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1.
Commun Biol ; 4(1): 999, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429502

RESUMO

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.


Assuntos
SARS-CoV-2/enzimologia , Dimerização , Humanos , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo
2.
Nat Struct Mol Biol ; 28(9): 740-746, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34381216

RESUMO

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.


Assuntos
COVID-19/prevenção & controle , Citidina/análogos & derivados , Hidroxilaminas/metabolismo , Mutagênese/genética , RNA Viral/genética , SARS-CoV-2/genética , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Sequência de Bases , COVID-19/tratamento farmacológico , COVID-19/virologia , Citidina/química , Citidina/metabolismo , Citidina/farmacologia , Humanos , Hidroxilaminas/química , Hidroxilaminas/farmacologia , Modelos Moleculares , Estrutura Molecular , Mutagênese/efeitos dos fármacos , Mutação/efeitos dos fármacos , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , RNA Viral/química , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
3.
Nucleic Acids Res ; 49(15): 8822-8835, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352100

RESUMO

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.


Assuntos
Nidovirales/enzimologia , Nucleotídeos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Difosfatos/farmacologia , Difosfonatos/farmacologia , Guanosina Trifosfato/metabolismo , Manganês , Modelos Moleculares , Nidovirales/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/metabolismo
4.
Nucleic Acids Res ; 49(15): 8811-8821, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365500

RESUMO

Viral RNA-dependent RNA polymerases (RdRPs) play central roles in the genome replication and transcription processes of RNA viruses. RdRPs initiate RNA synthesis either in primer-dependent or de novo mechanism, with the latter often assisted by a 'priming element' (PE) within the RdRP thumb domain. However, RdRP PEs exhibit high-level structural diversity, making it difficult to reconcile their conserved function in de novo initiation. Here we determined a 3.1-Å crystal structure of the Flaviviridae classical swine fever virus (CSFV) RdRP with a relative complete PE. Structure-based mutagenesis in combination with enzymology data further highlights the importance of a glycine residue (G671) and the participation of residues 665-680 in RdRP initiation. When compared with other representative Flaviviridae RdRPs, CSFV RdRP PE is structurally distinct but consistent in terminal initiation preference. Taken together, our work suggests that a conformational change in CSFV RdRP PE is necessary to fulfill de novo initiation, and similar 'induced-fit' mechanisms may be commonly taken by PE-containing de novo viral RdRPs.


Assuntos
Vírus da Febre Suína Clássica/enzimologia , RNA Polimerase Dependente de RNA/química , Iniciação da Transcrição Genética , Proteínas Virais/química , Cristalografia por Raios X , Flaviviridae/enzimologia , Modelos Moleculares , Mutação , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
5.
Nucleic Acids Res ; 49(15): 8796-8810, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379778

RESUMO

During RNA elongation, the influenza A viral (IAV) RNA-dependent RNA polymerase (RdRp) residues in the active site interact with the triphosphate moiety of nucleoside triphosphate (NTP) for catalysis. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation remain elusive. Here, we demonstrated through enzymology, virology and computational approaches that the R239 and K235 in the PB1 subunit of RdRp are critical to controlling the activity and fidelity of transcription. Contrary to common beliefs that high-fidelity RdRp variants exert a slower incorporation rate, we discovered a first-of-its-kind, single lysine-to-arginine mutation on K235 exhibited enhanced fidelity and activity compared with wild-type. In particular, we employed a single-turnover NTP incorporation assay for the first time on IAV RdRp to show that K235R mutant RdRp possessed a 1.9-fold increase in the transcription activity of the cognate NTP and a 4.6-fold increase in fidelity compared to wild-type. Our all-atom molecular dynamics simulations further elucidated that the higher activity is attributed to the shorter distance between K235R and the triphosphate moiety of NTP compared with wild-type. These results provide novel insights into NTP incorporation and fidelity control mechanisms, which lay the foundation for the rational design of IAV vaccine and antiviral targets.


Assuntos
Vírus da Influenza A/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico , Cães , Sequenciamento de Nucleotídeos em Larga Escala , Células Madin Darby de Rim Canino , Mutação , RNA Polimerase Dependente de RNA/genética , Alinhamento de Sequência , Proteínas Virais/genética
6.
Viruses ; 13(8)2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34452414

RESUMO

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5' end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.


