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1.
mBio ; 12(2)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727347

RESUMO

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins.IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.


Assuntos
Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Interferons/farmacologia , Espaço Intracelular/metabolismo , Proteínas de Membrana/genética , Mutação , Vírus de RNA/classificação , Vírus de RNA/metabolismo , Especificidade da Espécie , Ubiquitina-Proteína Ligases/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vírion/metabolismo , Replicação Viral
2.
Food Chem Toxicol ; 150: 112087, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33640537

RESUMO

Coronavirus disease-19 (COVID-19) is a complex disease that causes illness ranging from mild to severe respiratory problems. It is caused by a novel coronavirus SARS-CoV-2 (Severe acute respiratory syndrome coronavirus-2) that is an enveloped positive-sense single-stranded RNA (+ssRNA) virus belongs to coronavirus CoV family. It has a fast-spreading potential worldwide, which leads to high mortality regardless of lows death rates. Now some vaccines or a specific drug are approved but not available for every country for disease prevention and/or treatment. Therefore, it is a high demand to identify the known drugs and test them as a possible therapeutic approach. In this critical situation, one or more of these drugs may represent the only option to treat or reduce the severity of the disease, until some specific drugs or vaccines will be developed and/or approved for everyone in this pandemic. In this updated review, the available repurpose immunotherapeutic treatment strategies are highlighted, elucidating the crosstalk between the immune system and SARS-CoV-2. Despite the reasonable data availability, the effectiveness and safety of these drugs against SARS-CoV-2 needs further studies and validations aiming for a better clinical outcome.


Assuntos
Antivirais/farmacologia , Inflamação/etiologia , /efeitos dos fármacos , Corticosteroides/farmacologia , Corticosteroides/uso terapêutico , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antivirais/uso terapêutico , /imunologia , Citocinas/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Interferons/farmacologia , Interferons/uso terapêutico , Inibidores de Janus Quinases/farmacologia , Inibidores de Janus Quinases/uso terapêutico , /imunologia
3.
Viruses ; 13(1)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33478119

RESUMO

Respiratory viral infections constitute a global public health concern. Among prevalent respiratory viruses, two pneumoviruses can be life-threatening in high-risk populations. In young children, they constitute the first cause of hospitalization due to severe lower respiratory tract diseases. A better understanding of their pathogenesis is still needed as there are no approved efficient anti-viral nor vaccine against pneumoviruses. We studied Respiratory Syncytial virus (RSV) and human Metapneumovirus (HMPV) in single and dual infections in three-dimensional cultures, a highly relevant model to study viral respiratory infections of the airway epithelium. Our investigation showed that HMPV is less pathogenic than RSV in this model. Compared to RSV, HMPV replicated less efficiently, induced a lower immune response, did not block cilia beating, and was more sensitive to IFNs. In dual infections, RSV-infected epithelia were less permissive to HMPV. By neutralizing IFNs in co-infection assays, we partially prevented HMPV inhibition by RSV and significantly increased the number of co-infected cells in the tissue. This suggests that interference in dual infection would be at least partly mediated by the host immune response. In summary, this work provides new insight regarding virus-host and virus-virus interactions of pneumoviruses in the airway epithelium. This could be helpful for the proper handling of at-risk patients.


Assuntos
Técnicas de Cultura de Células , Coinfecção , Interações Hospedeiro-Patógeno , Metapneumovirus/fisiologia , Interações Microbianas , Vírus Sincicial Respiratório Humano/fisiologia , Replicação Viral , Linhagem Celular , Humanos , Interferon Tipo I/farmacologia , Interferons/farmacologia , Metapneumovirus/efeitos dos fármacos , Infecções por Paramyxoviridae/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Esferoides Celulares
4.
Cytokine ; 140: 155430, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33508651

RESUMO

In vitro interferon (IFN)α treatment of primary human upper airway basal cells has been shown to drive ACE2 expression, the receptor of SARS-CoV-2. The protease furin is also involved in mediating SARS-CoV-2 and other viral infections, although its association with early IFN response has not been evaluated yet. In order to assess the in vivo relationship between ACE2 and furin expression and the IFN response in nasopharyngeal cells, we first examined ACE2 and furin levels and their correlation with the well-known marker of IFNs' activation, ISG15, in children (n = 59) and adults (n = 48), during respiratory diseases not caused by SARS-CoV-2. A strong positive correlation was found between ACE2 expression, but not of furin, and ISG15 in all patients analyzed. In addition, type I and III IFN stimulation experiments were performed to examine the IFN-mediated activation of ACE2 isoforms (full-length and truncated) and furin in epithelial cell lines. Following all the IFNs treatments, only the truncated ACE2 levels, were upregulated significantly in the A549 and Calu3 cells, in particular by type I IFNs. If confirmed in vivo following IFNs' activation, the induction of the truncated ACE2 isoform only would not enhance the risk of SARS-CoV-2 infection in the respiratory tract.


