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1.
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
2.
PLoS One ; 15(8): e0236882, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790688

RESUMO

N6-methyladenosine (m6A) is the most prevalent type of RNA modification. METTL3 in the methyltransferase complex is the core enzyme responsible for methylation. METTL3 selectively catalyzes the adenosines centered in the RRAC motif. Functional studies established that m6A could enhance the translation efficiency (TE) of modified genes by recruiting reader protein YTHDF1 and other initiation factors. We downloaded the m6A peaks in HeLa cells from a previous study and defined the m6A modified genes and sites. Ancestral mutations in the genic region fixed in the HeLa cell samples were defined using their mRNA-Seq data and the alignment between human and mouse genomes. Furthermore, in the small interfering (si)-METTL3 sample, the calculated TE foldchange of all genes was compared to that in the negative control. The TE of m6A genes was globally down-regulated in si-METTL3 versus control compared to the non-m6A genes. In m6A modified genes, RRAC motif mutations were suppressed compared to mutations in non-motif regions or non-m6A genes. Among the m6A genes, a fraction RRAC motif mutations negatively correlated with the TE foldchange (si-METTL3 versus control). The TE of m6A modified genes was enhanced in HeLa cells. RRAC motif mutations could potentially prevent methylation of adenosines and consequently abolish the enhanced translation. Such mutations in the RRAC motif might be deleterious. Accordingly, we observed lower fractions of mutations in RRAC motifs than in other regions. This prevention of mutations in the RRAC motif could be a strategy adopted by cancer cells to maintain the elevated translation of particular genes.


Assuntos
Adenosina/análogos & derivados , Metiltransferases/genética , Adenosina/genética , Adenosina/metabolismo , Animais , Sequência de Bases , Células HeLa , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Camundongos , Mutação , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo
3.
DNA Cell Biol ; 39(10): 1767-1778, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32833542

RESUMO

N1-methyladenosine (m1A) is an important post-transcriptional modification in RNA, and plays critical roles in cellular functions. However, the relationship between m1A regulators and clinical significance of gynecological cancers remains unknown. In this study, we systematically analyzed RNA-seq and clinical data from several public database. Cell proliferation and migration assays were performed to verify the function of the m1A writer TRMT10C in cancer cells. We observed genetic alterations and dysregulated expressions of m1A regulators in gynecological cancer samples. We demonstrated that several m1A regulators could serve as prognostic biomarkers for gynecological cancer patients. The high correlations among the expression of m1A, N6-methyladenosine (m6A), and 5mC regulators were also revealed. Gene set enrichment analysis indicated that the mechanism of TRMT10C in regulating tumorigenesis was related to a variety of cancer-related pathways. Moreover, silencing TRMT10C suppressed the proliferation, colony formation, and migration of ovarian cancer and cervical cancer cells. In summary, our results highlight the importance of m1A regulators in regulating oncogenesis, and indicate that targeting specific m1A regulators might be a potential therapeutic strategy for gynecological cancers.


Assuntos
Biomarcadores Tumorais/genética , Metiltransferases/genética , Neoplasias Ovarianas/genética , Neoplasias do Colo do Útero/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Biomarcadores Tumorais/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Células HeLa , Humanos , Metiltransferases/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
4.
Life Sci ; 259: 118169, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32738360

