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1.
Talanta ; 236: 122868, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635250

RESUMO

Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: PEI-Ru@Ti3C2@AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials.


Assuntos
Sistemas CRISPR-Cas , RNA Polimerase Dependente de RNA/isolamento & purificação , SARS-CoV-2 , COVID-19 , DNA , Ouro , Humanos , Nanopartículas Metálicas , RNA Viral
2.
J Chem Inf Model ; 61(11): 5508-5523, 2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34730967

RESUMO

The lack of conformational sampling in virtual screening projects can lead to inefficient results because many of the potential drugs may not be able to bind to the target protein during the static docking simulations. Here, we performed ensemble docking for around 2000 United States Food and Drug Administration (FDA)-approved drugs with the RNA-dependent RNA polymerase (RdRp) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a target. The representative protein structures were generated by clustering classical molecular dynamics trajectories, which were evolved using three solvent scenarios, namely, pure water, benzene/water and phenol/water mixtures. The introduction of dynamic effects in the theoretical model showed improvement in docking results in terms of the number of strong binders and binding sites in the protein. Some of the discovered pockets were found only for the cosolvent simulations, where the nonpolar probes induced local conformational changes in the protein that lead to the opening of transient pockets. In addition, the selection of the ligands based on a combination of the binding free energy and binding free energy gap between the best two poses for each ligand provided more suitable binders than the selection of ligands based solely on one of the criteria. The application of cosolvent molecular dynamics to enhance the sampling of the configurational space is expected to improve the efficacy of virtual screening campaigns of future drug discovery projects.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , RNA Polimerase Dependente de RNA , Estados Unidos
3.
Enzymes ; 49: 149-193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696831

RESUMO

All RNA viruses encode an RNA-dependent RNA polymerase (RdRp) responsible for genome replication. It is now recognized that enzymes in general, and RdRps specifically, are dynamic macromolecular machines such that their moving parts, including active site loops, play direct functional roles. While X-ray crystallography has provided deep insight into structural elements important for RdRp function, this methodology generally provides only static snapshots, and so is limited in its ability to report on dynamic fluctuations away from the lowest energy conformation. Nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations and other biophysical techniques have brought new insight into RdRp function by their ability to characterize the trajectories, kinetics and thermodynamics of conformational motions. In particular, these methodologies have identified coordinated motions among conserved structural motifs necessary for nucleotide selection and incorporation. Disruption of these motions through amino acid substitutions or inhibitor binding impairs RdRp function. Understanding and re-engineering these motions thus provides exciting new avenues for anti-viral strategies. This chapter outlines the basics of these methodologies, summarizes the dynamic motions observed in different RdRps important for nucleotide selection and incorporation, and illustrates how this information can be leveraged towards rational vaccine strain development and anti-viral drug design.


Assuntos
Vírus de RNA , RNA Polimerase Dependente de RNA , Domínio Catalítico , Cristalografia por Raios X , Simulação de Dinâmica Molecular , RNA Polimerase Dependente de RNA/genética
4.
Enzymes ; 49: 215-233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696833

RESUMO

RNA-dependent RNA polymerases (RdRPs) encoded by RNA viruses represent a unique class of processive nucleic acid polymerases, carrying out DNA-independent replication/transcription processes. Although viral RdRPs have versatile global structures, they do share a structurally highly conserved active site comprising catalytic motifs A-G. In spite of different initiation modes, the nucleotide addition cycle (NAC) in the RdRP elongation phase probably follows consistent mechanisms. In this chapter, representative structures of picornavirus RdRP elongation complexes are used to illustrate RdRP NAC mechanisms. In the pre-chemistry part of the NAC, RdRPs utilize a unique palm domain-based active site closure that can be further decomposed into two sequential steps. In the post-chemistry part of the NAC, the translocation process is stringently controlled by the RdRP-specific motif G, resulting in asymmetric movements of the template-product RNA. Future efforts to elucidate regulation/intervention mechanisms by mismatched NTPs or nucleotide analog antivirals are necessary to achieve comprehensive understandings of viral RdRP NAC.


