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1.
Nat Commun ; 11(1): 5191, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060587

RESUMO

In grasses, phased small interfering RNAs (phasiRNAs), 21- or 24-nucleotide (nt) in length, are predominantly expressed in anthers and play a role in regulating male fertility. However, their targets and mode of action on the targets remain unknown. Here we profile phasiRNA expression in premeiotic and meiotic spikelets as well as in purified male meiocytes at early prophase I, tetrads and microspores in rice. We show that 21-nt phasiRNAs are most abundant in meiocytes at early prophase I while 24-nt phasiRNAs are more abundant in tetrads and microspores. By performing highly sensitive degradome sequencing, we find that 21-nt phasiRNAs direct target mRNA cleavage in male germ cells, especially in meiocytes at early prophase I. These targets include 435 protein-coding genes and 71 transposons that show an enrichment for carbohydrate biosynthetic and metabolic pathways. Our study provides strong evidence that 21-nt phasiRNAs act in a target-cleavage mode and may facilitate the progression of meiosis by fine-tuning carbohydrate biosynthesis and metabolism in male germ cells.


Assuntos
Células Germinativas/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Meiose/fisiologia , Nucleotídeos , Oryza/embriologia , Oryza/genética , Proteínas de Plantas/genética , RNA Replicase/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/metabolismo , Transcriptoma
2.
Nat Commun ; 11(1): 4682, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943628

RESUMO

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.


Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Pirazinas/farmacologia , Amidas/farmacocinética , Animais , Antivirais/farmacocinética , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Moleculares , Mutagênese/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Pirazinas/farmacocinética , RNA Replicase/química , RNA Replicase/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Análise de Sequência , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
3.
PLoS One ; 15(9): e0239899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32986763

RESUMO

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


Assuntos
Interações entre Hospedeiro e Microrganismos/genética , Domínios Proteicos , RNA Replicase/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Imunoprecipitação , Plasmídeos/genética , Ligação Proteica/genética , RNA Replicase/química , RNA Replicase/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Transfecção , Proteínas do Core Viral/genética , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral/genética
4.
Nat Commun ; 11(1): 4417, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887884

RESUMO

COVID-19 was declared a pandemic on March 11 by WHO, due to its great threat to global public health. The coronavirus main protease (Mpro, also called 3CLpro) is essential for processing and maturation of the viral polyprotein, therefore recognized as an attractive drug target. Here we show that a clinically approved anti-HCV drug, Boceprevir, and a pre-clinical inhibitor against feline infectious peritonitis (corona) virus (FIPV), GC376, both efficaciously inhibit SARS-CoV-2 in Vero cells by targeting Mpro. Moreover, combined application of GC376 with Remdesivir, a nucleotide analogue that inhibits viral RNA dependent RNA polymerase (RdRp), results in sterilizing additive effect. Further structural analysis reveals binding of both inhibitors to the catalytically active side of SARS-CoV-2 protease Mpro as main mechanism of inhibition. Our findings may provide critical information for the optimization and design of more potent inhibitors against the emerging SARS-CoV-2 virus.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Prolina/análogos & derivados , Inibidores de Proteases/farmacologia , Pirrolidinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacologia , Betacoronavirus/enzimologia , Sítios de Ligação/efeitos dos fármacos , Domínio Catalítico , Chlorocebus aethiops , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Pandemias , Prolina/farmacologia , RNA Replicase/antagonistas & inibidores , RNA Replicase/química , RNA Replicase/metabolismo , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
5.
PLoS Pathog ; 16(9): e1008825, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886709

RESUMO

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other's templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses.


Assuntos
Alphavirus , Genoma Viral , Conformação de Ácido Nucleico , RNA Replicase , RNA Viral , Proteínas Virais , Alphavirus/química , Alphavirus/genética , Alphavirus/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , RNA Replicase/química , RNA Replicase/genética , RNA Replicase/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
6.
Nat Commun ; 11(1): 3656, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694517

RESUMO

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.


