RESUMO
Highly transmissible Omicron variants of SARS-CoV-2 currently dominate globally. Here, we compare neutralization of Omicron BA.1, BA.1.1, and BA.2. BA.2 RBD has slightly higher ACE2 affinity than BA.1 and slightly reduced neutralization by vaccine serum, possibly associated with its increased transmissibility. Neutralization differences between sub-lineages for mAbs (including therapeutics) mostly arise from variation in residues bordering the ACE2 binding site; however, more distant mutations S371F (BA.2) and R346K (BA.1.1) markedly reduce neutralization by therapeutic antibody Vir-S309. In-depth structure-and-function analyses of 27 potent RBD-binding mAbs isolated from vaccinated volunteers following breakthrough Omicron-BA.1 infection reveals that they are focused in two main clusters within the RBD, with potent right-shoulder antibodies showing increased prevalence. Selection and somatic maturation have optimized antibody potency in less-mutated epitopes and recovered potency in highly mutated epitopes. All 27 mAbs potently neutralize early pandemic strains, and many show broad reactivity with variants of concern.
Assuntos
Anticorpos Monoclonais , Vacinas contra COVID-19/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Antivirais , COVID-19 , Vacinas contra COVID-19/administração & dosagem , Epitopos , Humanos , Testes de Neutralização , SARS-CoV-2/classificação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
The Omicron lineage of SARS-CoV-2, which was first described in November 2021, spread rapidly to become globally dominant and has split into a number of sublineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africa's Gauteng region uncovered two new sublineages, BA.4 and BA.5, which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences, and although closely related to BA.2, they contain further mutations in the receptor-binding domain of their spikes. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by the serum from individuals vaccinated with triple doses of AstraZeneca or Pfizer vaccine compared with BA.1 and BA.2. Furthermore, using the serum from BA.1 vaccine breakthrough infections, there are, likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.
Assuntos
COVID-19 , Vacinas Virais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Humanos , Testes de Neutralização , SARS-CoV-2/genética , África do SulRESUMO
The race to produce vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began when the first sequence was published, and this forms the basis for vaccines currently deployed globally. Independent lineages of SARS-CoV-2 have recently been reported: UK, B.1.1.7; South Africa, B.1.351; and Brazil, P.1. These variants have multiple changes in the immunodominant spike protein that facilitates viral cell entry via the angiotensin-converting enzyme-2 (ACE2) receptor. Mutations in the receptor recognition site on the spike are of great concern for their potential for immune escape. Here, we describe a structure-function analysis of B.1.351 using a large cohort of convalescent and vaccinee serum samples. The receptor-binding domain mutations provide tighter ACE2 binding and widespread escape from monoclonal antibody neutralization largely driven by E484K, although K417N and N501Y act together against some important antibody classes. In a number of cases, it would appear that convalescent and some vaccine serum offers limited protection against this variant.
Assuntos
Vacinas contra COVID-19/sangue , Vacinas contra COVID-19/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , COVID-19/imunologia , COVID-19/terapia , COVID-19/virologia , Chlorocebus aethiops , Ensaios Clínicos como Assunto , Células HEK293 , Humanos , Imunização Passiva , Modelos Moleculares , Mutação/genética , Testes de Neutralização , Ligação Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Células Vero , Soroterapia para COVID-19RESUMO
Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations, including P.1 from Brazil, B.1.351 from South Africa, and B.1.1.7 from the UK (12, 10, and 9 changes in the spike, respectively). All have mutations in the ACE2 binding site, with P.1 and B.1.351 having a virtually identical triplet (E484K, K417N/T, and N501Y), which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine-induced antibody responses than B.1.351, suggesting that changes outside the receptor-binding domain (RBD) impact neutralization. Monoclonal antibody (mAb) 222 neutralizes all three variants despite interacting with two of the ACE2-binding site mutations. We explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sítios de Ligação , COVID-19/terapia , COVID-19/virologia , Linhagem Celular , Humanos , Evasão da Resposta Imune , Imunização Passiva , Mutação , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/genética , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Vacinação , Vacinas/imunologia , Soroterapia para COVID-19RESUMO
Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the ßD-ßE loop and 110-112 in the ßF-ßG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.
