RESUMO
It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.
Assuntos
Infecções por Coronavirus , Coronavirus , Dipeptidil Peptidase 4 , Pangolins , Animais , Humanos , Camundongos , Quirópteros , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Endopeptidases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Peptídeo Hidrolases/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Coronavirus/fisiologiaRESUMO
Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
Assuntos
Infecções por Coronavirus/genética , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/imunologia , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Coronavirus/classificação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Células Vero , Internalização do VírusRESUMO
Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.
Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Betacoronavirus/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , COVID-19 , Camelídeos Americanos/imunologia , Infecções por Coronavirus/terapia , Reações Cruzadas , Imunoglobulina G/química , Imunoglobulina G/imunologia , Modelos Moleculares , Pandemias , Pneumonia Viral/terapia , Domínios Proteicos , Receptores Virais/química , SARS-CoV-2 , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.
Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Desenho Universal , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/química , COVID-19 , Vacinas contra COVID-19 , Linhagem Celular Tumoral , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , SARS-CoV-2 , Células Sf9 , Organismos Livres de Patógenos Específicos , Spodoptera , Transfecção , Vacinação/métodos , Células Vero , Vacinas ViraisRESUMO
Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.
Assuntos
Anticorpos Neutralizantes/química , Betacoronavirus/química , Infecções por Coronavirus/imunologia , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Betacoronavirus/imunologia , COVID-19 , Infecções por Coronavirus/sangue , Infecções por Coronavirus/terapia , Reações Cruzadas , Microscopia Crioeletrônica , Mapeamento de Epitopos , Epitopos , Humanos , Imunização Passiva , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Imunoglobulina G/sangue , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/ultraestrutura , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Modelos Moleculares , Pandemias , Pneumonia Viral/sangue , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologia , Soroterapia para COVID-19RESUMO
Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.
Assuntos
Coronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Coronavirus/metabolismo , Infecções por Coronavirus/imunologia , Células HEK293 , Humanos , Imunidade Humoral/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Mimetismo Molecular/imunologia , Ligação Proteica , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Glicoproteína da Espícula de Coronavírus/fisiologia , Células Vero , Internalização do VírusRESUMO
Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Hipermutação Somática de Imunoglobulina/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Sítios de Ligação , COVID-19 , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/imunologia , Humanos , Pandemias/prevenção & controle , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Ligação Proteica , Receptores Virais/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas Virais/imunologiaRESUMO
Single-particle cryoelectron microscopy (cryo-EM), whose full capabilities have been realized only within the past decade, has had a pivotal role in the fight against COVID-19. This is due to the technique's intrinsic power to depict both structural and dynamic features of molecules; in this case, of the spike protein of SARS-CoV-2. By now, numerous cryo-EM studies have furthered our understanding of spike protein-angiotensin-converting enzyme 2 (ACE2) receptor interactions, which has informed the design of effective vaccines, and have enabled the characterization of neutralizing antibody binding sites, which will lead to the design of novel therapeutics as the virus evolves.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Anticorpos Neutralizantes/metabolismo , Microscopia Crioeletrônica , Humanos , Ligação Proteica , SARS-CoV-2RESUMO
The nasal epithelium is the initial entry portal and primary barrier to infection by all human coronaviruses (HCoVs). We utilize primary human nasal epithelial cells grown at air-liquid interface, which recapitulate the heterogeneous cellular population as well as mucociliary clearance functions of the in vivo nasal epithelium, to compare lethal [Severe acute respiratory syndrome (SARS)-CoV-2 and Middle East respiratory syndrome-CoV (MERS-CoV)] and seasonal (HCoV-NL63 and HCoV-229E) HCoVs. All four HCoVs replicate productively in nasal cultures, though replication is differentially modulated by temperature. Infections conducted at 33 °C vs. 37 °C (reflective of temperatures in the upper and lower airway, respectively) revealed that replication of both seasonal HCoVs (HCoV-NL63 and -229E) is significantly attenuated at 37 °C. In contrast, SARS-CoV-2 and MERS-CoV replicate at both temperatures, though SARS-CoV-2 replication is enhanced at 33 °C late in infection. These HCoVs also diverge significantly in terms of cytotoxicity induced following infection, as the seasonal HCoVs as well as SARS-CoV-2 cause cellular cytotoxicity as well as epithelial barrier disruption, while MERS-CoV does not. Treatment of nasal cultures with type 2 cytokine IL-13 to mimic asthmatic airways differentially impacts HCoV receptor availability as well as replication. MERS-CoV receptor DPP4 expression increases with IL-13 treatment, whereas ACE2, the receptor used by SARS-CoV-2 and HCoV-NL63, is down-regulated. IL-13 treatment enhances MERS-CoV and HCoV-229E replication but reduces that of SARS-CoV-2 and HCoV-NL63, reflecting the impact of IL-13 on HCoV receptor availability. This study highlights diversity among HCoVs during infection of the nasal epithelium, which is likely to influence downstream infection outcomes such as disease severity and transmissibility.
