Zika virus (ZIKV) remains a persistent global public health threat. Despite antigenic similarities with dengue viruses, structural studies have suggested the extended CD-loop and hydrogen-bonding interaction network within the ZIKV envelope protein contribute to stability differences between the viral families. This enhanced stability may lead to the augmented infection, disease manifestation, and persistence in body fluids seen following ZIKV infection. To examine the role of these motifs in infection, we generated a series of ZIKV recombinant viruses that disrupted the hydrogen-bonding network (350A, 351A, and 350A/351A) or the CD-loop extension (Δ346). Our results demonstrate a key role for the ZIKV extended CD-loop in cell-type-dependent replication, virion stability, and in vivo pathogenesis. Importantly, the Δ346 mutant maintains similar antigenicity to wild-type virus, opening the possibility for its use as a live-attenuated vaccine platform for ZIKV and other clinically relevant flaviviruses.
Asunto(s)
Dominios y Motivos de Interacción de Proteínas , Proteínas del Envoltorio Viral/química , Infección por el Virus Zika/virología , Virus Zika/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Epítopos/inmunología , Femenino , Humanos , Enlace de Hidrógeno , Masculino , Ratones , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Relación Estructura-Actividad , Temperatura , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Replicación Viral , Virus Zika/patogenicidad , Virus Zika/ultraestructura , Infección por el Virus Zika/inmunologíaRESUMEN
Sulfolobus turreted icosahedral virus (STIV) was isolated in acidic hot springs where it infects the archeon Sulfolobus solfataricus. We determined the STIV structure using near-atomic resolution electron microscopy and X-ray crystallography allowing tracing of structural polypeptide chains and visualization of transmembrane proteins embedded in the viral membrane. We propose that the vertex complexes orchestrate virion assembly by coordinating interactions of the membrane and various protein components involved. STIV shares the same coat subunit and penton base protein folds as some eukaryotic and bacterial viruses, suggesting that they derive from a common ancestor predating the divergence of the three kingdoms of life. One architectural motif (ß-jelly roll fold) forms virtually the entire capsid (distributed in three different gene products), indicating that a single ancestral protein module may have been at the origin of its evolution.
Asunto(s)
Proteínas de la Membrana/química , Modelos Moleculares , Rudiviridae/química , Sulfolobus/virología , Microscopía por Crioelectrón , Cristalografía por Rayos XRESUMEN
2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H2O2. We show that the E. coli AhpC (EcAhpC, 187 residues) forms a decameric ring structure under reduced and close to physiological conditions, composed of five catalytic dimers. Single particle analysis of cryo-electron micrographs of C-terminal truncated (EcAhpC1 -172 and EcAhpC1 -182) and mutated forms of EcAhpC reveals the loss of decamer formation, indicating the importance of the very C-terminus of AhpC in dimer to decamer transition. The crystallographic structures of the truncated EcAhpC1 -172 and EcAhpC1 -182 demonstrate for the first time that, in contrast to the reduced form, the very C-terminus of the oxidized EcAhpC is oriented away from the AhpC dimer interface and away from the catalytic redox-center, reflecting structural rearrangements during redox-modulation and -oligomerization. Furthermore, using an ensemble of different truncated and mutated EcAhpC protein constructs the importance of the very C-terminus in AhpC activity and in AhpC-AhpF assembly has been demonstrated.
Asunto(s)
Proteínas de Escherichia coli/química , Estrés Oxidativo , Peroxirredoxinas/química , Biocatálisis , Microscopía por Crioelectrón , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutación , NAD/química , NAD/metabolismo , Oxidación-Reducción , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , EspectrofotometríaRESUMEN
Dengue virus (DENV), a mosquito-borne virus, is responsible for millions of cases of infections worldwide. There are four DENV serotypes (DENV1 to -4). After a primary DENV infection, the antibodies elicited confer lifetime protection against that DENV serotype. However, in a secondary infection with another serotype, the preexisting antibodies may cause antibody-dependent enhancement (ADE) of infection of macrophage cells, leading to the development of the more severe form of disease, dengue hemorrhagic fever. Thus, a safe vaccine should stimulate protection against all dengue serotypes simultaneously. To facilitate the development of a vaccine, good knowledge of different DENV serotype structures is crucial. Structures of DENV1 and DENV2 had been solved previously. Here we present a near-atomic resolution cryo-electron microscopy (cryo-EM) structure of mature DENV4. Comparison of the DENV4 structure with similar-resolution cryo-EM structures of DENV1 and DENV2 showed differences in surface charge distribution, which may explain their differences in binding to cellular receptors, such as heparin. Also, observed variations in amino acid residues involved in interactions between envelope and membrane proteins on the virus surface correlate with their ability to undergo structural changes at higher temperatures.
Asunto(s)
Microscopía por Crioelectrón/métodos , Virus del Dengue/ultraestructura , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunologíaRESUMEN
Dengue virus is a major human pathogen that has four serotypes (DENV1 to -4). Here we report the cryoelectron microscopy (cryo-EM) structures of immature and mature DENV1 at 6- and 4.5-Å resolution, respectively. The subnanometer-resolution maps allow accurate placement of all of the surface proteins. Although the immature and mature viruses showed vastly different surface protein organizations, the envelope protein transmembrane (E-TM) regions remain in similar positions. The pivotal role of the E-TM regions leads to the identification of the start and end positions of all surface proteins during maturation.