Assuntos
Vírus da Febre Aftosa/genética , Febre Aftosa/virologia , Modelos Moleculares , Vírion/metabolismo , Animais , Proteínas do Capsídeo/metabolismo , Cricetinae , Heparitina Sulfato/metabolismo , Simulação de Acoplamento Molecular/métodos , Receptores Virais/metabolismoRESUMO
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.
Assuntos
Antivirais/química , Antivirais/metabolismo , Ebolavirus/química , Toremifeno/química , Toremifeno/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Antivirais/farmacologia , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Ebolavirus/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ibuprofeno/química , Ibuprofeno/metabolismo , Ibuprofeno/farmacologia , Ligantes , Marburgvirus/química , Fusão de Membrana/efeitos dos fármacos , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Temperatura , Toremifeno/farmacologia , Proteínas do Envelope Viral/antagonistas & inibidores , Ligação Viral/efeitos dos fármacosRESUMO
The most frequently mutated protein in human cancer is p53, a transcription factor (TF) that regulates myriad genes instrumental in diverse cellular outcomes including growth arrest and cell death. Cell context-dependent p53 modulation is critical for this life-or-death balance, yet remains incompletely understood. Here we identify sequence signatures enriched in genomic p53-binding sites modulated by the transcription cofactor iASPP. Moreover, our p53-iASPP crystal structure reveals that iASPP displaces the p53 L1 loop-which mediates sequence-specific interactions with the signature-corresponding base-without perturbing other DNA-recognizing modules of the p53 DNA-binding domain. A TF commonly uses multiple structural modules to recognize its cognate DNA, and thus this mechanism of a cofactor fine-tuning TF-DNA interactions through targeting a particular module is likely widespread. Previously, all tumor suppressors and oncoproteins that associate with the p53 DNA-binding domain-except the oncogenic E6 from human papillomaviruses (HPVs)-structurally cluster at the DNA-binding site of p53, complicating drug design. By contrast, iASPP inhibits p53 through a distinct surface overlapping the E6 footprint, opening prospects for p53-targeting precision medicine to improve cancer therapy.
Assuntos
DNA/genética , DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Repressoras/metabolismo , Elementos de Resposta , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , DNA/química , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Motivos de Nucleotídeos , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/química , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/químicaRESUMO
Hepatitis A virus (HAV) remains enigmatic, despite 1.4 million cases worldwide annually. It differs radically from other picornaviruses, existing in an enveloped form and being unusually stable, both genetically and physically, but has proved difficult to study. Here we report high-resolution X-ray structures for the mature virus and the empty particle. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, whereas the empty particle harbours only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions that might correspond to receptor-binding sites. Peptide scanning data extend the previously reported VP3 antigenic site, while structure-based predictions suggest further epitopes. HAV contains no pocket factor and can withstand remarkably high temperature and low pH, and empty particles are even more robust than full particles. The virus probably uncoats via a novel mechanism, being assembled differently to other picornaviruses. It utilizes a VP2 'domain swap' characteristic of insect picorna-like viruses, and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between 'modern' picornaviruses and the more 'primitive' precursor insect viruses; for instance, HAV retains the ability to move from cell-to-cell by transcytosis.
Assuntos
Evolução Molecular , Vírus da Hepatite A/química , Picornaviridae/química , Animais , Capsídeo/química , Proteínas do Capsídeo/química , Cristalografia por Raios X , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Insetos/virologia , Modelos Moleculares , Filogenia , Transcitose , Vírion/química , Internalização do VírusRESUMO
We report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.