Assuntos
Picornaviridae , RNA Polimerase Dependente de RNA , Domínio Catalítico , Nucleotídeos , Picornaviridae/genética , RNA , RNA Polimerase Dependente de RNA/genética
5.
Enzymes ; 49: 265-303, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696835

RESUMO

Flaviviruses such as dengue, Japanese encephalitis, West Nile, Yellow Fever and Zika virus, cause viral hemorrhagic fever and encephalitis in humans. However, antiviral therapeutics to treat or prevent flavivirus infections are not yet available. Thus, there is pressing need to develop therapeutics and vaccines that target flavivirus infections. All flaviviruses carry a positive-sense single-stranded RNA genome, which encodes ten proteins; three structural proteins form the virus shell, and seven nonstructural (NS) proteins are involved in replication of the viral genome. While all NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are part of a functional membrane-bound replication complex, enzymatic activities required for flaviviral replication reside in only two NS proteins, NS3 and NS5. NS3 functions as a protease, helicase, and triphosphatase, and NS5 as a capping enzyme, methyltransferase, and RNA-dependent RNA polymerase. In this chapter, we provide an overview of viral replication focusing on the structure and function of NS3 and NS5 replicases. We further describe strategies and examples of current efforts to identify potential flavivirus inhibitors against NS3 and NS5 enzymatic activities that can be developed as therapeutic agents to combat flavivirus infections.


Assuntos
Inibidores Enzimáticos/farmacologia , Flavivirus , Proteínas não Estruturais Virais , Flavivirus/enzimologia , RNA Polimerase Dependente de RNA , Proteínas não Estruturais Virais/genética , Replicação Viral
6.
Enzymes ; 49: 1-37, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696829

RESUMO

The ongoing Covid-19 pandemic has spurred research in the biology of the nidovirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Much focus has been on the viral RNA synthesis machinery due to its fundamental role in viral propagation. The central and essential enzyme of the RNA synthesis process, the RNA-dependent RNA polymerase (RdRp), functions in conjunction with a coterie of viral-encoded enzymes that mediate crucial nucleic acid transactions. Some of these enzymes share common features with other RNA viruses, while others play roles unique to nidoviruses or CoVs. The RdRps are proven targets for viral pathogens, and many of the other nucleic acid processing enzymes are promising targets. The purpose of this review is to summarize recent advances in our understanding of the mechanisms of RNA synthesis in CoVs. By reflecting on these studies, we hope to emphasize the remaining gaps in our knowledge. The recent onslaught of structural information related to SARS-CoV-2 RNA synthesis, in combination with previous structural, genetic and biochemical studies, have vastly improved our understanding of how CoVs replicate and process their genomic RNA. Structural biology not only provides a blueprint for understanding the function of the enzymes and cofactors in molecular detail, but also provides a basis for drug design and optimization. The concerted efforts of researchers around the world, in combination with the renewed urgency toward understanding this deadly family of viruses, may eventually yield new and improved antivirals that provide relief to the current global devastation.


Assuntos
RNA Viral , SARS-CoV-2/genética , RNA Viral/biossíntese , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética
7.
Enzymes ; 49: 63-82, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696839