Assuntos
Proteínas Aviárias/ultraestrutura , Virus da Influenza A Subtipo H5N1/patogenicidade , Proteínas Nucleares/ultraestrutura , Domínios Proteicos/genética , RNA Replicase/ultraestrutura , Proteínas de Ligação a RNA/ultraestrutura , Proteínas Virais/ultraestrutura , Animais , Proteínas Aviárias/metabolismo , Aves/virologia , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Influenza Aviária/virologia , Influenza Humana/virologia , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , RNA Replicase/genética , RNA Replicase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Especificidade da Espécie , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
7.
Nat Commun ; 11(1): 3590, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681014

RESUMO

Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors.


Assuntos
Vírus La Crosse/enzimologia , RNA Replicase/química , RNA Replicase/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Cristalografia por Raios X , Vírus La Crosse/química , Vírus La Crosse/genética , Conformação Proteica , Domínios Proteicos , RNA Replicase/genética , Transcrição Genética , Proteínas Virais/genética
8.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641477

RESUMO

Positive-strand RNA [(+)RNA] viruses are important pathogens of humans, animals, and plants and replicate inside host cells by coopting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and coopted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with endoplasmic reticulum (ER)-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the double-stranded RNA (dsRNA) replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (-) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by phosphatidylinositol-3-phosphate [PI(3)P] and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 coopted replicase assembly factors, Vps34 phosphatidylinositol 3-kinase (PI3K) and the membrane-bending ESCRT factors, are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential coopted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication.IMPORTANCE Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals, and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and coopt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, we have developed an artificial giant unilamellar vesicle (GUV)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine, in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential coopted cellular factors.


Assuntos
RNA Replicase/genética , RNA de Cadeia Dupla/genética , Tombusvirus/genética , Lipossomas Unilamelares/metabolismo , Proteínas Virais/genética , Bioensaio , Linhagem Celular , Retículo Endoplasmático/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ergosterol/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , RNA Replicase/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Lipossomas Unilamelares/química , Proteínas Virais/metabolismo , Replicação Viral
9.
PLoS One ; 15(7): e0236481, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32716919

RESUMO

RNA-dependent RNA polymerase 6 (RDR6) is one of the key factors in plant defense responses and suppresses virus or viroid invasion into shoot apical meristem (SAM) in Nicotiana benthamiana. To evaluate the role of Solanum lycopersicum (Sl) RDR6 upon viroid infection, SlRDR6-suppressed (SlRDR6i) 'Moneymaker' tomatoes were generated by RNA interference and inoculated with intermediate or lethal strain of potato spindle tuber viroid (PSTVd). Suppression of SlRDR6 did not change disease symptoms of both PSTVd strains in 'Moneymaker' tomatoes. Analysis of PSTVd distribution in shoot apices by in situ hybridization revealed that both PSTVd strains similarly invade the basal part but not apical part including pluripotent stem cells of SAM in SlRDR6i plants at a low rate unlike a previous report in N. benthamiana. In addition, unexpectedly, amount of PSTVd accumulation was apparently lower in SlRDR6i plants than in control tomatoes transformed with empty cassette in early infection especially in the lethal strain. Meanwhile, SlRDR6 suppression did not affect the seed transmission rates of PSTVd. These results indicate that RDR6 generally suppresses PSTVd invasion into SAM in plants, while suppression of RDR6 does not necessarily elevate amount of PSTVd accumulation. Additionally, our results suggest that host factors such as RDR1 other than RDR6 may also be involved in the protection of SAM including pluripotent stem cells from PSTVd invasion and effective RNA silencing causing the decrease of PSTVd accumulation during early infection in tomato plants.


Assuntos
Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/virologia , Meristema/citologia , Meristema/virologia , Proteínas de Plantas/metabolismo , Células-Tronco Pluripotentes/virologia , RNA Replicase/metabolismo , Viroides/patogenicidade , Regulação da Expressão Gênica de Plantas , Genoma Viral , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Viroides/isolamento & purificação
10.
Cell Rep ; 31(11): 107774, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: covidwho-436427

RESUMO

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused a huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus (SARS-CoV-2). The viral polymerase is a promising antiviral target. Here, we describe the near-atomic-resolution structure of the SARS-CoV-2 polymerase complex consisting of the nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases and suggests a mechanism of activation by cofactors. Biochemical studies reveal reduced activity of the core polymerase complex and lower thermostability of individual subunits of SARS-CoV-2 compared with SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate adaptation of SARS-CoV-2 toward humans with a relatively lower body temperature than the natural bat hosts.


