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1.
Nat Commun ; 13(1): 2921, 2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35614127

RESUMO

Human coronavirus OC43 is a globally circulating common cold virus sustained by recurrent reinfections. How it persists in the population and defies existing herd immunity is unknown. Here we focus on viral glycoprotein S, the target for neutralizing antibodies, and provide an in-depth analysis of its antigenic structure. Neutralizing antibodies are directed to the sialoglycan-receptor binding site in S1A domain, but, remarkably, also to S1B. The latter block infection yet do not prevent sialoglycan binding. While two distinct neutralizing S1B epitopes are readily accessible in the prefusion S trimer, other sites are occluded such that their accessibility must be subject to conformational changes in S during cell-entry. While non-neutralizing antibodies were broadly reactive against a collection of natural OC43 variants, neutralizing antibodies generally displayed restricted binding breadth. Our data provide a structure-based understanding of protective immunity and adaptive evolution for this endemic coronavirus which emerged in humans long before SARS-CoV-2.


Assuntos
COVID-19 , Coronavirus Humano OC43 , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Coronavirus Humano OC43/metabolismo , Epitopos , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus
2.
Signal Transduct Target Ther ; 6(1): 414, 2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34873151

RESUMO

Azvudine (FNC) is a nucleoside analog that inhibits HIV-1 RNA-dependent RNA polymerase (RdRp). Recently, we discovered FNC an agent against SARS-CoV-2, and have taken it into Phase III trial for COVID-19 patients. FNC monophosphate analog inhibited SARS-CoV-2 and HCoV-OC43 coronavirus with an EC50 between 1.2 and 4.3 µM, depending on viruses or cells, and selective index (SI) in 15-83 range. Oral administration of FNC in rats revealed a substantial thymus-homing feature, with FNC triphosphate (the active form) concentrated in the thymus and peripheral blood mononuclear cells (PBMC). Treating SARS-CoV-2 infected rhesus macaques with FNC (0.07 mg/kg, qd, orally) reduced viral load, recuperated the thymus, improved lymphocyte profiles, alleviated inflammation and organ damage, and lessened ground-glass opacities in chest X-ray. Single-cell sequencing suggested the promotion of thymus function by FNC. A randomized, single-arm clinical trial of FNC on compassionate use (n = 31) showed that oral FNC (5 mg, qd) cured all COVID-19 patients, with 100% viral ribonucleic acid negative conversion in 3.29 ± 2.22 days (range: 1-9 days) and 100% hospital discharge rate in 9.00 ± 4.93 days (range: 2-25 days). The side-effect of FNC is minor and transient dizziness and nausea in 16.12% (5/31) patients. Thus, FNC might cure COVID-19 through its anti-SARS-CoV-2 activity concentrated in the thymus, followed by promoted immunity.


Assuntos
Antivirais/administração & dosagem , Azidas/administração & dosagem , COVID-19/tratamento farmacológico , Desoxicitidina/análogos & derivados , SARS-CoV-2/metabolismo , Timo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Coronavirus Humano OC43/metabolismo , Desoxicitidina/administração & dosagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Timo/metabolismo , Timo/virologia
3.
Int J Biol Macromol ; 183: 2248-2261, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34111482

RESUMO

The recent emergence of the novel coronavirus (SARS-CoV-2) has resulted in a devastating pandemic with global concern. However, to date, there are no regimens to prevent and treat SARS-CoV-2 virus. There is an urgent need to identify novel leads with anti-viral properties that impede viral pathogenesis in the host system. Esculentoside A (EsA), a saponin isolated from the root of Phytolacca esculenta, is known to exhibit diverse pharmacological properties, especially anti-inflammatory activity. To our knowledge, SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) to enter host cells. This is mediated through the proteins of SARS-CoV-2, especially the spike glycoprotein receptor binding domain. Thus, our primary goal is to prevent virus replication and binding to the host, which allows us to explore the efficiency of EsA on key surface drug target proteins using the computational biology paradigm approach. Here, the anti-coronavirus activity of EsA in vitro and its potential mode of inhibitory action on the S-protein of SARS-CoV-2 were investigated. We found that EsA inhibited the HCoV-OC43 coronavirus during the attachment and penetration stage. Molecular docking results showed that EsA had a strong binding affinity with the spike glycoprotein from SARS-CoV-2. The results of the molecular dynamics simulation revealed that EsA had higher stable binding with the spike protein. These results demonstrated that Esculentoside A can act as a spike protein blocker to inhibit SARS-CoV-2. Considering the poor bioavailability and low toxicity of EsA, it is suitable as novel lead for the inhibitor against binding interactions of SARS-CoV-2 of S-protein and ACE2.