Assuntos
Nucleotídeos/metabolismo , Vírus de RNA de Cadeia Positiva/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Caliciviridae/genética , Caliciviridae/metabolismo , Coronaviridae/genética , Coronaviridae/metabolismo , Genoma Viral , Nidovirales/genética , Nidovirales/metabolismo , Picornaviridae/genética , Picornaviridae/metabolismo , Vírus de RNA de Cadeia Positiva/genética , Potyviridae/genética , Potyviridae/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral
7.
Sci Rep ; 11(1): 16307, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381116

RESUMO

Structure-based drug design targeting the SARS-CoV-2 virus has been greatly facilitated by available virus-related protein structures. However, there is an urgent need for effective, safe small-molecule drugs to control the spread of the virus and variants. While many efforts are devoted to searching for compounds that selectively target individual proteins, we investigated the potential interactions between eight proteins related to SARS-CoV-2 and more than 600 compounds from a traditional Chinese medicine which has proven effective at treating the viral infection. Our original ensemble docking and cooperative docking approaches, followed by a total of over 16-micorsecond molecular simulations, have identified at least 9 compounds that may generally bind to key SARS-CoV-2 proteins. Further, we found evidence that some of these compounds can simultaneously bind to the same target, potentially leading to cooperative inhibition to SARS-CoV-2 proteins like the Spike protein and the RNA-dependent RNA polymerase. These results not only present a useful computational methodology to systematically assess the anti-viral potential of small molecules, but also point out a new avenue to seek cooperative compounds toward cocktail therapeutics to target more SARS-CoV-2-related proteins.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/farmacologia , Medicina Tradicional Chinesa , SARS-CoV-2/efeitos dos fármacos , Proteínas Virais/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/química , Antivirais/metabolismo , Gatos , Biologia Computacional , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/metabolismo , Flavonoides/metabolismo , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , RNA Polimerase Dependente de RNA/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-Atividade
8.
PLoS One ; 16(7): e0253364, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270554

RESUMO

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.


Assuntos
Antivirais/farmacologia , Inibidores de Proteases/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Anticorpos de Cadeia Única/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Células A549 , Complexo Antígeno-Anticorpo , Humanos , Concentração Inibidora 50 , RNA Polimerase Dependente de RNA/imunologia , RNA Polimerase Dependente de RNA/metabolismo , Anticorpos de Cadeia Única/imunologia , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo
9.
Int Immunopharmacol ; 98: 107831, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34247016

RESUMO

Explicit hindrance and blockade of the viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is considered one of the most promising and efficient approaches for developing highly potent remedies for COVID-19. However, almost all of the reported viral RdRp inhibitors (either repurposed or new antiviral drugs) lack specific selectivity against the novel coronaviral RdRp and still at a beginning phase of advancement. Herein, I discovered and introduce a new pyrazine derivative, (E)-N-(4-cyanobenzylidene)-6-fluoro-3-hydroxypyrazine-2-carboxamide (cyanorona-20), as the first potent SARS-CoV-2 RdRp inhibitor with very high selectivity (209- and 45-fold more potent than favipiravir and remdesivir, respectively). This promising selective specific anti-COVID-19 compound is also deemed to be the first distinctive derivative of favipiravir. Cyanorona-20, the unprecedented nucleoside/nucleotide analog, was designed, synthesized, characterized, computationally studied, and biologically evaluated for its anti-COVID-19 actions (through a precise in vitro anti-COVID-19 assay). The results of the biological assay displayed that cyanorona-20 surprisingly exhibited very high and largely significant anti-COVID-19 activities (anti-SARS-CoV-2 EC50 = 0.45 µM), and, in addition, it could be also a very promising guide and lead compound for the design and synthesis of new anti-SARS-CoV-2 and anti-COVID-19 agents through structural modifications and further computational studies. Further appraisal for the improvement of cyanorona-20 medication is a prerequisite requirement in the coming days. In a word, the ascent of the second member (cyanorona-20 "Corona Antidote") of the novel and promising class of anti-COVID-19 pyrazine derivatives would drastically make a medical uprising in the pharmacotherapeutic treatment regimens and protocols of the recently-emerged SARS-CoV-2 infection and its accompanying COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , COVID-19/diagnóstico , COVID-19/virologia , Desenho Assistido por Computador , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Interações Hospedeiro-Patógeno , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Terapia de Alvo Molecular , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/crescimento & desenvolvimento , Relação Estrutura-Atividade
10.
Artigo em Inglês | MEDLINE | ID: mdl-34263708

