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
Enveloped viruses enter cells by inducing fusion of viral and cellular membranes, a process catalyzed by a specialized membrane-fusion protein expressed on their surface. This review focuses on recent structural studies of viral fusion proteins with an emphasis on their metastable prefusion form and on interactions with neutralizing antibodies. The fusion glycoproteins have been difficult to study because they are present in a labile, metastable form at the surface of infectious virions. Such metastability is a functional requirement, allowing these proteins to refold into a lower energy conformation while transferring the difference in energy to catalyze the membrane fusion reaction. Structural studies have shown that stable immunogens presenting the same antigenic sites as the labile wild-type proteins efficiently elicit potently neutralizing antibodies, providing a framework with which to engineer the antigens for stability, as well as identifying key vulnerability sites that can be used in next-generation subunit vaccine design.
Asunto(s)
Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Virosis/inmunología , Virus/inmunología , Animales , Diseño de Fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/metabolismo , Virosis/virología , Virus/metabolismoRESUMEN
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
There are no vaccines or therapies to treat Epstein-Barr virus (EBV). Snijder et al. (2018) isolated a potent human antibody against EBV that blocks infection of both B cells and epithelial cells. Structural analysis of the antibody complexed with a viral surface protein complex identified a site that may be useful in vaccine development.
Asunto(s)
Anticuerpos , Herpesvirus Humano 4 , Linfocitos B , Células Epiteliales , HumanosRESUMEN
The flavivirus genus consists of several major human pathogens including dengue (DENV) and Zika viruses. The flavivirus nonstructural protein 1 (NS1) plays an important role in disease progression, for example, in the development of severe dengue disease. Anti-NS1 antibodies have been shown to confer protection, and two new studies by Biering et al. and Modhiran et al. on the structure of NS1:antibody complexes reveal their mechanism of neutralization.
Asunto(s)
Virus del Dengue , Dengue , Flavivirus , Infección por el Virus Zika , Virus Zika , Anticuerpos Antivirales , Virus del Dengue/inmunología , Humanos , Proteínas no Estructurales ViralesRESUMEN
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
UNLABELLED: Severe dengue virus (DENV)-associated diseases can occur in patients who have preexisting DENV antibodies (Abs) through antibody-dependent enhancement (ADE) of infection. It is well established that during ADE, DENV-antibody immune complexes (ICs) infect Fcγ receptor-bearing cells and increase the systemic viral burden that can be measured in the blood. For protection against infection with DENV serotypes 1 to 4, strongly neutralizing Abs must be elicited to overcome the effect of ADE. Clinical observations in infants who have maternal DENV Abs or recent phase II/III clinical trials with a leading tetravalent dengue vaccine suggested a lack of correlation between Ab neutralization and in vivo disease prevention. In addressing this gap in knowledge, we found that inoculation of ICs formed with serotype cross-reactive Abs that are more than 98% neutralized in vitro promotes high mortality in AG129 mice even though peak viremia was lower than that in direct virus infection. This suggests that the serum viremia level is not always correlated with disease severity. We further demonstrated that infection with the ICs resulted in increased vascular permeability, specifically in the small intestine, accompanied with increased tissue viral load and cytokine production, which can be suppressed by anti-tumor necrosis factor alpha (anti-TNF-α) Abs. Flow cytometric analysis identified increased infection in CD11b(int) CD11c(int/hi) CD103(-) antigen-presenting cells by IC inoculation, suggesting that these infected cells may be responsible for the increase in TNF-α production and vascular permeability in the small intestine that lead to mortality in mice. Our findings may have important implications for the development of dengue therapeutics. IMPORTANCE: We examined the relationship between the neutralizing level of Abs at the time of infection and subsequent disease progression in a mouse model in order to understand why patients who are shown to have a neutralizing quantity of Abs still allow sufficient DENV replication to induce severe dengue manifestations, which sometimes do not correlate with viremia level. Strikingly, we found that high mortality was induced in AG129 mice by the increase in TNF-α-induced vascular permeability accompanied by an increased viral load, specifically in the small intestine, even when the initial infection level is suppressed to less than 5% and the peak viremia level is not enhanced. This suggests that ADE overcomes the protective efficacy of Abs in a tissue-dependent manner that leads to severe small intestinal pathology. Our findings may serve to address the pathogenic role of Abs on severe dengue disease and also help to develop safe Ab-based therapeutic strategies.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Virus del Dengue/inmunología , Dengue/inmunología , Dengue/patología , Intestino Delgado/patología , Viremia , Animales , Acrecentamiento Dependiente de Anticuerpo , Permeabilidad Capilar , Citocinas/metabolismo , Dengue/mortalidad , Modelos Animales de Enfermedad , Intestino Delgado/virología , Subgrupos Linfocitarios/virología , Ratones , Análisis de Supervivencia , Carga ViralRESUMEN
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
RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7)GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.