Assuntos
Enzima de Conversão de Angiotensina 2 , Antivirais , COVID-19/tratamento farmacológico , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ácido Oleanólico/análogos & derivados , SARS-CoV-2 , Saponinas , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , Antivirais/farmacologia , Linhagem Celular Tumoral , Coronavirus Humano OC43/química , Coronavirus Humano OC43/metabolismo , Humanos , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , SARS-CoV-2/química , SARS-CoV-2/fisiologia , Saponinas/química , Saponinas/farmacologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
Mol Cell Proteomics ; 20: 100120, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34186245

RESUMO

Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold-like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, nonstructural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) data set for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance, nuclear protein import for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , SARS-CoV-2/patogenicidade , Coronavirus Humano 229E/metabolismo , Coronavirus Humano 229E/patogenicidade , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/patogenicidade , Proteases Semelhantes à Papaína de Coronavírus/genética , Degradação Associada com o Retículo Endoplasmático , Células HEK293 , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Mapas de Interação de Proteínas , SARS-CoV-2/metabolismo , Resposta a Proteínas não Dobradas , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
5.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33789998

RESUMO

The COVID-19 pandemic poses a serious global health threat. The rapid global spread of SARS-CoV-2 highlights an urgent need to develop effective therapeutics for blocking SARS-CoV-2 infection and spread. Stimulator of Interferon Genes (STING) is a chief element in host antiviral defense pathways. In this study, we examined the impact of the STING signaling pathway on coronavirus infection using the human coronavirus OC43 (HCoV-OC43) model. We found that HCoV-OC43 infection did not stimulate the STING signaling pathway, but the activation of STING signaling effectively inhibits HCoV-OC43 infection to a much greater extent than that of type I interferons (IFNs). We also discovered that IRF3, the key STING downstream innate immune effector, is essential for this anticoronavirus activity. In addition, we found that the amidobenzimidazole (ABZI)-based human STING agonist diABZI robustly blocks the infection of not only HCoV-OC43 but also SARS-CoV-2. Therefore, our study identifies the STING signaling pathway as a potential therapeutic target that could be exploited for developing broad-spectrum antiviral therapeutics against multiple coronavirus strains in order to face the challenge of future coronavirus outbreaks.IMPORTANCE The highly infectious and lethal SARS-CoV-2 is posing an unprecedented threat to public health. Other coronaviruses are likely to jump from a nonhuman animal to humans in the future. Novel broad-spectrum antiviral therapeutics are therefore needed to control known pathogenic coronaviruses such as SARS-CoV-2 and its newly mutated variants, as well as future coronavirus outbreaks. STING signaling is a well-established host defense pathway, but its role in coronavirus infection remains unclear. In the present study, we found that activation of the STING signaling pathway robustly inhibits infection of HCoV-OC43 and SARS-CoV-2. These results identified the STING pathway as a novel target for controlling the spread of known pathogenic coronaviruses, as well as emerging coronavirus outbreaks.