Assuntos
/genética , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Interferons/farmacologia , /efeitos dos fármacos , Células A549 , Adulto , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular Tumoral , Criança , Citocinas/genética , Células Epiteliais/metabolismo , Humanos , Interferons/metabolismo , Pulmão/citologia , Pessoa de Meia-Idade , Ubiquitinas/genética
6.
Ann Palliat Med ; 10(1): 707-720, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33440983

RESUMO

The whole world is battling through coronavirus disease 2019 (COVID-19) which is a fatal pandemic. In the early 2020, the World Health Organization (WHO) declared it as a global health emergency without definitive treatments and preventive approaches. In the absence of definitive therapeutic agents, this thorough review summarizes and outlines the potency and safety of all molecules and therapeutics which may have potential antiviral effects. A number of molecules and therapeutics licensed or being tested for some other conditions were found effective in different in vitro studies as well as in many small sample-sized clinical trials and independent case studies. However, in those clinical trials, there were some limitations which need to be overcome to find the most promising antiviral against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In conclusion, many of above-mentioned antivirals seems to have some therapeutic effects but none of them have been shown to have a strong evidence for their proper recommendation and approval in the treatment of COVID-19. Constantly evolving new evidences, exclusive adult data, language barrier, and type of study (observational, retrospective, small-sized clinical trials, or independent case series) resulted to the several limitations of this review. The need for multicentered, large sample-sized, randomized, placebo-controlled trials on COVID-19 patients to reach a proper conclusion on the most promising antiviral agent is warranted.


Assuntos
Antivirais/uso terapêutico , /terapia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Amidas/farmacologia , Amidas/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Azetidinas/farmacologia , Azetidinas/uso terapêutico , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Combinação de Medicamentos , Humanos , Hidroxicloroquina/farmacologia , Hidroxicloroquina/uso terapêutico , Imunização Passiva , Indóis/farmacologia , Indóis/uso terapêutico , Interferons/farmacologia , Interferons/uso terapêutico , Ivermectina/farmacologia , Ivermectina/uso terapêutico , Lopinavir/farmacologia , Lopinavir/uso terapêutico , Oseltamivir/farmacologia , Oseltamivir/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ribavirina/farmacologia , Ribavirina/uso terapêutico , Ritonavir/farmacologia , Ritonavir/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Tiazóis/farmacologia , Tiazóis/uso terapêutico
7.
Gene ; 771: 145368, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33346100

RESUMO

Coronavirus disease-2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has become an immense threat to global public health. In this study, we performed complete genome sequencing of a SARS-CoV-2 isolate. More than 67,000 genome sequences were further inspected from Global Initiative on Sharing All Influenza Data (GISAID). Using several in silico techniques, we proposed prospective therapeutics against this virus. Through meticulous analysis, several conserved and therapeutically suitable regions of SARS-CoV-2 such as RNA-dependent RNA polymerase (RdRp), Spike (S) and Membrane glycoprotein (M) coding genes were selected. Both S and M were chosen for the development of a chimeric vaccine that can generate memory B and T cells. siRNAs were also designed for S and M gene silencing. Moreover, six new drug candidates were suggested that might inhibit the activity of RdRp. Since SARS-CoV-2 and SARS-CoV-1 have 82.30% sequence identity, a Gene Expression Omnibus (GEO) dataset of Severe Acute Respiratory Syndrome (SARS) patients were analyzed. In this analysis, 13 immunoregulatory genes were found that can be used to develop type 1 interferon (IFN) based therapy. The proposed vaccine, siRNAs, drugs and IFN based analysis of this study will accelerate the development of new treatments.


