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1.
Sci Rep ; 10(1): 19395, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33173052

RESUMO

An incomplete understanding of the molecular mechanisms behind impairment of lung pathobiology by COVID-19 complicates its clinical management. In this study, we analyzed the gene expression pattern of cells obtained from biopsies of COVID-19-affected patient and compared to the effects observed in typical SARS-CoV-2 and SARS-CoV-infected cell-lines. We then compared gene expression patterns of COVID-19-affected lung tissues and SARS-CoV-2-infected cell-lines and mapped those to known lung-related molecular networks, including hypoxia induced responses, lung development, respiratory processes, cholesterol biosynthesis and surfactant metabolism; all of which are suspected to be downregulated following SARS-CoV-2 infection based on the observed symptomatic impairments. Network analyses suggest that SARS-CoV-2 infection might lead to acute lung injury in COVID-19 by affecting surfactant proteins and their regulators SPD, SPC, and TTF1 through NSP5 and NSP12; thrombosis regulators PLAT, and EGR1 by ORF8 and NSP12; and mitochondrial NDUFA10, NDUFAF5, and SAMM50 through NSP12. Furthermore, hypoxia response through HIF-1 signaling might also be targeted by SARS-CoV-2 proteins. Drug enrichment analysis of dysregulated genes has allowed us to propose novel therapies, including lung surfactants, respiratory stimulants, sargramostim, and oseltamivir. Our study presents a distinct mechanism of probable virus induced lung damage apart from cytokine storm.


Assuntos
Infecções por Coronavirus/genética , Infecções por Coronavirus/metabolismo , Perfilação da Expressão Gênica , Pulmão/metabolismo , Terapia de Alvo Molecular , Pneumonia Viral/genética , Pneumonia Viral/metabolismo , Surfactantes Pulmonares/metabolismo , Biologia de Sistemas , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Epigênese Genética , Humanos , Pulmão/efeitos dos fármacos , Especificidade de Órgãos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Proteínas Virais/metabolismo
2.
Nat Commun ; 11(1): 5043, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028863

RESUMO

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Integrases/ultraestrutura , Proteína Fosfatase 2/ultraestrutura , Vírus Linfotrópico T Tipo 1 de Símios/enzimologia , Proteínas Virais/ultraestrutura , Integração Viral , Motivos de Aminoácidos/genética , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Viral/metabolismo , DNA Viral/ultraestrutura , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Integrases/genética , Integrases/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Paraparesia Espástica Tropical/patologia , Paraparesia Espástica Tropical/virologia , Multimerização Proteica , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Vírus Linfotrópico T Tipo 1 de Símios/genética , Imagem Individual de Molécula , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
mBio ; 11(5)2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082264

RESUMO

An accurate diagnostic test for early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the key weapon to control the coronavirus disease 2019 (COVID-19) pandemic. We previously reported that the SARS-CoV-2 genome contains a unique orf8 accessory gene absent from other human-pathogenic coronaviruses. Here, we characterized the SARS-CoV-2 orf8 as a novel immunogenic secreted protein and utilized it for the accurate diagnosis of COVID-19. Extracellular orf8 protein was detected in cell culture supernatant and in sera of COVID-19 patients. In addition, orf8 was found highly immunogenic in COVID-19 patients, who showed early seropositivity for anti-orf8 IgM, IgG, and IgA. We hypothesize that orf8 secretion during SARS-CoV-2 infection facilitates early mounting of B cell response. The serological test detecting anti-orf8 IgG antibody can be used for the early and accurate diagnosis of COVID-19.IMPORTANCE Current commercially available serological tests for COVID-19 patients are detecting antibodies against SARS-CoV-2 nucleoprotein and spike glycoprotein. The antinucleoprotein and antispike antibodies can be accurately detected in patients during the mid or late stage of infection, and therefore, these assays have not been widely used for early diagnosis of COVID-19. In this study, we characterized the secretory property of a SARS-CoV-2 orf8 protein and proposed that orf8 secretion during infection facilitates early mounting of the B cell response. We demonstrated the presence of anti-orf8 antibodies in both symptomatic and asymptomatic patients during the early stage of infection, while the anti-N antibody is not detected. Our serological test detecting anti-orf8 antibodies may facilitate the development of early and accurate diagnosis for COVID-19.