Assuntos
COVID-19/metabolismo , Coronavirus Humano OC43/metabolismo , Proteínas de Membrana/metabolismo , SARS-CoV-2/metabolismo , Transdução de Sinais , Células A549 , Animais , COVID-19/genética , Chlorocebus aethiops , Coronavirus Humano OC43/genética , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , SARS-CoV-2/genética , Células Vero
6.
Artigo em Inglês | MEDLINE | ID: mdl-33558299

RESUMO

The impact of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, is global and unprecedented. Although remdesivir has recently been approved by the FDA to treat SARS-CoV-2 infection, no oral antiviral is available for outpatient treatment. AT-527, an orally administered double prodrug of a guanosine nucleotide analog, was previously shown to be highly efficacious and well tolerated in hepatitis C virus (HCV)-infected subjects. Here, we report the potent in vitro activity of AT-511, the free base of AT-527, against several coronaviruses, including SARS-CoV-2. In normal human airway epithelial cells, the concentration of AT-511 required to inhibit replication of SARS-CoV-2 by 90% (EC90) was 0.47 µM, very similar to its EC90 against human coronavirus (HCoV)-229E, HCoV-OC43, and SARS-CoV in Huh-7 cells. Little to no cytotoxicity was observed for AT-511 at concentrations up to 100 µM. Substantial levels of the active triphosphate metabolite AT-9010 were formed in normal human bronchial and nasal epithelial cells incubated with 10 µM AT-511 (698 ± 15 and 236 ± 14 µM, respectively), with a half-life of at least 38 h. Results from steady-state pharmacokinetic and tissue distribution studies of nonhuman primates administered oral doses of AT-527, as well as pharmacokinetic data from subjects given daily oral doses of AT-527, predict that twice daily oral doses of 550 mg AT-527 will produce AT-9010 trough concentrations in human lung that exceed the EC90 observed for the prodrug against SARS-CoV-2 replication. This suggests that AT-527 may be an effective treatment option for COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Guanosina Monofosfato/análogos & derivados , Guanosina/farmacologia , Fosforamidas/farmacologia , Pró-Fármacos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Administração Oral , Animais , COVID-19/virologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Coronavirus Humano 229E/metabolismo , Coronavirus Humano OC43/metabolismo , Cricetinae , Células Epiteliais/virologia , Guanosina Monofosfato/farmacologia , Humanos , Pulmão/virologia , SARS-CoV-2/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos
7.
Int J Mol Sci ; 22(4)2021 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-33572480

RESUMO

BACKGROUND: Preexisting immunity to SARS-CoV-2 could be related to cross-reactive antibodies to common human-coronaviruses (HCoVs). This study aimed to evaluate whether human milk antibodies against to S1 and S2 subunits SARS-CoV-2 are cross-reactive to S1 and S2 subunits HCoV-OC43 and HCoV-229E in mothers with a confirmed COVID-19 PCR test, in mothers with previous viral symptoms during COVID-19 pandemic, and in unexposed mothers; Methods: The levels of secretory IgA (SIgA)/IgA, secretory IgM (SIgM)/IgM, and IgG specific to S1 and S2 SARS-CoV-2, and reactive to S1 + S2 HCoV-OC43, and HCoV-229E were measured in milk from 7 mothers with a confirmed COVID-19 PCR test, 20 mothers with viral symptoms, and unexposed mothers (6 Ctl1-2018 and 16 Ctl2-2018) using ELISA; Results: The S2 SARS-CoV-2 IgG levels were higher in the COVID-19 PCR (p = 0.014) and viral symptom (p = 0.040) groups than in the Ctl1-2018 group. We detected a higher number of positive correlations between the antigens and secretory antibodies in the COVID-19 PCR group than in the viral symptom and Ctl-2018 groups. S1 + S2 HCoV-OC43-reactive IgG was higher in the COVID-19 group than in the control group (p = 0.002) but did not differ for the other antibodies; Conclusions: Mothers with a confirmed COVID-19 PCR and mothers with previous viral symptoms had preexisting human milk antibodies against S2 subunit SARS-CoV-2. Human milk IgG were more specific to S2 subunit SARS-CoV-2 than other antibodies, whereas SIgA and SIgM were polyreactive and cross-reactive to S1 or S2 subunit SARS-CoV-2.


Assuntos
Anticorpos Antivirais/imunologia , COVID-19/patologia , Coronavirus Humano 229E/metabolismo , Coronavirus Humano OC43/metabolismo , Leite Humano/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Reações Antígeno-Anticorpo , COVID-19/virologia , Reações Cruzadas , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina M/imunologia , Mães , Reação em Cadeia da Polimerase , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(41): 25759-25770, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32994342

RESUMO

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.