Asunto(s)
Virus del Dengue/crecimiento & desarrollo , Virus del Dengue/genética , Virus del Dengue/ultraestructura , Modelos Moleculares , Conformación Proteica , Proteínas Virales/metabolismo , Microscopía por Crioelectrón , Serotipificación , Proteínas Virales/químicaRESUMEN
Previous binding studies of antibodies that recognized a partially or fully hidden epitope suggest that insect cell-derived dengue virus undergoes structural changes at an elevated temperature. This was confirmed by our cryo-electron microscopy images of dengue virus incubated at 37°C, where viruses change their surface from smooth to rough. Here we present the cryo-electron microscopy structures of dengue virus at 37°C. Image analysis showed four classes of particles. The three-dimensional (3D) map of one of these classes, representing half of the imaged virus population, shows that the E protein shell has expanded and there is a hole at the 3-fold vertices. Fitting E protein structures into the map suggests that all of the interdimeric and some intradimeric E protein interactions are weakened. The accessibility of some previously found cryptic epitopes on this class of particles is discussed.
Asunto(s)
Virus del Dengue/química , Virus del Dengue/ultraestructura , Modelos Moleculares , Conformación Proteica , Temperatura , Proteínas del Envoltorio Viral/ultraestructura , Animales , Línea Celular , Microscopía por Crioelectrón , Culicidae , Electroforesis en Gel de Poliacrilamida , Colorantes de RosanilinaRESUMEN
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern such as Omicron hampered efforts in controlling the ongoing coronavirus disease 2019 pandemic due to their ability to escape neutralizing antibodies induced by vaccination or prior infection, highlighting the need to develop broad-spectrum vaccines and therapeutics. Most human monoclonal antibodies (mAbs) reported to date have not demonstrated true pan-sarbecovirus neutralizing breadth especially against animal sarbecoviruses. Here, we report the isolation and characterization of highly potent mAbs targeting the receptor binding domain (RBD) of huACE2-dependent sarbecovirus from a SARS-CoV survivor vaccinated with BNT162b2. Among the six mAbs identified, one (E7) showed better huACE2-dependent sarbecovirus neutralizing potency and breadth than any other mAbs reported to date. Mutagenesis and cryo-electron microscopy studies indicate that these mAbs have a unique RBD contact footprint and that E7 binds to a quaternary structure-dependent epitope.
Asunto(s)
COVID-19 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Animales , Humanos , Anticuerpos Antivirales , Pruebas de Neutralización , Vacuna BNT162 , Anticuerpos Monoclonales/química , Microscopía por Crioelectrón , COVID-19/prevención & control , SARS-CoV-2RESUMEN
Dengue virus infection can cause dengue hemorrhagic fever (DHF). Dengue NS1 is multifunctional. The intracellular dimeric NS1 (iNS1) forms part of the viral replication complex. Previous studies suggest the extracellular secreted NS1 (sNS1), which is a major factor contributing to DHF, exists as hexamers. The structure of the iNS1 is well-characterised but not that of sNS1. Here we show by cryoEM that the recombinant sNS1 exists in multiple oligomeric states: the tetrameric (stable and loose conformation) and hexameric structures. Stability of the stable and loose tetramers is determined by the conformation of their N-terminal domain - elongated ß-sheet or ß-roll. Binding of an anti-NS1 Fab breaks the loose tetrameric and hexameric sNS1 into dimers, whereas the stable tetramer remains largely unbound. Our results show detailed quaternary organization of different oligomeric states of sNS1 and will contribute towards the design of dengue therapeutics.
Asunto(s)
Virus del Dengue , Dengue , Síndrome de Noonan , Dengue Grave , Humanos , Virus del Dengue/metabolismo , Proteínas no Estructurales Virales/químicaRESUMEN
Structures of flavivirus (dengue virus and Zika virus) particles are known to near-atomic resolution and show detailed structure and arrangement of their surface proteins (E and prM in immature virus or M in mature virus). By contrast, the arrangement of the capsid proteins:RNA complex, which forms the core of the particle, is poorly understood, likely due to inherent dynamics. Here, we stabilize immature Zika virus via an antibody that binds across the E and prM proteins, resulting in a subnanometer resolution structure of capsid proteins within the virus particle. Fitting of the capsid protein into densities shows the presence of a helix previously thought to be removed via proteolysis. This structure illuminates capsid protein quaternary organization, including its orientation relative to the lipid membrane and the genomic RNA, and its interactions with the transmembrane regions of the surface proteins. Results show the capsid protein plays a central role in the flavivirus assembly process.
