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1.
Nat Commun ; 11(1): 5066, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033255

RESUMO

The inducible co-stimulator (ICOS) is a member of the CD28/B7 superfamily, and delivers a positive co-stimulatory signal to activated T cells upon binding to its ligand (ICOS-L). Dysregulation of this pathway has been implicated in autoimmune diseases and cancer, and is currently under clinical investigation as an immune checkpoint blockade. Here, we describe the molecular interactions of the ICOS/ICOS-L immune complex at 3.3 Å resolution. A central FDPPPF motif and residues within the CC' loop of ICOS are responsible for the specificity of the interaction with ICOS-L, with a distinct receptor binding orientation in comparison to other family members. Furthermore, our structure and binding data reveal that the ICOS N110 N-linked glycan participates in ICOS-L binding. In addition, we report crystal structures of ICOS and ICOS-L in complex with monoclonal antibodies under clinical evaluation in immunotherapy. Strikingly, antibody paratopes closely mimic receptor-ligand binding core interactions, in addition to contacting peripheral residues to confer high binding affinities. Our results uncover key molecular interactions of an immune complex central to human adaptive immunity and have direct implications for the ongoing development of therapeutic interventions targeting immune checkpoint receptors.


Assuntos
Anticorpos/uso terapêutico , Complexo Antígeno-Anticorpo/química , Ligante Coestimulador de Linfócitos T Induzíveis/química , Proteína Coestimuladora de Linfócitos T Induzíveis/química , Mimetismo Molecular/imunologia , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/metabolismo , Antígenos CD28/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Multimerização Proteica
2.
Nat Commun ; 11(1): 4955, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009385

RESUMO

The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960 nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82 Å resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the αß-polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the αß-polypeptides that are hydrogen-bonded to BChl a. The Ca2+-facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of α- and ß-polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside.


Assuntos
Cálcio/metabolismo , Microscopia Crioeletrônica , Complexos de Proteínas Captadores de Luz/ultraestrutura , Peptídeos/metabolismo , Fotossíntese , Sequência de Aminoácidos , Bacterioclorofila A/metabolismo , Sítios de Ligação , Chromatiaceae/metabolismo , Detergentes/química , Dimerização , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Lipídeos/química , Peptídeos/química , Quinonas/química
3.
Nat Commun ; 11(1): 4940, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009411

RESUMO

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Éxons/genética , Genoma , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , NAD/metabolismo , Proteínas Nucleares/química , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transcrição Genética
4.
Front Immunol ; 11: 2008, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013857

RESUMO

Coronavirus disease (COVID-19), caused by the virus SARS-CoV-2, is already responsible for more than 4.3 million confirmed cases and 295,000 deaths worldwide as of May 15, 2020. Ongoing efforts to control the pandemic include the development of peptide-based vaccines and diagnostic tests. In these approaches, HLA allelic diversity plays a crucial role. Despite its importance, current knowledge of HLA allele frequencies in South America is very limited. In this study, we have performed a literature review of datasets reporting HLA frequencies of South American populations, available in scientific literature and/or in the Allele Frequency Net Database. This allowed us to enrich the current scenario with more than 12.8 million data points. As a result, we are presenting updated HLA allelic frequencies based on country, including 91 alleles that were previously thought to have frequencies either under 5% or of an unknown value. Using alleles with an updated frequency of at least ≥5% in any South American country, we predicted epitopes in SARS-CoV-2 proteins using NetMHCpan (I and II) and MHC flurry. Then, the best predicted epitopes (class-I and -II) were selected based on their binding to South American alleles (Coverage Score). Class II predicted epitopes were also filtered based on their three-dimensional exposure. We obtained 14 class-I and four class-II candidate epitopes with experimental evidence (reported in the Immune Epitope Database and Analysis Resource), having good coverage scores for South America. Additionally, we are presenting 13 HLA-I and 30 HLA-II novel candidate epitopes without experimental evidence, including 16 class-II candidates in highly exposed conserved areas of the NTD and RBD regions of the Spike protein. These novel candidates have even better coverage scores for South America than those with experimental evidence. Finally, we show that recent similar studies presenting candidate epitopes also predicted some of our candidates but discarded them in the selection process, resulting in candidates with suboptimal coverage for South America. In conclusion, the candidate epitopes presented provide valuable information for the development of epitope-based strategies against SARS-CoV-2, such as peptide vaccines and diagnostic tests. Additionally, the updated HLA allelic frequencies provide a better representation of South America and may impact different immunogenetic studies.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Epitopos de Linfócito T/imunologia , Frequência do Gene , Antígenos HLA/genética , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Proteínas do Envelope Viral/imunologia , Alelos , Sequência de Aminoácidos , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Variação Genética , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , América do Sul/epidemiologia , Vacinas de Subunidades/imunologia , Vacinas Virais/imunologia
5.
Front Immunol ; 11: 565278, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013929

