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1.
J Inorg Biochem ; 231: 111777, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35255411

RESUMO

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is currently the major challenge to global public health. Two proteases, papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro or Mpro), are indispensable for SARS-CoV-2 replication, making them attractive targets for antiviral therapy development. Here we screened a panel of essential metal ions using a proteolytic assay and identified that zinc gluconate, a widely-used zinc supplement, strongly inhibited the proteolytic activities of the two proteases in vitro. Biochemical and crystallographic data reveal that zinc gluconate exhibited the inhibitory function via binding to the protease catalytic site residues. We further show that treatment of zinc gluconate in combination with a small molecule ionophore hinokitiol, could lead to elevated intracellular Zn2+ level and thereby significantly impaired the two protease activities in cellulo. Particularly, this approach could also be applied to rescue SARS-CoV-2 infected mammalian cells, indicative of potential application to combat coronavirus infections. Our studies provide the direct experimental evidence that elevated intracellular zinc concentration directly inhibits SARS-CoV-2 replication and suggest the potential benefits to use the zinc supplements for coronavirus disease 2019 (COVID-19) treatment.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Antivirais/química , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Gluconatos , Mamíferos/metabolismo , Monoterpenos , Peptídeo Hidrolases/metabolismo , Tropolona/análogos & derivados , Zinco/farmacologia
2.
Autophagy ; : 1-17, 2022 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-35239449

RESUMO

SARS-CoV-2 infections have resulted in a very large number of severe cases of COVID-19 and deaths worldwide. However, knowledge of SARS-CoV-2 infection, pathogenesis and therapy remains limited, emphasizing the urgent need for fundamental studies and drug development. Studies have shown that induction of macroautophagy/autophagy and hijacking of the autophagic machinery are essential for the infection and replication of SARS-CoV-2; however, the mechanism of this manipulation and the function of autophagy during SARS-CoV-2 infection remain unclear. In the present study, we identified ORF3a as an inducer of autophagy (in particular reticulophagy) and revealed that ORF3a localizes to the ER and induces RETREG1/FAM134B-related reticulophagy through the HMGB1-BECN1 (beclin 1) pathway. As a consequence, ORF3a induces ER stress and inflammatory responses through reticulophagy and then sensitizes cells to the acquisition of an ER stress-related early apoptotic phenotype and facilitates SARS-CoV-2 infection, suggesting that SARS-CoV-2 ORF3a hijacks reticulophagy and then disrupts ER homeostasis to induce ER stress and inflammatory responses during SARS-CoV-2 infection. These findings reveal the sequential induction of reticulophagy, ER stress and acute inflammatory responses during SARS-CoV-2 infection and imply the therapeutic potential of reticulophagy and ER stress-related drugs for COVID-19.

3.
Nat Metab ; 4(1): 29-43, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34992299

RESUMO

Severe cases of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are associated with elevated blood glucose levels and metabolic complications. However, the molecular mechanisms for how SARS-CoV-2 infection alters glycometabolic control are incompletely understood. Here, we connect the circulating protein GP73 with enhanced hepatic gluconeogenesis during SARS-CoV-2 infection. We first demonstrate that GP73 secretion is induced in multiple tissues upon fasting and that GP73 stimulates hepatic gluconeogenesis through the cAMP/PKA signaling pathway. We further show that GP73 secretion is increased in cultured cells infected with SARS-CoV-2, after overexpression of SARS-CoV-2 nucleocapsid and spike proteins and in lungs and livers of mice infected with a mouse-adapted SARS-CoV-2 strain. GP73 blockade with an antibody inhibits excessive glucogenesis stimulated by SARS-CoV-2 in vitro and lowers elevated fasting blood glucose levels in infected mice. In patients with COVID-19, plasma GP73 levels are elevated and positively correlate with blood glucose levels. Our data suggest that GP73 is a glucogenic hormone that likely contributes to SARS-CoV-2-induced abnormalities in systemic glucose metabolism.


