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1.
BMC Bioinformatics ; 21(1): 431, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33008363

RESUMO

BACKGROUND: This paper describes a web based tool that uses a combination of sonification and an animated display to inquire into the SARS-CoV-2 genome. The audio data is generated in real time from a variety of RNA motifs that are known to be important in the functioning of RNA. Additionally, metadata relating to RNA translation and transcription has been used to shape the auditory and visual displays. Together these tools provide a unique approach to further understand the metabolism of the viral RNA genome. This audio provides a further means to represent the function of the RNA in addition to traditional written and visual approaches. RESULTS: Sonification of the SARS-CoV-2 genomic RNA sequence results in a complex auditory stream composed of up to 12 individual audio tracks. Each auditory motive is derived from the actual RNA sequence or from metadata. This approach has been used to represent transcription or translation of the viral RNA genome. The display highlights the real-time interaction of functional RNA elements. The sonification of codons derived from all three reading frames of the viral RNA sequence in combination with sonified metadata provide the framework for this display. Functional RNA motifs such as transcription regulatory sequences and stem loop regions have also been sonified. Using the tool, audio can be generated in real-time from either genomic or sub-genomic representations of the RNA. Given the large size of the viral genome, a collection of interactive buttons has been provided to navigate to regions of interest, such as cleavage regions in the polyprotein, untranslated regions or each gene. These tools are available through an internet browser and the user can interact with the data display in real time. CONCLUSION: The auditory display in combination with real-time animation of the process of translation and transcription provide a unique insight into the large body of evidence describing the metabolism of the RNA genome. Furthermore, the tool has been used as an algorithmic based audio generator. These audio tracks can be listened to by the general community without reference to the visual display to encourage further inquiry into the science.


Assuntos
Betacoronavirus/genética , Genoma Viral , Software , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Genômica , Humanos , Fases de Leitura Aberta/genética , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo
2.
Folia Biol (Praha) ; 66(3): 91-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069188

RESUMO

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.


Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Acetiltransferases/deficiência , Acetiltransferases/genética , Acetiltransferases/fisiologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Cálcio/fisiologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/patogenicidade , Cátions/farmacologia , Adesão Celular , Ciclo Celular , Parede Celular/efeitos dos fármacos , Quitinases/farmacologia , Dano ao DNA , Proteínas Fúngicas/genética , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/farmacologia , Hifas/crescimento & desenvolvimento , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Fases de Leitura Aberta , Reprodução Assexuada , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/fisiologia , Virulência/genética
3.
Med Hypotheses ; 143: 110203, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33017912

RESUMO

MicroRNAs (miRNAs) naturally occur in plants and all living organisms. They play an important role in gene regulation through binding toa specific region in open reading frames (ORFs) and/or untranslated regions (UTRs) to block the translation processes through either degrading or blocking mRNA resulting in knocking down or suppression of targeted genes. Plants and many organisms protect themselves from viruses through the production of miRNAs, which are complementary to 3UTR of viruses resulting in degrading the viral mRNA or block the translation on ribosomes. As pandemic, COVID-19, and its consequences on the global economy, we hypothesized a new approach for the treatment of COVID-19 paints. This approach includes designing a mix of miRNAs targeting several regions on COVID-19 open reading frame (ORF) and 3 UTR and suitable delivery system targeting respiratory system tissues. These synthesized miRNAs may be delivered to humansinnon-viral delivery systems such as liposomes like exosome (extracellular vesicle), polymer-based carriers, or inorganic nanoparticles, which are considered to be more suitable for human use.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/terapia , MicroRNAs/uso terapêutico , Pneumonia Viral/terapia , Regiões 3' não Traduzidas , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Sistemas de Liberação de Medicamentos , Exossomos , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genoma Viral , Humanos , Lipossomos/química , Nanopartículas/química , Fases de Leitura Aberta , Pandemias , Pneumonia Viral/virologia , Polímeros/química
4.
Int J Mol Sci ; 21(19)2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33019591