Assuntos
Betacoronavirus/enzimologia , Microscopia Crioeletrônica , RNA Replicase/química , Proteínas não Estruturais Virais/química , Substituição de Aminoácidos , Escherichia coli/genética , Evolução Molecular , Modelos Moleculares , Complexos Multiproteicos/química , RNA Replicase/metabolismo , Vírus da SARS/enzimologia , Proteínas não Estruturais Virais/metabolismo
11.
Cell Rep ; 31(11): 107774, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: covidwho-594914

RESUMO

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused a huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus (SARS-CoV-2). The viral polymerase is a promising antiviral target. Here, we describe the near-atomic-resolution structure of the SARS-CoV-2 polymerase complex consisting of the nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases and suggests a mechanism of activation by cofactors. Biochemical studies reveal reduced activity of the core polymerase complex and lower thermostability of individual subunits of SARS-CoV-2 compared with SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate adaptation of SARS-CoV-2 toward humans with a relatively lower body temperature than the natural bat hosts.


Assuntos
Betacoronavirus/enzimologia , Microscopia Crioeletrônica , RNA Replicase/química , Proteínas não Estruturais Virais/química , Substituição de Aminoácidos , Escherichia coli/genética , Evolução Molecular , Modelos Moleculares , Complexos Multiproteicos/química , RNA Replicase/metabolismo , Vírus da SARS/enzimologia , Proteínas não Estruturais Virais/metabolismo
13.
Arch Virol ; 165(9): 2105-2109, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32556598

RESUMO

Here, we describe the molecular characterization of a novel mycovirus isolated from a phytopathogenic fungus, Alternaria dianthicola, which we have named "Alternaria dianthicola dsRNA virus 1" (AdRV1). AdRV1 has a genome of 3,014 bp that contains two non-overlapping open reading frames (ORF1 and 2) coding for a hypothetical protein and an RNA-dependent RNA polymerase (RdRp), respectively. Based on the RdRp, AdRV1 is phylogenetically related to some unclassified dsRNA mycoviruses, including Alternaria longipes dsRNA virus 1, and shows a distant relationship to members of the family Partitiviridae. To the best of our knowledge, this is the first report of mycovirus infecting A. dianthicola.


Assuntos
Alternaria/virologia , Micovírus/isolamento & purificação , Doenças das Plantas/microbiologia , Vírus de RNA/isolamento & purificação , Alternaria/genética , Alternaria/fisiologia , Sequência de Aminoácidos , Micovírus/classificação , Micovírus/genética , Genoma Viral , Fases de Leitura Aberta , Filogenia , RNA Replicase/química , RNA Replicase/genética , RNA Replicase/metabolismo , Vírus de RNA/classificação , Vírus de RNA/genética , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
Nature ; 582(7811): 283-288, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499657

RESUMO

Mobile genetic elements threaten genome integrity in all organisms. RDE-3 (also known as MUT-2) is a ribonucleotidyltransferase that is required for transposon silencing and RNA interference in Caenorhabditis elegans1-4. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3' termini of these RNAs (designated poly(UG) or pUG tails)5. Here we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to targets of RNA interference, as well as to transposon RNAs. RNA fragments attached to pUG tails with more than 16 perfectly alternating 3' U and G nucleotides become gene-silencing agents. pUG tails promote gene silencing by recruiting RNA-dependent RNA polymerases, which use pUG-tailed RNAs (pUG RNAs) as templates to synthesize small interfering RNAs (siRNAs). Our results show that cycles of pUG RNA-templated siRNA synthesis and siRNA-directed pUG RNA biogenesis underlie double-stranded-RNA-directed transgenerational epigenetic inheritance in the C. elegans germline. We speculate that this pUG RNA-siRNA silencing loop enables parents to inoculate progeny against the expression of unwanted or parasitic genetic elements.


Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/parasitologia , Epigênese Genética/genética , Genoma/genética , Hereditariedade , Poli G/genética , Poli U/genética , RNA Mensageiro/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Nucleotidiltransferases/metabolismo , Interferência de RNA , RNA Replicase/metabolismo , RNA Interferente Pequeno/genética , Moldes Genéticos
15.
Molecules ; 25(10)2020 May 17.
Artigo em Inglês | MEDLINE | ID: covidwho-276849

RESUMO

Remdesivir is a nucleotide prodrug that is currently undergoing extensive clinical trials for the treatment of COVID-19. The prodrug is metabolized to its active triphosphate form and interferes with the action of RNA-dependent RNA polymerase of SARS-COV-2. Herein, we report the antiviral activity of remdesivir against human coronavirus 229E (HCoV-229E) compared to known anti-HIV agents. These agents included tenofovir (TFV), 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), alovudine (FLT), lamivudine (3TC), and emtricitabine (FTC), known as nucleoside reverse-transcriptase inhibitors (NRTIs), and a number of 5'-O-fatty acylated anti-HIV nucleoside conjugates. The anti-HIV nucleosides interfere with HIV RNA-dependent DNA polymerase and/or act as chain terminators. Normal human fibroblast lung cells (MRC-5) were used to determine the cytotoxicity of the compounds. The study revealed that remdesivir exhibited an EC50 value of 0.07 µM against HCoV-229E with TC50 of > 2.00 µM against MRC-5 cells. Parent NRTIs were found to be inactive against (HCoV-229E) at tested concentrations. Among all the NRTIs and 5'-O-fatty acyl conjugates of NRTIs, 5'-O-tetradecanoyl ester conjugate of FTC showed modest activity with EC50 and TC50 values of 72.8 µM and 87.5 µM, respectively. These data can be used for the design of potential compounds against other coronaviruses.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Fármacos Anti-HIV/farmacologia , Coronavirus Humano 229E/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Fármacos Anti-HIV/química , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Linhagem Celular , Coronavirus Humano 229E/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , RNA Replicase/metabolismo , Inibidores da Transcriptase Reversa/química
16.
Nature ; 581(7808): 252-255, 2020 05.
Artigo em Inglês | MEDLINE | ID: covidwho-265650

Assuntos
Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/imunologia , Desenho de Fármacos , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Vacinas Virais , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Antivirais/química , Azóis/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , China , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Microscopia Crioeletrônica , Cristalização , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Avaliação Pré-Clínica de Medicamentos , Alemanha , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , National Institutes of Health (U.S.)/economia , National Institutes of Health (U.S.)/organização & administração , Compostos Organosselênicos/farmacologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Inibidores de Proteases/farmacologia , RNA Replicase/antagonistas & inibidores , RNA Replicase/química , RNA Replicase/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Síncrotrons , Fatores de Tempo , Reino Unido , Estados Unidos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/imunologia , Vacinas Virais/química , Vacinas Virais/imunologia
17.
J Transl Med ; 18(1): 185, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: covidwho-175840

RESUMO

A new human coronavirus named SARS-CoV-2 was identified in several cases of acute respiratory syndrome in Wuhan, China in December 2019. On March 11 2020, WHO declared the SARS-CoV-2 infection to be a pandemic, based on the involvement of 169 nations. Specific drugs for SARS-CoV-2 are obviously not available. Currently, drugs originally developed for other viruses or parasites are currently in clinical trials based on empiric data. In the quest of an effective antiviral drug, the most specific target for an RNA virus is the RNA-dependent RNA-polymerase (RdRp) which shows significant differences between positive-sense and negative-sense RNA viruses. An accurate evaluation of RdRps from different viruses may guide the development of new drugs or the repositioning of already approved antiviral drugs as treatment of SARS-CoV-2. This can accelerate the containment of the SARS-CoV-2 pandemic and, hopefully, of future pandemics due to other emerging zoonotic RNA viruses.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , RNA Replicase/antagonistas & inibidores , RNA Replicase/química , Sequência de Aminoácidos , Betacoronavirus/isolamento & purificação , Sequência Conservada , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/transmissão , Reposicionamento de Medicamentos , Humanos , Modelos Moleculares , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/transmissão , RNA Replicase/metabolismo , Alinhamento de Sequência , Replicação Viral/efeitos dos fármacos , Eliminação de Partículas Virais/efeitos dos fármacos
18.
J Phys Chem Lett ; 11(11): 4430-4435, 2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: covidwho-233085