Assuntos
COVID-19/complicações , COVID-19/virologia , Glucose/metabolismo , Hiperglicemia/etiologia , Hiperglicemia/metabolismo , Proteínas de Membrana/metabolismo , SARS-CoV-2 , Animais , Biomarcadores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Jejum , Expressão Gênica , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/genética , Interações Hospedeiro-Patógeno , Humanos , Hiperglicemia/sangue , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Especificidade de Órgãos/genética
4.
Cell Death Differ ; 2022 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-35022571

RESUMO

Deaths caused by coronavirus disease 2019 (COVID-19) are largely due to the lungs edema resulting from the disruption of the lung alveolo-capillary barrier, induced by SARS-CoV-2-triggered pulmonary cell apoptosis. However, the molecular mechanism underlying the proapoptotic role of SARS-CoV-2 is still unclear. Here, we revealed that SARS-CoV-2 membrane (M) protein could induce lung epithelial cells mitochondrial apoptosis. Notably, M protein stabilized B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) via inhibiting its ubiquitination and promoted BOK mitochondria translocation. The endodomain of M protein was required for its interaction with BOK. Knockout of BOK by CRISPR/Cas9 increased cellular resistance to M protein-induced apoptosis. BOK was rescued in the BOK-knockout cells, which led to apoptosis induced by M protein. M protein induced BOK to trigger apoptosis in the absence of BAX and BAK. Furthermore, the BH2 domain of BOK was required for interaction with M protein and proapoptosis. In vivo M protein recombinant lentivirus infection induced caspase-associated apoptosis and increased alveolar-capillary permeability in the mouse lungs. BOK knockdown improved the lung edema due to lentivirus-M protein infection. Overall, M protein activated the BOK-dependent apoptotic pathway and thus exacerbated SARS-CoV-2 associated lung injury in vivo. These findings proposed a proapoptotic role for M protein in SARS-CoV-2 pathogenesis, which may provide potential targets for COVID-19 treatments.

5.
Signal Transduct Target Ther ; 7(1): 22, 2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-35075101

RESUMO

As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I-MAVS complex to attenuate the RIG-I-mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.


Assuntos
Proteases 3C de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteína DEAD-box 58/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/genética , Receptores Imunológicos/genética , SARS-CoV-2/genética , /genética , Animais , Chlorocebus aethiops , Proteases 3C de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteína DEAD-box 58/imunologia , DNA Helicases/imunologia , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Evasão da Resposta Imune , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Poli I-C/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Ligação Proteica , RNA Helicases/imunologia , Proteínas com Motivo de Reconhecimento de RNA/imunologia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , Proteínas de Ligação a RNA/imunologia , Receptores Imunológicos/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Vírus Sendai/genética , Vírus Sendai/imunologia , Transdução de Sinais , /imunologia , Células Vero , Vesiculovirus/genética , Vesiculovirus/imunologia
6.
Cell Chem Biol ; 29(1): 5-18.e6, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34672954

RESUMO

The global epidemic caused by the coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in the infection of over 200 million people. To extend the knowledge of interactions between SARS-CoV-2 and humans, we systematically investigate the interactome of 29 viral proteins in human cells by using an antibody-based TurboID assay. In total, 1,388 high-confidence human proximal proteins with biotinylated sites are identified. Notably, we find that SARS-CoV-2 manipulates the antiviral and immune responses. We validate that the membrane protein ITGB1 associates angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 entry. Moreover, we reveal that SARS-CoV-2 proteins inhibit activation of the interferon pathway through the mitochondrial protein mitochondrial antiviral-signaling protein (MAVS) and the methyltransferase SET domain containing 2, histone lysine methyltransferase (SETD2). We propose 111 potential drugs for the clinical treatment of coronavirus disease 2019 (COVID-19) and identify three compounds that significantly inhibit the replication of SARS-CoV-2. The proximity labeling map of SARS-CoV-2 and humans provides a resource for elucidating the mechanisms of viral infection and developing drugs for COVID-19 treatment.


Assuntos
Anticorpos/imunologia , Antivirais/imunologia , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Antivirais/farmacologia , COVID-19/tratamento farmacológico , COVID-19/imunologia , Humanos , Integrina beta1/imunologia , Testes de Sensibilidade Microbiana
7.
Sci Adv ; 7(50): eabi6802, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34878838