RESUMO

Tom70 is a versatile adaptor protein of 70 kDa anchored in the outer membrane of mitochondria in metazoa, fungi and amoeba. The tertiary structure was resolved for the Tom70 of yeast, showing 26 α-helices, most of them participating in the formation of 11 tetratricopeptide repeat (TPR) motifs. Tom70 serves as a docking site for cytosolic chaperone proteins and co-chaperones and is thereby involved in the uptake of newly synthesized chaperone-bound proteins in mitochondrial biogenesis. In yeast, Tom70 additionally mediates ER-mitochondria contacts via binding to sterol transporter Lam6/Ltc1. In mammalian cells, TOM70 promotes endoplasmic reticulum (ER) to mitochondria Ca2+ transfer by association with the inositol-1,4,5-triphosphate receptor type 3 (IP3R3). TOM70 is specifically targeted by the Bcl-2-related protein MCL-1 that acts as an anti-apoptotic protein in macrophages infected by intracellular pathogens, but also in many cancer cells. By participating in the recruitment of PINK1 and the E3 ubiquitin ligase Parkin, TOM70 can be implicated in the development of Parkinson's disease. TOM70 acts as receptor of the mitochondrial antiviral-signaling protein (MAVS) and thereby participates in the corresponding system of innate immunity against viral infections. The protein encoded by Orf9b in the genome of SARS-CoV-2 binds to TOM70, probably compromising the synthesis of type I interferons.


Assuntos
Imunidade Inata , Proteínas de Transporte da Membrana Mitocondrial/química , Animais , Betacoronavirus/genética , Sítios de Ligação , Humanos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fases de Leitura Aberta , Ligação Proteica , Transporte Proteico
5.
Nat Commun ; 11(1): 4676, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938922

RESUMO

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.


Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética
6.
Nat Commun ; 11(1): 4677, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938929

RESUMO

The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estresse do Retículo Endoplasmático/genética , Fatores de Iniciação em Eucariotos/genética , Biossíntese de Proteínas , Fatores de Transcrição/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Linhagem Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Mutação , Fases de Leitura Aberta , Interferência de RNA , Degeneração Retiniana/genética , Fatores de Transcrição/metabolismo
7.
Sci Signal ; 13(651)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994211

RESUMO

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/química , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Virais/genética , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Fases de Leitura Aberta/genética , Pandemias , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
8.
Virology ; 550: 70-77, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32890979

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent RNA virus that spread around the planet in about 4 months. The consequences of this rapid dispersion are under investigation. In this work, we analyzed thousands of genomes and protein sequences from Africa, America, Asia, Europe, and Oceania. We provide statistically significant evidence that SARS-CoV-2 phylogeny is spatially structured. Remarkably, the virus phylogeographic patterns were correlated with ancestral amino acidic substitutions, suggesting that such mutations emerged along colonization events. We hypothesize that geographic structuring is the result of founder effects occurring as a consequence of, and local evolution occurring after, long-distance dispersion. Based on previous studies, the possibility that this could significantly affect the virus biology is not remote.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Surtos de Doenças , Variação Genética , Genoma Viral , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , África/epidemiologia , América/epidemiologia , Ásia/epidemiologia , Betacoronavirus/classificação , Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Europa (Continente)/epidemiologia , Evolução Molecular , Humanos , Oceania/epidemiologia , Fases de Leitura Aberta , Pandemias , Filogenia , Filogeografia , Pneumonia Viral/diagnóstico , Proteínas Virais/genética
9.
BMC Bioinformatics ; 21(Suppl 8): 201, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938407