Assuntos
Antígenos Virais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Proteínas Virais/imunologia , Anticorpos Antivirais/sangue , Antígenos Virais/sangue , Antígenos Virais/metabolismo , Linhagem Celular , Infecções por Coronavirus/sangue , Diagnóstico Precoce , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Pandemias , Pneumonia Viral/sangue , Proteínas Virais/sangue , Proteínas Virais/metabolismo
4.
Sci Rep ; 10(1): 17772, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082395

RESUMO

SARS-CoV-2 can infiltrate the lower respiratory tract, resulting in severe respiratory failure and a high death rate. Normally, the airway and alveolar epithelium can be rapidly reconstituted by multipotent stem cells after episodes of infection. Here, we analyzed published RNA-seq datasets and demonstrated that cells of four different lung epithelial stem cell types express SARS-CoV-2 entry factors, including Ace2. Thus, stem cells can be potentially infected by SARS-CoV-2, which may lead to defects in regeneration capacity partially accounting for the severity of SARS-CoV-2 infection and its consequences.


Assuntos
Betacoronavirus/fisiologia , Proteínas Virais/metabolismo , Internalização do Vírus , Betacoronavirus/isolamento & purificação , Diferenciação Celular , Linhagem Celular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Pulmão/citologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Serina Endopeptidases/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/virologia , Proteínas Virais/genética
5.
Immun Inflamm Dis ; 8(4): 753-762, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33124193

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) is a single-stranded RNA virus responsible for the global pandemic of the coronavirus disease-2019 (COVID-19). To date, there are still no effective approaches for the prevention and treatment of COVID-19. OBJECTIVE: The present study aims to explore the possible mechanisms of SARS-CoV-2 infection in human lung cells. METHODS: Data interpretation was conducted by recruiting bioinformatics analysis, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways analysis using downloaded data from the NCBI Gene Expression Omnibus database. RESULTS: The present study demonstrated that SARS-CoV-2 infection induces the upregulation of 14 interferon-stimulated genes, indicative of immune, and interferon responses to the virus. Notably, genes for pyrimidine metabolism and steroid hormone biosynthesis are selectively enriched in human lung cells after SARS-CoV-2 infection, suggesting that altered pyrimidine metabolism and steroid biosynthesis are remarkable, and perhaps druggable features after SARS-CoV-2 infection. Besides, there is a strong positive correlation between viral ORF1ab, ORF6, and angiotensin-converting enzyme 2 (ACE2) expression in human lung cells, implying that ACE2 facilitates SARS-CoV-2 infection and replication in host cells probably through the induction of ORF1ab and ORF6.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/etiologia , Interferons/metabolismo , Pulmão/patologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/etiologia , Betacoronavirus/metabolismo , Biologia Computacional , Infecções por Coronavirus/patologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Pulmão/citologia , Pulmão/imunologia , Pulmão/virologia , Pandemias , Pneumonia Viral/patologia , Pirimidinas/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Transdução de Sinais/imunologia , Esteroides/biossíntese , Regulação para Cima/imunologia , Proteínas Virais/metabolismo
6.
J Proteome Res ; 19(11): 4355-4363, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33006287

