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2.
PLoS Pathog ; 16(9): e1008852, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960931

RESUMO

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridium difficile , Enterocolite Pseudomembranosa , Glucosiltransferases , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridium difficile/enzimologia , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Cricetinae , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/enzimologia , Enterocolite Pseudomembranosa/genética , Enterocolite Pseudomembranosa/patologia , Feminino , Deleção de Genes , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Masculino , Camundongos
3.
Sci Rep ; 10(1): 15917, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985513

RESUMO

SARS-CoV-2 is the novel coronavirus responsible for the outbreak of COVID-19, a disease that has spread to over 100 countries and, as of the 26th July 2020, has infected over 16 million people. Despite the urgent need to find effective therapeutics, research on SARS-CoV-2 has been affected by a lack of suitable animal models. To facilitate the development of medical approaches and novel treatments, we compared the ACE2 receptor, and TMPRSS2 and Furin proteases usage of the SARS-CoV-2 Spike glycoprotein in human and in a panel of animal models, i.e. guinea pig, dog, cat, rat, rabbit, ferret, mouse, hamster and macaque. Here we showed that ACE2, but not TMPRSS2 or Furin, has a higher level of sequence variability in the Spike protein interaction surface, which greatly influences Spike protein binding mode. Using molecular docking simulations we compared the SARS-CoV and SARS-CoV-2 Spike proteins in complex with the ACE2 receptor and showed that the SARS-CoV-2 Spike glycoprotein is compatible to bind the human ACE2 with high specificity. In contrast, TMPRSS2 and Furin are sufficiently similar in the considered hosts not to drive susceptibility differences. Computational analysis of binding modes and protein contacts indicates that macaque, ferrets and hamster are the most suitable models for the study of inhibitory antibodies and small molecules targeting the SARS-CoV-2 Spike protein interaction with ACE2. Since TMPRSS2 and Furin are similar across species, our data also suggest that transgenic animal models expressing human ACE2, such as the hACE2 transgenic mouse, are also likely to be useful models for studies investigating viral entry.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/veterinária , Pandemias/veterinária , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/veterinária , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos/genética , Animais , Gatos , Biologia Computacional/métodos , Infecções por Coronavirus/patologia , Cricetinae , Modelos Animais de Doenças , Cães , Furões , Furina/genética , Furina/metabolismo , Cobaias , Humanos , Macaca fascicularis , Camundongos , Simulação de Acoplamento Molecular , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Coelhos , Ratos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
4.
Emerg Microbes Infect ; 9(1): 2105-2113, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32893735

RESUMO

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Pandemias , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
5.
Zhonghua Yu Fang Yi Xue Za Zhi ; 54(9): 942-946, 2020 Sep 06.
Artigo em Chinês | MEDLINE | ID: mdl-32907282

RESUMO

Objective: Aanalysis the effect of booster one dose of hepatitis B vaccine after 21-32 years of primary immunization in Zhengding Country of Hebei Province. Methods: A total of 322 participants who were born between 1986 and 1996, received a full course of primary vaccination with plasma-derived hepatitis B vaccine (HepB), had no experience with booster vaccination, were HBsAg, anti-HBcnegative, had anti-HBs<10 mIU/ml, completed the booster and had laboratory results were enrolled between August 2017 to February 2018. A simple random method was uesd to randomly assigned 322 subjects to two groups, receiving a booster dose of HepB derived from either Saccharomyces cerevisiae ï¼»HepB (SC), (151 cases)ï¼½ or Chinese hamster ovary-derived HepB ï¼»HepB (CHO), (171 cases)ï¼½, the dose was 20 µg. Blood samples were collected 30 days after boosting and quantitatively tested for the geometric mean concentration (GMC) of anti-HBs to assess immunological effect. The related influencing factors of GMC and seroconversion rates of anti-HBs were analyzed by multiple linear regression and multivariate logistic regression models. Results: The 266 subjects (82.61%) had anti-HBs≥ 10 mIU/ml, and GMC was (131.63±12.94) mIU/ml.The seroconversion rates of anti-HBs in the anti-HBs<2.5 mIU/ml group and 2.5-10 mIU/ml group were 74.54% (161 cases) and 99.06% (105 cases), respectively (P<0.001).The seroconversion rates of anti-HBs after one dose of HepB (CHO) was higher than that of one dose of HepB (SC), the seroconversion rates were 87.13% (149 cases) and 77.48% (117 cases), respectively (P=0.023). Participants boostered with HepB (CHO) was the factor influencing the effect of strengthening immunization compared with boostered with HepB (SC), and OR (95%CI) was 1.91 (1.02-3.56) (P=0.042).Compared with anti-HBs<2.5 mIU/ml, prebooster anti-HBs was between 2.5 mIU/ml and 10 mIU/ml was the related factor of seroconversion rates of anti-HBs after booster immunization, and OR (95%CI) was 36.15 (4.91-266.02) (P<0.001). Conclusion: Participants boostered withone dose of HepB had a good immune response. Pre-booster anti-HBs concentration and a variety of vaccine were related factors of immune response.