RESUMO

MicroRNA are small non-coding RNAs that post-transcriptionally regulate the expression levels of messenger RNAs. MicroRNA regulation activity depends on the recognition of binding sites located on mRNA molecules. ComiR is a web tool realized to predict the targets of a set of microRNAs, starting from their expression profile. ComiR was trained with the information regarding binding sites in the 3'utr region, by using a reliable dataset containing the targets of endogenously expressed microRNA in D. melanogaster S2 cells. This dataset was obtained by comparing the results from two different experimental approaches, i.e., inhibition, and immunoprecipitation of the AGO1 protein--a component of the microRNA induced silencing complex.In this work, we tested whether including coding region binding sites in ComiR algorithm improves the performance of the tool in predicting microRNA targets. We focused the analysis on the D. melanogaster species and updated the ComiR underlying database with the currently available releases of mRNA and microRNA sequences. As a result, we find that ComiR algorithm trained with the information related to the coding regions is more efficient in predicting the microRNA targets, with respect to the algorithm trained with 3'utr information. On the other hand, we show that 3'utr based predictions can be seen as complementary to the coding region based predictions, which suggests that both predictions, from 3'utr and coding regions, should be considered in comprehensive analysis.Furthermore, we observed that the lists of targets obtained by analyzing data from one experimental approach only, that is, inhibition or immunoprecipitation of AGO1, are not reliable enough to test the performance of our microRNA target prediction algorithm. Further analysis will be conducted to investigate the effectiveness of the tool with data from other species, provided that validated datasets, as obtained from the comparison of RISC proteins inhibition and immunoprecipitation experiments, will be available for the same samples. Finally, we propose to upgrade the existing ComiR web-tool by including the coding region based trained model, available together with the 3'utr based one.


Assuntos
Drosophila melanogaster/genética , MicroRNAs/genética , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , Algoritmos , Animais , Humanos
10.
PLoS One ; 15(9): e0233197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946445

RESUMO

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons-the ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.


Assuntos
Códon de Iniciação/genética , Biossíntese de Proteínas/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Composição de Bases/genética , Códon de Iniciação/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Simulação de Dinâmica Molecular , Fases de Leitura Aberta/genética , Proteína-Arginina N-Metiltransferases/biossíntese , Proteína-Arginina N-Metiltransferases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
12.
Lancet ; 396(10251): 603-611, 2020 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-32822564

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with a 382-nucleotide deletion (∆382) in the open reading frame 8 (ORF8) region of the genome have been detected in Singapore and other countries. We investigated the effect of this deletion on the clinical features of infection. METHODS: We retrospectively identified patients who had been screened for the ∆382 variant and recruited to the PROTECT study-a prospective observational cohort study conducted at seven public hospitals in Singapore. We collected clinical, laboratory, and radiological data from patients' electronic medical records and serial blood and respiratory samples taken during hospitalisation and after discharge. Individuals infected with the ∆382 variant were compared with those infected with wild-type SARS-CoV-2. Exact logistic regression was used to examine the association between the infection groups and the development of hypoxia requiring supplemental oxygen (an indicator of severe COVID-19, the primary endpoint). Follow-up for the study's primary endpoint is completed. FINDINGS: Between Jan 22 and March 21, 2020, 278 patients with PCR-confirmed SARS-CoV-2 infection were screened for the ∆382 deletion and 131 were enrolled onto the study, of whom 92 (70%) were infected with the wild-type virus, ten (8%) had a mix of wild-type and ∆382-variant viruses, and 29 (22%) had only the ∆382 variant. Development of hypoxia requiring supplemental oxygen was less frequent in the ∆382 variant group (0 [0%] of 29 patients) than in the wild-type only group (26 [28%] of 92; absolute difference 28% [95% CI 14-28]). After adjusting for age and presence of comorbidities, infection with the ∆382 variant only was associated with lower odds of developing hypoxia requiring supplemental oxygen (adjusted odds ratio 0·07 [95% CI 0·00-0·48]) compared with infection with wild-type virus only. INTERPRETATION: The ∆382 variant of SARS-CoV-2 seems to be associated with a milder infection. The observed clinical effects of deletions in ORF8 could have implications for the development of treatments and vaccines. FUNDING: National Medical Research Council Singapore.


Assuntos
Infecções por Coronavirus/virologia , Deleção de Genes , Genoma Viral/genética , Pneumonia Viral/virologia , Adulto , Idoso , Betacoronavirus , Infecções por Coronavirus/complicações , Infecções por Coronavirus/epidemiologia , Humanos , Hipóxia/etiologia , Hipóxia/terapia , Pessoa de Meia-Idade , Fases de Leitura Aberta , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/epidemiologia , Estudos Prospectivos , Terapia Respiratória , Índice de Gravidade de Doença , Singapura/epidemiologia , Replicação Viral
13.
Virol J ; 17(1): 131, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32854725