Assuntos
Algoritmos , Cristalografia por Raios X/métodos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Vírus/ultraestrutura , Reprodutibilidade dos Testes , Tamanho da Amostra , Sensibilidade e EspecificidadeRESUMO
The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/ß-catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N-[2-(5-fluoro-1H-indol-3-yl)ethyl]acetamide that is structurally similar to melatonin came to our attention. We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high-resolution structures (≤1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid, and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µmol/L), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which upregulation of Wnt signalling may be beneficial.
Assuntos
Inibidores Enzimáticos/química , Esterases/antagonistas & inibidores , Esterases/química , Melatonina/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Relação Estrutura-AtividadeRESUMO
Hepatitis A virus (HAV) infects â¼1.4 million people annually and, although there is a vaccine, there are no licensed therapeutic drugs. HAV is unusually stable (making disinfection problematic) and little is known of how it enters cells and releases its RNA. Here we report a potent HAV-specific monoclonal antibody, R10, which neutralizes HAV infection by blocking attachment to the host cell. High-resolution cryo-EM structures of HAV full and empty particles and of the complex of HAV with R10 Fab reveal the atomic details of antibody binding and point to a receptor recognition site at the pentamer interface. These results, together with our observation that the R10 Fab destabilizes the capsid, suggest the use of a receptor mimic mechanism to neutralize virus infection, providing new opportunities for therapeutic intervention.
Assuntos
Anticorpos Neutralizantes/imunologia , Vírus da Hepatite A/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação/imunologia , Capsídeo/imunologia , Proteínas do Capsídeo/imunologia , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The quasi-envelopment of hepatitis A virus (HAV) capsids in exosome-like virions (eHAV) is an important but incompletely understood aspect of the hepatovirus life cycle. This process is driven by recruitment of newly assembled capsids to endosomal vesicles into which they bud to form multivesicular bodies with intraluminal vesicles that are later released at the plasma membrane as eHAV. The endosomal sorting complexes required for transport (ESCRT) are key to this process, as is the ESCRT-III-associated protein, ALIX, which also contributes to membrane budding of conventional enveloped viruses. YPX1or3L late domains in the structural proteins of these viruses mediate interactions with ALIX, and two such domains exist in the HAV VP2 capsid protein. Mutational studies of these domains are confounded by the fact that the Tyr residues (important for interactions of YPX1or3L peptides with ALIX) are required for efficient capsid assembly. However, single Leu-to-Ala substitutions within either VP2 YPX3L motif (L1-A and L2-A mutants) were well tolerated, albeit associated with significantly reduced eHAV release. In contrast, simultaneous substitutions in both motifs (L1,2-A) eliminated virus release but did not inhibit assembly of infectious intracellular particles. Immunoprecipitation experiments suggested that the loss of eHAV release was associated with a loss of ALIX recruitment. Collectively, these data indicate that HAV YPX3L motifs function as redundant late domains during quasi-envelopment and viral release. Since these motifs present little solvent-accessible area in the crystal structure of the naked extracellular capsid, the capsid structure may be substantially different during quasi-envelopment.IMPORTANCE Nonlytic release of hepatitis A virus (HAV) as exosome-like quasi-enveloped virions is a unique but incompletely understood aspect of the hepatovirus life cycle. Several lines of evidence indicate that the host protein ALIX is essential for this process. Tandem YPX3L "late domains" in the VP2 capsid protein could be sites of interaction with ALIX, but they are not accessible on the surface of an X-ray model of the extracellular capsid, raising doubts about this putative late domain function. Here, we describe YPX3L domain mutants that assemble capsids normally but fail to bind ALIX and be secreted as quasi-enveloped eHAV. Our data support late domain function for the VP2 YPX3L motifs and raise questions about the structure of the HAV capsid prior to and following quasi-envelopment.