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an outbreak of a pandemic worldwide. For better understanding the viral spike (S) protein variations and its potential effects on the interaction with the host immune system and also in vaccine development, the cell epitopes, glycosylation profile and their changes during the global transmission course were characterized and compared with SARS-CoV for their glycosylation profile. We analyzed totally 7,813 sequences screened from 8,897 whole genome sequences on GISAID database up to April 26, and 18 S protein amino acid variations with relatively high frequency (≥10-3) were identified. A total of 228 sequences of variants had multiple variations, of note, most of them harboring the D614G mutation. Among the predicted 69 linear B cell epitopes, 175 discontinuous B cell epitopes and 41 cytotoxic T lymphocyte epitopes in the viral S protein, we found that the protein structure and its potential function of some sites changed, such as the linear epitope length shortened and discontinuous epitope disappeared of G476S. In addition, we detected 9 predicted N-glycosylation sites and 3 O-glycosylation sites unique to SARS-CoV-2, but no evidently observed variation of the glycan sites so far. Our findings provided an important snapshot of temporal and geographical distributions on SARS-CoV-2 S protein cell epitopes and glycosylation sites, which would be an essential basis for the selection of vaccine candidates.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/transmissão , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/transmissão , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Infecções por Coronavirus/virologia , Genoma Viral/genética , Glicosilação , Interações Hospedeiro-Patógeno/imunologia , Humanos , Glicoproteínas de Membrana/imunologia , Pandemias , Pneumonia Viral/virologia , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química , Sequenciamento Completo do Genoma
6.
Front Immunol ; 11: 565278, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-796802

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an outbreak of a pandemic worldwide. For better understanding the viral spike (S) protein variations and its potential effects on the interaction with the host immune system and also in vaccine development, the cell epitopes, glycosylation profile and their changes during the global transmission course were characterized and compared with SARS-CoV for their glycosylation profile. We analyzed totally 7,813 sequences screened from 8,897 whole genome sequences on GISAID database up to April 26, and 18 S protein amino acid variations with relatively high frequency (≥10-3) were identified. A total of 228 sequences of variants had multiple variations, of note, most of them harboring the D614G mutation. Among the predicted 69 linear B cell epitopes, 175 discontinuous B cell epitopes and 41 cytotoxic T lymphocyte epitopes in the viral S protein, we found that the protein structure and its potential function of some sites changed, such as the linear epitope length shortened and discontinuous epitope disappeared of G476S. In addition, we detected 9 predicted N-glycosylation sites and 3 O-glycosylation sites unique to SARS-CoV-2, but no evidently observed variation of the glycan sites so far. Our findings provided an important snapshot of temporal and geographical distributions on SARS-CoV-2 S protein cell epitopes and glycosylation sites, which would be an essential basis for the selection of vaccine candidates.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/transmissão , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/transmissão , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Infecções por Coronavirus/virologia , Genoma Viral/genética , Glicosilação , Interações Hospedeiro-Patógeno/imunologia , Humanos , Glicoproteínas de Membrana/imunologia , Pandemias , Pneumonia Viral/virologia , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química , Sequenciamento Completo do Genoma
7.
Front Immunol ; 11: 2008, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-781995