RESUMO

AIMS: The recent outbreak of pandemic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led the world towards a global health emergency. Currently, no proper medicine or effective treatment strategies are available; therefore, repurposing of FDA approved drugs may play an important role in overcoming the situation. MATERIALS AND METHODS: The SARS-CoV-2 genome encodes for 2-O-methyltransferase (2'OMTase), which plays a key role in methylation of viral RNA for evading host immune system. In the present study, the protein sequence of 2'OMTase of SARS-CoV-2 was analyzed, and its structure was modeled by a comparative modeling approach and validated. The library of 3000 drugs was screened against the active site of 2'OMTase followed by re-docking analysis. The apo and ligand-bound 2'OMTase were further validated and analyzed by using molecular dynamics simulation. KEY FINDINGS: The modeled structure displayed the conserved characteristic fold of class I MTase family. The quality assessment analysis by SAVES server reveals that the modeled structure follows protein folding rules and of excellent quality. The docking analysis displayed that the active site of 2'OMTase accommodates an array of drugs, which includes alkaloids, antivirals, cardiac glycosides, anticancer, steroids, and other drugs. The redocking and MD simulation analysis of the best 5 FDA approved drugs reveals that these drugs form a stable conformation with the 2'OMTase. SIGNIFICANCE: The results suggested that these drugs may be used as potential inhibitors for 2'OMTase for combating the SARS-CoV-2 infection.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/antagonistas & inibidores , Pneumonia Viral/tratamento farmacológico , Antivirais/química , Antivirais/farmacologia , Biologia Computacional/métodos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Metilação/efeitos dos fármacos , Metiltransferases/química , Metiltransferases/metabolismo , Metiltransferases/ultraestrutura , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Pandemias , Pneumonia Viral/virologia , Homologia de Sequência de Aminoácidos
5.
Proc Natl Acad Sci U S A ; 117(33): 20159-20170, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747553

RESUMO

Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB. N6 -methylation of adenosine (m6A) in RNA regulates many pathophysiological processes. Here, we show that deletion of the m6A demethylase Alkbh5 sensitized tumors to cancer immunotherapy. Alkbh5 has effects on m6A density and splicing events in tumors during ICB. Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells. Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy. Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy. Our results suggest that m6A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Vacinas Anticâncer/imunologia , Lactatos/metabolismo , Melanoma/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Reguladores/fisiologia , Homólogo AlkB 5 da RNA Desmetilase/genética , Anticorpos , Citocinas/genética , Citocinas/metabolismo , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/terapia , Metiltransferases/genética , Metiltransferases/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Células Supressoras Mieloides/fisiologia , Sítios de Splice de RNA , Processamento de RNA , Simportadores/genética , Simportadores/metabolismo , Transcriptoma , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Cell ; 182(6): 1560-1573.e13, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32783916

RESUMO

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.


Assuntos
Metiltransferases/química , RNA Helicases/química , RNA Replicase/química , Proteínas não Estruturais Virais/química , Replicação Viral , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Holoenzimas/química , Holoenzimas/metabolismo , Magnésio/metabolismo , Metiltransferases/metabolismo , Ligação Proteica , RNA Helicases/metabolismo , RNA Replicase/metabolismo , RNA Viral/química , Proteínas não Estruturais Virais/metabolismo
7.
Int J Mol Sci ; 21(16)2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32824072

RESUMO

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a severe global health crisis. In this paper, we used docking and simulation methods to identify potential targets and the mechanism of action of chloroquine (CQ) and hydroxychloroquine (HCQ) against SARS-CoV-2. Our results showed that both CQ and HCQ influenced the functionality of the envelope (E) protein, necessary in the maturation processes of the virus, due to interactions that modify the flexibility of the protein structure. Furthermore, CQ and HCQ also influenced the proofreading and capping of viral RNA in SARS-CoV-2, performed by nsp10/nsp14 and nsp10/nsp16. In particular, HCQ demonstrated a better energy binding with the examined targets compared to CQ, probably due to the hydrogen bonding of the hydroxyl group of HCQ with polar amino acid residues.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Cloroquina/farmacologia , Exorribonucleases/metabolismo , Hidroxicloroquina/farmacologia , Metiltransferases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Proteínas do Envelope Viral/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos
8.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Nucleic Acids Res ; 48(14): 7924-7943, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652011

RESUMO

Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.


Assuntos
Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , RNA Ribossômico 16S/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/enzimologia , Linhagem Celular , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Metilação , Metiltransferases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologia , Ribossomos Mitocondriais/metabolismo , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Fosforilação Oxidativa , Biossíntese de Proteínas , RNA Ribossômico 16S/química , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Fatores de Transcrição/metabolismo
10.
Nat Commun ; 11(1): 3717, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709887

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.