Assuntos
Antivirais/farmacologia , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Sequenciamento Completo do Genoma/métodos , Antivirais/uso terapêutico , Simulação por Computador , Sequência Conservada , Desenho de Fármacos , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Interferons/farmacologia , Pessoa de Meia-Idade , Estudos Prospectivos , /classificação , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/genética
8.
Medicine (Baltimore) ; 99(46): e23155, 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33181688

RESUMO

BACKGROUND: There are currently no available standard drugs treating human papillomavirus (HPV) infection, especially for patients with low-grade cervical lesion. Several therapies are explored but the results are inconclusive. The objective of this study was to evaluate the efficacy of reported non-invasive treatments in patients with HPV infection and cervical lesions by meta-analysis. METHODS: A comprehensive search of prospective and randomized studies published from April 2000 to April 2020 was conducted in electronic databases. The statistical analyses of the pooled risk ratios (RRs) and the corresponding 95% confidence intervals (95% CIs) were performed using the Revman 5.2 software. RESULTS: Twelve articles including 12 randomized controlled studies and 1 prospective controlled randomized pilot study were enrolled. Therapeutic medications included biological and herbal regimen, interferon regimen and probiotics. The meta-analysis showed the experimental treatments had a statistically significant improvement in HPV clearance rate compared with the controls (RR = 0.71, 95% CI [0.63, 0.80], P < .00001); subgroup analyses stratified by regimen categories were consistent with results in the overall group. Treatment using biological and herbal regimen, interferon regimen or probiotics also resulted in a beneficial outcome in regression rate of cervical lesions compared with the controls (RR = 0.55, 95% CI [0.39, 0.79], P = .001). The trend was more favorable in the probiotics than that in the biological and herbal regimen (RR 0.48 vs 0.72). CONCLUSION: Treatment of biological and herbal regimen, interferon regimen and probiotics benefit patients who have HPV infection and cervical lesions. Both the clearance of HPV and regression of cervical lesions are significant. More studies with less heterogeneity are needed to draw a concrete conclusion.


Assuntos
Produtos Biológicos/farmacologia , Neoplasia Intraepitelial Cervical/terapia , Interferons/farmacologia , Infecções por Papillomavirus/terapia , Preparações de Plantas/farmacologia , Antivirais/farmacologia , Neoplasia Intraepitelial Cervical/virologia , Feminino , Humanos , Infecções por Papillomavirus/complicações , Resultado do Tratamento
9.
mBio ; 11(5)2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067384

RESUMO

Recent evidence shows that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is sensitive to interferons (IFNs). However, the most effective types of IFNs and the underlying antiviral effectors remain to be defined. Here, we show that zinc finger antiviral protein (ZAP), which preferentially targets CpG dinucleotides in viral RNA sequences, restricts SARS-CoV-2. We further demonstrate that ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells. Type I, II, and III IFNs all strongly inhibited SARS-CoV-2 and further induced ZAP expression. Comprehensive sequence analyses revealed that SARS-CoV-2 and its closest relatives from horseshoe bats showed the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, endogenous ZAP expression restricted SARS-CoV-2 replication in human lung cells, particularly upon treatment with IFN-α or IFN-γ. Both the long and the short isoforms of human ZAP reduced SARS-CoV-2 RNA expression levels, but the former did so with greater efficiency. Finally, we show that the ability to restrict SARS-CoV-2 is conserved in ZAP orthologues of the reservoir bat and potential intermediate pangolin hosts of human coronaviruses. Altogether, our results show that ZAP is an important effector of the innate response against SARS-CoV-2, although this pandemic pathogen emerged from zoonosis of a coronavirus that was preadapted to the low-CpG environment in humans.IMPORTANCE Although interferons inhibit SARS-CoV-2 and have been evaluated for treatment of coronavirus disease 2019 (COVID-19), the most effective types and antiviral effectors remain to be defined. Here, we show that IFN-γ is particularly potent in restricting SARS-CoV-2 and in inducing expression of the antiviral factor ZAP in human lung cells. Knockdown experiments revealed that endogenous ZAP significantly restricts SARS-CoV-2. We further show that CpG dinucleotides which are specifically targeted by ZAP are strongly suppressed in the SARS-CoV-2 genome and that the two closest horseshoe bat relatives of SARS-CoV-2 show the lowest genomic CpG content of all coronavirus sequences available from this reservoir host. Nonetheless, both the short and long isoforms of human ZAP reduced SARS-CoV-2 RNA levels, and this activity was conserved in horseshoe bat and pangolin ZAP orthologues. Our findings indicating that type II interferon is particularly efficient against SARS-CoV-2 and that ZAP restricts this pandemic viral pathogen might promote the development of effective immune therapies against COVID-19.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Ilhas de CpG , Pneumonia Viral/virologia , Proteínas de Ligação a RNA/metabolismo , Animais , Betacoronavirus/classificação , Betacoronavirus/genética , Betacoronavirus/metabolismo , Linhagem Celular , Coronavirus/classificação , Coronavirus/genética , Coronavirus/fisiologia , Expressão Gênica/efeitos dos fármacos , Genoma Viral , Humanos , Interferons/farmacologia , Pandemias , Filogenia , Isoformas de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Replicação Viral/efeitos dos fármacos
10.
mBio ; 11(5)2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913009