Assuntos
Coronavirus/fisiologia , Hemaglutininas Virais/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais de Fusão/genética , Vírion/metabolismo , Animais , Evolução Biológica , Linhagem Celular , Coronavirus/genética , Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/fisiologia , Coronavirus Bovino/genética , Coronavirus Bovino/metabolismo , Coronavirus Bovino/fisiologia , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Camundongos , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , Seleção Genética , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Vírion/genética , Ligação Viral , Liberação de Vírus
9.
J Microbiol Biotechnol ; 30(10): 1495-1499, 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-32807752

RESUMO

The study of climate and respiratory viral infections using big data may enable the recognition and interpretation of relationships between disease occurrence and climatic variables. In this study, realtime reverse transcription quantitative PCR (qPCR) methods were used to identify Human respiratory coronaviruses (HCoV). infections in patients below 10 years of age with respiratory infections who visited Dankook University Hospital in Cheonan, South Korea, from January 1, 2012, to December 31, 2018. Out of the 9010 patients who underwent respiratory virus real-time reverse transcription qPCR test, 364 tested positive for HCoV infections. Among these 364 patients, 72.8% (n = 265) were below 10 years of age. Data regarding the frequency of infections was used to uncover the seasonal pattern of the two viral strains, which was then compared with local meteorological data for the same time period. HCoV-229E and HCoV-OC43 showed high infection rates in patients below 10 years of age. There was a negative relationship between HCoV-229E and HCoV-OC43 infections with air temperature and wind-chill temperatures. Both HCoV-229E and HCoV-OC43 rates of infection were positively related to atmospheric pressure, while HCoV-229E was also positively associated with particulate matter concentrations. Our results suggest that climatic variables affect the rate in which children below 10 years of age are infected with HCoV. These findings may help to predict when prevention strategies may be most effective.


Assuntos
Clima , Infecções por Coronavirus/epidemiologia , Coronavirus Humano OC43 , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Criança , Pré-Escolar , Coronavirus/genética , Coronavirus Humano 229E/isolamento & purificação , Coronavirus Humano 229E/metabolismo , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/isolamento & purificação , Coronavirus Humano OC43/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , República da Coreia/epidemiologia , Estudos Retrospectivos
10.
Infect Genet Evol ; 84: 104440, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32622082

RESUMO

SARS-CoV-2, a new coronavirus strain responsible for COVID-19, has emerged in Wuhan City, China, and continuing its global pandemic nature. The availability of the complete gene sequences of the virus helps to know about the origin and molecular characteristics of this virus. In the present study, we performed bioinformatic analysis of the available gene sequence data of SARS-CoV-2 for the understanding of evolution and molecular characteristics and immunogenic resemblance of the circulating viruses. Phylogenetic analysis was performed for four types of representative viral proteins (spike, membrane, envelope and nucleoprotein) of SARS-CoV-2, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, HKU4, HKU5 and BufCoV-HKU26. The findings demonstrated that SARS-CoV-2 exhibited convergent evolutionary relation with previously reported SARS-CoV. It was also depicted that SARS-CoV-2 proteins were highly similar and identical to SARS-CoV proteins, though proteins from other coronaviruses showed a lower level of resemblance. The cross-checked conservancy analysis of SARS-CoV-2 antigenic epitopes showed significant conservancy with antigenic epitopes derived from SARS-CoV. Descriptive epidemiological analysis on several epidemiological indices was performed on available epidemiological outbreak information from several open databases on COVID-19 (SARS-CoV-2). Satellite-derived imaging data have been employed to understand the role of temperature in the environmental persistence of the virus. Findings of the descriptive analysis were used to describe the global impact of newly emerged SARS-CoV-2, and the risk of an epidemic in Bangladesh.


