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1.
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
2.
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
3.
Sci Transl Med ; 12(541)2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32253226

RESUMO

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog ß-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (ß-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.


Assuntos
Antivirais/administração & dosagem , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Ribonucleosídeos/administração & dosagem , Replicação Viral/efeitos dos fármacos , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/análogos & derivados , Alanina/administração & dosagem , Alanina/análogos & derivados , Animais , Antibioticoprofilaxia , Betacoronavirus/fisiologia , Linhagem Celular , Infecções por Coronavirus/patologia , Citidina/administração & dosagem , Citidina/análogos & derivados , Modelos Animais de Doenças , Farmacorresistência Viral , Humanos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Modelos Moleculares , Mutação/efeitos dos fármacos , Pandemias , Pneumonia Viral/patologia , Cultura Primária de Células , RNA Replicase/química , RNA Replicase/genética , RNA Viral , Distribuição Aleatória , Sistema Respiratório/citologia
4.
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
5.
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
6.
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
7.
Nucleic Acids Res ; 48(3): 1392-1405, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31863580

RESUMO

The enterovirus 71 (EV71) 3Dpol is an RNA-dependent RNA polymerase (RdRP) that plays the central role in the viral genome replication, and is an important target in antiviral studies. Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5'-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity (KM,NTP). We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.


Assuntos
Enterovirus Humano A/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , RNA Replicase/ultraestrutura , Antivirais/química , Sítios de Ligação , Cristalografia por Raios X , Enterovirus Humano A/química , Enterovirus Humano A/genética , Genoma Viral , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Nucleotídeos/química , RNA Replicase/química , RNA Viral/química , RNA Viral/ultraestrutura , Replicação Viral/genética
8.
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
9.
Nat Struct Mol Biol ; 26(11): 1023-1034, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31695188

RESUMO

Endogenous RNA transcription characterizes double-stranded RNA (dsRNA) viruses in the Reoviridae, a family that is exemplified by its simple, single-shelled member cytoplasmic polyhedrosis virus (CPV). Because of the lack of in situ structures of the intermediate stages of RNA-dependent RNA polymerase (RdRp) during transcription, it is poorly understood how RdRp detects environmental cues and internal transcriptional states to initiate and coordinate repeated cycles of transcript production inside the capsid. Here, we captured five high-resolution (2.8-3.5 Å) RdRp-RNA in situ structures-representing quiescent, initiation, early elongation, elongation and abortive states-under seven experimental conditions of CPV. We observed the 'Y'-form initial RNA fork in the initiation state and the complete transcription bubble in the elongation state. These structures reveal that de novo RNA transcription involves three major conformational changes during state transitions. Our results support an ouroboros model for endogenous conservative transcription in dsRNA viruses.


Assuntos
RNA de Cadeia Dupla/genética , RNA Viral/genética , Reoviridae/genética , Transcrição Genética , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , RNA Replicase/química , RNA Replicase/ultraestrutura , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/ultraestrutura , RNA Viral/química , RNA Viral/ultraestrutura , Reoviridae/química , Reoviridae/ultraestrutura , Infecções por Reoviridae/virologia , Proteínas Virais/química , Proteínas Virais/ultraestrutura
10.
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
11.
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
12.
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
13.
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
14.
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
15.
Molecules ; 24(13)2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31247979

RESUMO

Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the Picornaviridae family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5'-end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed uridylylation, is catalyzed by the viral RNA-dependent RNA polymerase named 3Dpol. 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg uridylylation. Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg uridylylation by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with 3Dpol. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of 3Dpol in the presence of VPg1-FUMP showed an 8 Å movement of the ß9-α11 loop of the polymerase towards the active site cavity relative to the complex of 3Dpol with VPg1-UMP. The conformational rearrangement of this loop preceding the 3Dpol B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the uridylylation of their VPg proteins to initiate the viral RNA synthesis.


Assuntos
Vírus da Febre Aftosa/metabolismo , Peptídeos/química , Proteínas Virais/química , Sequência de Aminoácidos , Modelos Moleculares , Conformação Molecular , Engenharia de Proteínas , RNA Replicase/síntese química , RNA Replicase/química , RNA Replicase/metabolismo , Relação Estrutura-Atividade , Uridina Monofosfato/química , Proteínas Virais/síntese química , Proteínas Virais/metabolismo
16.
PLoS Pathog ; 15(4): e1007656, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30951555

RESUMO

Zika virus (ZIKV), a member of the Flaviviridae family, has emerged as a major public health threat, since ZIKV infection has been connected to microcephaly and other neurological disorders. Flavivirus genome replication is driven by NS5, an RNA-dependent RNA polymerase (RdRP) that also contains a N-terminal methyltransferase domain essential for viral mRNA capping. Given its crucial roles, ZIKV NS5 has become an attractive antiviral target. Here, we have used integrated structural biology approaches to characterize the supramolecular arrangement of the full-length ZIKV NS5, highlighting the assembly and interfaces between NS5 monomers within a dimeric structure, as well as the dimer-dimer interactions to form higher order fibril-like structures. The relative orientation of each monomer within the dimer provides a model to explain the coordination between MTase and RdRP domains across neighboring NS5 molecules and mutational studies underscore the crucial role of the MTase residues Y25, K28 and K29 in NS5 dimerization. The basic residue K28 also participates in GTP binding and competition experiments indicate that NS5 dimerization is disrupted at high GTP concentrations. This competition represents a first glimpse at a molecular level explaining how dimerization might regulate the capping process.