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is a recently emerged respiratory coronavirus that has infected >23 million people worldwide with >800,000 deaths. Few COVID-19 therapeutics are available, and the basis for severe infections is poorly understood. Here, we investigated properties of type I (ß), II (γ), and III (λ1) interferons (IFNs), potent immune cytokines that are normally produced during infection and that upregulate IFN-stimulated gene (ISG) effectors to limit virus replication. IFNs are already in clinical trials to treat COVID-19. However, recent studies highlight the potential for IFNs to enhance expression of host angiotensin-converting enzyme 2 (ACE2), suggesting that IFN therapy or natural coinfections could exacerbate COVID-19 by upregulating this critical virus entry receptor. Using a cell line model, we found that beta interferon (IFN-ß) strongly upregulated expression of canonical antiviral ISGs, as well as ACE2 at the mRNA and cell surface protein levels. Strikingly, IFN-λ1 upregulated antiviral ISGs, but ACE2 mRNA was only marginally elevated and did not lead to detectably increased ACE2 protein at the cell surface. IFN-γ induced the weakest ISG response but clearly enhanced surface expression of ACE2. Importantly, all IFN types inhibited SARS-CoV-2 replication in a dose-dependent manner, and IFN-ß and IFN-λ1 exhibited potent antiviral activity in primary human bronchial epithelial cells. Our data imply that type-specific mechanisms or kinetics shape IFN-enhanced ACE2 transcript and cell surface levels but that the antiviral action of IFNs against SARS-CoV-2 counterbalances any proviral effects of ACE2 induction. These insights should aid in evaluating the benefits of specific IFNs, particularly IFN-λ, as repurposed therapeutics.IMPORTANCE Repurposing existing, clinically approved, antiviral drugs as COVID-19 therapeutics is a rapid way to help combat the SARS-CoV-2 pandemic. Interferons (IFNs) usually form part of the body's natural innate immune defenses against viruses, and they have been used with partial success to treat previous new viral threats, such as HIV, hepatitis C virus, and Ebola virus. Nevertheless, IFNs can have undesirable side effects, and recent reports indicate that IFNs upregulate the expression of host ACE2 (a critical entry receptor for SARS-CoV-2), raising the possibility that IFN treatments could exacerbate COVID-19. Here, we studied the antiviral- and ACE2-inducing properties of different IFN types in both a human lung cell line model and primary human bronchial epithelial cells. We observed differences between IFNs with respect to their induction of antiviral genes and abilities to enhance the cell surface expression of ACE2. Nevertheless, all the IFNs limited SARS-CoV-2 replication, suggesting that their antiviral actions can counterbalance increased ACE2.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Interferons/farmacologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Idoso , Animais , Betacoronavirus/imunologia , Linhagem Celular , Chlorocebus aethiops , Feminino , Humanos , Imunoterapia/métodos , Interferon Tipo I/efeitos adversos , Interferon gama/efeitos adversos , Interferons/efeitos adversos , Pandemias , Peptidil Dipeptidase A/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Virais/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Regulação para Cima/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos
11.
Biochimie ; 177: 50-52, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32805303

RESUMO

Various interferon (IFN)-inducible transmembrane (IFITM) proteins are known to be expressed in human tissues though only IFITM 1-3 are inducible by IFN. Numerous studies have shown that activation of IFITM3 could suppress infection by influenza and coronaviruses such as the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). In view of the potential application of IFITM proteins' induction to target SARS-CoV-2 infection that causes COVID-19, this article layout insights into the known antiviral mechanisms and therapeutic agents related to IFITM. Blocking viral entry through various mechanisms and the potential application of the FDA approved immunosuppressant agent, mycophenolic acid, as inducer of IFITM3 are among those discussed.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Interferons/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Ácido Micofenólico/farmacologia , Pneumonia Viral/tratamento farmacológico , Proteínas de Ligação a RNA/efeitos dos fármacos , Animais , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Humanos , Imunossupressores/farmacologia , Proteínas de Membrana/imunologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Proteínas de Ligação a RNA/imunologia
12.
Nature ; 586(7830): 560-566, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32854108

