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1.
Molecules ; 25(10)2020 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-32429580

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
2.
J Transl Med ; 18(1): 185, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32370758

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
3.
J Transl Med ; 18(1): 179, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321524

RESUMO

BACKGROUND: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance. METHODS: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance. RESULTS: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on February 9th, 2020 while a different mutation in RdRp changing its amino acid composition emerged on February 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001). CONCLUSIONS: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Evolução Molecular , Genoma Viral/genética , Mutação , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Replicase/genética , Adulto , Ásia/epidemiologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/mortalidade , Farmacorresistência Viral/genética , Europa (Continente)/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , América do Norte/epidemiologia , Oceania/epidemiologia , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/mortalidade , RNA Replicase/antagonistas & inibidores , RNA Replicase/metabolismo
4.
Science ; 368(6492): 779-782, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32277040

RESUMO

A novel coronavirus [severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase [(RdRp), also named nsp12] is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo-electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified ß-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.


Assuntos
Betacoronavirus/enzimologia , RNA Replicase/química , RNA Replicase/ultraestrutura , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/ultraestrutura , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/metabolismo , Alanina/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Domínio Catalítico , Microscopia Crioeletrônica , Desenho de Fármacos , Modelos Moleculares , Conformação Proteica em Folha beta , Domínios Proteicos , RNA Replicase/antagonistas & inibidores , RNA Replicase/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
5.
Life Sci ; 248: 117477, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32119961

RESUMO

AIMS: A newly emerged Human Coronavirus (HCoV) is reported two months ago in Wuhan, China (COVID-19). Until today >2700 deaths from the 80,000 confirmed cases reported mainly in China and 40 other countries. Human to human transmission is confirmed for COVID-19 by China a month ago. Based on the World Health Organization (WHO) reports, SARS HCoV is responsible for >8000 cases with confirmed 774 deaths. Additionally, MERS HCoV is responsible for 858 deaths out of about 2500 reported cases. The current study aims to test anti-HCV drugs against COVID-19 RNA dependent RNA polymerase (RdRp). MATERIALS AND METHODS: In this study, sequence analysis, modeling, and docking are used to build a model for Wuhan COVID-19 RdRp. Additionally, the newly emerged Wuhan HCoV RdRp model is targeted by anti-polymerase drugs, including the approved drugs Sofosbuvir and Ribavirin. KEY FINDINGS: The results suggest the effectiveness of Sofosbuvir, IDX-184, Ribavirin, and Remidisvir as potent drugs against the newly emerged HCoV disease. SIGNIFICANCE: The present study presents a perfect model for COVID-19 RdRp enabling its testing in silico against anti-polymerase drugs. Besides, the study presents some drugs that previously proved its efficiency against the newly emerged viral infection.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/química , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Guanosina Monofosfato/análogos & derivados , Pneumonia Viral/tratamento farmacológico , RNA Replicase/antagonistas & inibidores , Ribavirina/química , Sofosbuvir/química , Proteínas Virais/antagonistas & inibidores , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/química , Alanina/metabolismo , Alphacoronavirus/enzimologia , Alphacoronavirus/genética , Sequência de Aminoácidos , Antivirais/metabolismo , Betacoronavirus/genética , Domínio Catalítico , Biologia Computacional/métodos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos/métodos , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Replicase/química , RNA Replicase/metabolismo , Ribavirina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sofosbuvir/metabolismo , Termodinâmica , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
6.
Chembiochem ; 21(5): 730-738, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32022370

RESUMO

With the current trajectory of the 2019-nCoV outbreak unknown, public health and medicinal measures will both be needed to contain spreading of the virus and to optimize patient outcomes. Although little is known about the virus, an examination of the genome sequence shows strong homology with its better-studied cousin, SARS-CoV. The spike protein used for host cell infection shows key nonsynonymous mutations that might hamper the efficacy of previously developed therapeutics but remains a viable target for the development of biologics and macrocyclic peptides. Other key drug targets, including RNA-dependent RNA polymerase and coronavirus main proteinase (3CLpro), share a strikingly high (>95 %) homology to SARS-CoV. Herein, we suggest four potential drug candidates (an ACE2-based peptide, remdesivir, 3CLpro-1 and a novel vinylsulfone protease inhibitor) that could be used to treat patients suffering with the 2019-nCoV. We also summarize previous efforts into drugging these targets and hope to help in the development of broad-spectrum anti-coronaviral agents for future epidemics.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus , Infecções por Coronavirus/prevenção & controle , Pneumonia Viral/prevenção & controle , Antivirais/química , Betacoronavirus/enzimologia , Betacoronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/transmissão , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Desenho de Fármacos , Humanos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/transmissão , RNA Replicase/química , RNA Replicase/genética , RNA Replicase/metabolismo
7.
Nat Commun ; 11(1): 368, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953395