Assuntos
COVID-19 , Coronaviridae , Coronavirus Humano 229E , Humanos , Interleucina-13/metabolismo , Estações do Ano , SARS-CoV-2 , Células EpiteliaisRESUMO
Alternative splicing (AS) is an essential post-transcriptional mechanism that regulates many biological processes. However, identifying comprehensive types of AS events without guidance from a reference genome is still a challenge. Here, we proposed a novel method, MkcDBGAS, to identify all seven types of AS events using transcriptome alone, without a reference genome. MkcDBGAS, modeled by full-length transcripts of human and Arabidopsis thaliana, consists of three modules. In the first module, MkcDBGAS, for the first time, uses a colored de Bruijn graph with dynamic- and mixed- kmers to identify bubbles generated by AS with precision higher than 98.17% and detect AS types overlooked by other tools. In the second module, to further classify types of AS, MkcDBGAS added the motifs of exons to construct the feature matrix followed by the XGBoost-based classifier with the accuracy of classification greater than 93.40%, which outperformed other widely used machine learning models and the state-of-the-art methods. Highly scalable, MkcDBGAS performed well when applied to Iso-Seq data of Amborella and transcriptome of mouse. In the third module, MkcDBGAS provides the analysis of differential splicing across multiple biological conditions when RNA-sequencing data is available. MkcDBGAS is the first accurate and scalable method for detecting all seven types of AS events using the transcriptome alone, which will greatly empower the studies of AS in a wider field.
Assuntos
Processamento Alternativo , Arabidopsis , Animais , Humanos , Camundongos , Transcriptoma , Splicing de RNA , Análise de Sequência de RNA/métodos , RNA , Arabidopsis/genética , Perfilação da Expressão Gênica/métodosRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathwaysinterferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lungderived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.
Assuntos
COVID-19 , Infecções por Coronavirus , Coronavírus da Síndrome Respiratória do Oriente Médio , Infecções por Coronavirus/imunologia , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células Epiteliais/metabolismo , Humanos , Imunidade Inata , Pulmão/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Mucosa Nasal , SARS-CoV-2/patogenicidade , Endorribonucleases Específicas de UridilatoRESUMO
Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The modified vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of 3 candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S-specific CD8+ T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8+ T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity.
Assuntos
Linfócitos T CD8-Positivos , Epitopos de Linfócito T , Coronavírus da Síndrome Respiratória do Oriente Médio , Glicoproteína da Espícula de Coronavírus , Vacinas Virais , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Epitopos de Linfócito T/imunologia , Linfócitos T CD8-Positivos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , VacinaçãoRESUMO
Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.
Assuntos
COVID-19 , Interferons , Humanos , Peptídeo Hidrolases/metabolismo , SARS-CoV-2 , Ubiquitina/metabolismo , Antivirais , Poliproteínas , Imunidade , Citocinas/metabolismo , Ubiquitinas/genética , Ubiquitina TiolesteraseRESUMO
Middle East respiratory syndrome coronavirus (MERS CoV) and severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) are RNA viruses from the Betacoronavirus family that cause serious respiratory illness in humans. One of the conserved non-structural proteins encoded for by the coronavirus family is non-structural protein 9 (nsp9). Nsp9 plays an important role in the RNA capping process of the viral genome, where it is covalently linked to viral RNA (known as RNAylation) by the conserved viral polymerase, nsp12. Nsp9 also directly binds to RNA; we have recently revealed a distinct RNA recognition interface in the SARS CoV-2 nsp9 by using a combination of nuclear magnetic resonance spectroscopy and biolayer interferometry. In this study, we have utilized a similar methodology to determine a structural model of RNA binding of the related MERS CoV. Based on these data, we uncover important similarities and differences to SARS CoV-2 nsp9 and other coronavirus nsp9 proteins. Our findings that replacing key RNA binding residues in MERS CoV nsp9 affects RNAylation efficiency indicate that recognition of RNA may play a role in the capping process of the virus.
Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , RNA/metabolismoRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in dromedaries in Africa, but camel-to-human transmission is limited. Sustained 12-month sampling of dromedaries in a Kenya abattoir hub showed biphasic MERS-CoV incidence; peak detections occurred in October 2022 and February 2023. Dromedary-exposed abattoir workers (7/48) had serologic signs of previous MERS-CoV exposure.
Assuntos
Camelus , Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , Animais , Quênia/epidemiologia , Incidência , MatadourosRESUMO
For identification of marker-trait associations (MTAs) for complex traits in animals and plants, thousands of genome-wide association studies (GWAS) were conducted during the past two decades. This involved regular improvement in methodology. Initially, a reference genome and SNPs were used; more recently pan-genomes and the markers structural variations (SVs)/k-mers are also being used.
