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1.
Proc Natl Acad Sci U S A ; 118(48)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34782481

RESUMO

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in ß-propiolactone-inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.


Assuntos
SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus/química , Motivos de Aminoácidos , Animais , Chlorocebus aethiops , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Glicosilação , Domínios Proteicos , Multimerização Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
2.
Natl Sci Rev ; 8(3): nwaa297, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34676096

RESUMO

Receptor recognition and subsequent membrane fusion are essential for the establishment of successful infection by SARS-CoV-2. Halting these steps can cure COVID-19. Here we have identified and characterized a potent human monoclonal antibody, HB27, that blocks SARS-CoV-2 attachment to its cellular receptor at sub-nM concentrations. Remarkably, HB27 can also prevent SARS-CoV-2 membrane fusion. Consequently, a single dose of HB27 conferred effective protection against SARS-CoV-2 in two established mouse models. Rhesus macaques showed no obvious adverse events when administrated with 10 times the effective dose of HB27. Cryo-EM studies on complex of SARS-CoV-2 trimeric S with HB27 Fab reveal that three Fab fragments work synergistically to occlude SARS-CoV-2 from binding to the ACE2 receptor. Binding of the antibody also restrains any further conformational changes of the receptor binding domain, possibly interfering with progression from the prefusion to the postfusion stage. These results suggest that HB27 is a promising candidate for immuno-therapies against COVID-19.

3.
Natl Sci Rev ; 8(8): nwab053, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34676098

RESUMO

Mutations and transient conformational movements of the receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable present immune escape routes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting the N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, a combination of RBD-targeting NAbs and NTD-binding NAbs, FC05, enhanced the neutralization potency in cell-based assays and an animal model. Results of competitive surface plasmon resonance assays and cryo-electron microscopy (cryo-EM) structures of antigen-binding fragments bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in the NTD and RBD of S, when immunized in rabbits and macaques, elicited potent protective immune responses against SARS-CoV-2. More importantly, two immunizations of this combination of NTD and RBD immunogens provided complete protection in macaques against a SARS-CoV-2 challenge, without observable antibody-dependent enhancement of infection. These results provide a proof of concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD.

4.
Nat Commun ; 12(1): 5654, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580297

RESUMO

There is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.


Assuntos
COVID-19/diagnóstico , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Sítios de Ligação/genética , COVID-19/mortalidade , COVID-19/virologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Ligação Proteica/genética , Domínios Proteicos/genética , SARS-CoV-2/genética , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/genética
5.
J Hazard Mater ; 420: 126549, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34252665

RESUMO

Arsenic calcium residue (ACR) generated from the As-bearing wastewater treatment is highly hazardous due to high content of available As, which was seeking a suitable method for safe disposal such as stabilization treatment. In this study, the stabilization of available As in ACR was performed by combined treatment with FeSO4 and H2SO4. After stabilization treatment, the As leaching concentrations extracted by China Standard Leaching Test (CSLT, HJ/T299-2007) decreased significantly from 162 mg/L to less than the Chinese regulation limit of 1.2 mg/L. And FeSO4-H2SO4 treated ACR could maintain good long-term stability even after cured for 365 days. The stabilization mechanism for available As in ACR using leaching tests, sequential extraction analysis, XPS, XRD, and SEM-EDS was investigated. H+ from H2SO4 and Fe(Ⅱ) hydrolysis was committed to the full release of available As. Reactive oxygen species (ROSs) produced from Fe(Ⅱ) oxygenation drove the oxidation of As(Ⅲ) to As(Ⅴ). The release As was stabilized by forming stable Fe-O-As complexes (FeAsO4·xFe(OH)3). Moreover, this study also presented an effective and feasible method for ACR disposal.


Assuntos
Arsênio , Purificação da Água , Arsênio/análise , Cálcio , China , Compostos Ferrosos
6.
Nat Commun ; 12(1): 3917, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168138

RESUMO

SARS-CoV-2 carries the largest single-stranded RNA genome and is the causal pathogen of the ongoing COVID-19 pandemic. How the SARS-CoV-2 RNA genome is folded in the virion remains unknown. To fill the knowledge gap and facilitate structure-based drug development, we develop a virion RNA in situ conformation sequencing technology, named vRIC-seq, for probing viral RNA genome structure unbiasedly. Using vRIC-seq data, we reconstruct the tertiary structure of the SARS-CoV-2 genome and reveal a surprisingly "unentangled globule" conformation. We uncover many long-range duplexes and higher-order junctions, both of which are under purifying selections and contribute to the sequential package of the SARS-CoV-2 genome. Unexpectedly, the D614G and the other two accompanying mutations may remodel duplexes into more stable forms. Lastly, the structure-guided design of potent small interfering RNAs can obliterate the SARS-CoV-2 in Vero cells. Overall, our work provides a framework for studying the genome structure, function, and dynamics of emerging deadly RNA viruses.