RESUMO

Coronavirus disease (COVID-19), caused by the virus SARS-CoV-2, is already responsible for more than 4.3 million confirmed cases and 295,000 deaths worldwide as of May 15, 2020. Ongoing efforts to control the pandemic include the development of peptide-based vaccines and diagnostic tests. In these approaches, HLA allelic diversity plays a crucial role. Despite its importance, current knowledge of HLA allele frequencies in South America is very limited. In this study, we have performed a literature review of datasets reporting HLA frequencies of South American populations, available in scientific literature and/or in the Allele Frequency Net Database. This allowed us to enrich the current scenario with more than 12.8 million data points. As a result, we are presenting updated HLA allelic frequencies based on country, including 91 alleles that were previously thought to have frequencies either under 5% or of an unknown value. Using alleles with an updated frequency of at least ≥5% in any South American country, we predicted epitopes in SARS-CoV-2 proteins using NetMHCpan (I and II) and MHC flurry. Then, the best predicted epitopes (class-I and -II) were selected based on their binding to South American alleles (Coverage Score). Class II predicted epitopes were also filtered based on their three-dimensional exposure. We obtained 14 class-I and four class-II candidate epitopes with experimental evidence (reported in the Immune Epitope Database and Analysis Resource), having good coverage scores for South America. Additionally, we are presenting 13 HLA-I and 30 HLA-II novel candidate epitopes without experimental evidence, including 16 class-II candidates in highly exposed conserved areas of the NTD and RBD regions of the Spike protein. These novel candidates have even better coverage scores for South America than those with experimental evidence. Finally, we show that recent similar studies presenting candidate epitopes also predicted some of our candidates but discarded them in the selection process, resulting in candidates with suboptimal coverage for South America. In conclusion, the candidate epitopes presented provide valuable information for the development of epitope-based strategies against SARS-CoV-2, such as peptide vaccines and diagnostic tests. Additionally, the updated HLA allelic frequencies provide a better representation of South America and may impact different immunogenetic studies.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Epitopos de Linfócito T/imunologia , Frequência do Gene , Antígenos HLA/genética , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Proteínas do Envelope Viral/imunologia , Alelos , Sequência de Aminoácidos , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Variação Genética , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , América do Sul/epidemiologia , Vacinas de Subunidades/imunologia , Vacinas Virais/imunologia
8.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2361-2367, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018481

RESUMO

Deep learning has proven to be a useful tool for modelling protein properties. However, given the variability in the length of proteins, it can be difficult to summarise the sequence of amino acids effectively. In many cases, as a result of using fixed-length representations, information about long proteins can be lost through truncation, or model training can be slow due to the use of excessive padding. In this work, we aim to overcome these problems by expanding upon the original vocabulary used to represent the protein sequence. To this end, we utilise two prominent subword algorithms that have been previously used to reach state-of-the-art results in various Natural Language Processing tasks. The algorithms are used to encode the original protein sequence into a set of subsequences before they are analysed by a Doc2Vec model. The pre-trained encodings produced by each algorithm are tested on a variety of downstream tasks: four protein property prediction tasks (plasma membrane localization, thermostability, peak absorption wavelength, enantioselectivity) as well as drug-target affinity prediction tasks over two datasets. Our results significantly improve on the state-of-the-art for these tasks, demonstrating the benefits of using subword compression algorithms for modelling proteins.


Assuntos
Algoritmos , Vocabulário , Sequência de Aminoácidos , Processamento de Linguagem Natural , Proteínas
9.
Int J Antimicrob Agents ; 56(2): 106020, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32862840

RESUMO

The emergence of SARS-coronavirus-2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19). Hydroxychloroquine (CLQ-OH)/azithromycin (ATM) combination therapy is currently being tested for the treatment of COVID-19, with promising results. However, the molecular mechanism of action of this combination is not yet established. Using molecular dynamics (MD) simulations, this study shows that the drugs act in synergy to prevent any close contact between the virus and the plasma membrane of host cells. Unexpected molecular similarity is shown between ATM and the sugar moiety of GM1, a lipid raft ganglioside acting as a host attachment cofactor for respiratory viruses. Due to this mimicry, ATM interacts with the ganglioside-binding domain of SARS-CoV-2 spike protein. This binding site shared by ATM and GM1 displays a conserved amino acid triad Q-134/F-135/N-137 located at the tip of the spike protein. CLQ-OH molecules are shown to saturate virus attachment sites on gangliosides in the vicinity of the primary coronavirus receptor, angiotensin-converting enzyme-2 (ACE-2). Taken together, these data show that ATM is directed against the virus, whereas CLQ-OH is directed against cellular attachment cofactors. We conclude that both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane. This is consistent with a synergistic antiviral mechanism at the plasma membrane level, where therapeutic intervention is likely to be most efficient. This molecular mechanism may explain the beneficial effects of CLQ-OH/ATM combination therapy in patients with COVID-19. Incidentally, the data also indicate that the conserved Q-134/F-135/N-137 triad could be considered as a target for vaccine strategies.