RESUMO

Coronaviruses are prone to transmission to new host species, as recently demonstrated by the spread to humans of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic1. Small animal models that recapitulate SARS-CoV-2 disease are needed urgently for rapid evaluation of medical countermeasures2,3. SARS-CoV-2 cannot infect wild-type laboratory mice owing to inefficient interactions between the viral spike protein and the mouse orthologue of the human receptor, angiotensin-converting enzyme 2 (ACE2)4. Here we used reverse genetics5 to remodel the interaction between SARS-CoV-2 spike protein and mouse ACE2 and designed mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA), a recombinant virus that can use mouse ACE2 for entry into cells. SARS-CoV-2 MA was able to replicate in the upper and lower airways of both young adult and aged BALB/c mice. SARS-CoV-2 MA caused more severe disease in aged mice, and exhibited more clinically relevant phenotypes than those seen in Hfh4-ACE2 transgenic mice, which express human ACE2 under the control of the Hfh4 (also known as Foxj1) promoter. We demonstrate the utility of this model using vaccine-challenge studies in immune-competent mice with native expression of mouse ACE2. Finally, we show that the clinical candidate interferon-λ1a (IFN-λ1a) potently inhibits SARS-CoV-2 replication in primary human airway epithelial cells in vitro-both prophylactic and therapeutic administration of IFN-λ1a diminished SARS-CoV-2 replication in mice. In summary, the mouse-adapted SARS-CoV-2 MA model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN-λ1a as a treatment for human COVID-196.


Assuntos
Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Interferons/farmacologia , Interferons/uso terapêutico , Interleucinas/farmacologia , Interleucinas/uso terapêutico , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Envelhecimento/imunologia , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Interferon-alfa/administração & dosagem , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , Interferons/administração & dosagem , Interleucinas/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Moleculares , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/genética , Pneumonia Viral/imunologia , Receptores Virais/genética , Receptores Virais/metabolismo
13.
PLoS One ; 15(7): e0235705, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32649682

RESUMO

Mutations of the SWI/SNF chromatin remodeling complex occur in 20% of all human cancers, including ovarian cancer. Approximately half of ovarian clear cell carcinomas (OCCC) carry mutations in the SWI/SNF subunit ARID1A, while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) presents with inactivating mutations of the SWI/SNF ATPase SMARCA4 alongside epigenetic silencing of the ATPase SMARCA2. Loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A), which regulates the chromatin landscape and gene expression by demethylating proteins such as histone H3. Cross-cancer analysis of the TCGA database shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. SCCOHT and OCCC cell lines have shown sensitivity to the reversible LSD1 inhibitor SP-2577 (Seclidemstat), suggesting that SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Moreover, it has been shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of endogenous retroviral elements and may thereby overcome resistance to checkpoint blockade. In this study, we investigated the ability of SP-2577 to promote anti-tumor immunity and T-cell infiltration in SCCOHT and OCCC cell lines. We found that SP-2577 stimulated IFN-dependent anti-tumor immunity in SCCOHT and promoted the expression of PD-L1 in both SCCOHT and OCCC. Together, these findings suggest that the combination therapy of SP-2577 with checkpoint inhibitors may induce or augment immunogenic responses of SWI/SNF-mutated ovarian cancers and warrants further investigation.


Assuntos
Antineoplásicos/farmacologia , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Linfócitos T/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Humanos , Interferons/farmacologia , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Fatores de Transcrição/metabolismo
14.
J Biol Chem ; 295(41): 13958-13964, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32587093