RESUMO

The therapeutic targeting of the nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) of the Hepatitis C Virus (HCV) with nucleotide analogs led to a deep understanding of this enzymes structure, function and substrate specificity. Unlike previously studied DNA polymerases including the reverse transcriptase of Human Immunodeficiency Virus, development of biochemical assays for HCV RdRp proved challenging due to low solubility of the full-length protein and inefficient acceptance of exogenous primer/templates. Despite the poor apparent specific activity, HCV RdRp was found to support rapid and processive transcription once elongation is initiated in vitro consistent with its high level of viral replication in the livers of patients. Understanding of the substrate specificity of HCV RdRp led to the discovery of the active triphosphate of sofosbuvir as a nonobligate chain-terminator of viral RNA transcripts. The ternary crystal structure of HCV RdRp, primer/template, and incoming nucleotide showed the interaction between the nucleotide analog and the 2'-hydroxyl binding pocket and how an unfit mutation of serine 282 to threonine results in resistance by interacting with the uracil base and modified 2'-position of the analog. Host polymerases mediate off-target toxicity of nucleotide analogs and the active metabolite of sofosbuvir was found to not be efficiently incorporated by host polymerases including the mitochondrial RNA polymerase (POLRMT). Knowledge from studying inhibitors of HCV RdRp serves to advance antiviral drug discovery for other emerging RNA viruses including the discovery of remdesivir as an inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), the virus that causes COVID-19.


Assuntos
Hepacivirus , Sofosbuvir/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , RNA Viral , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2
8.
Enzymes ; 49: 315-354, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696837

RESUMO

The treatment of viral infections remains challenging, in particular in the face of emerging pathogens. Broad-spectrum antiviral drugs could potentially be used as a first line of defense. The RNA-dependent RNA polymerase (RdRp) of RNA viruses serves as a logical target for drug discovery and development efforts. Herein we discuss compounds that target RdRp of poliovirus, hepatitis C virus, influenza viruses, respiratory syncytial virus, and the growing data on coronaviruses. We focus on nucleotide analogs and mechanisms of action and resistance.


Assuntos
Antivirais/farmacologia , Nucleotídeos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas do Complexo da Replicase Viral/antagonistas & inibidores , Vírus/enzimologia , Replicação Viral
9.
Int J Mol Sci ; 22(20)2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34681689

RESUMO

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has infected >235 million people and killed over 4.8 million individuals worldwide. Although vaccines have been developed for prophylactic management, there are no clinically proven antivirals to treat the viral infection. Continuous efforts are being made all over the world to develop effective drugs but these are being delayed by periodic outbreak of mutated SARS-CoV-2 and a lack of knowledge of molecular mechanisms underlying viral pathogenesis and post-infection complications. In this regard, the involvement of Annexin A2 (AnxA2), a lipid-raft related phospholipid-binding protein, in SARS-CoV-2 attachment, internalization, and replication has been discussed. In addition to the evidence from published literature, we have performed in silico docking of viral spike glycoprotein and RNA-dependent RNA polymerase with human AnxA2 to find the molecular interactions. Overall, this review provides the molecular insights into a potential role of AnxA2 in the SARS-CoV-2 pathogenesis and post-infection complications, especially thrombosis, cytokine storm, and insulin resistance.


Assuntos
Anexina A2/metabolismo , COVID-19/patologia , Anexina A2/química , COVID-19/virologia , Síndrome da Liberação de Citocina/metabolismo , Síndrome da Liberação de Citocina/patologia , Humanos , Simulação de Acoplamento Molecular , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Trombose/metabolismo , Trombose/patologia , Internalização do Vírus
10.
Int J Mol Sci ; 22(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34681802

RESUMO

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new pathogen from the family of Coronaviridae that caused a global pandemic of COVID-19 disease. In the absence of effective antiviral drugs, research of novel therapeutic targets such as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) becomes essential. This viral protein is without a human counterpart and thus represents a unique prospective drug target. However, in vitro biological evaluation testing on RdRp remains difficult and is not widely available. Therefore, we prepared a database of commercial small-molecule compounds and performed an in silico high-throughput virtual screening on the active site of the SARS-CoV-2 RdRp using ensemble docking. We identified a novel thioether-amide or guanidine-linker class of potential RdRp inhibitors and calculated favorable binding free energies of representative hits by molecular dynamics simulations coupled with Linear Interaction Energy calculations. This innovative procedure maximized the respective phase-space sampling and yielded non-covalent inhibitors representing small optimizable molecules that are synthetically readily accessible, commercially available as well as suitable for further biological evaluation and mode of action studies.