Assuntos
Motivos de Aminoácidos , Proteínas do Capsídeo/metabolismo , Capsídeo/fisiologia , Carcinoma Hepatocelular/metabolismo , Vírus da Hepatite A/fisiologia , Vírion/fisiologia , Replicação Viral , Substituição de Aminoácidos , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos , Hepatite A/genética , Hepatite A/metabolismo , Hepatite A/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Corpos Multivesiculares , Mutação , Conformação Proteica , Células Tumorais Cultivadas , Liberação de VírusRESUMO
Foot-and-mouth disease virus (FMDV) belongs to the Aphthovirus genus of the Picornaviridae, a family of small, icosahedral, non-enveloped, single-stranded RNA viruses. It is a highly infectious pathogen and is one of the biggest hindrances to the international trade of animals and animal products. FMDV capsids (which are unstable below pH6.5) release their genome into the host cell from an acidic compartment, such as that of an endosome, and in the process dissociate into pentamers. Whilst other members of the family (enteroviruses) have been visualized to form an expanded intermediate capsid with holes from which inner capsid proteins (VP4), N-termini (VP1) and RNA can be released, there has been no visualization of any such state for an aphthovirus, instead the capsid appears to simply dissociate into pentamers. Here we present the 8-Å resolution structure of isolated dissociated pentamers of FMDV, lacking VP4. We also found these pentamers to re-associate into a rigid, icosahedrally symmetric assembly, which enabled their structure to be solved at higher resolution (5.2 Å). In this assembly, the pentamers unexpectedly associate 'inside out', but still with their exposed hydrophobic edges buried. Stabilizing interactions occur between the HI loop of VP2 and its symmetry related partners at the icosahedral 3-fold axes, and between the BC and EF loops of VP3 with the VP2 ßB-strand and the CD loop at the 2-fold axes. A relatively extensive but subtle structural rearrangement towards the periphery of the dissociated pentamer compared to that in the mature virus provides insight into the mechanism of dissociation of FMDV and the marked difference in antigenicity.
Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Vírus da Febre Aftosa/química , Vírion/química , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Vírus da Febre Aftosa/metabolismo , Modelos Moleculares , RNA Viral/metabolismo , Vírion/genética , Vírion/metabolismoRESUMO
Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for the potential use of such mutants in improving the stability of SAT2 vaccines in countries where FMD is endemic, which rely heavily on the maintenance of the cold chain, with potential improvement to the duration of immune responses.
Assuntos
Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/fisiologia , Vacinas Virais/genética , Substituição de Aminoácidos , Animais , Vírus da Febre Aftosa/imunologia , Instabilidade Genômica , Concentração de Íons de Hidrogênio , Imunogenicidade da Vacina , Íons , Cinética , Mutação , Sorogrupo , Cloreto de Sódio/farmacologia , Temperatura , Potência de Vacina , Vacinas Virais/químicaRESUMO
The replication of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), which are the major cause of hand, foot and mouth disease (HFMD) in children, can be inhibited by the capsid binder GPP3. Here, we present the crystal structure of CVA16 in complex with GPP3, which clarifies the role of the key residues involved in interactions with the inhibitor. Based on this model, in silico docking was performed to investigate the interactions with the two next-generation capsid binders NLD and ALD, which we show to be potent inhibitors of a panel of enteroviruses with potentially interesting pharmacological properties. A meta-analysis was performed using the available structural information to obtain a deeper insight into those structural features required for capsid binders to interact effectively and also those that confer broad-spectrum anti-enterovirus activity.
Assuntos
Antivirais/farmacologia , Enterovirus Humano A/química , Enterovirus Humano A/efeitos dos fármacos , Enterovirus Humano A/ultraestrutura , Modelos Moleculares , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Infecções por Coxsackievirus/prevenção & controle , Cristalografia por Raios X , HumanosRESUMO
UNLABELLED: Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) that affect more than a million children in China each year and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, the development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The structural details of the expanded forms of the capsids, which possess nonnative antigenicity, are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. Here, we remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant viruslike particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilize the native antigenic state that is different from the one used in a natural virus infection. As expected, the mature CVA16 virus is similar to EV71; however, structural and immunogenic comparisons highlight differences that may have implications for vaccine production. IMPORTANCE: Hand-foot-and-mouth disease is a serious public health threat to children in Asian-Pacific countries, resulting in millions of cases. EV71 and CVA16 are the two dominant causative agents of the disease that, while usually mild, can cause severe neurological complications, leading to hundreds of deaths. EV71 vaccines do not provide protection against CVA16. A CVA16 vaccine or bivalent EV71/CVA16 vaccine is therefore urgently needed. We report atomic structures for the mature CVA16 virus, a natural empty particle, and a recombinant CVA16 virus-like particle that does not contain the viral genome. All three particles have similar structures and identical antigenicity. The recombinant particles, produced in insect cells (a system suitable for making vaccine antigen), are stabilized by recruiting from the insect cells a small molecule that is different from that used by the virus in a normal infection. We present structural and immunogenic comparisons with EV71 to facilitate structure-based drug design and vaccine development.