RESUMO

Limited understanding of T cell responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impeded vaccine development and drug discovery for coronavirus disease 2019 (COVID-19). We found that triggering receptor expressed on myeloid cells 2 (TREM-2) was induced in T cells in the blood and lungs of patients with COVID-19. After binding to SARS-CoV-2 membrane (M) protein through its immunoglobulin domain, TREM-2 then activated the CD3ζ/ZAP70 complex, leading to STAT1 phosphorylation and T-bet transcription. In vitro stimulation with M protein-reconstituted pseudovirus or recombinant M protein, and TREM-2 promoted the T helper cell 1 (TH1) cytokines interferon-γ and tumor necrosis factor. In vivo infection of CD4­TREM-2 conditional knockout mice with murine coronavirus mouse hepatitis virus A-59 showed that intrinsic TREM-2 in T cells enhanced TH1 response and viral clearance, thus aggravating lung destruction. These findings demonstrate a previously unidentified role for TREM-2 in SARS-CoV-2 infection, and suggest potential strategies for drug discovery and clinical management of COVID-19.

8.
Artigo em Inglês | MEDLINE | ID: mdl-34905492

RESUMO

Geodesics measure the shortest distance between points on curved surfaces and are a fundamental tool in digital geometry processing. Although there are many algorithms for computing geodesic paths, most of them are designed for a single type of geometric domain (typically a triangle mesh), thus diminishing its practical usage. In this paper, we propose a general framework for computing geodesic paths for various geometric domains, including meshes, point clouds, implicit surfaces and parametric surfaces. Our key idea is to decouple geodesic computation from the type of the input surfaces. Discretizing the initial path by a sequence of points{x_i}, i=1,2,…,n that can only move on the surface, we minimize H(||x_i-x_(i+1)||), i=1,2,…,n. We show that the point sequence yielded by our approach is also a minimizer of the traditional geodesic length function, and the points are evenly spaced along the path. Our method encodes 3D geometry by signed distance functions (SDF) and can work for all types of input. We also propose a simple yet effective strategy to generate initial paths. We demonstrate the advantages of our methods over the conventional geodesic path algorithms in terms of accuracy, performance and scalability. Finally, we show that our method can be extended to solve general minimal-cost path problem.

9.
Chem Sci ; 12(42): 14098-14102, 2021 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-34760193

RESUMO

The SARS-CoV-2 3-chymotrypsin-like protease (3CLpro or Mpro) is a key cysteine protease for viral replication and transcription, making it an attractive target for antiviral therapies to combat the COVID-19 disease. Here, we demonstrate that bismuth drug colloidal bismuth subcitrate (CBS) is a potent inhibitor for 3CLpro in vitro and in cellulo. Rather than targeting the cysteine residue at the catalytic site, CBS binds to an allosteric site and results in dissociation of the 3CLpro dimer and proteolytic dysfunction. Our work provides direct evidence that CBS is an allosteric inhibitor of SARS-CoV-2 3CLpro.

11.
Cell Res ; 31(10): 1047-1060, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34465913

RESUMO

The outbreak of SARS-CoV-2 (SARS2) has caused a global COVID-19 pandemic. The spike protein of SARS2 (SARS2-S) recognizes host receptors, including ACE2, to initiate viral entry in a complex biomechanical environment. Here, we reveal that tensile force, generated by bending of the host cell membrane, strengthens spike recognition of ACE2 and accelerates the detachment of spike's S1 subunit from the S2 subunit to rapidly prime the viral fusion machinery. Mechanistically, such mechano-activation is fulfilled by force-induced opening and rotation of spike's receptor-binding domain to prolong the bond lifetime of spike/ACE2 binding, up to 4 times longer than that of SARS-S binding with ACE2 under 10 pN force application, and subsequently by force-accelerated S1/S2 detachment which is up to ~103 times faster than that in the no-force condition. Interestingly, the SARS2-S D614G mutant, a more infectious variant, shows 3-time stronger force-dependent ACE2 binding and 35-time faster force-induced S1/S2 detachment. We also reveal that an anti-S1/S2 non-RBD-blocking antibody that was derived from convalescent COVID-19 patients with potent neutralizing capability can reduce S1/S2 detachment by 3 × 106 times under force. Our study sheds light on the mechano-chemistry of spike activation and on developing a non-RBD-blocking but S1/S2-locking therapeutic strategy to prevent SARS2 invasion.