Assuntos
COVID-19/patologia , RNA Viral/química , SARS-CoV-2/genética , Análise de Sequência de RNA/métodos , Vírion/genética , Animais , COVID-19/genética , COVID-19/virologia , Células Cultivadas , Chlorocebus aethiops , Genoma Viral , Humanos , Conformação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade , Vírion/química , Vírion/metabolismo
7.
J Virol ; 95(16): e0017721, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34011545

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals that causes a significant economic burden globally. Vaccination is the most effective FMD control strategy. However, FMD virus (FMDV) particles are prone to dissociate when appropriate physical or chemical conditions are unavailable, such as an incomplete cold chain. Such degraded vaccines result in compromised herd vaccination. Therefore, thermostable FMD particles are needed for use in vaccines. This study generated thermostable FMDV mutants (M3 and M10) by serial passages at high temperature, subsequent amplification, and purification. Both mutants contained an alanine-to-threonine mutation at position 13 in VP1 (A1013T), although M3 contained 3 additional mutations. The selected mutants showed improved stability and immunogenicity in neutralizing antibody titers, compared with the wild-type (wt) virus. The sequencing analysis and cryo-electron microscopy showed that the mutation of alanine to threonine at the 13th amino acid in the VP1 protein (A1013T) is critical for the capsid stability of FMDV. Virus-like particles containing A1013T (VLPA1013T) also showed significantly improved stability to heat treatment. This study demonstrated that Thr at the 13th amino acid of VP1 could stabilize the capsid of FMDV. Our findings will facilitate the development of a stable vaccine against FMDV serotype O. IMPORTANCE Foot-and-mouth disease (FMD) serotype O is one of the global epidemic serotypes and causes significant economic loss. Vaccination plays a key role in the prevention and control of FMD. However, the success of vaccination mainly depends on the quality of the vaccine. Here, the thermostable FMD virus (FMDV) mutants (M3 and M10) were selected through thermal screening at high temperatures with improved stability and immunogenicity compared with the wild-type virus. The results of multisequence alignment and cryo-electron microscopy (cryo-EM) analysis showed that the Thr substitution at the 13th amino acid in the VP1 protein is critical for the capsid stability of FMDV. For thermolabile type O FMDV, this major discovery will aid the development of its thermostable vaccine.


Assuntos
Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Vírus da Febre Aftosa/imunologia , Vacinas Virais/imunologia , Substituição de Aminoácidos , Animais , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Microscopia Crioeletrônica , Febre Aftosa/prevenção & controle , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/metabolismo , Cobaias , Temperatura Alta , Imunogenicidade da Vacina , Mutação , Estabilidade Proteica , Sorogrupo , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Virologia
13.
Cell Host Microbe ; 29(2): 222-235.e4, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33388094

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an unprecedented public health crisis. Evidence suggests that SARS-CoV-2 infection causes dysregulation of the immune system. However, the unique signature of early immune responses remains elusive. We characterized the transcriptome of rhesus macaques and mice infected with SARS-CoV-2. Alarmin S100A8 was robustly induced in SARS-CoV-2-infected animal models as well as in COVID-19 patients. Paquinimod, a specific inhibitor of S100A8/A9, could rescue the pneumonia with substantial reduction of viral loads in SARS-CoV-2-infected mice. Remarkably, Paquinimod treatment resulted in almost 100% survival in a lethal model of mouse coronavirus infection using the mouse hepatitis virus (MHV). A group of neutrophils that contributes to the uncontrolled pathological damage and onset of COVID-19 was dramatically induced by coronavirus infection. Paquinimod treatment could reduce these neutrophils and regain anti-viral responses, unveiling key roles of S100A8/A9 and aberrant neutrophils in the pathogenesis of COVID-19, highlighting new opportunities for therapeutic intervention.


Assuntos
Alarminas/farmacologia , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/metabolismo , COVID-19/virologia , Modelos Animais de Doenças , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Transcriptoma , Carga Viral
14.
Cell Rep ; 34(5): 108699, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33485405