Assuntos
Antivirais/química , Inibidores Enzimáticos/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/enzimologia , Proteínas Virais/antagonistas & inibidores , Amidas/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/virologia , Domínio Catalítico , Bases de Dados de Compostos Químicos , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Guanidina/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/isolamento & purificação , Relação Estrutura-Atividade , Sulfetos/química , Termodinâmica , Proteínas Virais/metabolismo
11.
Cell Rep ; 37(4): 109882, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34653416

RESUMO

Remdesivir (RDV), a nucleotide analog with broad-spectrum features, has exhibited effectiveness in COVID-19 treatment. However, the precise working mechanism of RDV when targeting the viral RNA-dependent RNA polymerase (RdRP) has not been fully elucidated. Here, we solve a 3.0-Å structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRP elongation complex (EC) and assess RDV intervention in polymerase elongation phase. Although RDV could induce an "i+3" delayed termination in meta-stable complexes, only pausing and subsequent elongation are observed in the EC. A comparative investigation using an enterovirus RdRP further confirms similar delayed intervention and demonstrates that steric hindrance of the RDV-characteristic 1'-cyano at the -4 position is responsible for the "i+3" intervention, although two representative Flaviviridae RdRPs do not exhibit similar behavior. A comparison of representative viral RdRP catalytic complex structures indicates that the product RNA backbone encounters highly conserved structural elements, highlighting the broad-spectrum intervention potential of 1'-modified nucleotide analogs in anti-RNA virus drug development.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , RNA Polimerase Dependente de RNA/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Proteínas Virais/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , COVID-19/tratamento farmacológico , Microscopia Crioeletrônica , Humanos , RNA Viral/química , RNA Viral/efeitos dos fármacos , RNA Polimerase Dependente de RNA/química , SARS-CoV-2/química , Proteínas Virais/química , Replicação Viral/efeitos dos fármacos
12.
J Gen Virol ; 102(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34661516

RESUMO

The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml-1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50 %:50 % mixture inoculum evolved to mean WT/I38T ratios of 71 %:29 % and 66.4 %:33.6 % on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.


Assuntos
Vírus da Influenza A Subtipo H3N2/fisiologia , Infecções por Orthomyxoviridae/virologia , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Células A549 , Substituição de Aminoácidos , Animais , Antivirais/farmacologia , Dibenzotiepinas/farmacologia , Cães , Farmacorresistência Viral , Cobaias , Humanos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/patogenicidade , Células Madin Darby de Rim Canino , Morfolinas/farmacologia , Nariz/virologia , Infecções por Orthomyxoviridae/transmissão , Piridonas/farmacologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Genética Reversa , Triazinas/farmacologia , Carga Viral , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral
13.
Front Cell Infect Microbiol ; 11: 746926, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34604118

RESUMO

Due to lacking a proofreading mechanism in their RNA-dependent RNA polymerases (RdRp), RNA viruses generally possess high mutation frequencies, making them evolve rapidly to form viral quasispecies during serial passages in cells, especially treated with mutagens, like ribavirin. Canine distemper virus (CDV) belongs to the genus Morbillivirus. Its L protein functions as an RdRp during viral replication. In this study, a recombinant enhanced green fluorescence protein-tagged CDV (rCDV-eGFP) was rescued from its cDNA clone, followed by viral identification and characterization at passage-7 (P7). This recombinant was independently subjected to extra 40 serial passages (P8 to 47) in ribavirin- and non-treated cells. Two viral progenies, undergoing passages in ribavirin- and non-treated VDS cells, were named rCDV-eGFP-R and -N, respectively. Both progenies were simultaneously subjected to next-generation sequencing (NGS) at P47 for comparing their quasispecies diversities with each other. The rCDV-eGFP-R and -N showed 62 and 23 single-nucleotide mutations (SNMs) in individual antigenomes, respectively, suggesting that the ribavirin conferred a mutagenic effect on the rCDV-eGFP-R. The spectrum of 62 SNMs contained 26 missense and 36 silent mutations, and that of 23 SNMs was composed of 17 missense and 6 silent mutations. Neither the rCDV-eGFP-R nor -N exhibited nonsense mutation in individual antigenomes. We speculate that the rCDV-eGFP-R may contain at least one P47 sub-progeny characterized by high-fidelity replication in cells. If such a sub-progeny can be purified from the mutant swarm, its L protein would elucidate a molecular mechanism of CDV high-fidelity replication.