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic of the coronavirus disease in 2019. RNA-dependent RNA polymerase (RdRp) plays an essential role in RNA replication and transcription in SARS-CoV-2. In this study, we focused on the RNA template component of viral RdRp structure and analyzed human microRNAs (miRNAs) targeting specific sequences in this RNA. By examining miRNA databases and using an in vitro RNA-RNA interaction assay, we observed hsa-miR-15b-5p interacting with the RNA component of viral RdRp. Our findings provide evidence that hsa-miR15b-5p may suppresses viral infection and proliferation by targeting the RNA template component of SARS-CoV-2 RdRp.


Assuntos
COVID-19/metabolismo , Regulação Viral da Expressão Gênica , MicroRNAs/fisiologia , RNA Polimerase Dependente de RNA/metabolismo , Biomarcadores/metabolismo , COVID-19/enzimologia , COVID-19/genética , COVID-19/virologia , Bases de Dados Genéticas , Humanos , Técnicas In Vitro , SARS-CoV-2 , Transcrição Genética
11.
Molecules ; 26(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205768

RESUMO

Since December 2019, novel coronavirus disease 2019 (COVID-19) pandemic has caused tremendous economic loss and serious health problems worldwide. In this study, we investigated 14 natural compounds isolated from Amphimedon sp. via a molecular docking study, to examine their ability to act as anti-COVID-19 agents. Moreover, the pharmacokinetic properties of the most promising compounds were studied. The docking study showed that virtually screened compounds were effective against the new coronavirus via dual inhibition of SARS-CoV-2 RdRp and the 3CL main protease. In particular, nakinadine B (1), 20-hepacosenoic acid (11) and amphimedoside C (12) were the most promising compounds, as they demonstrated good interactions with the pockets of both enzymes. Based on the analysis of the molecular docking results, compounds 1 and 12 were selected for molecular dynamics simulation studies. Our results showed Amphimedon sp. to be a rich source for anti-COVID-19 metabolites.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Proteases 3C de Coronavírus/química , Poríferos/química , Poríferos/metabolismo , RNA Polimerase Dependente de RNA/química , SARS-CoV-2/efeitos dos fármacos , Amino Açúcares/química , Amino Açúcares/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Sítios de Ligação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacocinética , COVID-19/tratamento farmacológico , Biologia Computacional , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Piridinas/química , Piridinas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/metabolismo
12.
Nat Commun ; 12(1): 4176, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234134

RESUMO

Mammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor µ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase µ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.


Assuntos
Proteínas do Capsídeo/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Orthoreovirus/ultraestrutura , RNA Viral/metabolismo , Fatores de Transcrição/metabolismo , Regulação Alostérica , Animais , Proteínas do Capsídeo/ultraestrutura , Linhagem Celular , Microscopia Crioeletrônica , Regulação Viral da Expressão Gênica , Genoma Viral , Macaca mulatta , Nucleosídeo-Trifosfatase/ultraestrutura , Orthoreovirus/genética , Orthoreovirus/metabolismo , Multimerização Proteica , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/ultraestrutura , RNA Mensageiro/metabolismo , RNA Viral/ultraestrutura , RNA Polimerase Dependente de RNA/metabolismo , Fatores de Transcrição/ultraestrutura , Ativação Transcricional , Montagem de Vírus/genética
13.
Biomed Pharmacother ; 138: 111544, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34311539