RESUMO

The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activities - nucleotide polymerization, cap addition, and cap methylation. How RSV L and P coordinate these activities is poorly understood. Here, we present a 3.67 Å cryo-EM structure of the RSV polymerase (L:P) complex. The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (POD) and C-terminal domain (PCTD) of a tetramer of P. The density of the methyltransferase (MT) domain of L and the N-terminal domain of P (PNTD) is missing. Further analysis and comparison with other RNA polymerases at different stages suggest the structure we obtained is likely to be at an elongation-compatible stage. Together, these data provide enriched insights into the interrelationship, the inhibitors, and the evolutionary implications of the RSV polymerase.


Assuntos
Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/química , RNA Replicase/química , Vírus Sincicial Respiratório Humano/enzimologia , Proteínas Virais/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Fosfoproteínas/química , Conformação Proteica , Domínios Proteicos , RNA Replicase/genética , RNA Replicase/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Estruturas Virais
8.
Proc Natl Acad Sci U S A ; 117(3): 1731-1741, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31896581

RESUMO

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral-host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.


Assuntos
Vírus da Hepatite E/genética , Vírus da Hepatite E/fisiologia , Hepatite E/metabolismo , Hepatócitos/virologia , Animais , Antivirais/farmacologia , Carcinoma Hepatocelular , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Genótipo , Células Hep G2 , Hepatite E/virologia , Vírus da Hepatite E/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , RNA Replicase/genética , RNA Replicase/metabolismo , Replicon , Ribavirina/metabolismo , Suínos , Carga Viral , Replicação Viral
9.
PLoS Negl Trop Dis ; 13(11): e0007894, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738758

RESUMO

Dengue is a mosquito-borne viral infection that has spread globally in recent years. Around half of the world's population, especially in the tropics and subtropics, is at risk of infection. Every year, 50-100 million clinical cases are reported, and more than 500,000 patients develop the symptoms of severe dengue infection: dengue haemorrhagic fever and dengue shock syndrome, which threaten life in Asia and Latin America. No antiviral drug for dengue is available. The dengue virus (DENV) non-structural protein 5 (NS5), which possesses the RNA-dependent RNA polymerase (RdRp) activity and is responsible for viral replication and transcription, is an attractive target for anti-dengue drug development. In the present study, 16,240 small-molecule compounds in a fragment library were screened for their capabilities to inhibit the DENV type 2 (DENV2) RdRp activities in vitro. Based on in cellulo antiviral and cytotoxity assays, we selected the compound RK-0404678 with the EC50 value of 6.0 µM for DENV2. Crystallographic analyses revealed two unique binding sites for RK-0404678 within the RdRp, which are conserved in flavivirus NS5 proteins. No resistant viruses emerged after nine rounds of serial passage of DENV2 in the presence of RK-0404678, suggesting the high genetic barrier of this compound to the emergence of a resistant virus. Collectively, RK-0404678 and its binding sites provide a new framework for antiviral drug development.


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , RNA Replicase/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Testes de Sensibilidade Microbiana , Ligação Proteica , RNA Replicase/química , RNA Replicase/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
10.
Emerg Microbes Infect ; 8(1): 1465-1478, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31608791

RESUMO

The ANP32A is responsible for mammalian-restricted influenza virus polymerase activity. However, the mechanism of ANP32A modulation of polymerase activity remains poorly understood. Here, we report that chicken ANP32A (chANP32A) -X1 and -X2 stimulated mammalian-restricted PB2 627E polymerase activity in a dose-dependent manner. Distinct effects of ANP32A constructs suggested that the 180VK181 residues within chANP32A-X1 are necessary but not sufficient to stimulate PB2 627E polymerase activity. The PB2 N567D, T598V, A613V or F636L mutations promoted PB2 627E polymerase activity and chANP32A-X1 showed additive effects, providing further support that species-specific regulation of ANP32A might be only relevant with the PB2 E627K mutation. Rescue of cycloheximide-mediated inhibition showed that ANP32A is species-specific for modulation of vRNA but not mRNA and cRNA, demonstrating chANP32A-X1 compensated for defective cRNPs produced by PB2 627E virus in mammalian cells. The promoter mutations of cRNA enhanced the restriction of PB2 627E polymerase in mammalian cells, which could be restored by chANP32A-X1, indicating that ANP32A is likely to regulate the interaction of viral polymerase with RNA promoter. Coimmunoprecipitation showed that ANP32A did not affect the primary cRNPs assembly. We propose a model that chANP32A-X1 regulates PB2 627E polymerase for suitable interaction with cRNA promoter for vRNA replication.