RESUMO

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the devastating COVID-19 lung disease pandemic. Here, we tested the inhibitory activities of the antiviral interferons of type I (IFN-α) and type III (IFN-λ) against SARS-CoV-2 and compared them with those against SARS-CoV-1, which emerged in 2003. Using two mammalian epithelial cell lines (human Calu-3 and simian Vero E6), we found that both IFNs dose-dependently inhibit SARS-CoV-2. In contrast, SARS-CoV-1 was restricted only by IFN-α in these cell lines. SARS-CoV-2 generally exhibited a broader IFN sensitivity than SARS-CoV-1. Moreover, ruxolitinib, an inhibitor of IFN-triggered Janus kinase/signal transducer and activator of transcription signaling, boosted SARS-CoV-2 replication in the IFN-competent Calu-3 cells. We conclude that SARS-CoV-2 is sensitive to exogenously added IFNs. This finding suggests that type I and especially the less adverse effect-prone type III IFN are good candidates for the management of COVID-19.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Interferon Tipo I/farmacologia , Interferons/farmacologia , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Janus Quinases/metabolismo , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Pirazóis/farmacologia , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos
15.
Gene ; 754: 144858, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32531455

RESUMO

Not only are autophagy-related (ATG) proteins the essential orchestrators of the autophagy machinery, but also they regulate many other cellular pathways. Here, we demonstrated that ATG13 exerted an obviously antiviral activity against the infection of peste des petits ruminants virus (PPRV) in cell culture model. We found that PPRV infection or the treatment with interferon (IFN) against PPRV infection significantly induced ATG13 expression. Mechanistically, ATG13 stimulated interferon expression and the subsequent activation of the JAK-STAT cascade. These activations triggered the transcription of interferon-stimulated genes (ISGs) to exert antiviral activity. Conversely, the loss of ATG13 significantly attenuated the potency of RIG-IN in activating IFN responses. In summary, we have demonstrated that basal ATG13 was involved in host antiviral activities against PPRV infection and the over-expression of ATG13 activated IFN production to inhibit PPRV replication in an unconventional fashion.


Assuntos
Antivirais/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interferons/farmacologia , Peste dos Pequenos Ruminantes/tratamento farmacológico , Vírus da Peste dos Pequenos Ruminantes/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Autofagia , Proteínas Relacionadas à Autofagia , Células HEK293 , Humanos , Peste dos Pequenos Ruminantes/imunologia , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , Transdução de Sinais
16.
Science ; 369(6504): 712-717, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32527928

RESUMO

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α and IFN-ß) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α and IFN-ß responses can lead to harmful proinflammatory effects, whereas IFN-λ mainly signals in epithelia, thereby inducing localized antiviral immunity. In this work, we show that IFN signaling interferes with lung repair during influenza recovery in mice, with IFN-λ driving these effects most potently. IFN-induced protein p53 directly reduces epithelial proliferation and differentiation, which increases disease severity and susceptibility to bacterial superinfections. Thus, excessive or prolonged IFN production aggravates viral infection by impairing lung epithelial regeneration. Timing and duration are therefore critical parameters of endogenous IFN action and should be considered carefully for IFN therapeutic strategies against viral infections such as influenza and coronavirus disease 2019 (COVID-19).


Assuntos
Células Epiteliais Alveolares/patologia , Citocinas/metabolismo , Interferon Tipo I/metabolismo , Interferons/metabolismo , Pulmão/patologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Células Epiteliais Alveolares/imunologia , Animais , Apoptose , Líquido da Lavagem Broncoalveolar/imunologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citocinas/administração & dosagem , Citocinas/imunologia , Feminino , Vírus da Influenza A Subtipo H3N2 , Interferon Tipo I/administração & dosagem , Interferon Tipo I/farmacologia , Interferon-alfa/administração & dosagem , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Interferon beta/administração & dosagem , Interferon beta/metabolismo , Interferon beta/farmacologia , Interferons/administração & dosagem , Interferons/farmacologia , Masculino , Camundongos , Infecções por Orthomyxoviridae/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
17.
Nat Commun ; 11(1): 2953, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528005

RESUMO

The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferon-stimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus.


Assuntos
Proteínas de Transporte/metabolismo , Ebolavirus/patogenicidade , Interferons/farmacologia , Animais , Western Blotting , Proteínas de Transporte/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Proteínas do Citoesqueleto , Ebolavirus/metabolismo , Imunofluorescência , Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Proteínas Virais/metabolismo , Internalização do Vírus , Replicação Viral/genética , Replicação Viral/fisiologia
18.
PLoS Pathog ; 16(4): e1008515, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32353085