RESUMO

The RNA-dependent RNA polymerase (RdRp) and 3C-like protease (3CLpro) from SARS-CoV-2 play crucial roles in the viral life cycle and are considered the most promising targets for drug discovery against SARS-CoV-2. In this study, FDA-approved drugs were screened to identify the probable anti-RdRp and 3CLpro inhibitors by molecular docking approach. The number of ligands selected from the PubChem database of NCBI for screening was 1760. Ligands were energy minimized using Open Babel. The RdRp and 3CLpro protein sequences were retrieved from the NCBI database. For Homology Modeling predictions, we used the Swiss model server. Their structure was then energetically minimized using SPDB viewer software and visualized in the CHIMERA UCSF software. Molecular dockings were performed using AutoDock Vina, and candidate drugs were selected based on binding affinity (∆G). Hydrogen bonding and hydrophobic interactions between ligands and proteins were visualized using Ligplot and the Discovery Studio Visualizer v3.0 software. Our results showed 58 drugs against RdRp, which had binding energy of - 8.5 or less, and 69 drugs to inhibit the 3CLpro enzyme with a binding energy of - 8.1 or less. Six drugs based on binding energy and number of hydrogen bonds were chosen for the next step of molecular dynamics (MD) simulations to investigate drug-protein interactions (including Nilotinib, Imatinib and dihydroergotamine for 3clpro and Lapatinib, Dexasone and Relategravir for RdRp). Except for Lapatinib, other drugs-complexes were stable during MD simulation. Raltegravir, an anti-HIV drug, was observed to be the best compound against RdRp based on docking binding energy (-9.5 kcal/mole) and MD results. According to the MD results and binding energy, dihydroergotamine is a suitable candidate for 3clpro inhibition (-9.6 kcal/mol). These drugs were classified into several categories, including antiviral, antibacterial, anti-inflammatory, anti-allergic, cardiovascular, anticoagulant, BPH and impotence, antipsychotic, antimigraine, anticancer, and so on. The common prescription-indications for some of these medication categories appeared somewhat in line with manifestations of COVID-19. We hope that they can be beneficial for patients with certain specific symptoms of SARS-CoV-2 infection, but they can also probably inhibit viral enzymes. We recommend further experimental evaluations in vitro and in vivo on these FDA-approved drugs to assess their potential antiviral effect on SARS-CoV-2.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Reposicionamento de Medicamentos , Inibidores Enzimáticos/uso terapêutico , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/efeitos adversos , COVID-19/virologia , Proteases 3C de Coronavírus/metabolismo , Di-Hidroergotamina/uso terapêutico , Aprovação de Drogas , Interações Hospedeiro-Patógeno , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , RNA Polimerase Dependente de RNA/metabolismo , Raltegravir Potássico/uso terapêutico , SARS-CoV-2/enzimologia , Estados Unidos , United States Food and Drug Administration
14.
Nat Microbiol ; 6(7): 921-931, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34127846

RESUMO

Zoonotic arenaviruses can lead to life-threating diseases in humans. These viruses encode a large (L) polymerase that transcribes and replicates the viral genome. At the late stage of replication, the multifunctional Z protein interacts with the L polymerase to shut down RNA synthesis and initiate virion assembly. However, the mechanism by which the Z protein regulates the activity of L polymerase is unclear. Here, we used cryo-electron microscopy to resolve the structures of both Lassa and Machupo virus L polymerases in complex with their cognate Z proteins, and viral RNA, to 3.1-3.9 Å resolutions. These structures reveal that Z protein binding induces conformational changes in two catalytic motifs of the L polymerase, and restrains their conformational dynamics to inhibit RNA synthesis, which is supported by hydrogen-deuterium exchange mass spectrometry analysis. Importantly, we show, by in vitro polymerase reactions, that Z proteins of Lassa and Machupo viruses can cross-inhibit their L polymerases, albeit with decreased inhibition efficiencies. This cross-reactivity results from a highly conserved determinant motif at the contacting interface, but is affected by other variable auxiliary motifs due to the divergent evolution of Old World and New World arenaviruses. These findings could provide promising targets for developing broad-spectrum antiviral drugs.