Assuntos
Azitromicina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Gangliosídeo G(M1)/metabolismo , Hidroxicloroquina/farmacologia , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Alinhamento de Sequência
10.
Nat Commun ; 11(1): 4420, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887876

RESUMO

SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the 'up' and 'down' conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Betacoronavirus/efeitos dos fármacos , Camelídeos Americanos/imunologia , Infecções por Coronavirus/tratamento farmacológico , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Anticorpos de Domínio Único/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Sítios de Ligação , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Epitopos/imunologia , Epitopos/metabolismo , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/virologia , Ligação Proteica , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
11.
J Transl Med ; 18(1): 329, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867854

RESUMO

BACKGROUND: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. METHODS: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. RESULTS: We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. CONCLUSIONS: Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Betacoronavirus/patogenicidade , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/epidemiologia , Frequência do Gene , Genoma Viral , Geografia , Humanos , Lisina/genética , Modelos Moleculares , Pandemias/estatística & dados numéricos , Fenilalanina/genética , Pneumonia Viral/epidemiologia , Domínios Proteicos/genética , Serina/genética , Proteínas não Estruturais Virais/química , Virulência/genética , Replicação Viral/genética
12.
Viruses ; 12(9)2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32971895

RESUMO

Coronaviruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding.


Assuntos
Vírus da Hepatite Murina/química , Peptídeos/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Infecções por Coronavirus , Humanos , Membranas Intracelulares/metabolismo , Camundongos , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/metabolismo , Mutação , Peptídeos/síntese química , Peptídeos/metabolismo , Células Sf9 , Spodoptera , Lipossomas Unilamelares/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
13.
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
14.
PLoS One ; 15(8): e0238256, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866176

RESUMO

In recent years, the binary definition of sex is being challenged by repetitive reports about individuals with ambiguous sexual identity from various animal groups. This has created an urge to decode the molecular mechanism underlying sexual development. However, sexual ambiguities are extremely uncommon in nature, limiting their experimental value. Here, we report the establishment of a genetically modified clone of Daphnia magna from which intersex daphniids can be readily generated. By mutating the conserved central sex determining factor Doublesex1, body-wide feminization of male daphniid could be achieved. Comparative transcriptomic analysis also revealed a genetic network correlated with Doublesex1 activity which may account for the establishment of sexual identity in D. magna. We found that Dsx1 repressed genes related to growth and promoted genes related to signaling. We infer that different intersex phenotypes are the results of fluctuation in activity of these Dsx1 downstream factors. Our results demonstrated that the D. magna genome is capable of expressing sex in a continuous array, supporting the idea that sex is actually a spectrum.


Assuntos
Daphnia/genética , Daphnia/fisiologia , Transtornos do Desenvolvimento Sexual/genética , Redes Reguladoras de Genes/genética , Desenvolvimento Sexual/genética , Sequência de Aminoácidos , Animais , Genoma/genética , Fenótipo , Transcriptoma/genética
15.
Vet Q ; 40(1): 243-249, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32921279

RESUMO

Several cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection transmitted from human owners to their dogs have recently been reported. The first ever case of SARS-CoV-2 transmission from a human owner to a domestic cat was confirmed on March 27, 2020. A tiger from a zoo in New York, USA, was also reportedly infected with SARS-CoV-2. It is believed that SARS-CoV-2 was transmitted to tigers from their caretakers, who were previously infected with this virus. On May 25, 2020, the Dutch Minister of Agriculture, Nature and Food Quality reported that two employees were infected with SARS-CoV-2 transmitted from minks. These reports have influenced us to perform a comparative analysis among angiotensin-converting enzyme 2 (ACE2) homologous proteins for verifying the conservation of specific protein regions. One of the most conserved peptides is represented by the peptide "353-KGDFR-357 (H. sapiens ACE2 residue numbering), which is located on the surface of the ACE2 molecule and participates in the binding of SARS-CoV-2 spike receptor binding domain (RBD). Multiple sequence alignments of the ACE2 proteins by ClustalW, whereas the three-dimensional structure of its binding region for the spike glycoprotein of SARS-CoV-2 was assessed by means of Spanner, a structural homology modeling pipeline method. In addition, evolutionary phylogenetic tree analysis by ETE3 was used. ACE2 works as a receptor for the SARS-CoV-2 spike glycoprotein between humans, dogs, cats, tigers, minks, and other animals, except for snakes. The three-dimensional structure of the KGDFR hosting protein region involved in direct interactions with SARS-CoV-2 spike RBD of the mink ACE2 appears to form a loop structurally related to the human ACE2 corresponding protein loop, despite of the reduced available protein length (401 residues of the mink ACE2 available sequence vs 805 residues of the human ACE2). The multiple sequence alignments of the ACE2 proteins shows high homology and complete conservation of the five amino acid residues: 353-KGDFR-357 with humans, dogs, cats, tigers, minks, and other animals, except for snakes. Where the information revealed from our examinations can support precision vaccine design and the discovery of antiviral therapeutics, which will accelerate the development of medical countermeasures, the World Health Organization recently reported on the possible risks of reciprocal infections regarding SARS-CoV-2 transmission from animals to humans.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/transmissão , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/transmissão , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Animais , Betacoronavirus/genética , Gatos , Infecções por Coronavirus/prevenção & controle , Cães , Humanos , Vison , Pandemias/prevenção & controle , Peptidil Dipeptidase A/química , Filogenia , Pneumonia Viral/prevenção & controle , Receptores Virais/química , Receptores Virais/genética , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Tigres
16.
PLoS One ; 15(9): e0238089, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903266