Assuntos
Vacinas contra Hepatite B , Hepatite B/prevenção & controle , Animais , Células CHO , Cricetinae , Cricetulus , Seguimentos , Anticorpos Anti-Hepatite B , Antígenos de Superfície da Hepatite B , Imunização Secundária , Vacinação
6.
Front Immunol ; 11: 2163, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32983181

RESUMO

Introduction: The response to the SARS-CoV-2 coronavirus epidemic requires increased research efforts to expand our knowledge of the disease. Questions related to infection rates and mechanisms, the possibility of reinfection, and potential therapeutic approaches require us not only to use the experimental models previously employed for the SARS-CoV and MERS-CoV coronaviruses but also to generate new models to respond to urgent questions. Development: We reviewed the different experimental models used in the study of central nervous system (CNS) involvement in COVID-19 both in different cell lines that have enabled identification of the virus' action mechanisms and in animal models (mice, rats, hamsters, ferrets, and primates) inoculated with the virus. Specifically, we reviewed models used to assess the presence and effects of SARS-CoV-2 on the CNS, including neural cell lines, animal models such as mouse hepatitis virus CoV (especially the 59 strain), and the use of brain organoids. Conclusion: Given the clear need to increase our understanding of SARS-CoV-2, as well as its potential effects on the CNS, we must endeavor to obtain new information with cellular or animal models, with an appropriate resemblance between models and human patients.


Assuntos
Betacoronavirus , Infecções do Sistema Nervoso Central/complicações , Infecções do Sistema Nervoso Central/imunologia , Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Modelos Animais de Doenças , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Animais , Linhagem Celular Tumoral , Infecções do Sistema Nervoso Central/virologia , Infecções por Coronavirus/virologia , Cricetinae , Células HEK293 , Humanos , Camundongos , Organoides , Pandemias , Pneumonia Viral/virologia
7.
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
8.
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
9.
PLoS Pathog ; 16(9): e1008875, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898162

RESUMO

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrPSc, onto the cell-expressed conformer, PrPC. Here we study the conversion of PrPC to PrPSc using a recombinant mouse PrPSc conformer (mouse protein-only recPrPSc) as a unique tool that can convert bank vole but not mouse PrPC substrates in vitro. Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrPC substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrPSc. Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrPC substrates to be converted by Mo protein-only recPrPSc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion.


Assuntos
Encéfalo/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Animais , Arvicolinae , Encéfalo/patologia , Cricetinae , Mesocricetus , Camundongos , Camundongos Transgênicos , Proteínas PrPC/genética , Proteínas PrPSc/genética , Doenças Priônicas/genética , Doenças Priônicas/patologia , Domínios Proteicos
10.
PLoS Negl Trop Dis ; 14(9): e0008133, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925939

RESUMO

The emergence and spread of tick-borne arboviruses pose an increased challenge to human and animal health. In Europe this is demonstrated by the increasingly wide distribution of tick-borne encephalitis virus (TBEV, Flavivirus, Flaviviridae), which has recently been found in the United Kingdom (UK). However, much less is known about other tick-borne flaviviruses (TBFV), such as the closely related louping ill virus (LIV), an animal pathogen which is endemic to the UK and Ireland, but which has been detected in other parts of Europe including Scandinavia and Russia. The emergence and potential spatial overlap of these viruses necessitates improved understanding of LIV genomic diversity, geographic spread and evolutionary history. We sequenced a virus archive composed of 22 LIV isolates which had been sampled throughout the UK over a period of over 80 years. Combining this dataset with published virus sequences, we detected no sign of recombination and found low diversity and limited evidence for positive selection in the LIV genome. Phylogenetic analysis provided evidence of geographic clustering as well as long-distance movement, including movement events that appear recent. However, despite genomic data and an 80-year time span, we found that the data contained insufficient temporal signal to reliably estimate a molecular clock rate for LIV. Additional analyses revealed that this also applied to TBEV, albeit to a lesser extent, pointing to a general problem with phylogenetic dating for TBFV. The 22 LIV genomes generated during this study provide a more reliable LIV phylogeny, improving our knowledge of the evolution of tick-borne flaviviruses. Our inability to estimate a molecular clock rate for both LIV and TBEV suggests that temporal calibration of tick-borne flavivirus evolution should be interpreted with caution and highlight a unique aspect of these viruses which may be explained by their reliance on tick vectors.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Evolução Molecular , Genoma Viral , Animais , Linhagem Celular , Cricetinae , Vírus da Encefalite Transmitidos por Carrapatos/classificação , Encefalite Transmitida por Carrapatos/virologia , Genética Populacional , Metagenômica , Filogenia , Análise de Sequência de RNA , Ovinos , Reino Unido
11.
mBio ; 11(5)2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978313

RESUMO

Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.