Assuntos
Antígenos Virais/genética , Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Genoma Viral , Pandemias , Pneumonia Viral/epidemiologia , Vírus da SARS/genética , Glicoproteína da Espícula de Coronavírus/química , Alphacoronavirus/classificação , Alphacoronavirus/genética , Alphacoronavirus/metabolismo , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/metabolismo , Bangladesh/epidemiologia , Sequência de Bases , Betacoronavirus/classificação , Betacoronavirus/metabolismo , Sítios de Ligação , COVID-19 , Quirópteros/virologia , Biologia Computacional , Coronavirus Humano 229E/classificação , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/metabolismo , Infecções por Coronavirus/virologia , Coronavirus Humano NL63/classificação , Coronavirus Humano NL63/genética , Coronavirus Humano NL63/metabolismo , Coronavirus Humano OC43/classificação , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/classificação , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Modelos Moleculares , Mutação , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Filogenia , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/classificação , Vírus da SARS/metabolismo , SARS-CoV-2 , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
11.
Acta Chim Slov ; 67(3): 949-956, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33533428

RESUMO

Due to the current spreading of the new disease CoViD-19, the World Health Organization formally declared a world pandemic on March 11, 2020. The present trends indicate that the pandemic will have an enormous clinical and economic impact on population health. Infections are initiated by the transmembrane spike (S) glycoproteins of human coronavirus (hCoV) binding to host receptors. Ongoing research and therapeutic product development are of vital importance for the successful treatment of CoViD-19. To contribute somewhat to the overall effort, herein, single point mutations (SPMs) of the binding site residues in hCoV-OC43 S that recognizes cellular surface components containing 9-O-acetylated sialic acid (9-O-Ac-Sia) are explored using an in silico protein engineering approach, while their effects on the binding of 9-O-Ac-Sia and Hidroxychloroquine (Hcq) are evaluated using molecular docking simulations. Thr31Met and Val84Arg are predicted to be the critical - most likely SPMs in hCoV-OC43 S for the binding of 9-O-Ac-Sia and Hcq, respectively, even though Thr31Met is a very likely SPM in the case of Hcq too. The corresponding modes of interaction indicate a comparable strength of the Thr31Met/9-O-Ac-Sia and Val84Arg/Hcq (or Thr31Met/Hcq) complexes. Given that the binding site is conserved in all CoV S glycoproteins that associate with 9-O-acetyl-sialoglycans, the high hydrophobic affinity of Hcq to hCoV-OC43 S speaks in favor of its ability to competitively inhibit rapid S-mediated virion attachment in high-density receptor environments, but its considerably low specificity to hCoV-OC43 S may be one of the key obstacles in considering the potential of Hcq to become a drug candidate.


Assuntos
Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Hidroxicloroquina/metabolismo , Mutação Puntual , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Sítios de Ligação , COVID-19/virologia , Infecções por Coronavirus/metabolismo , Coronavirus Humano OC43/química , Coronavirus Humano OC43/metabolismo , Humanos , Simulação de Acoplamento Molecular/métodos , Engenharia de Proteínas , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
12.
J Virol ; 93(12)2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30918074

RESUMO

Coronaviruses (CoVs) act as cross-species viruses and have the potential to spread rapidly into new host species and cause epidemic diseases. Despite the severe public health threat of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome CoV (MERS-CoV), there are currently no drugs available for their treatment; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are urgently needed. To search for effective inhibitory agents, we performed high-throughput screening (HTS) of a 2,000-compound library of approved drugs and pharmacologically active compounds using the established genetically engineered human CoV OC43 (HCoV-OC43) strain expressing Renilla luciferase (rOC43-ns2Del-Rluc) and validated the inhibitors using multiple genetically distinct CoVs in vitro We screened 56 hits from the HTS data and validated 36 compounds in vitro using wild-type HCoV-OC43. Furthermore, we identified seven compounds (lycorine, emetine, monensin sodium, mycophenolate mofetil, mycophenolic acid, phenazopyridine, and pyrvinium pamoate) as broad-spectrum inhibitors according to their strong inhibition of replication by four CoVs in vitro at low-micromolar concentrations. Additionally, we found that emetine blocked MERS-CoV entry according to pseudovirus entry assays and that lycorine protected BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This represents the first demonstration of in vivo real-time bioluminescence imaging to monitor the effect of lycorine on the spread and distribution of HCoV-OC43 in a mouse model. These results offer critical information supporting the development of an effective therapeutic strategy against CoV infection.IMPORTANCE Currently, there is no approved therapy to treat coronavirus infection; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are needed. Based on our high-throughput screening assay using a compound library, we identified seven compounds with broad-spectrum efficacy against the replication of four CoVs in vitro Additionally, one compound (lycorine) was found to protect BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This inhibitor might offer promising therapeutic possibilities for combatting novel CoV infections in the future.