Assuntos
COVID-19/diagnóstico , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Resistência à Tração , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , COVID-19/terapia , COVID-19/virologia , Humanos , Concentração de Íons de Hidrogênio , Imunização Passiva , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos/imunologia , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Subunidades Proteicas/metabolismo , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Internalização do Vírus
12.
Gene ; 804: 145902, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34403773

RESUMO

BACKGROUNDS: Osteonecrosis of the femoral head (ONFH) is one of the common and complicated diseases in the orthopedic clinic. Previous studies indicate that genetic factors play a crucial role in the occurrence of ONFH. This case-control study aimed to investigate the associations of MIR137HG genetic polymorphisms with the alcohol-induced ONFH risk. METHODS: A total of 731 participants were recruited to detect the effect of MIR137HG SNPs on the alcohol-induced ONFH risk in a Chinese male population. Odds ratios (OR) and 95% confidence intervals (CI) were calculated to evaluate the associations. Multifactor dimensionality reduction (MDR) was used to analyze the SNP-SNP interaction with the alcohol-induced ONFH risk. RESULTS: Our study showed that rs7549905 played a protective role in alcohol-induced ONFH risk (OR 0.57, p = 0.045). Stratified analysis indicated that rs9440302 was associated with an increased risk of patients aged >45 years (OR 2.00, p = 0.038), and rs7549905 showed a reduced risk in patients aged ≤ 45 years (OR 0.43, p = 0.023). In addition, we found that rs9440302 and rs7554283 exhibited a significantly increased susceptibility of III-IV grade alcohol-induced ONFH patients (OR 2.34, p = 0.003; OR 2.13, p = 0.011, respectively). We also observed that rs12138817 was related to an increased risk in patients with >21 months of course (OR 1.77, p = 0.043). Interestingly, rs17371457 showed a significant correlation with low-density lipoprotein-cholesterol (p = 0.040). CONCLUSION: Our study suggests that MIR137HG genetic variants are associated with the alcohol-induced ONFH susceptibility in a Chinese male population, which may give scientific evidence for exploring molecular mechanisms of the alcohol-induced ONFH.


Assuntos
Necrose da Cabeça do Fêmur/genética , MicroRNAs/genética , Adolescente , Adulto , Idoso , Consumo de Bebidas Alcoólicas/efeitos adversos , Transtornos Induzidos por Álcool/epidemiologia , Transtornos Induzidos por Álcool/genética , Transtornos Induzidos por Álcool/metabolismo , Estudos de Casos e Controles , Criança , China/epidemiologia , Cabeça do Fêmur/metabolismo , Cabeça do Fêmur/patologia , Necrose da Cabeça do Fêmur/epidemiologia , Necrose da Cabeça do Fêmur/patologia , Frequência do Gene , Predisposição Genética para Doença , Haplótipos , Humanos , Desequilíbrio de Ligação , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Nucleotídeo Único
13.
Biochim Biophys Acta Mol Basis Dis ; 1867(12): 166260, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34461258

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection-induced inflammatory responses are largely responsible for the death of novel coronavirus disease 2019 (COVID-19) patients. However, the mechanism by which SARS-CoV-2 triggers inflammatory responses remains unclear. Here, we aimed to explore the regulatory role of SARS-CoV-2 spike protein in infected cells and attempted to elucidate the molecular mechanism of SARS-CoV-2-induced inflammation. METHODS: SARS-CoV-2 spike pseudovirions (SCV-2-S) were generated using the spike-expressing virus packaging system. Western blot, mCherry-GFP-LC3 labeling, immunofluorescence, and RNA-seq were performed to examine the regulatory mechanism of SCV-2-S in autophagic response. The effects of SCV-2-S on apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Western blot, and flow cytometry analysis. Enzyme-linked immunosorbent assay (ELISA) was carried out to examine the mechanism of SCV-2-S in inflammatory responses. RESULTS: Angiotensin-converting enzyme 2 (ACE2)-mediated SCV-2-S infection induced autophagy and apoptosis in human bronchial epithelial and microvascular endothelial cells. Mechanistically, SCV-2-S inhibited the PI3K/AKT/mTOR pathway by upregulating intracellular reactive oxygen species (ROS) levels, thus promoting the autophagic response. Ultimately, SCV-2-S-induced autophagy triggered inflammatory responses and apoptosis in infected cells. These findings not only improve our understanding of the mechanism underlying SARS-CoV-2 infection-induced pathogenic inflammation but also have important implications for developing anti-inflammatory therapies, such as ROS and autophagy inhibitors, for COVID-19 patients.