RESUMO

BACKGROUND: The Covid19 infection is caused by the SARS-CoV-2 virus, a novel member of the coronavirus (CoV) family. CoV genomes code for a ORF1a / ORF1ab polyprotein and four structural proteins widely studied as major drug targets. The genomes also contain a variable number of open reading frames (ORFs) coding for accessory proteins that are not essential for virus replication, but appear to have a role in pathogenesis. The accessory proteins have been less well characterized and are difficult to predict by classical bioinformatics methods. METHODS: We propose a computational tool GOFIX to characterize potential ORFs in virus genomes. In particular, ORF coding potential is estimated by searching for enrichment in motifs of the X circular code, that is known to be over-represented in the reading frames of viral genes. RESULTS: We applied GOFIX to study the SARS-CoV-2 and related genomes including SARS-CoV and SARS-like viruses from bat, civet and pangolin hosts, focusing on the accessory proteins. Our analysis provides evidence supporting the presence of overlapping ORFs 7b, 9b and 9c in all the genomes and thus helps to resolve some differences in current genome annotations. In contrast, we predict that ORF3b is not functional in all genomes. Novel putative ORFs were also predicted, including a truncated form of the ORF10 previously identified in SARS-CoV-2 and a little known ORF overlapping the Spike protein in Civet-CoV and SARS-CoV. CONCLUSIONS: Our findings contribute to characterizing sequence properties of accessory genes of SARS coronaviruses, and especially the newly acquired genes making use of overlapping reading frames.


Assuntos
Betacoronavirus/genética , Genoma Viral , Fases de Leitura Aberta , Vírus da SARS/genética , Proteínas Virais Reguladoras e Acessórias/genética , Animais , Códon , Biologia Computacional , Evolução Molecular , Genes Virais , Humanos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Proteínas da Matriz Viral/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais Reguladoras e Acessórias/química
14.
Nat Commun ; 11(1): 3810, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32733001

RESUMO

The pandemic of COVID-19 has posed an unprecedented threat to global public health. However, the interplay between the viral pathogen of COVID-19, SARS-CoV-2, and host innate immunity is poorly understood. Here we show that SARS-CoV-2 induces overt but delayed type-I interferon (IFN) responses. By screening 23 viral proteins, we find that SARS-CoV-2 NSP1, NSP3, NSP12, NSP13, NSP14, ORF3, ORF6 and M protein inhibit Sendai virus-induced IFN-ß promoter activation, whereas NSP2 and S protein exert opposite effects. Further analyses suggest that ORF6 inhibits both type I IFN production and downstream signaling, and that the C-terminus region of ORF6 is critical for its antagonistic effect. Finally, we find that IFN-ß treatment effectively blocks SARS-CoV-2 replication. In summary, our study shows that SARS-CoV-2 perturbs host innate immune response via both its structural and nonstructural proteins, and thus provides insights into the pathogenesis of SARS-CoV-2.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Evasão da Resposta Imune , Interferon Tipo I/metabolismo , Pneumonia Viral/virologia , Transdução de Sinais , Betacoronavirus/genética , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Linhagem Celular , Infecções por Coronavirus/imunologia , Humanos , Imunidade Inata , Interferon beta/genética , Interferon beta/metabolismo , Interferon beta/farmacologia , Mutação , Fases de Leitura Aberta , Pandemias , Pneumonia Viral/imunologia , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
15.
Sci Rep ; 10(1): 14004, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814791