RESUMO

Several potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have been identified. However, antibody-dependent enhancement (ADE) has not been comprehensively studied for SARS-CoV-2, and the relationship between enhancing versus neutralizing activities and antibody epitopes remains unknown. Here, we select a convalescent individual with potent IgG neutralizing activity and characterize his antibody response. Monoclonal antibodies isolated from memory B cells target four groups of five non-overlapping receptor-binding domain (RBD) epitopes. Antibodies to one group of these RBD epitopes mediate ADE of entry in Raji cells via an Fcγ receptor-dependent mechanism. In contrast, antibodies targeting two other distinct epitope groups neutralize SARS-CoV-2 without ADE, while antibodies against the fourth epitope group are poorly neutralizing. One antibody, XG014, potently cross-neutralizes SARS-CoV-2 variants, as well as SARS-CoV-1, with respective IC50 (50% inhibitory concentration) values as low as 5.1 and 23.7 ng/mL, while not exhibiting ADE. Therefore, neutralization and ADE of human SARS-CoV-2 antibodies correlate with non-overlapping RBD epitopes.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Facilitadores , Epitopos/imunologia , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/uso terapêutico , Reações Antígeno-Anticorpo , COVID-19/tratamento farmacológico , COVID-19/imunologia , COVID-19/virologia , Linhagem Celular , Criança , Análise por Conglomerados , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Pessoa de Meia-Idade , Domínios Proteicos/imunologia , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
15.
Cell Res ; 31(1): 25-36, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262452

RESUMO

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19 , Epitopos/imunologia , SARS-CoV-2/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , Chlorocebus aethiops , Modelos Animais de Doenças , Humanos , Anticorpos de Cadeia Única/farmacologia , Células Vero
17.
Lancet Infect Dis ; 21(2): 181-192, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33217362

RESUMO

BACKGROUND: With the unprecedented morbidity and mortality associated with the COVID-19 pandemic, a vaccine against COVID-19 is urgently needed. We investigated CoronaVac (Sinovac Life Sciences, Beijing, China), an inactivated vaccine candidate against COVID-19, containing inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for its safety, tolerability and immunogenicity. METHODS: In this randomised, double-blind, placebo-controlled, phase 1/2 clinical trial, healthy adults aged 18-59 years were recruited from the community in Suining County of Jiangsu province, China. Adults with SARS-CoV-2 exposure or infection history, with axillary temperature above 37·0°C, or an allergic reaction to any vaccine component were excluded. The experimental vaccine for the phase 1 trial was manufactured using a cell factory process (CellSTACK Cell Culture Chamber 10, Corning, Wujiang, China), whereas those for the phase 2 trial were produced through a bioreactor process (ReadyToProcess WAVE 25, GE, Umea, Sweden). The phase 1 trial was done in a dose-escalating manner. At screening, participants were initially separated (1:1), with no specific randomisation, into two vaccination schedule cohorts, the days 0 and 14 vaccination cohort and the days 0 and 28 vaccination cohort, and within each cohort the first 36 participants were assigned to block 1 (low dose CoronaVac [3 µg per 0·5 mL of aluminium hydroxide diluent per dose) then another 36 were assigned to block 2 (high-dose Coronavc [6 µg per 0·5 mL of aluminium hydroxide diluent per dse]). Within each block, participants were randomly assigned (2:1), using block randomisation with a block size of six, to either two doses of CoronaVac or two doses of placebo. In the phase 2 trial, at screening, participants were initially separated (1:1), with no specific randomisation, into the days 0 and 14 vaccination cohort and the days 0 and 28 vaccination cohort, and participants were randomly assigned (2:2:1), using block randomisation with a block size of five, to receive two doses of either low-dose CoronaVac, high-dose CoronaVac, or placebo. Participants, investigators, and laboratory staff were masked to treatment allocation. The primary safety endpoint was adverse reactions within 28 days after injection in all participants who were given at least one dose of study drug (safety population). The primary immunogenic outcome was seroconversion rates of neutralising antibodies to live SARS-CoV-2 at day 14 after the last dose in the days 0 and 14 cohort, and at day 28 after the last dose in the days 0 and 28 cohort in participants who completed their allocated two-dose vaccination schedule (per-protocol population). This trial is registered with ClinicalTrials.gov, NCT04352608, and is closed to accrual. FINDINGS: Between April 16 and April 25, 2020, 144 participants were enrolled in the phase 1 trial, and between May 3 and May 5, 2020, 600 participants were enrolled in the phase 2 trial. 743 participants received at least one dose of investigational product (n=143 for phase 1 and n=600 for phase 2; safety population). In the phase 1 trial, the incidence of adverse reactions for the days 0 and 14 cohort was seven (29%) of 24 participants in the 3 ug group, nine (38%) of 24 in the 6 µg group, and two (8%) of 24 in the placebo group, and for the days 0 and 28 cohort was three (13%) of 24 in the 3 µg group, four (17%) of 24 in the 6 µg group, and three (13%) of 23 in the placebo group. The seroconversion of neutralising antibodies on day 14 after the days 0 and 14 vaccination schedule was seen in 11 (46%) of 24 participants in the 3 µg group, 12 (50%) of 24 in the 6 µg group, and none (0%) of 24 in the placebo group; whereas at day 28 after the days 0 and 28 vaccination schedule, seroconversion was seen in 20 (83%) of 24 in the 3 µg group, 19 (79%) of 24 in the 6 µg group, and one (4%) of 24 in the placebo group. In the phase 2 trial, the incidence of adverse reactions for the days 0 and 14 cohort was 40 (33%) of 120 participants in the 3 µg group, 42 (35%) of 120 in the 6 µg group, and 13 (22%) of 60 in the placebo group, and for the days 0 and 28 cohort was 23 (19%) of 120 in the 3 µg group, 23 (19%) of 120 in the 6 µg group, and 11 (18%) of 60 for the placebo group. Seroconversion of neutralising antibodies was seen for 109 (92%) of 118 participants in the 3 µg group, 117 (98%) of 119 in the 6 µg group, and two (3%) of 60 in the placebo group at day 14 after the days 0 and 14 schedule; whereas at day 28 after the days 0 and 28 schedule, seroconversion was seen in 114 (97%) of 117 in the 3 µg group, 118 (100%) of 118 in the 6 µg group, and none (0%) of 59 in the placebo group. INTERPRETATION: Taking safety, immunogenicity, and production capacity into account, the 3 µg dose of CoronaVac is the suggested dose for efficacy assessment in future phase 3 trials. FUNDING: Chinese National Key Research and Development Program and Beijing Science and Technology Program.