Assuntos
Antígenos Virais/química , Proteínas do Capsídeo/química , Capsídeo/química , Enterovirus Humano A/química , Enterovirus/química , Vírion/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Antígenos Virais/genética , Antígenos Virais/imunologia , Baculoviridae/genética , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Chlorocebus aethiops , Cristalografia por Raios X , Enterovirus/genética , Enterovirus/imunologia , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Células Vero , Vírion/genética , Vírion/imunologiaRESUMO
Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.
Assuntos
Engenharia Biomédica/métodos , Febre Aftosa/prevenção & controle , Picornaviridae/genética , Vacinas Virais/biossíntese , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Chlorocebus aethiops , Cristalografia por Raios X , Febre Aftosa/imunologia , Vetores Genéticos/química , Vetores Genéticos/genética , Células HEK293 , Humanos , Modelos Moleculares , Picornaviridae/metabolismo , Picornaviridae/fisiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Vacinação/efeitos adversos , Vacinação/métodos , Vacinação/veterinária , Vaccinia virus/química , Vaccinia virus/genética , Vaccinia virus/imunologia , Vacinas Virais/efeitos adversos , Vacinas Virais/química , Vacinas Virais/genéticaRESUMO
A departure from a linear or an exponential intensity decay in the diffracting power of protein crystals as a function of absorbed dose is reported. The observation of a lag phase raises the possibility of collecting significantly more data from crystals held at room temperature before an intolerable intensity decay is reached. A simple model accounting for the form of the intensity decay is reintroduced and is applied for the first time to high frame-rate room-temperature data collection.
Assuntos
Cristalografia por Raios X/métodos , Cristalografia por Raios X/instrumentação , Enterovirus Bovino/química , Vírus da Febre Aftosa/química , Modelos Teóricos , Proteínas/química , TemperaturaRESUMO
The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form ß-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6â Å resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.
Assuntos
Regiões Determinantes de Complementaridade , Fragmentos Fab das Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina , Modelos Moleculares , Animais , Bovinos , Cadeias Pesadas de Imunoglobulinas/química , Cristalografia por Raios X , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Regiões Determinantes de Complementaridade/química , Fragmentos Fab das Imunoglobulinas/química , Sequência de Aminoácidos , Conformação ProteicaRESUMO
BA.2.87.1 represents a major shift in the BA.2 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is unusual in having two lengthy deletions of polypeptide in the spike (S) protein, one of which removes a beta-strand. Here we investigate its neutralization by a variety of sera from infected and vaccinated individuals and determine its spike (S) ectodomain structure. The BA.2.87.1 receptor binding domain (RBD) is structurally conserved and the RBDs are tightly packed in an "all-down" conformation with a small rotation relative to the trimer axis as compared to the closest previously observed conformation. The N-terminal domain (NTD) maintains a remarkably similar structure overall; however, the rearrangements resulting from the deletions essentially destroy the so-called supersite epitope and eliminate one glycan site, while a mutation creates an additional glycan site, effectively shielding another NTD epitope. BA.2.87.1 is relatively easily neutralized but acquisition of additional mutations in the RBD could increase antibody escape allowing it to become a dominant sub-lineage.