Assuntos
Coronavirus Humano OC43/efeitos dos fármacos , Coronavirus/efeitos dos fármacos , Alcaloides de Amaryllidaceae/farmacologia , Animais , Antivirais/farmacologia , Linhagem Celular , Coronavirus/patogenicidade , Coronavirus Humano OC43/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Emetina/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Fenantridinas/farmacologia , Vírus da SARS/efeitos dos fármacos
13.
J Virol ; 91(1)2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27795420

RESUMO

Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic, and neurovirulent properties, highlighting the importance of studying the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death, which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/Us183-241) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determined if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death, as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process, reflecting the severity of neuropathogenesis. IMPORTANCE: Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection.


Assuntos
Infecções por Coronavirus/genética , Coronavirus Humano OC43/patogenicidade , Proteínas Ativadoras de GTPase/genética , Interações Hospedeiro-Patógeno , Proteínas Quinases/genética , Glicoproteína da Espícula de Coronavírus/genética , Animais , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Embrião de Mamíferos , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Neurônios/patologia , Neurônios/virologia , Cultura Primária de Células , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/metabolismo , Análise de Sobrevida , Virulência , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
14.
J Virol ; 88(1): 54-65, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24109243

RESUMO

The human coronavirus OC43 is a major contributor to the common cold worldwide, though due to its low mortality rate, little research has focused on this human pathogen. The nucleocapsid is an essential structural protein with conserved functions across the coronavirus family. While a multitude of studies have examined nucleocapsid function, none have described the effects of OC43 nucleocapsid on the transcription factor NF-κB. We report that the nucleocapsid protein of OC43 causes potentiation of NF-κB activation. This prolonged activation is the direct result of the ability of the nucleocapsid to bind RNA, specifically microRNA 9 (miR-9), which is a negative regulator of NF-κB. This previously undescribed interaction between virus and host is a potential mechanism of immune evasion in RNA viruses.


Assuntos
Coronavirus Humano OC43/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Imunoprecipitação , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real
15.
Biochim Biophys Acta ; 1834(6): 1054-62, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23501675

RESUMO

Human coronavirus OC43 (HCoV-OC43) is a causative agent of the common cold. The nucleocapsid (N) protein, which is a major structural protein of CoVs, binds to the viral RNA genome to form the virion core and results in the formation of the ribonucleoprotein (RNP) complex. We have solved the crystal structure of the N-terminal domain of HCoV-OC43 N protein (N-NTD) (residues 58 to 195) to a resolution of 2.0Å. The HCoV-OC43 N-NTD is a single domain protein composed of a five-stranded ß-sheet core and a long extended loop, similar to that observed in the structures of N-NTDs from other coronaviruses. The positively charged loop of the HCoV-OC43 N-NTD contains a structurally well-conserved positively charged residue, R106. To assess the role of R106 in RNA binding, we undertook a series of site-directed mutagenesis experiments and docking simulations to characterize the interaction between R106 and RNA. The results show that R106 plays an important role in the interaction between the N protein and RNA. In addition, we showed that, in cells transfected with plasmids that encoded the mutant (R106A) N protein and infected with virus, the level of the matrix protein gene was decreased by 7-fold compared to cells that were transfected with the wild-type N protein. This finding suggests that R106, by enhancing binding of the N protein to viral RNA plays a critical role in the viral replication. The results also indicate that the strength of N protein/RNA interactions is critical for HCoV-OC43 replication.


Assuntos
Coronavirus Humano OC43/química , Coronavirus Humano OC43/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Proteínas do Nucleocapsídeo de Coronavírus , Coronavirus Humano OC43/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/métodos , Proteínas do Nucleocapsídeo/genética , Estrutura Terciária de Proteína , RNA Viral/química , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência
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