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a novel evolutionary divergent RNA virus, is responsible for the present devastating COVID-19 pandemic. To explore the genomic signatures, we comprehensively analyzed 2,492 complete and/or near-complete genome sequences of SARS-CoV-2 strains reported from across the globe to the GISAID database up to 30 March 2020. Genome-wide annotations revealed 1,516 nucleotide-level variations at different positions throughout the entire genome of SARS-CoV-2. Moreover, nucleotide (nt) deletion analysis found twelve deletion sites throughout the genome other than previously reported deletions at coding sequence of the ORF8 (open reading frame), spike, and ORF7a proteins, specifically in polyprotein ORF1ab (n = 9), ORF10 (n = 1), and 3´-UTR (n = 2). Evidence from the systematic gene-level mutational and protein profile analyses revealed a large number of amino acid (aa) substitutions (n = 744), demonstrating the viral proteins heterogeneous. Notably, residues of receptor-binding domain (RBD) showing crucial interactions with angiotensin-converting enzyme 2 (ACE2) and cross-reacting neutralizing antibody were found to be conserved among the analyzed virus strains, except for replacement of lysine with arginine at 378th position of the cryptic epitope of a Shanghai isolate, hCoV-19/Shanghai/SH0007/2020 (EPI_ISL_416320). Furthermore, our results of the preliminary epidemiological data on SARS-CoV-2 infections revealed that frequency of aa mutations were relatively higher in the SARS-CoV-2 genome sequences of Europe (43.07%) followed by Asia (38.09%), and North America (29.64%) while case fatality rates remained higher in the European temperate countries, such as Italy, Spain, Netherlands, France, England and Belgium. Thus, the present method of genome annotation employed at this early pandemic stage could be a promising tool for monitoring and tracking the continuously evolving pandemic situation, the associated genetic variants, and their implications for the development of effective control and prophylaxis strategies.


Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Heterogeneidade Genética , Genoma Viral/genética , Estudo de Associação Genômica Ampla/métodos , Saúde Global , Pneumonia Viral/epidemiologia , Sequência de Aminoácidos/genética , Anticorpos Neutralizantes/imunologia , Pareamento Incorreto de Bases , Sequência de Bases/genética , Clima , Infecções por Coronavirus/virologia , Humanos , Fases de Leitura Aberta/genética , Pandemias , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/virologia , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
16.
Artigo em Inglês | MEDLINE | ID: mdl-32850499

RESUMO

The spread of the novel coronavirus (SARS-CoV-2) has triggered a global emergency, that demands urgent solutions for detection and therapy to prevent escalating health, social, and economic impacts. The spike protein (S) of this virus enables binding to the human receptor ACE2, and hence presents a prime target for vaccines preventing viral entry into host cells. The S proteins from SARS and SARS-CoV-2 are similar, but structural differences in the receptor binding domain (RBD) preclude the use of SARS-specific neutralizing antibodies to inhibit SARS-CoV-2. Here we used comparative pangenomic analysis of all sequenced reference Betacoronaviruses, complemented with functional and structural analyses. This analysis reveals that, among all core gene clusters present in these viruses, the envelope protein E shows a variant cluster shared by SARS and SARS-CoV-2 with two completely-conserved key functional features, namely an ion-channel, and a PDZ-binding motif (PBM). These features play a key role in the activation of the inflammasome causing the acute respiratory distress syndrome, the leading cause of death in SARS and SARS-CoV-2 infections. Together with functional pangenomic analysis, mutation tracking, and previous evidence, on E protein as a determinant of pathogenicity in SARS, we suggest E protein as an alternative therapeutic target to be considered for further studies to reduce complications of SARS-CoV-2 infections in COVID-19.


Assuntos
Betacoronavirus/química , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Infecções por Coronavirus/virologia , Genes Essenciais , Genes Virais , Genoma Viral , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Mutação , Fases de Leitura Aberta , Domínios PDZ , Pandemias , Pneumonia Viral/virologia , Domínios Proteicos , Vírus da SARS/química
17.
Int J Mol Sci ; 21(15)2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32759818