Assuntos
Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Imunogenicidade da Vacina , SARS-CoV-2/imunologia , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologia , Adolescente , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais , COVID-19/epidemiologia , Vacinas contra COVID-19/administração & dosagem , China/epidemiologia , Feminino , Voluntários Saudáveis , Humanos , Esquemas de Imunização , Imunoglobulina G , Imunoglobulina M , Masculino , Pessoa de Meia-Idade , Soroconversão , Vacinação , Vacinas de Produtos Inativados/administração & dosagem , Adulto Jovem
18.
Nature ; 2021 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-35016194

RESUMO

The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) requires immediate investigation. Here, we used high-throughput yeast display screening1,2 to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs and showed that the NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications3-5. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlap with ACE2-binding motif, are largely escaped by K417N, G446S, E484A, and Q493R. Group E (S309 site)6 and F (CR3022 site)7 NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, N440K, and S371L. Furthermore, Omicron pseudovirus neutralization showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, the neutralization potency of LY-CoV016/LY-CoV555, REGN10933/REGN10987, AZD1061/AZD8895, and BRII-196 were greatly reduced by Omicron, while VIR-7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron would cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.

19.
Protein Sci ; 29(12): 2363-2374, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33007128

RESUMO

Human ATP-binding cassette transporter 6 of subfamily B (ABCB6) is an ABC transporter involved in the translocation toxic metals and anti-cancer drugs. Using cryo-electron microscopy, we determined the molecular structure of full-length ABCB6 in an apo state. The structure of ABCB6 unravels the architecture of a full-length ABCB transporter that harbors two N-terminal transmembrane domains which is indispensable for its ATPase activity in our in vitro assay. A slit-like substrate binding pocket of ABCB6 may accommodate the planar shape of porphyrins, and the existence of a secondary cavity near the mitochondrial intermembrane space side would further facilitate substrate release. Furthermore, the ATPase activity of ABCB6 stimulated with a variety of porphyrin substrates showed different profiles in the presence of glutathione (GSH), suggesting the action of a distinct substrate translocation mechanism depending on the use of GSH as a cofactor.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Microscopia Crioeletrônica , Transportadores de Cassetes de Ligação de ATP/metabolismo , Glutationa/química , Glutationa/metabolismo , Células HEK293 , Humanos , Porfirinas/química , Porfirinas/metabolismo , Domínios Proteicos
20.
Nat Commun ; 11(1): 4421, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887891

RESUMO

Receptor usage that determines cell tropism and drives viral classification closely correlates with the virus structure. Enterovirus B (EV-B) consists of several subgroups according to receptor usage, among which echovirus 30 (E30), a leading causative agent for human aseptic meningitis, utilizes FcRn as an uncoating receptor. However, receptors for many EVs remain unknown. Here we analyzed the atomic structures of E30 mature virion, empty- and A-particles, which reveals serotype-specific epitopes and striking conformational differences between the subgroups within EV-Bs. Of these, the VP1 BC loop markedly distinguishes E30 from other EV-Bs, indicative of a role as a structural marker for EV-B. By obtaining cryo-electron microscopy structures of E30 in complex with its receptor FcRn and CD55 and comparing its homologs, we deciphered the underlying molecular basis for receptor recognition. Together with experimentally derived viral receptor identifications, we developed a structure-based in silico algorithm to inform a rational prediction for EV receptor usage.


Assuntos
Complexo Antígeno-Anticorpo/ultraestrutura , Enterovirus Humano B/ultraestrutura , Antígenos Virais/ultraestrutura , Antígenos CD55/imunologia , Microscopia Crioeletrônica , Enterovirus Humano B/imunologia , Epitopos/ultraestrutura , Humanos , Receptores Fc/imunologia , Vírion/ultraestrutura
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