Assuntos
Betacoronavirus/genética , Cromossomos Artificiais Bacterianos/genética , Animais , Betacoronavirus/patogenicidade , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Cricetinae , DNA Complementar/genética , Genoma Viral/genética , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Genética Reversa , Células Vero , Replicação Viral
12.
Nat Commun ; 11(1): 4137, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811827

RESUMO

The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas-G/química , Receptores dos Hormônios Gastrointestinais/química , Secretina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Cricetinae , Microscopia Crioeletrônica , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Insetos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos/genética , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Acoplados a Proteínas-G/ultraestrutura , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores dos Hormônios Gastrointestinais/ultraestrutura , Secretina/metabolismo
13.
Nat Commun ; 11(1): 4160, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814772

RESUMO

Ghrelin is a gastric peptide hormone with important physiological functions. The unique feature of ghrelin is its Serine 3 acyl-modification, which is essential for ghrelin's activity. However, it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity. To address these questions, we solved the crystal structure of the ghrelin receptor bound to antagonist. The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E124 and R283. A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap (crevasse) between the TM6 and TM7 bundles that is rich in hydrophobic amino acids, including a cluster of phenylalanine residues. Mutagenesis analyses suggest that the interaction between the gap structure and the acyl acid moiety of ghrelin may participate in transforming the ghrelin receptor into an active conformation.


Assuntos
Grelina/metabolismo , Fenilalanina/metabolismo , Receptores de Grelina/metabolismo , Animais , Sítios de Ligação/genética , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Grelina/química , Grelina/genética , Células HEK293 , Humanos , Ligantes , Camundongos Endogâmicos MRL lpr , Mutagênese Sítio-Dirigida , Fenilalanina/química , Fenilalanina/genética , Ligação Proteica , Conformação Proteica , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/genética , Células Sf9 , Spodoptera
15.
J Chromatogr A ; 1627: 461378, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823092

RESUMO

Downstream processing (DSP) of large bionanoparticles is still a challenge. The present study aims to systematically compare some of the most commonly used DSP strategies for capture and purification of enveloped viruses and virus-like particles (eVLPs) by using the same staring material and analytical tools. As a model, Human Immunodeficiency Virus-1 (HIV-1) gag VLPs produced in CHO cells were used. Four different DSP strategies were tested. An anion-exchange monolith and a membrane adsorber, for direct capture and purification of eVLPs, and a polymer-grafted anion-exchange resin and a heparin-affinity resin for eVLP purification after a first flow-through step to remove small impurities. All tested strategies were suitable for capture and purification of eVLPs. The performance of the different strategies was evaluated regarding its binding capacity, ability to separate different particle populations and product purity. The highest binding capacity regarding total particles was obtained using the anion exchange membrane adsorber (5.3 × 1012 part/mL membrane), however this method did not allow the separation of different particle populations. Despite having a lower binding capacity (1.5 × 1011 part/mL column) and requiring a pre-processing step with flow-through chromatography, Heparin-affinity chromatography showed the best performance regarding separation of different particle populations, allowing not only the separation of HIV-1 gag VLPs from host cell derived bionanoparticles but also from chromatin. This work additionally shows the importance of thorough sample characterization combining several biochemical and biophysical methods in eVLP DSP.


Assuntos
Convecção , HIV-1/isolamento & purificação , Adsorção , Animais , Ânions , Células CHO , Cromatina/metabolismo , Cromatografia de Afinidade , Cricetinae , Cricetulus , HIV-1/ultraestrutura , Histonas/metabolismo , Humanos , Microesferas , Nanopartículas/química , Nanopartículas/ultraestrutura , Polímeros/química , Porosidade , Vírion/isolamento & purificação , Vírion/ultraestrutura
16.
PLoS One ; 15(8): e0237930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841274

RESUMO

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismo
17.
Parasitol Res ; 119(9): 3041-3051, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32779021