Assuntos
Vírus da Influenza A Subtipo H1N1/enzimologia , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Vírus da Influenza A Subtipo H9N2/enzimologia , Influenza Aviária/metabolismo , Influenza Humana/metabolismo , Doenças das Aves Domésticas/metabolismo , RNA Replicase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Aviária/genética , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/virologia , Mutação , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Ligação Proteica , RNA Replicase/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Especificidade da Espécie , Proteínas Virais/genética , Replicação Viral
11.
PLoS Pathog ; 15(10): e1008034, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31581279

RESUMO

The influenza A virus RNA-dependent RNA polymerase complex consists in three subunits, PB2, PB1 and PA, that perform transcription and replication of the viral genome through very distinct mechanisms. Biochemical and structural studies have revealed that the polymerase can adopt multiple conformations and form oligomers. However so far it remained unclear whether the available oligomeric crystal structures represent a functional state of the polymerase. Here we gained new insights into this question, by investigating the incompatibility between non-cognate subunits of influenza polymerase brought together through genetic reassortment. We observed that a 7:1 reassortant virus whose PB2 segment derives from the A/WSN/33 (WSN) virus in an otherwise A/PR/8/34 (PR8) backbone is attenuated, despite a 97% identity between the PR8-PB2 and WSN-PB2 proteins. Independent serial passages led to the selection of phenotypic revertants bearing distinct second-site mutations on PA, PB1 and/or PB2. The constellation of mutations present on one revertant virus was studied extensively using reverse genetics and cell-based reconstitution of the viral polymerase. The PA-E349K mutation appeared to play a major role in correcting the initial defect in replication (cRNA -> vRNA) of the PR8xWSN-PB2 reassortant. Strikingly the PA-E349K mutation, and also the PB2-G74R and PB1-K577G mutations present on other revertants, are located at a dimerization interface of the polymerase. All three restore wild-type-like polymerase activity in a minigenome assay while decreasing the level of polymerase dimerization. Overall, our data show that the polymerase subunits co-evolve to ensure not only optimal inter-subunit interactions within the heterotrimer, but also proper levels of dimerization of the heterotrimer which appears to be essential for efficient viral RNA replication. Our findings point to influenza polymerase dimerization as a feature that is controlled by a complex interplay of genetic determinants, can restrict genetic reassortment, and could become a target for antiviral drug development.


Assuntos
Vírus da Influenza A/enzimologia , Influenza Humana/virologia , Mutação , Multimerização Proteica , RNA Replicase/química , RNA Replicase/genética , Vírus Reordenados/genética , Células A549 , Células HEK293 , Humanos , Influenza Humana/genética , Conformação Proteica , Subunidades Proteicas , RNA Replicase/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
12.
PLoS Pathog ; 15(8): e1007995, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381607

RESUMO

Measles virus (MeV) is a highly contagious, re-emerging, major human pathogen. Replication requires a viral RNA-dependent RNA polymerase (RdRP) consisting of the large (L) polymerase protein complexed with the homo-tetrameric phosphoprotein (P). In addition, P mediates interaction with the nucleoprotein (N)-encapsidated viral RNA genome. The nature of the P:L interface and RdRP negotiation of the ribonucleoprotein template are poorly understood. Based on biochemical interface mapping, swapping of the central P tetramerization domain (OD) for yeast GCN4, and functional assays, we demonstrate that the MeV P-to-L interface is bipartite, comprising a coiled-coil microdomain proximal to the OD and an unoccupied face of the triangular prism-shaped C-terminal P X-domain (P-XD), which is distinct from the known P-XD face that binds N-tail. Mixed null-mutant P tetramers regained L-binding competence in a ratio-dependent manner and fully reclaimed bioactivity in minireplicon assays and recombinant MeV, demonstrating that the individual L-binding interface elements are physically and mechanistically distinct. P-XD binding competence to L and N was likewise trans-complementable, which, combined with mathematical modeling, enabled the mechanistic characterization of P through two- and stoichiometrically-controlled three-way complementations. Only one each of the four XDs per P tetramer must be L or N binding-competent for bioactivity, but interaction of the same P-XD with L and N was mutually exclusive, and L binding superseded engaging N. Mixed P tetramers with a single, designated L binding-competent P-XD caused significant RdRP hyperactivity, outlining a model of iterative resolution and reformation of the P-XD:L interface regulating polymerase mobility.