Assuntos
Conformação Proteica , Multimerização Proteica , RNA Replicase/química , Proteínas não Estruturais Virais/química , Zika virus/enzimologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , RNA Replicase/metabolismo , Proteínas não Estruturais Virais/metabolismo
17.
J Biol Chem ; 294(19): 7573-7587, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30867194

RESUMO

RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modeling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that at least some of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses.


Assuntos
Guanosina Trifosfato/química , Hepacivirus/enzimologia , Simulação de Dinâmica Molecular , RNA Replicase/química , Uridina Trifosfato/química , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Domínio Catalítico , Guanosina Trifosfato/metabolismo , RNA Replicase/metabolismo , Uridina Trifosfato/metabolismo , Proteínas não Estruturais Virais/metabolismo
18.
Arch Virol ; 164(4): 971-982, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30721364

RESUMO

The RNA-dependent RNA polymerase (RdRp) of sesbania mosaic virus (SeMV) was previously shown to interact with the viral protein P10, which led to enhanced polymerase activity. In the present investigation, the equilibrium dissociation constant for the interaction between the two proteins was determined to be 0.09 µM using surface plasmon resonance, and the disordered C-terminal domain of RdRp was shown to be essential for binding to P10. The association with P10 brought about a change in the oligomeric state of RdRp, resulting in reduced aggregation and increased polymerase activity. Interestingly, unlike the wild-type RdRp, C-terminal deletion mutants (C del 43 and C del 72) were found to exist predominantly as monomers and were as active as the RdRp-P10 complex. Thus, either the deletion of the C-terminal disordered domain or its masking by binding to P10 results in the activation of polymerase activity. Further, deletion of the C-terminal 85 residues of RdRp resulted in complete loss of activity. Mutation of a conserved tyrosine (RdRp Y480) within motif E, located between 72 and 85 residues from the C-terminus of RdRp, rendered the protein inactive, demonstrating the importance of motif E in RNA synthesis in vitro.


Assuntos
Vírus de Plantas/enzimologia , RNA Replicase/química , RNA Replicase/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Vírus de Plantas/química , Vírus de Plantas/genética , Ligação Proteica , Domínios Proteicos , RNA Replicase/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/genética
19.
Structure ; 27(4): 660-668.e4, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30799076

RESUMO

Nipah virus is a highly lethal zoonotic pathogen found in Southeast Asia that has caused human encephalitis outbreaks with 40%-70% mortality. NiV encodes its own RNA-dependent RNA polymerase within the large protein, L. Efficient polymerase activity requires the phosphoprotein, P, which tethers L to its template, the viral nucleocapsid. P is a multifunctional protein with modular domains. The central P multimerization domain is composed of a long, tetrameric coiled coil. We investigated the importance of structural features found in this domain for polymerase function using a newly constructed NiV bicistronic minigenome assay. We identified a conserved basic patch and central kink in the coiled coil that are important for polymerase function, with R555 being absolutely essential. This basic patch and central kink are conserved in the related human pathogens measles and mumps viruses, suggesting that this mechanism may be conserved.


Assuntos
RNA Polimerases Dirigidas por DNA/química , Genoma Viral , Vírus Nipah/química , Fosfoproteínas/química , RNA Replicase/química , Proteínas Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Vírus do Sarampo/química , Vírus do Sarampo/enzimologia , Vírus do Sarampo/genética , Modelos Moleculares , Vírus da Caxumba/química , Vírus da Caxumba/enzimologia , Vírus da Caxumba/genética , Vírus Nipah/enzimologia , Vírus Nipah/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , RNA Replicase/genética , RNA Replicase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
Arch Virol ; 164(3): 787-798, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30666459

RESUMO

The subunits PA, PB1, and PB2 of influenza A virus RNA polymerase are essential for efficient viral RNA synthesis and virus replication because of their role in recruiting multiple nuclear proteins. ANP32A is an acidic leucine-rich nuclear phosphoprotein 32 (ANP32) family member and a crucial cellular protein that determines the species specificity of the influenza virus RNA polymerase activity. However, how ANP32A modulates polymerase activity remains largely unknown. In this study, we showed that viral RNA synthesis was increased in A549 cells overexpressing ANP32A and decreased after treatment with ANP32A RNAi. This decrease in RNA synthesis was reversed by rescued ANP32A expression. The results of docking modeling, co-immunoprecipitation, and yeast two-hybrid assays showed that PB2 was the only subunit of the three that interacted with ANP32A. The C-terminal portion of ANP32A and the middle domains (residues 307-534) of PB2 were required for PB2-ANP32A interaction. Glu189 and Glu196 in ANP32A and Gly450 and Gln447 in PB2 were essential for interaction between ANP32A and PB2. These residues were located in conserved regions of the ANP32A or PB2 protein sequences. These data suggest that ANP32A is recruited to the polymerase through direct interaction with PB2 via critical amino acid residue interactions and promotes viral RNA synthesis. Our findings might provide new insights into the molecular mechanisms underlying influenza virus RNA synthesis and replication in infected human cells.


Assuntos
Vírus da Influenza A Subtipo H1N1/enzimologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , RNA Replicase/metabolismo , RNA Viral/genética , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Simulação de Acoplamento Molecular , Ligação Proteica , RNA Replicase/química , RNA Replicase/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA , Proteínas Virais/química , Proteínas Virais/genética
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