RESUMO

Our objective was to investigate clinical progression, presence of parasites and DNAs, parasite loads, and histological alterations in BALB/c mice and Syrian golden hamsters after intraperitoneal inoculation with Leishmania (Mundinia) martiniquensis promastigotes with a goal to choosing an appropriate animal model for visceral leishmaniasis. Infections were monitored for 16 weeks. Infected BALB/c mice were asymptomatic during the infection course. Parasite DNAs were detected in the liver at week 8 of infection, followed by clearance in most animals at week 16; whereas in the spleen, parasite DNAs were detected until week 16. These results are correlated to those obtained measuring parasite loads in both organs. No parasite DNA and no alteration in the bone marrow were observed indicating that no dissemination occurred. These results suggest the control of visceralization of L. martiniquensis by BALB/c mice. In hamsters, weight loss, cachexia, and fatigue were observed after week 11. Leishmania martiniquensis parasites were observed in tissue smears of the liver, spleen, and bone marrow by week 16. Parasite loads correlated with those from the presence of parasites and DNAs in the examined tissues. Alterations in the liver with nuclear destruction and cytoplasmic degeneration of infected hepatocytes, presence of inflammatory infiltrates, necrosis of hepatocytes, and changes in splenic architecture and reduction and deformation of white pulp in the spleen were noted. These results indicate a chronic form of visceral leishmaniasis indicating that the hamster is a suitable animal model for the study of pathological features of chronic visceral leishmaniasis caused by L. martiniquensis.


Assuntos
Leishmania/fisiologia , Leishmaniose Visceral/parasitologia , Animais , Cricetinae , Modelos Animais de Doenças , Humanos , Leishmania/genética , Fígado/parasitologia , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Baço/parasitologia
18.
Nat Commun ; 11(1): 4070, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792502

RESUMO

Human astroviruses are small non-enveloped viruses with positive-sense single-stranded RNA genomes. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. It is still unknown how astrovirus particles exit infected cells following replication. Through comparative genomic analysis and ribosome profiling we here identify and confirm the expression of a conserved alternative-frame ORF, encoding the protein XP. XP-knockout astroviruses are attenuated and pseudo-revert on passaging. Further investigation into the function of XP revealed plasma and trans Golgi network membrane-associated roles in virus assembly and/or release through a viroporin-like activity. XP-knockout replicons have only a minor replication defect, demonstrating the role of XP at late stages of infection. The discovery of XP advances our knowledge of these important human viruses and opens an additional direction of research into their life cycle and pathogenesis.


Assuntos
Canais Iônicos/metabolismo , Mamastrovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Genômica/métodos , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Canais Iônicos/genética , Mamastrovirus/genética , Microscopia de Fluorescência , Plasmídeos/genética , Ribossomos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologia
19.
J Med Life ; 13(2): 241-248, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32742521

RESUMO

Cell culture is one of the most commonly used techniques in the production of biological products. Many physical and chemical parameters may affect cell growth and proliferation. This study was conducted to investigate the effect of chemical components as supplements using the experimental design method, which aimed at reducing the number of experiments. For this purpose, supplements including chemical components using four levels, with three replications in suspension and batch culture conditions, were examined for 72 hours using the Taguchi experimental design method. From the experiments, it was concluded that the culture media composition had a significant impact on final cell count and pH. High concentrations of different media composition alone were insufficient to ensure higher cell count. According to the results, this insufficiency was associated with an increase of 20% in the number of final cells. In the majority of cultures, the number of final cells at 48 hours increased relative to the number of final cells at 24 hours after culturing the cells.


Assuntos
Técnicas de Cultura de Células/métodos , Vírus da Febre Aftosa/imunologia , Rim/citologia , Vacinas Virais/imunologia , Aminoácidos/farmacologia , Animais , Contagem de Células , Células Cultivadas , Cricetinae , Vírus da Febre Aftosa/efeitos dos fármacos , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Polietilenoglicóis/química , Proteínas/farmacologia , Vitaminas/farmacologia
20.
PLoS Negl Trop Dis ; 14(8): e0008469, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32764759

RESUMO

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus, which has rapidly spread around the globe thereby causing millions of infections. CHIKV is an enveloped virus belonging to the Togaviridae family and enters its host cell primarily via clathrin-mediated endocytosis. Upon internalization, the endocytic vesicle containing the virus particle moves through the cell and delivers the virus to early endosomes where membrane fusion is observed. Thereafter, the nucleocapsid dissociates and the viral RNA is translated into proteins. In this study, we examined the importance of the microtubule network during the early steps of infection and dissected the intracellular trafficking behavior of CHIKV particles during cell entry. We observed two distinct CHIKV intracellular trafficking patterns prior to membrane hemifusion. Whereas half of the CHIKV virions remained static during cell entry and fused in the cell periphery, the other half showed fast-directed microtubule-dependent movement prior to delivery to Rab5-positive early endosomes and predominantly fused in the perinuclear region of the cell. Disruption of the microtubule network reduced the number of infected cells. At these conditions, membrane hemifusion activity was not affected yet fusion was restricted to the cell periphery. Furthermore, follow-up experiments revealed that disruption of the microtubule network impairs the delivery of the viral genome to the cell cytosol. We therefore hypothesize that microtubules may direct the particle to a cellular location that is beneficial for establishing infection or aids in nucleocapsid uncoating.


Assuntos
Vírus Chikungunya/fisiologia , Genoma Viral , Microtúbulos/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Nocodazol/farmacologia , Moduladores de Tubulina/farmacologia
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