Assuntos
Vírus do Sarampo/enzimologia , Fosfoproteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas , RNA Replicase/metabolismo , Replicação Viral , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Modelos Teóricos , Fosfoproteínas/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA Replicase/química , Homologia de Sequência
13.
Nucleic Acids Res ; 47(17): 9037-9052, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31372633

RESUMO

RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV's largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica de Plantas , Motivos de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Metilação de DNA/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Inativação Gênica , Genoma de Planta , Plantas Geneticamente Modificadas , Domínios Proteicos/genética , RNA Replicase/metabolismo , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Retroelementos/genética
14.
Nucleic Acids Res ; 47(17): 9104-9114, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31372641

RESUMO

Spontaneous post-transcriptional silencing of sense transgenes (S-PTGS) is established in each generation and is accompanied by DNA methylation, but the pathway of PTGS-dependent DNA methylation is unknown and so is its role. Here we show that CHH and CHG methylation coincides spatially and temporally with RDR6-dependent products derived from the central and 3' regions of the coding sequence, and requires the components of the RNA-directed DNA methylation (RdDM) pathway NRPE1, DRD1 and DRM2, but not CLSY1, NRPD1, RDR2 or DCL3, suggesting that RDR6-dependent products, namely long dsRNAs and/or siRNAs, trigger PTGS-dependent DNA methylation. Nevertheless, none of these RdDM components are required to establish S-PTGS or produce a systemic silencing signal. Moreover, preventing de novo DNA methylation in non-silenced transgenic tissues grafted onto homologous silenced tissues does not inhibit the triggering of PTGS. Overall, these data indicate that gene body DNA methylation is a consequence, not a cause, of PTGS, and rule out the hypothesis that a PTGS-associated DNA methylation signal is transmitted independent of a PTGS signal.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , Inativação Gênica , RNA Replicase/genética , Proteínas de Arabidopsis/metabolismo , DNA-Citosina Metilases/genética , DNA-Citosina Metilases/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Genéticos , Plantas Geneticamente Modificadas/genética , RNA Replicase/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo
15.
Nucleic Acids Res ; 47(17): 9024-9036, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31329950

RESUMO

In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV's largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.


Assuntos
Proteínas de Arabidopsis/genética , Metilação de DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica de Plantas/genética , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citosina/química , Citosina/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Inativação Gênica , Loci Gênicos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metiltransferases/metabolismo , Plantas Geneticamente Modificadas , Domínios Proteicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Replicase/metabolismo , Sequenciamento Completo do Genoma
16.
PLoS Pathog ; 15(7): e1007919, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31265471

RESUMO

Avian influenza virus H9N2 has been endemic in birds in the Middle East, in particular in Egypt with multiple cases of human infections since 1998. Despite concerns about the pandemic threat posed by H9N2, little is known about the biological properties of H9N2 in this epicentre of infection. Here, we investigated the evolutionary dynamics of H9N2 in the Middle East and identified phylogeny-associated PB2 mutations that acted cooperatively to increase H9N2 replication/transcription in human cells. The accumulation of PB2 mutations also correlated with an increase in H9N2 virus growth in the upper and lower airways of mice and in virulence. These mutations clustered on a solvent-exposed region in the PB2-627 domain in proximity to potential interfaces with host factors. These PB2 mutations have been found at high prevalence during evolution of H9N2 in the field, indicating that they have provided a selective advantage for viral adaptation to infect poultry. Therefore, continuous prevalence of H9N2 virus in the Middle East has generated a far more fit or optimized replication phenotype, leading to an expanded viral host range, including to mammals, which may pose public health risks beyond the current outbreaks.