RESUMO

Type III interferons (IFN-lambdas(λ)) are important cytokines that inhibit viruses and modulate immune responses by acting through a unique IFN-λR1/IL-10RB heterodimeric receptor. Until now, the primary antiviral function of IFN-λs has been proposed to be at anatomical barrier sites. Here, we examine the regulation of IFN-λR1 expression and measure the downstream effects of IFN-λ3 stimulation in primary human blood immune cells, compared with lung or liver epithelial cells. IFN-λ3 directly bound and upregulated IFN-stimulated gene (ISG) expression in freshly purified human B cells and CD8+ T cells, but not monocytes, neutrophils, natural killer cells, and CD4+ T cells. Despite similar IFNLR1 transcript levels in B cells and lung epithelial cells, lung epithelial cells bound more IFN-λ3, which resulted in a 50-fold greater ISG induction when compared to B cells. The reduced response of B cells could be explained by higher expression of the soluble variant of IFN-λR1 (sIFN-λR1), which significantly reduced ISG induction when added with IFN-λ3 to peripheral blood mononuclear cells or liver epithelial cells. T-cell receptor stimulation potently, and specifically, upregulated membrane-bound IFNLR1 expression in CD4+ T cells, leading to greater antiviral gene induction, and inhibition of human immunodeficiency virus type 1 infection. Collectively, our data demonstrate IFN-λ3 directly interacts with the human adaptive immune system, unlike what has been previously shown in published mouse models, and that type III IFNs could be potentially utilized to suppress both mucosal and blood-borne viral infections.


Assuntos
Interferons/farmacologia , Receptores de Interferon/biossíntese , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Células Epiteliais/metabolismo , Expressão Gênica , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Interferon alfa-2/farmacologia , Interferons/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fígado/metabolismo , Fígado/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Processamento de RNA , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Viroses/genética , Viroses/imunologia , Viroses/metabolismo
19.
Nat Commun ; 11(1): 2449, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415096

RESUMO

A comprehensive examination of protein-protein interactions (PPIs) is fundamental for the understanding of cellular machineries. However, limitations in current methodologies often prevent the detection of PPIs with low abundance proteins. To overcome this challenge, we develop a mRNA display with library of even-distribution (md-LED) method that facilitates the detection of low abundance binders with high specificity and sensitivity. As a proof-of-principle, we apply md-LED to IAV NS1 protein. Complementary to AP-MS, md-LED enables us to validate previously described PPIs as well as to identify novel NS1 interactors. We show that interacting with FASN allows NS1 to directly regulate the synthesis of cellular fatty acids. We also use md-LED to identify a mutant of NS1, D92Y, results in a loss of interaction with CPSF1. The use of high-throughput sequencing as the readout for md-LED enables sensitive quantification of interactions, ultimately enabling massively parallel experimentation for the investigation of PPIs.


Assuntos
Biblioteca Gênica , Vírus da Influenza A/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células A549 , Ácido Graxo Sintase Tipo I/metabolismo , Ontologia Genética , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Interferons/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Mutação/genética , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
20.
J Gen Virol ; 101(6): 573-586, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32375950

RESUMO

Bone marrow stromal cell antigen-2 (BST-2), also known as tetherin, is an interferon-inducible membrane-associated protein. It effectively targets enveloped viruses at the release step of progeny viruses from host cells, thereby restricting the further spread of viral infection. Junin virus (JUNV) is a member of Arenaviridae, which causes Argentine haemorrhagic fever that is associated with a high rate of mortality. In this study, we examined the effect of human BST-2 on the replication and propagation of JUNV. The production of JUNV Z-mediated virus-like particles (VLPs) was significantly inhibited by over-expression of BST-2. Electron microscopy analysis revealed that BST-2 functions by forming a physical link that directly retains VLPs on the cell surface. Infection using JUNV showed that infectious JUNV production was moderately inhibited by endogenous or exogenous BST-2. We also observed that JUNV infection triggers an intense interferon response, causing an upregulation of BST-2, in infected cells. However, the expression of cell surface BST-2 was reduced upon infection. Furthermore, the expression of JUNV nucleoprotein (NP) partially recovered VLP production from BST-2 restriction, suggesting that the NP functions as an antagonist against antiviral effect of BST-2. We further showed that JUNV NP also rescued the production of Ebola virus VP40-mediated VLP from BST-2 restriction as a broad spectrum BST-2 antagonist. To our knowledge, this is the first report showing that an arenavirus protein counteracts the antiviral function of BST-2.


Assuntos
Antígenos CD/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Vírus Junin/fisiologia , Nucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Liberação de Vírus/fisiologia , Células A549 , Antivirais/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferons/farmacologia , Vírus Junin/efeitos dos fármacos , Liberação de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
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