RESUMO

A model that predicts levels of coronavirus (CoV) respiratory and fecal-oral transmission potentials based on the shell disorder has been built using neural network (artificial intelligence, AI) analysis of the percentage of disorder (PID) in the nucleocapsid, N, and membrane, M, proteins of the inner and outer viral shells, respectively. Using primarily the PID of N, SARS-CoV-2 is grouped as having intermediate levels of both respiratory and fecal-oral transmission potentials. Related studies, using similar methodologies, have found strong positive correlations between virulence and inner shell disorder among numerous viruses, including Nipah, Ebola, and Dengue viruses. There is some evidence that this is also true for SARS-CoV-2 and SARS-CoV, which have N PIDs of 48% and 50%, and case-fatality rates of 0.5-5% and 10.9%, respectively. The underlying relationship between virulence and respiratory potentials has to do with the viral loads of vital organs and body fluids, respectively. Viruses can spread by respiratory means only if the viral loads in saliva and mucus exceed certain minima. Similarly, a patient is likelier to die when the viral load overwhelms vital organs. Greater disorder in inner shell proteins has been known to play important roles in the rapid replication of viruses by enhancing the efficiency pertaining to protein-protein/DNA/RNA/lipid bindings. This paper suggests a novel strategy in attenuating viruses involving comparison of disorder patterns of inner shells (N) of related viruses to identify residues and regions that could be ideal for mutation. The M protein of SARS-CoV-2 has one of the lowest M PID values (6%) in its family, and therefore, this virus has one of the hardest outer shells, which makes it resistant to antimicrobial enzymes in body fluid. While this is likely responsible for its greater contagiousness, the risks of creating an attenuated virus with a more disordered M are discussed.


Assuntos
Inteligência Artificial , Betacoronavirus , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Vacinas Virais , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Desenvolvimento de Medicamentos/métodos , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Carga Viral , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
7.
EBioMedicine ; 61: 103036, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33045467

RESUMO

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.


Assuntos
Proteínas de Bactérias/metabolismo , Betacoronavirus/genética , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endodesoxirribonucleases/metabolismo , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Cavidade Nasal/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
J Proteome Res ; 19(11): 4553-4566, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33103435

RESUMO

While the COVID-19 pandemic is causing important loss of life, knowledge of the effects of the causative SARS-CoV-2 virus on human cells is currently limited. Investigating protein-protein interactions (PPIs) between viral and host proteins can provide a better understanding of the mechanisms exploited by the virus and enable the identification of potential drug targets. We therefore performed an in-depth computational analysis of the interactome of SARS-CoV-2 and human proteins in infected HEK 293 cells published by Gordon et al. (Nature 2020, 583, 459-468) to reveal processes that are potentially affected by the virus and putative protein binding sites. Specifically, we performed a set of network-based functional and sequence motif enrichment analyses on SARS-CoV-2-interacting human proteins and on PPI networks generated by supplementing viral-host PPIs with known interactions. Using a novel implementation of our GoNet algorithm, we identified 329 Gene Ontology terms for which the SARS-CoV-2-interacting human proteins are significantly clustered in PPI networks. Furthermore, we present a novel protein sequence motif discovery approach, LESMoN-Pro, that identified 9 amino acid motifs for which the associated proteins are clustered in PPI networks. Together, these results provide insights into the processes and sequence motifs that are putatively implicated in SARS-CoV-2 infection and could lead to potential therapeutic targets.


Assuntos
Betacoronavirus , Infecções por Coronavirus , Interações Hospedeiro-Patógeno/genética , Pandemias , Pneumonia Viral , Mapas de Interação de Proteínas , Algoritmos , Motivos de Aminoácidos , Betacoronavirus/química , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , Análise por Conglomerados , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Ontologia Genética , Células HEK293 , Humanos , Anotação de Sequência Molecular , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Mapas de Interação de Proteínas/genética , Mapas de Interação de Proteínas/fisiologia , Proteínas/química , Proteínas/classificação , Proteínas/genética , Proteínas/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
9.
Arch Virol ; 165(12): 2857-2867, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33034763