Assuntos
Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Humana/virologia , Mutação , RNA Replicase/genética , Proteínas Virais/genética , Animais , Evolução Molecular , Feminino , Células HEK293 , Especificidade de Hospedeiro/genética , Humanos , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Humana/epidemiologia , Mamíferos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Oriente Médio/epidemiologia , Modelos Moleculares , Infecções por Orthomyxoviridae/virologia , Filogenia , RNA Replicase/química , RNA Replicase/metabolismo , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Vírus Reordenados/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Virulência/genética , Replicação Viral/genética , Zoonoses/virologia
17.
Elife ; 82019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31159925

RESUMO

Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that the gene currently designated as avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV relies solely on chicken ANP32A to support its replication in chicken cells. Amino acids 129I and 130N, accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resilient genome edited chickens.


Assuntos
Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Vírus da Influenza A/crescimento & desenvolvimento , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular , Galinhas , Humanos , Vírus da Influenza A/enzimologia , RNA Replicase/metabolismo , Replicação Viral
18.
Nat Struct Mol Biol ; 26(6): 460-470, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160782

RESUMO

Influenza virus RNA-dependent RNA polymerase uses unique mechanisms to transcribe its single-stranded genomic viral RNA (vRNA) into messenger RNA. The polymerase is initially bound to a promoter comprising the partially base-paired 3' and 5' extremities of the RNA. A short, capped primer, 'cap-snatched' from a nascent host polymerase II transcript, is directed towards the polymerase active site to initiate RNA synthesis. Here we present structural snapshots, as determined by X-ray crystallography and cryo-electron microscopy, of actively initiating influenza polymerase as it transitions towards processive elongation. Unexpected conformational changes unblock the active site cavity to allow establishment of a nine-base-pair template-product RNA duplex before the strands separate into distinct exit channels. Concomitantly, as the template translocates, the promoter base pairs are broken and the template entry region is remodeled. These structures reveal details of the influenza polymerase active site that will help optimize nucleoside analogs or other compounds that directly inhibit viral RNA synthesis.


Assuntos
Influenzavirus B/enzimologia , RNA Replicase/química , Proteínas Virais/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Influenza Humana/virologia , Influenzavirus B/química , Influenzavirus B/genética , Influenzavirus B/metabolismo , Modelos Moleculares , Conformação Proteica , RNA Replicase/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Elongação da Transcrição Genética , Iniciação da Transcrição Genética , Proteínas Virais/metabolismo
19.
PLoS Comput Biol ; 15(6): e1007094, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170146

RESUMO

The emergence of replicases that can replicate themselves is a central issue in the origin of life. Recent experiments suggest that such replicases can be realized if an RNA polymerase ribozyme is divided into fragments short enough to be replicable by the ribozyme and if these fragments self-assemble into a functional ribozyme. However, the continued self-replication of such replicases requires that the production of every essential fragment be balanced and sustained. Here, we use mathematical modeling to investigate whether and under what conditions fragmented replicases achieve continued self-replication. We first show that under a simple batch condition, the replicases fail to display continued self-replication owing to positive feedback inherent in these replicases. This positive feedback inevitably biases replication toward a subset of fragments, so that the replicases eventually fail to sustain the production of all essential fragments. We then show that this inherent instability can be resolved by small rates of random content exchange between loose compartments (i.e., horizontal transfer). In this case, the balanced production of all fragments is achieved through negative frequency-dependent selection operating in the population dynamics of compartments. The horizontal transfer also ensures the presence of all essential fragments in each compartment, sustaining self-replication. Taken together, our results underline compartmentalization and horizontal transfer in the origin of the first self-replicating replicases.


Assuntos
Evolução Molecular , RNA Replicase , RNA Catalítico , Biologia Computacional , Modelos Moleculares , RNA Replicase/genética , RNA Replicase/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo
20.
Nat Microbiol ; 4(10): 1750-1759, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31209309

RESUMO

The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the conserved viral genomic RNA (vRNA) or complementary RNA (cRNA) promoter. Here, we determined the apo and promoter-bound influenza D polymerase structures using cryo-electron microscopy and found the polymerase has an evolutionarily conserved stable core structure with inherently flexible peripheral domains. Strikingly, two conformations (mode A and B) of the vRNA promoter were observed where the 3'-vRNA end can bind at two different sites, whereas the cRNA promoter only binds in the mode B conformation. Functional studies confirmed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. This work advances our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for antiviral drug design.


Assuntos
RNA Replicase/metabolismo , RNA Viral/biossíntese , RNA Viral/química , Thogotovirus/enzimologia , Microscopia Crioeletrônica , Modelos Biológicos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , RNA Replicase/química , RNA Replicase/genética , RNA Complementar/biossíntese , RNA Complementar/química , Thogotovirus/ultraestrutura , Transcrição Genética , Replicação Viral
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