RESUMO

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.


Assuntos
Infecções por Coronavirus/patologia , Epitopos/química , Pneumonia Viral/patologia , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Mapeamento de Epitopos , Epitopos/sangue , Epitopos/imunologia , Furina/metabolismo , Humanos , Pandemias , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Pneumonia Viral/virologia , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
17.
Open Biol ; 10(9): 200209, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898469

RESUMO

Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and contribute to establish the basis to tackle the pathogenesis of SARS-CoV-2.


Assuntos
Betacoronavirus/metabolismo , Eucariotos/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Betacoronavirus/isolamento & purificação , Membrana Celular/metabolismo , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Eucariotos/citologia , Humanos , Microssomos/metabolismo , Mutação , Pandemias , Filogenia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/classificação , Proteínas do Envelope Viral/genética
18.
Sci Rep ; 10(1): 15917, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: covidwho-800800

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
19.
Viruses ; 12(9)2020 09 22.
Artigo em Inglês | MEDLINE | ID: covidwho-797048

RESUMO

Coronaviruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding.


Assuntos
Vírus da Hepatite Murina/química , Peptídeos/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Infecções por Coronavirus , Humanos , Membranas Intracelulares/metabolismo , Camundongos , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/metabolismo , Mutação , Peptídeos/síntese química , Peptídeos/metabolismo , Células Sf9 , Spodoptera , Lipossomas Unilamelares/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
20.
J Chem Phys ; 153(11): 115101, 2020 Sep 21.
Artigo em Inglês | MEDLINE | ID: covidwho-796705

RESUMO

Broad-spectrum antiviral drugs are urgently needed to stop the Coronavirus Disease 2019 pandemic and prevent future ones. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is related to the SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused the previous outbreaks. The papain-like protease (PLpro) is an attractive drug target due to its essential roles in the viral life cycle. As a cysteine protease, PLpro is rich in cysteines and histidines, and their protonation/deprotonation modulates catalysis and conformational plasticity. Here, we report the pKa calculations and assessment of the proton-coupled conformational dynamics of SARS-CoV-2 in comparison to SARS-CoV and MERS-CoV PLpros using the recently developed graphical processing unit (GPU)-accelerated implicit-solvent continuous constant pH molecular dynamics method with a new asynchronous replica-exchange scheme, which allows computation on a single GPU card. The calculated pKa's support the catalytic roles of the Cys-His-Asp triad. We also found that several residues can switch protonation states at physiological pH among which is C270/271 located on the flexible blocking loop 2 (BL2) of SARS-CoV-2/CoV PLpro. Simulations revealed that the BL2 can open and close depending on the protonation state of C271/270, consistent with the most recent crystal structure evidence. Interestingly, despite the lack of an analogous cysteine, BL2 in MERS-CoV PLpro is also very flexible, challenging a current hypothesis. These findings are supported by the all-atom fixed-charge simulations and provide a starting point for more detailed studies to assist the structure-based design of broad-spectrum inhibitors against CoV PLpros.


Assuntos
Antivirais/farmacologia , Betacoronavirus/enzimologia , Desenho de Fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Simulação de Dinâmica Molecular , Papaína/química , Papaína/metabolismo , Prótons , Sequência de Aminoácidos , Histidina , Concentração de Íons de Hidrogênio , Papaína/antagonistas & inibidores , Domínios Proteicos
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