RESUMO

Rabies is an important zoonotic disease in Iran. Autophagy is a process that maintains homeostasis and can be used as an innate defense mechanism against viruses. Apoptosis is the process of programmed cell death induced by physiological and pathological conditions. The crosstalk of autophagy and apoptosis plays a key role in rabies virus infection. In the current study, NMRI mice intra-cranially received 3-Methyl Adenine (3-MA), rapamycin, street rabies virus (SRABV) and drugs plus SRABV. SRABV and Map1lc3, Beclin-1, Atg5 gene expression were assayed by real-time PCR. Immunohistochemistry was carried out via LC3 protein staining as an autophagy marker, and apoptotic cell death was measured using a TUNEL assay. Map1lc3, Beclin-1 and Atg5 genes expression was significantly increased in drug-plus-SRBV-treated tissues compared to control at 24 hpi. Map1lc3 and Atg5 gene expression showed a slight change in the drugs-plus-virus group compared with the control at 72 hpi. The presence of LC3 in the tissues of the group treated with rapamycin plus SRBV confirmed induction of autophagy, but it was not present in the tissues treated with 3-MA plus SRBV. Our data revealed that apoptosis was induced only in the groups receiving the SRBV or rapamycin or both at 24 hpi. Apoptosis was observed after 72 hours, when the drugs' effect had disappeared in all but the autophagy inhibitor group. Understanding the interaction of SRABV with autophagy pathway genes and its effect on host cell apoptosis may open a new horizon for human intervention and allow a deeper understanding of rabies infections.


Assuntos
Apoptose , Autofagia , Encéfalo/patologia , Neurônios/citologia , Vírus da Raiva/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Encéfalo/virologia , Modelos Animais de Doenças , Técnica Direta de Fluorescência para Anticorpo , Camundongos , Neurônios/virologia , Raiva/patologia , Raiva/virologia , Vírus da Raiva/genética , Sirolimo/farmacologia , Proteínas Virais/metabolismo , Replicação Viral
10.
PLoS One ; 15(9): e0239899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32986763

RESUMO

Influenza A virus controls replication and transcription of its genome through the tight regulation of interaction between the ribonucleoprotein (RNP) complex subunits. The helical scaffold of RNP is maintained by nucleoprotein (NP). Previous studies have revealed that NP interacts with both PB2 N-terminal and C-terminal regions, with both regions sharing similar affinity to NP as revealed in co-immunoprecipitation assay. Our work here suggests that the interaction between NP and PB2 N-terminal region lies in the cap-binding domain (residue 320-483). By co-immunoprecipitation assay, the interaction was found to involve RNA. On the other hand, the cap-binding activity was not essential in the interaction. As shown by the NHS pull-down assay, a specific RNA sequence was not required. Among the cap-binding domain, residues K331 and R332 of PB2 play a role in RNP function so that polymerase activity was reduced when these residues were mutated, while K331 was found to be more crucial in the NP interaction. Collectively, our findings suggest a new binding mode between NP and PB2 which was mediated by RNA, and such interaction may provide a novel interacting site for influenza drug development.


Assuntos
Interações entre Hospedeiro e Microrganismos/genética , Domínios Proteicos , RNA Replicase/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Imunoprecipitação , Plasmídeos/genética , Ligação Proteica/genética , RNA Replicase/química , RNA Replicase/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Transfecção , Proteínas do Core Viral/genética , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral/genética
11.
Nat Commun ; 11(1): 4894, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994400

RESUMO

Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increasing the chance to preferentially detect viral proteomes. We identify nine previously cryptic orphan protein coding sequences whose translated products are expressed in HSV-1-infected cells. Functional characterization of one identified protein, designated piUL49, shows that it is critical for HSV-1 neurovirulence in vivo by regulating the activity of virally encoded dUTPase, a key enzyme that maintains accurate DNA replication. Our results demonstrate that cryptic orphan protein coding genes of HSV-1, and probably other large DNA viruses, remain to be identified.