Assuntos
Betacoronavirus/enzimologia , Metiltransferases/química , Proteínas não Estruturais Virais/química , Proteínas Virais Reguladoras e Acessórias/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Domínio Catalítico , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , Capuzes de RNA , Estabilidade de RNA , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
11.
Nat Commun ; 11(1): 3718, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709886

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.


Assuntos
Metiltransferases/química , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/química , Proteínas Virais Reguladoras e Acessórias/química , Betacoronavirus , Infecções por Coronavirus/virologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Difração de Raios X
12.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: covidwho-595042

RESUMO

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Interferons/antagonistas & inibidores , Interferons/metabolismo , Metiltransferases/metabolismo , Pneumonia Viral/metabolismo , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Exorribonucleases/genética , Interações Hospedeiro-Patógeno , Humanos , Interferons/genética , Metiltransferases/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Helicases/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
13.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32529952

RESUMO

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Interferons/antagonistas & inibidores , Interferons/metabolismo , Metiltransferases/metabolismo , Pneumonia Viral/metabolismo , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Exorribonucleases/genética , Interações Hospedeiro-Patógeno , Humanos , Interferons/genética , Metiltransferases/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Helicases/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
14.
Nucleic Acids Res ; 48(13): 7027-7040, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542340

RESUMO

Methylation of miRNAs at the 2'-hydroxyl group on the ribose at 3'-end (2'-O-methylation, 2'Ome) is critical for miRNA function in plants and Drosophila. Whether this methylation phenomenon exists for mammalian miRNA remains unknown. Through LC-MS/MS analysis, we discover that majority of miR-21-5p isolated from human non-small cell lung cancer (NSCLC) tissue possesses 3'-terminal 2'Ome. Predominant 3'-terminal 2'Ome of miR-21-5p in cancer tissue is confirmed by qRT-PCR and northern blot after oxidation/ß-elimination procedure. Cancerous and the paired non-cancerous lung tissue miRNAs display different pattern of 3'-terminal 2'Ome. We further identify HENMT1 as the methyltransferase responsible for 3'-terminal 2'Ome of mammalian miRNAs. Compared to non-methylated miR-21-5p, methylated miR-21-5p is more resistant to digestion by 3'→5' exoribonuclease polyribonucleotide nucleotidyltransferase 1 (PNPT1) and has higher affinity to Argonaute-2, which may contribute to its higher stability and stronger inhibition on programmed cell death protein 4 (PDCD4) translation, respectively. Our findings reveal HENMT1-mediated 3'-terminal 2'Ome of mammalian miRNAs and highlight its role in enhancing miRNA's stability and function.


Assuntos
Proteínas Argonauta/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Exorribonucleases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Metilação , Proteínas de Ligação a RNA/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(25): 14251-14258, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513732

RESUMO

Nearly 50% of mouse and human genomes are composed of repetitive sequences. Transcription of these sequences is tightly controlled during development to prevent genomic instability, inappropriate gene activation and other maladaptive processes. Here, we demonstrate an integral role for H1 linker histones in silencing repetitive elements in mouse embryonic stem cells. Strong H1 depletion causes a profound de-repression of several classes of repetitive sequences, including major satellite, LINE-1, and ERV. Activation of repetitive sequence transcription is accompanied by decreased H3K9 trimethylation of repetitive sequence chromatin. H1 linker histones interact directly with Suv39h1, Suv39h2, and SETDB1, the histone methyltransferases responsible for H3K9 trimethylation of chromatin within these regions, and stimulate their activity toward chromatin in vitro. However, we also implicate chromatin compaction mediated by H1 as an additional, dominant repressive mechanism for silencing of repetitive major satellite sequences. Our findings elucidate two distinct, H1-mediated pathways for silencing heterochromatin.


Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Sequências Repetitivas de Ácido Nucleico/fisiologia , Animais , Epigenômica , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Metilação , Metiltransferases/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Repressoras/metabolismo
16.
Eur J Med Chem ; 201: 112557, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32563813

RESUMO

The spreading of new viruses is known to provoke global human health threat. The current COVID-19 pandemic caused by the recently emerged coronavirus SARS-CoV-2 is one significant and unfortunate example of what the world will have to face in the future with emerging viruses in absence of appropriate treatment. The discovery of potent and specific antiviral inhibitors and/or vaccines to fight these massive outbreaks is an urgent research priority. Enzymes involved in the capping pathway of viruses and more specifically RNA N7- or 2'O-methyltransferases (MTases) are now admitted as potential targets for antiviral chemotherapy. We designed bisubstrate inhibitors by mimicking the transition state of the 2'-O-methylation of the cap RNA in order to block viral 2'-O MTases. This work resulted in the synthesis of 16 adenine dinucleosides with both adenosines connected by various nitrogen-containing linkers. Unexpectedly, all the bisubstrate compounds were barely active against 2'-O MTases of several flaviviruses or SARS-CoV but surprisingly, seven of them showed efficient and specific inhibition against SARS-CoV N7-MTase (nsp14) in the micromolar to submicromolar range. The most active nsp14 inhibitor identified is as potent as but particularly more specific than the broad-spectrum MTase inhibitor, sinefungin. Molecular docking suggests that the inhibitor binds to a pocket formed by the S-adenosyl methionine (SAM) and cap RNA binding sites, conserved among SARS-CoV nsp14. These dinucleoside SAM analogs will serve as starting points for the development of next inhibitors for SARS-CoV-2 nsp14 N7-MTase.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Nucleosídeos/química , Pneumonia Viral/tratamento farmacológico , Capuzes de RNA/metabolismo , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Adenina/química , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Exorribonucleases/metabolismo , Humanos , Metilação , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(27): 15609-15619, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571953

RESUMO

Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Escherichia coli ΔrrmJ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from ΔrrmJ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the ΔrrmJ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the ΔrrmJ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.


Assuntos
Escherichia coli/fisiologia , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , RNA Ribossômico 23S/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Técnicas de Inativação de Genes , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Uridina/metabolismo
18.
Virol Sin ; 35(3): 321-329, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32500504

RESUMO

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.


Assuntos
Betacoronavirus/metabolismo , Bismuto/farmacologia , Metiltransferases/metabolismo , Nucleosídeo-Trifosfatase/efeitos dos fármacos , RNA Helicases/efeitos dos fármacos , Sais/farmacologia , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Betacoronavirus/enzimologia , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Humanos , Metiltransferases/genética , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Pandemias , Pneumonia Viral/virologia , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes , Síndrome Respiratória Aguda Grave , Proteínas não Estruturais Virais/genética , Replicação Viral
19.
Nucleic Acids Res ; 48(14): 8022-8034, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32573735

RESUMO

Mammalian mitochondrial ribosomes contain a set of modified nucleotides, which is distinct from that of the cytosolic ribosomes. Nucleotide m4C840 of the murine mitochondrial 12S rRNA is equivalent to the dimethylated m4Cm1402 residue of Escherichia coli 16S rRNA. Here we demonstrate that mouse METTL15 protein is responsible for the formation of m4C residue of the 12S rRNA. Inactivation of Mettl15 gene in murine cell line perturbs the composition of mitochondrial protein biosynthesis machinery. Identification of METTL15 interaction partners revealed that the likely substrate for this RNA methyltransferase is an assembly intermediate of the mitochondrial small ribosomal subunit containing an assembly factor RBFA.


Assuntos
Metiltransferases/metabolismo , Mitocôndrias/enzimologia , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/enzimologia , Animais , Células Cultivadas , Metilação , Camundongos , Mitocôndrias/metabolismo , RNA Mitocondrial/química , RNA Mitocondrial/metabolismo , RNA Ribossômico/química , RNA Ribossômico 28S/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
20.
Cells ; 9(5)2020 05 20.
Artigo em Inglês | MEDLINE | ID: covidwho-324261

RESUMO

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/metabolismo , Replicação Viral/fisiologia , Animais , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Genoma Viral/genética , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Pandemias , Conformação Proteica em alfa-Hélice , RNA Helicases/química , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo
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