Assuntos
Estudo de Associação Genômica Ampla/métodos , Estudo de Associação Genômica Ampla/normas , Animais , Genoma/genética , Humanos , Fenótipo , Plantas/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Recurrent epidemics of coronaviruses have posed significant threats to human life and health. The mortality rate of patients infected with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is 35 %. The main protease (Mpro) plays a crucial role in the MERS-CoV life cycle, and Mpro exhibited a high degree of conservation among different coronaviruses. Therefore inhibition of Mpro has become an effective strategy for the development of broad-spectrum anti-coronaviral drugs. The inhibition of SARS-CoV-2 Mpro by the anti-tumor drug carmofur has been revealed, but structural studies of carmofur in complex with Mpro from other types of coronavirus have not been reported. Hence, we revealed the structure of the MERS-CoV Mpro-carmofur complex, analysed the structural basis for the binding of carmofur to MERS-CoV Mpro in detail, and compared the binding patterns of carmofur to Mpros of two different coronaviruses, MERS-CoV and SARS-CoV-2. Considering the importance of Mpros for coronavirus therapy, structural understanding of Mpro inhibition by carmofur could contribute to the design and development of novel antiviral drugs with safe and broad-spectrum efficacy.
RESUMO
IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.
Assuntos
Infecções por Coronavirus , Coronavírus da Síndrome Respiratória do Oriente Médio , Proteínas não Estruturais Virais , Animais , Humanos , Camundongos , Substituição de Aminoácidos , Camelus , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Mutação , Proteínas não Estruturais Virais/genéticaRESUMO
In vitro investigations of host-virus interactions are reliant on suitable cell and tissue culture models. Results are only as good as the model they are generated in. However, choosing cell models for in vitro work often depends on availability and previous use alone. Despite the vast increase in coronavirus research over the past few years, scientists are still heavily reliant on: non-human, highly heterogeneous or not fully differentiated, or naturally unsusceptible cells requiring overexpression of receptors and other accessory factors. Complex primary or stem cell models are highly representative of human tissues but are expensive and time-consuming to develop and maintain with limited suitability for high-throughput experiments.Using tissue-specific expression patterns, we identified human kidney cells as an ideal target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and broader coronavirus infection. We show the use of the well-characterized human kidney cell line Caki-1 for infection with three human coronaviruses (hCoVs): Betacoronaviruses SARS-CoV-2 and Middle Eastern respiratory syndrome coronavirus and Alphacoronavirus hCoV 229E. Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cell lines for the cultivation of the respective virus. Antibody staining against SARS-CoV-2 N protein shows comparable replication rates. A panel of 26 custom antibodies shows the location of SARS-CoV-2 proteins during replication using immunocytochemistry. In addition, Caki-1 cells were found to be susceptible to two other human respiratory viruses, influenza A virus and respiratory syncytial virus, making them an ideal model for cross-comparison for a broad range of respiratory viruses. IMPORTANCE Cell lines remain the backbone of virus research, but results are only as good as their originating model. Despite increased research into human coronaviruses following the COVID-19 pandemic, researchers continue to rely on suboptimal cell line models of: non-human origin, incomplete differentiation, or lacking active interferon responses. We identified the human kidney Caki-1 cell line as a potential target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This cell line could be shown to be infectable with a wide range of coronaviruses including common cold virus hCoV-229E, epidemic virus MERS-CoV, and SARS-CoV-2 as well as other important respiratory viruses influenza A virus and respiratory syncytial virus. We could show the localization of 26 SARS-CoV-2 proteins in Caki-1 cells during natural replication and the cells are competent of forming a cellular immune response. Together, this makes Caki-1 cells a unique tool for cross-virus comparison in one cell line.
Assuntos
Linhagem Celular , Infecções por Coronaviridae , Coronaviridae , Humanos , Coronaviridae/fisiologia , Rim/citologia , Pandemias , Infecções por Coronaviridae/patologia , Infecções por Coronaviridae/virologiaRESUMO
This study evaluated the potential for antibody-dependent enhancement (ADE) in serum samples from patients exposed to Middle East respiratory syndrome coronavirus (MERS-CoV). Furthermore, we evaluated the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination on ADE in individuals with a MERS infection history. We performed ADE assay in sera from MERS recovered and SARS-CoV-2-vaccinated individuals using BHK cells expressing FcgRIIa, SARS-CoV-2, and MERS-CoV pseudoviruses (PVs). Further, we analyzed the association of ADE to serum IgG levels and neutralization. Out of 16 MERS patients, nine demonstrated ADE against SARS-CoV-2 PV, however, none of the samples demonstrated ADE against MERS-CoV PV. Furthermore, out of the seven patients exposed to SARS-CoV-2 vaccination after MERS-CoV infection, only one patient (acutely infected with MERS-CoV) showed ADE for SARS-CoV-2 PV. Further analysis indicated that IgG1, IgG2, and IgG3 against SARS-CoV-2 S1 and RBD subunits, IgG1 and IgG2 against the MERS-CoV S1 subunit, and serum neutralizing activity were low in ADE-positive samples. In summary, samples from MERS-CoV-infected patients exhibited ADE against SARS-CoV-2 and was significantly associated with low levels of neutralizing antibodies. Subsequent exposure to SARS-CoV-2 vaccination resulted in diminished ADE activity while the PV neutralization assay demonstrated a broadly reactive antibody response in some patient samples.