Assuntos
Encefalite por Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Pirofosfatases/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Replicação do DNA , Modelos Animais de Doenças , Encefalite por Herpes Simples/patologia , Feminino , Genes Virais/genética , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Camundongos , Biossíntese de Proteínas , Proteômica/métodos , Células Vero , Proteínas Virais/genética , Fatores de Virulência/genética , Replicação Viral
12.
PLoS Negl Trop Dis ; 14(9): e0008555, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976538

RESUMO

Junin virus (JUNV) is a New World arenavirus that is the causative agent of Argentine hemorrhagic fever (AHF). Candid#1 (Can) is a live-attenuated vaccine strain of JUNV that since its introduction has resulted in a marked decrease in AHF incidence within the endemic regions of the Pampas in Argentina. Originally, the viral determinants and mechanisms of Can attenuation were not well understood. Recent work has identified the glycoprotein as the major attenuating factor for Can. The establishment of attenuating strategies based on any of the other viral proteins, however, has not been pursued. Here, we document the role of Can Z resulting in incompatibilities with wild type JUNV that results in decreased growth in vitro. In addition, this incompatibility results in attenuation of the virus in the guinea pig model. Further, we identify a single mutation (V64G) in the Z protein that is able to confer this demonstrated attenuation. By establishing and characterizing a novel attenuation strategy for New World mammarenaviruses, we hope to aid future vaccine development for related emerging pathogens including Machupo virus (MACV), Guanarito virus (GTOV), and Sabia virus (SABV).


Assuntos
Glicoproteínas/genética , Febre Hemorrágica Americana/virologia , Vírus Junin/genética , Mutação , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Feminino , Glicoproteínas/metabolismo , Cobaias , Vírus Junin/crescimento & desenvolvimento , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo
13.
PLoS Negl Trop Dis ; 14(9): e0008602, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886656

RESUMO

Besides the common Fc receptor (FcR)-mediated mechanism of antibody-dependent enhancement (ADE), Ebola virus (EBOV) is known to utilize the complement component C1q for ADE of infection. This mechanism is FcR-independent and mediated by cross-linking of virus-antibody-C1q complexes to cell surface C1q receptors, leading to enhanced viral entry into cells. Using confocal microscopy, we found that virus-like particles (VLPs) consisting of EBOV glycoprotein, nucleoprotein, and matrix protein attached to the surface of human kidney 293 cells more efficiently in the presence of an ADE monoclonal antibody and C1q than with the antibody or C1q alone, and that there was no significant difference in the efficiency of VLP uptake into endosomes between the C1q-mediated ADE and non-ADE entry. Accordingly, both ADE and non-ADE infection were similarly decreased by inhibitors of the signaling pathways known to be required for endocytosis. These results suggest that C1q-mediated ADE of EBOV infection is simply caused by increased attachment of virus particles to the cell surface, which is distinct from the mechanism of FcR-mediated ADE requiring intracellular signaling to promote phagocytosis/macropinocytosis.


Assuntos
Anticorpos Facilitadores/imunologia , Complemento C1q/metabolismo , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Glicoproteínas de Membrana/metabolismo , Receptores de Complemento/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Ebolavirus/patogenicidade , Endocitose/imunologia , Células HEK293 , Doença pelo Vírus Ebola/patologia , Humanos , Células Vero , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Ligação Viral
14.
J Proteome Res ; 19(11): 4637-4648, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32893632

RESUMO

The pandemic caused by SARS-CoV-2 is currently representing a major health and economic threat to humanity. So far, no specific treatment to this viral infection has been developed and the emergency still requires an efficient intervention. In this work, we used virtual screening to facilitate drug repurposing against SARS-CoV-2, targeting viral main proteinase and spike protein with 3000 existing drugs. We used a protocol based on a docking step followed by a short molecular dynamic simulation and rescoring by the Nwat-MMGBSA approach. Our results provide suggestions for prioritizing in vitro and/or in vivo tests of already available compounds.