RESUMO

The current COronaVIrus Disease 2019 (COVID-19) pandemic started in December 2019. COVID-19 cases are confirmed by the detection of SARS-CoV-2 RNA in biological samples by RT-qPCR. However, limited numbers of SARS-CoV-2 genomes were available when the first RT-qPCR methods were developed in January 2020 for initial in silico specificity evaluation and to verify whether the targeted loci are highly conserved. Now that more whole genome data have become available, we used the bioinformatics tool SCREENED and a total of 4755 publicly available SARS-CoV-2 genomes, downloaded at two different time points, to evaluate the specificity of 12 RT-qPCR tests (consisting of a total of 30 primers and probe sets) used for SARS-CoV-2 detection and the impact of the virus' genetic evolution on four of them. The exclusivity of these methods was also assessed using the human reference genome and 2624 closely related other respiratory viral genomes. The specificity of the assays was generally good and stable over time. An exception is the first method developed by the China Center for Disease Control and prevention (CDC), which exhibits three primer mismatches present in 358 SARS-CoV-2 genomes sequenced mainly in Europe from February 2020 onwards. The best results were obtained for the assay of Chan et al. (2020) targeting the gene coding for the spiking protein (S). This demonstrates that our user-friendly strategy can be used for a first in silico specificity evaluation of future RT-qPCR tests, as well as verifying that the former methods are still capable of detecting circulating SARS-CoV-2 variants.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Genoma Viral , Pneumonia Viral/diagnóstico , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Bases de Dados Genéticas , Humanos , Fases de Leitura Aberta/genética , Pandemias , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , RNA Replicase/genética , RNA Viral/análise , Sensibilidade e Especificidade , Sequenciamento Completo do Genoma
18.
PLoS One ; 15(8): e0237559, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780783

RESUMO

BACKGROUND: The world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV-2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5' and 3' untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon. METHODS: To have an idea of the global changes in 5' and 3'-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV-2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by 'RBPmap' and 'RNA22 v2' respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue. RESULTS: We identified 28 unique variants in SARS-CoV-2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5'-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3'UTR among most of the continents. Furthermore, we found that despite the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs. CONCLUSION: Our results, shows for the first time in SARS-Cov-2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Betacoronavirus/genética , Infecções por Coronavirus/metabolismo , Genoma Viral/genética , Instabilidade Genômica/genética , Interações Hospedeiro-Patógeno/genética , MicroRNAs/metabolismo , Pneumonia Viral/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Infecções por Coronavirus/virologia , Humanos , Fases de Leitura Aberta/genética , Pandemias , Pneumonia Viral/virologia , Ligação Proteica/genética
19.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32763951

RESUMO

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Assuntos
Betacoronavirus/genética , Fases de Leitura Aberta/genética , Betacoronavirus/isolamento & purificação , Clonagem Molecular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Humanos , Pandemias , Plasmídeos/genética , Plasmídeos/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Potyvirus/genética
20.
J Med Microbiol ; 69(9): 1151-1168, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32840477

RESUMO

Introduction. Enterococcus faecalis is a facultative, anaerobic, opportunistic pathogen associated with medical and dental diseases. Bacterial phenotypic traits and pathogenesis are often influenced by lysogeny.Aim. The aim of this study was to characterize both the morphology and complete genome sequences of induced prophages purified from E. faecalis clinical isolates.Methodology. E. faecalis isolates were recovered from the roots of teeth of patients attending an endodontic clinic. The morphological features of isolated phage were characterized using transmission electron microscopy (TEM). DNA sequencing was performed using the Illumina MiSeq platform.Results. TEM indicated that the isolated φEf-vB1 prophage belongs to the family Siphoviridae. The φEf-vB1 prophage was stable over a wide range of temperatures and pH. Sequencing of φEf-vB1 DNA revealed that the phage genome is 37 561 bp in length with a G+C content of 37.6mol% and contained 53 ORFs. Comparison with previously predicted prophage genomes using blast revealed that φEf-vB1 has a high sequence similarity to previously characterized phage genomes. The lysogenic E. faecalis strain exhibited a higher biofilm formation capacity relative to the non-lysogenic strain.Conclusion. The current findings highlight the role of lysogeny in modification of E. faecalis properties and reveal the potential importance of prophages in E. faecalis biology and pathogenesis.


Assuntos
Bacteriófagos/fisiologia , Enterococcus faecalis/fisiologia , Enterococcus faecalis/virologia , Prófagos/fisiologia , Siphoviridae/isolamento & purificação , Composição de Bases , Cavidade Pulpar/microbiologia , Enterococcus faecalis/genética , Enterococcus faecalis/isolamento & purificação , Genoma Viral , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Lisogenia , Fases de Leitura Aberta , Periodontite , Prófagos/classificação , Prófagos/genética , Prófagos/isolamento & purificação , Siphoviridae/classificação , Siphoviridae/genética , Siphoviridae/fisiologia
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