Assuntos
Arenavirus do Novo Mundo/química , Vírus Lassa/química , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , Motivos de Aminoácidos , Antivirais/farmacologia , Arenavirus do Novo Mundo/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Vírus Lassa/metabolismo , Mutação , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , RNA Viral/química , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
Virus Res ; 302: 198484, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34146608

RESUMO

Novel corona virus SARS-CoV-2, causing coronavirus disease 2019 (COVID-19), has become a global health challenge particularly for developing countries like Pakistan where overcrowded cities, inadequate sanitation, little health awareness and poor socioeconomic conditions exist. The SARS-CoV-2 has been known to spread primarily through direct contact and respiratory droplets. However, detection of SARS-CoV-2 in stool and sewage have raised the possibility of fecal-oral mode of transmission. Currently, quantitative reverse-transcriptase PCR (qRT-PCR) is the only method being used for SARS-CoV-2 detection, which requires expensive instrumentation, dedicated laboratory setup, highly skilled staff, and several hours to report results. Considering the high transmissibility and rapid spread, a robust, sensitive, specific and cheaper assay for rapid SARS-CoV-2 detection is highly needed. Herein, we report a novel colorimetric RT-LAMP assay for naked-eye detection of SARS-COV-2 in clinical as well as sewage samples. Our SARS-CoV-2 RdRp-based LAMP assay could successfully detect the virus RNA in 26/28 (93%) of RT-PCR positive COVID-19 clinical samples with 100% specificity (n = 7) within 20 min. We also tested the effect of various additives on the performance of LAMP assay and found that addition of 1 mg/ml bovine serum albumin (BSA) could increase the sensitivity of assay up to 101 copies of target sequence. Moreover, we also successfully applied this assay to detect SARS-CoV-2 in sewage waters collected from those areas of Lahore, a city of Punjab province of Pakistan, declared as virus hotspots by local government. Our optimized LAMP assay could provide a sensitive first tier strategy for SARS-CoV-2 screening and can potentially help diagnostic laboratories in better handling of high sample turnout during pandemic situation. By providing rapid naked-eye SARS-CoV-2 detection in sewage samples, this assay may support pandemic readiness and emergency response to any possible virus outbreaks in future.


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , SARS-CoV-2/isolamento & purificação , Esgotos/virologia , COVID-19/virologia , Teste para COVID-19 , Colorimetria , Fezes/virologia , Humanos , Programas de Rastreamento , Paquistão/epidemiologia , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/genética , Sensibilidade e Especificidade
16.
J Gen Virol ; 102(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34061017

RESUMO

Avian H9N2 influenza viruses in East Asia are genetically diversified and multiple genotypes (A-W) have been established in poultry. Genotype S strains are currently the most prevalent strains, have caused many human infections and pose a public health threat. In this study, human adaptation mutations in the PB2 polymerase in genotype S strains were identified by database screening. Several PB2 double mutations were identified that acted cooperatively to produce higher genotype S virus polymerase activity and replication in human cells than in avian cells and to increase viral growth and virulence in mice. These mutations were chronologically and phylogenetically clustered in a new group within genotype S viruses. Most of the relevant human virus isolates carry the PB2-A588V mutation together with another PB2 mutation (i.e. K526R, E627V or E627K), indicating a host adaptation advantage for these double mutations. The prevalence of PB2 double mutations in human H9N2 virus isolates has also been found in genetically related human H7N9 and H10N8 viruses. These results suggested that PB2 double mutations in viruses in the field acted cooperatively to increase human adaptation of the currently prevalent H9N2 genotype S strains. This may have contributed to the recent surge of H9N2 infections and may be applicable to the human adaptation of several other avian influenza viruses. Our study provides a better understanding of the human adaptation pathways of genetically related H9N2, H7N9 and H10N8 viruses in nature.