Assuntos
Antivirais , Reposicionamento de Medicamentos/métodos , Peptídeo Hidrolases , Glicoproteína da Espícula de Coronavírus , Antivirais/química , Antivirais/metabolismo , Betacoronavirus/química , Betacoronavirus/enzimologia , Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
15.
J Proteome Res ; 19(11): 4698-4705, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32946692

RESUMO

The coronavirus disease of 2019 (COVID-19) pandemic speaks to the need for drugs that not only are effective but also remain effective given the mutation rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To this end, we describe structural binding-site insights for facilitating COVID-19 drug design when targeting RNA-dependent RNA polymerase (RDRP), a common conserved component of RNA viruses. We combined an RDRP structure data set, including 384 RDRP PDB structures and all corresponding RDRP-ligand interaction fingerprints, thereby revealing the structural characteristics of the active sites for application to RDRP-targeted drug discovery. Specifically, we revealed the intrinsic ligand-binding modes and associated RDRP structural characteristics. Four types of binding modes with corresponding binding pockets were determined, suggesting two major subpockets available for drug discovery. We screened a drug data set of 7894 compounds against these binding pockets and presented the top-10 small molecules as a starting point in further exploring the prevention of virus replication. In summary, the binding characteristics determined here help rationalize RDRP-targeted drug discovery and provide insights into the specific binding mechanisms important for containing the SARS-CoV-2 virus.


Assuntos
Betacoronavirus , Infecções por Coronavirus/virologia , Descoberta de Drogas/métodos , Pneumonia Viral/virologia , RNA Replicase , Proteínas Virais , Betacoronavirus/química , Betacoronavirus/metabolismo , Sítios de Ligação , Humanos , Simulação de Acoplamento Molecular , Pandemias , Ligação Proteica , RNA Replicase/química , RNA Replicase/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
16.
PLoS Pathog ; 16(9): e1008767, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32903273

RESUMO

Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.


Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica , Lyssavirus/imunologia , Infecções por Rhabdoviridae/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Virais/metabolismo , Células HEK293 , Células HeLa , Humanos , Interleucina-6/metabolismo , Infecções por Rhabdoviridae/metabolismo , Infecções por Rhabdoviridae/virologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Transativadores , Proteínas Virais/genética
17.
Nat Commun ; 11(1): 4775, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963221

RESUMO

Enterovirus 71 (EV71) poses serious threats to human health, particularly in Southeast Asia, and no drugs or vaccines are available. Previous work identified the stem loop II structure of the EV71 internal ribosomal entry site as vital to viral translation and a potential target. After screening an RNA-biased library using a peptide-displacement assay, we identify DMA-135 as a dose-dependent inhibitor of viral translation and replication with no significant toxicity in cell-based studies. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein, thus repressing translation. This mechanism is supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.


Assuntos
Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Sítios Internos de Entrada Ribossomal/fisiologia , Replicação Viral/fisiologia , Regiões 5' não Traduzidas , Linhagem Celular , Infecções por Enterovirus/virologia , Regulação Viral da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , RNA Viral/química , Proteínas Virais/metabolismo
18.
Cell Rep ; 33(1): 108234, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32979938

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and host immune response determine coronavirus disease 2019 (COVID-19), but studies evaluating viral evasion of immune response are lacking. Here, we use unbiased screening to identify SARS-CoV-2 proteins that antagonize type I interferon (IFN-I) response. We found three proteins that antagonize IFN-I production via distinct mechanisms: nonstructural protein 6 (nsp6) binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, nsp13 binds and blocks TBK1 phosphorylation, and open reading frame 6 (ORF6) binds importin Karyopherin α 2 (KPNA2) to inhibit IRF3 nuclear translocation. We identify two sets of viral proteins that antagonize IFN-I signaling through blocking signal transducer and activator of transcription 1 (STAT1)/STAT2 phosphorylation or nuclear translocation. Remarkably, SARS-CoV-2 nsp1 and nsp6 suppress IFN-I signaling more efficiently than SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Thus, when treated with IFN-I, a SARS-CoV-2 replicon replicates to a higher level than chimeric replicons containing nsp1 or nsp6 from SARS-CoV or MERS-CoV. Altogether, the study provides insights on SARS-CoV-2 evasion of IFN-I response and its potential impact on viral transmission and pathogenesis.