RESUMO

The pandemic outbreak of a new coronavirus (CoV), SARS-CoV-2, has captured the world's attention, demonstrating that CoVs represent a continuous global threat. As this is a highly contagious virus, it is imperative to understand RNA-dependent-RNA-polymerase (RdRp), the key component in virus replication. Although the SARS-CoV-2 genome shares 80% sequence identity with severe acute respiratory syndrome SARS-CoV, their RdRps and nucleotidyl-transferases (NiRAN) share 98.1% and 93.2% identity, respectively. Sequence alignment of six coronaviruses demonstrated higher identity among their RdRps (60.9%-98.1%) and lower identity among their Spike proteins (27%-77%). Thus, a 3D structural model of RdRp, NiRAN, non-structural protein 7 (nsp7), and nsp8 of SARS-CoV-2 was generated by modeling starting from the SARS counterpart structures. Furthermore, we demonstrate the binding poses of three viral RdRp inhibitors (Galidesivir, Favipiravir, and Penciclovir), which were recently reported to have clinical significance for SARS-CoV-2. The network of interactions established by these drug molecules affirms their efficacy to inhibit viral RNA replication and provides an insight into their structure-based rational optimization for SARS-CoV-2 inhibition.


Assuntos
Betacoronavirus/enzimologia , Nucleotidiltransferases/química , RNA Replicase/química , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Amidas/química , Amidas/metabolismo , Antivirais/química , Antivirais/metabolismo , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Simulação de Acoplamento Molecular , Nucleotidiltransferases/metabolismo , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Pirazinas/química , Pirazinas/metabolismo , Pirrolidinas/química , Pirrolidinas/metabolismo , RNA Replicase/metabolismo
19.
PLoS Pathog ; 16(5): e1008484, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32357182

RESUMO

The flaviviruses pose serious threats to human health. Being a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP), NS5 is the most conserved flavivirus protein and an important antiviral target. Previously reported NS5 structures represented by those from the Japanese encephalitis virus (JEV) and Dengue virus serotype 3 (DENV3) exhibit two apparently different global conformations, defining two sets of intra-molecular MTase-RdRP interactions. However, whether these NS5 conformations are conserved in flaviviruses and their specific functions remain elusive. Here we report two forms of DENV serotype 2 (DENV2) NS5 crystal structures representing two conformational states with defined analogies to the JEV-mode and DENV3-mode conformations, respectively, demonstrating the conservation of both conformation modes and providing clues for how different conformational states may be interconnected. Data from in vitro polymerase assays further demonstrate that perturbing the JEV-mode but not the DENV3-mode intra-molecular interactions inhibits catalysis only at initiation, while the cell-based virological analysis suggests that both modes of interactions are important for virus proliferation. Our work highlights the role of MTase as a unique intra-molecular initiation factor specifically only through the JEV-mode conformation, providing an example of conformation-based crosstalk between naturally fused protein functional modules.


Assuntos
Vírus da Dengue/química , Vírus da Encefalite Japonesa (Espécie)/química , RNA Replicase/química , Proteínas não Estruturais Virais/química , Animais , Cricetinae , Cristalografia por Raios X , Vírus da Dengue/metabolismo , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Humanos , Domínios Proteicos , RNA Replicase/metabolismo , Proteínas não Estruturais Virais/metabolismo
20.
Nat Commun ; 11(1): 2605, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451382

RESUMO

Each polymerase nucleotide addition cycle is associated with two primary conformational changes of the catalytic complex: the pre-chemistry active site closure and post-chemistry translocation. While active site closure is well interpreted by numerous crystallographic snapshots, translocation intermediates are rarely captured. Here we report three types of intermediate structures in an RNA-dependent RNA polymerase (RdRP). The first two types, captured in forward and reverse translocation events, both highlight the role of RdRP-unique motif G in restricting the RNA template movement, corresponding to the rate-limiting step in translocation. By mutating two critical residues in motif G, we obtain the third type of intermediates that may mimic the transition state of this rate-limiting step, demonstrating a previously unidentified movement of the template strand. We propose that a similar strategy may be utilized by other classes of nucleic acid polymerases to ensure templating nucleotide positioning for efficient catalysis through restricting interactions with template RNA.


Assuntos
RNA Replicase/química , RNA Replicase/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Transporte Biológico Ativo , Domínio Catalítico , Cristalografia por Raios X , Enterovirus Humano A/enzimologia , Enterovirus Humano A/genética , Genes Virais , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Conformação Proteica , RNA Replicase/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Elongação da Transcrição Genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
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