Asunto(s)
Proteínas de la Cápside/metabolismo , Ensamble de Virus , Infección por el Virus Zika/virología , Virus Zika/fisiología , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Humanos , ARN Viral/genética , ARN Viral/metabolismo , Virus Zika/química , Virus Zika/genéticaRESUMEN
Human antibody SIgN-3C neutralizes dengue virus (DENV) and Zika virus (ZIKV) differently. DENV:SIgN-3C Fab and ZIKV:SIgN-3C Fab cryoelectron microscopy (cryo-EM) complex structures show Fabs crosslink E protein dimers at extracellular pH 8.0 condition and also when further incubated at acidic endosomal conditions (pH 8.0-6.5). We observe Fab binding to DENV (pH 8.0-5.0) prevents virus fusion, and the number of bound Fabs increase (from 120 to 180). For ZIKV, although there are already 180 copies of Fab at pH 8.0, virus structural changes at pH 5.0 are not inhibited. The immunoglobulin G (IgG):DENV structure at pH 8.0 shows both Fab arms bind to epitopes around the 2-fold vertex. On ZIKV, an additional Fab around the 5-fold vertex at pH 8.0 suggests one IgG arm would engage with an epitope, although the other may bind to other viruses, causing aggregation. For DENV2 at pH 5.0, a similar scenario would occur, suggesting DENV2:IgG complex would aggregate in the endosome. Hence, a single antibody employs different neutralization mechanisms against different flaviviruses.
Asunto(s)
Flavivirus/patogenicidad , Pruebas de Neutralización/métodos , HumanosRESUMEN
Previous flavivirus (dengue and Zika viruses) studies showed largely spherical particles either with smooth or bumpy surfaces. Here, we demonstrate flavivirus particles have high structural plasticity by the induction of a non-spherical morphology at elevated temperatures: the club-shaped particle (clubSP), which contains a cylindrical tail and a disc-like head. Complex formation of DENV and ZIKV with Fab C10 stabilize the viruses allowing cryoEM structural determination to ~10 Å resolution. The caterpillar-shaped (catSP) Fab C10:ZIKV complex shows Fabs locking the E protein raft structure containing three E dimers. However, compared to the original spherical structure, the rafts have rotated relative to each other. The helical tail structure of Fab C10:DENV3 clubSP showed although the Fab locked an E protein dimer, the dimers have shifted laterally. Morphological diversity, including clubSP and the previously identified bumpy and smooth-surfaced spherical particles, may help flavivirus survival and immune evasion.
Asunto(s)
Anticuerpos Antivirales/metabolismo , Virus del Dengue/ultraestructura , Proteínas del Envoltorio Viral/metabolismo , Virus Zika/ultraestructura , Aedes , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/inmunología , Línea Celular , Microscopía por Crioelectrón , Dengue/inmunología , Dengue/terapia , Dengue/virología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Virus del Dengue/metabolismo , Evasión Inmune , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Mesocricetus , Multimerización de Proteína , Propiedades de Superficie , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/ultraestructura , Acoplamiento Viral , Virus Zika/inmunología , Virus Zika/metabolismo , Infección por el Virus ZikaRESUMEN
Dengue virus (DENV) particles are released from cells in different maturation states. Fully immature DENV (immDENV) is generally non-infectious, but can become infectious when complexed with anti-precursor membrane (prM) protein antibodies. It is unknown how anti-prM antibody-coated particles can undergo membrane fusion since the prM caps the envelope (E) protein fusion loop. Here, we determined cryoelectron microscopy (cryo-EM) maps of the immDENV:anti-prM complex at different pH values, mimicking the extracellular (pH 8.0) or endosomal (pH 5.0) environments. At pH 5.0, there are two structural classes with fewer antibodies bound than at pH 8.0. These classes may represent different maturation states. Molecular simulations, together with the measured high-affinity pr:antibody interaction (versus the weak pr:E interaction) and also the low pH cryo-EM structures, suggest how antibody:pr complex can dislodge from the E protein at low pH. This exposes the E protein fusion loop enhancing virus interaction with endosomes.
Asunto(s)
Virus del Dengue/fisiología , Endosomas/virología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Proteínas Virales/inmunología , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Línea Celular , Microscopía por Crioelectrón , Virus del Dengue/química , Virus del Dengue/inmunología , Endosomas/química , Endosomas/inmunología , Humanos , Concentración de Iones de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/química , Modelos Moleculares , Células THP-1 , Acoplamiento ViralRESUMEN
The rapid spread of Zika virus (ZIKV), which causes microcephaly and Guillain-Barré syndrome, signals an urgency to identify therapeutics. Recent efforts to rescreen dengue virus human antibodies for ZIKV cross-neutralization activity showed antibody C10 as one of the most potent. To investigate the ability of the antibody to block fusion, we determined the cryoEM structures of the C10-ZIKV complex at pH levels mimicking the extracellular (pH8.0), early (pH6.5) and late endosomal (pH5.0) environments. The 4.0 Å resolution pH8.0 complex structure shows that the antibody binds to E proteins residues at the intra-dimer interface, and the virus quaternary structure-dependent inter-dimer and inter-raft interfaces. At pH6.5, antibody C10 locks all virus surface E proteins, and at pH5.0, it locks the E protein raft structure, suggesting that it prevents the structural rearrangement of the E proteins during the fusion event-a vital step for infection. This suggests antibody C10 could be a good therapeutic candidate.