Assuntos
Proteínas do Capsídeo/metabolismo , Infecções por Coronavirus/imunologia , Evasão da Resposta Imune , Interferon Tipo I/metabolismo , Metiltransferases/metabolismo , Pneumonia Viral/imunologia , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Células A549 , Animais , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Cricetinae , Cricetulus , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Pandemias , Pneumonia Viral/virologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , alfa Carioferinas/metabolismo
19.
Mol Biol Evol ; 37(9): 2463-2464, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32893295

RESUMO

Identifying the origin of SARS-CoV-2, the etiological agent of the current COVID-19 pandemic, may help us to avoid future epidemics of coronavirus and other zoonoses. Several theories about the zoonotic origin of SARS-CoV-2 have recently been proposed. Although Betacoronavirus found in Rhinolophus bats from China have been broadly implicated, their genetic dissimilarity to SARS-CoV-2 is so high that they are highly unlikely to be its direct ancestors. Thus, an intermediary host is suspected to link bat to human coronaviruses. Based on genomic CpG dinucleotide patterns in different coronaviruses from different hosts, it was suggested that SARS-CoV-2 might have evolved in a canid gastrointestinal tract prior to transmission to humans. However, similar CpG patterns are now reported in coronaviruses from other hosts, including bats themselves and pangolins. Therefore, reduced genomic CpG alone is not a highly predictive biomarker, suggesting a need for additional biomarkers to reveal intermediate hosts or tissues. The hunt for the zoonotic origin of SARS-CoV-2 continues.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Genoma Viral , Pandemias , Pneumonia Viral/epidemiologia , Proteínas Virais/genética , Zoonoses/epidemiologia , Animais , Betacoronavirus/classificação , Betacoronavirus/patogenicidade , Quirópteros/virologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Ilhas de CpG , Eutérios/virologia , Evolução Molecular , Expressão Gênica , Mutação , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Vírus Reordenados/classificação , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Recombinação Genética , Proteínas Virais/metabolismo , Zoonoses/transmissão , Zoonoses/virologia
20.
Nat Commun ; 11(1): 4571, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917871

RESUMO

Early therapeutic interventions are essential to prevent Alzheimer Disease (AD). The association of several inflammation-related genetic markers with AD and the early activation of pro-inflammatory pathways in AD suggest inflammation as a plausible therapeutic target. Inflammatory Caspase-1 has a significant impact on AD-like pathophysiology and Caspase-1 inhibitor, VX-765, reverses cognitive deficits in AD mouse models. Here, a one-month pre-symptomatic treatment of Swedish/Indiana mutant amyloid precursor protein (APPSw/Ind) J20 and wild-type mice with VX-765 delays both APPSw/Ind- and age-induced episodic and spatial memory deficits. VX-765 delays inflammation without considerably affecting soluble and aggregated amyloid beta peptide (Aß) levels. Episodic memory scores correlate negatively with microglial activation. These results suggest that Caspase-1-mediated inflammation occurs early in the disease and raise hope that VX-765, a previously Food and Drug Administration-approved drug for human CNS clinical trials, may be a useful drug to prevent the onset of cognitive deficits and brain inflammation in AD.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Disfunção Cognitiva/metabolismo , Serpinas/metabolismo , Proteínas Virais/metabolismo , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Animais , Comportamento Animal , Disfunção Cognitiva/tratamento farmacológico , Citocinas/metabolismo , Dipeptídeos/sangue , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Encefalite/metabolismo , Encefalite/patologia , Feminino , Humanos , Inflamação/metabolismo , Masculino , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Serpinas/sangue , Serpinas/farmacologia , Memória Espacial/fisiologia , Proteínas Virais/sangue , Proteínas Virais/farmacologia , para-Aminobenzoatos/sangue , para-Aminobenzoatos/farmacologia
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