Assuntos
Adaptação ao Hospedeiro , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Humana/virologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , Animais , Aves , Linhagem Celular , Genes Virais , Genótipo , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H9N2/classificação , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/virologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Infecções por Orthomyxoviridae/virologia , Filogenia , Aves Domésticas , RNA Polimerase Dependente de RNA/química , Vírus Reordenados/genética , Proteínas Virais/química , Zoonoses Virais , Virulência/genética
17.
Viruses ; 13(5)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066055

RESUMO

Arthropod-borne flaviviruses, such as Zika virus (ZIKV), Usutu virus (USUV), and West Nile virus (WNV), are a growing cause of human illness and death around the world. Presently, no licensed antivirals to control them are available and, therefore, search for broad-spectrum antivirals, including host-directed compounds, is essential. The PI3K/Akt pathway controls essential cellular functions involved in cell metabolism and proliferation. Moreover, Akt has been found to participate in modulating replication in different viruses including the flaviviruses. In this work we studied the interaction of flavivirus NS5 polymerases with the cellular kinase Akt. In vitro NS5 phosphorylation experiments with Akt showed that flavivirus NS5 polymerases are phosphorylated and co-immunoprecipitate by Akt. Polymerase activity assays of Ala- and Glu-generated mutants for the Akt-phosphorylated residues also indicate that Glu mutants of ZIKV and USUV NS5s present a reduced primer-extension activity that was not observed in WNV mutants. Furthermore, treatment with Akt inhibitors (MK-2206, honokiol and ipatasertib) reduced USUV and ZIKV titers in cell culture but, except for honokiol, not WNV. All these findings suggest an important role for Akt in flavivirus replication although with specific differences among viruses and encourage further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral potential target.


Assuntos
Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Flavivirus/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Flavivirus/efeitos dos fármacos , Genoma Viral , Humanos , Mutação , Fases de Leitura Aberta , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Proteoma , Proteômica/métodos , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/genética , Vírus do Nilo Ocidental/fisiologia , Zika virus/fisiologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia
18.
Molecules ; 26(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071102

RESUMO

Dengue virus (DENV) infection causes mild to severe illness in humans that can lead to fatality in severe cases. Currently, no specific drug is available for the treatment of DENV infection. Thus, the development of an anti-DENV drug is urgently required. Cordycepin (3'-deoxyadenosine), which is a major bioactive compound in Cordyceps (ascomycete) fungus that has been used for centuries in Chinese traditional medicine, was reported to exhibit antiviral activity. However, the anti-DENV activity of cordycepin is unknown. We hypothesized that cordycepin exerts anti-DENV activity and that, as an adenosine derivative, it inhibits DENV replication. To test this hypothesis, we investigated the anti-DENV activity of cordycepin in DENV-infected Vero cells. Cordycepin treatment significantly decreased DENV protein at a half-maximal effective concentration (EC50) of 26.94 µM. Moreover, DENV RNA was dramatically decreased in cordycepin-treated Vero cells, indicating its effectiveness in inhibiting viral RNA replication. Via in silico molecular docking, the binding of cordycepin to DENV non-structural protein 5 (NS5), which is an important enzyme for RNA synthesis, at both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, was predicted. The results of this study demonstrate that cordycepin is able to inhibit DENV replication, which portends its potential as an anti-dengue therapy.


Assuntos
Vírus da Dengue/efeitos dos fármacos , Desoxiadenosinas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Chlorocebus aethiops , Dengue/tratamento farmacológico , Vírus da Dengue/metabolismo , Desoxiadenosinas/metabolismo , Simulação de Acoplamento Molecular , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Células Vero/virologia , Proteínas não Estruturais Virais/metabolismo
19.
Nat Commun ; 12(1): 3492, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108460

RESUMO

In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with translating ribosomes, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Uso do Códon , Biossíntese de Proteínas , RNA Interferente Pequeno/biossíntese , Animais , Proteínas Argonauta/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Catálise , Citosol/metabolismo , Mutação , Oogônios/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Ribossomos/metabolismo
20.
J Virol ; 95(16): e0059421, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34037421

RESUMO

Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction caused by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction, and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensures the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help us to understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. IMPORTANCE It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help us to understand the polymerase function of negative-stranded RNA viruses.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Vesiculovirus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Animais , Células Cultivadas , Peixes , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Fosfoproteínas/metabolismo , Estabilidade Proteica , RNA Viral/biossíntese , Infecções por Rhabdoviridae/veterinária , Infecções por Rhabdoviridae/virologia , Vesiculovirus/metabolismo
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