Assuntos
COVID-19/metabolismo , Inflamação/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Apoptose/imunologia , Autofagia/fisiologia , Linhagem Celular , Chlorocebus aethiops , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Inflamação/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , SARS-CoV-2/patogenicidade , Transdução de Sinais/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células Vero
14.
Immunol Lett ; 237: 33-41, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34228987

RESUMO

OBJECTIVE: In this study, we focused on the interaction between SARS-CoV-2 and host Type I Interferon (IFN) response, so as to identify whether IFN effects could be influenced by the products of SARS-CoV-2. METHODS: All the structural and non-structural proteins of SARS-CoV-2 were transfected and overexpressed in the bronchial epithelial cell line BEAS-2B respectively, and typical antiviral IFN-stimulated gene (ISG) ISG15 expression was detected by qRT-PCR. RNA-seq based transcriptome analysis was performed between control and Spike (S) protein-overexpressed BEAS-2B cells. The expression of ACE2 and IFN effector JAK-STAT signaling activation were detected in control and S protein-overexpressed BEAS-2B cells by qRT-PCR or/and Western blot respectively. The interaction between S protein with STAT1 and STAT2, and the association between JAK1 with downstream STAT1 and STAT2 were measured in BEAS-2B cells by co-immunoprecipitation (co-IP). RESULTS: S protein could activate IFN effects and downstream ISGs expression. By transcriptome analysis, overexpression of S protein induced a set of genes expression, including series of ISGs and the SARS-CoV-2 receptor ACE2. Mechanistically, S protein enhanced the association between the upstream JAK1 and downstream STAT1 and STAT2, so as to promote STAT1 and STAT2 phosphorylation and ACE2 expression. CONCLUSION: SARS-CoV-2 S protein enhances ACE2 expression via facilitating IFN effects, which may help its infection.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Brônquios/efeitos dos fármacos , COVID-19/virologia , Células Epiteliais/efeitos dos fármacos , Interferon alfa-2/farmacologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Brônquios/enzimologia , Brônquios/virologia , COVID-19/enzimologia , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Janus Quinase 1/metabolismo , Fosforilação , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Ubiquitinas/genética , Ubiquitinas/metabolismo , Regulação para Cima
15.
J Virol ; 95(17): e0074721, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34133897

RESUMO

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus , Interferon beta , Regiões Promotoras Genéticas , SARS-CoV-2 , RNA-Polimerase RNA-Dependente de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/antagonistas & inibidores , Interferon beta/biossíntese , Interferon beta/genética , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo
18.
J Med Virol ; 93(9): 5376-5389, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33913550

RESUMO

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.


Assuntos
Proteína DEAD-box 58/imunologia , Evasão da Resposta Imune/genética , Interferons/imunologia , Nucleotidiltransferases/imunologia , Receptores Imunológicos/imunologia , SARS-CoV-2/imunologia , Receptor 3 Toll-Like/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteína DEAD-box 58/genética , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Interferons/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , /imunologia , Receptores Imunológicos/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/genética , Transfecção , Células Vero , Replicação Viral/imunologia
19.
J Virol ; 2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658349

RESUMO

Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, the fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL (∼0.043 nM) and 3.18 ng/mL (∼0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnostic reagents for COVID-19.ImportanceTo date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics, including SARS-CoV-2 targeting antibodies, remains critical. Due to their small size (13-15 kDa), high solubility, and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multivalent formats than the conventional antibody. Here, we report a series of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.

20.
Stem Cell Res ; 53: 102197, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33771478

RESUMO

Excessive prostaglandin E2 (PGE2) is the key pathological basis for COVID-19 and a Celebrex treatment of hospitalized COVID-19 patients with comorbidities led to 100% discharged rate and zero death (Hong et al. 2020). It is also suggested that SARS-CoV-2 infected multiple organs and the SARS-CoV nucleocapsid (N) protein transcriptionally drives the expression of the host COX-2 gene. In order to test whether SARS-CoV-2 N protein activates COX-2 transcription in multiple human relevant cell types, an expression inducible human embryonic stem cell line was generated by piggyBac transposon system. This cell line maintained its pluripotency, differentiation potentials, normal morphology and karyotype.


Assuntos
COVID-19 , Células-Tronco Embrionárias Humanas , Linhagem Celular , Humanos , Proteínas do Nucleocapsídeo/genética , SARS-CoV-2
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