Assuntos
Vírus da Cinomose Canina , Animais , Vírus da Cinomose Canina/genética , Mutação , RNA Polimerase Dependente de RNA , Ribavirina/farmacologia , Inoculações Seriadas
14.
Virology ; 564: 33-38, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34619630

RESUMO

Endemic seasonal coronaviruses cause morbidity and mortality in a subset of patients, but no specific treatment is available. Molnupiravir is a promising pipeline antiviral drug for treating SARS-CoV-2 infection potentially by targeting RNA-dependent RNA polymerase (RdRp). This study aims to evaluate the potential of repurposing molnupiravir for treating seasonal human coronavirus (HCoV) infections. Molecular docking revealed that the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC), has similar binding affinity to RdRp of SARS-CoV-2 and seasonal HCoV-NL63, HCoV-OC43 and HCoV-229E. In cell culture models, treatment of molnupiravir effectively inhibited viral replication and production of infectious viruses of the three seasonal coronaviruses. A time-of-drug-addition experiment indicates the specificity of molnupiravir in inhibiting viral components. Furthermore, combining molnupiravir with the protease inhibitor GC376 resulted in enhanced antiviral activity. Our findings highlight that the great potential of repurposing molnupiravir for treating seasonal coronavirus infected patients.


Assuntos
Coronavirus Humano 229E/genética , Infecções por Coronavirus/tratamento farmacológico , Coronavirus Humano NL63/genética , Coronavirus Humano OC43/genética , Citidina/análogos & derivados , Hidroxilaminas/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Resfriado Comum/tratamento farmacológico , Coronavirus Humano 229E/efeitos dos fármacos , Coronavirus Humano 229E/fisiologia , Coronavirus Humano NL63/efeitos dos fármacos , Coronavirus Humano NL63/fisiologia , Coronavirus Humano OC43/efeitos dos fármacos , Coronavirus Humano OC43/fisiologia , Citidina/farmacologia , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Pirrolidinas/farmacologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Estações do Ano , Ácidos Sulfônicos/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
15.
Phys Chem Chem Phys ; 23(38): 21399-21406, 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34550142

RESUMO

In recent years, it has been experimentally established that transcription, a fundamental biological process that involves the synthesis of messenger RNA molecules from DNA templates, does not proceed continuously as was expected. Rather, it exhibits a distinct dynamic behavior of alternating between productive phases when RNA molecules are actively synthesized and inactive phases when there is no RNA production at all. The bimodal transcriptional dynamics is now confirmed to be present in most living systems. This phenomenon is known as transcriptional bursting and it attracts significant amounts of attention from researchers in different fields. However, despite multiple experimental and theoretical investigations, the microscopic origin and biological functions of the transcriptional bursting remain unclear. Here we discuss the recent developments in uncovering the underlying molecular mechanisms of transcriptional bursting and our current understanding of them. Our analysis presents a physicochemical view of the processes that govern transcriptional bursting in living cells.


Assuntos
RNA Polimerase Dependente de RNA/genética , RNA/genética , RNA/química , RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Ativação Transcricional/genética
16.
Nat Commun ; 12(1): 5233, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475387

RESUMO

Measles virus (MeV) is a highly contagious pathogen that enters the human host via the respiratory route. Besides acute pathologies including fever, cough and the characteristic measles rash, the infection of lymphocytes leads to substantial immunosuppression that can exacerbate the outcome of infections with additional pathogens. Despite the availability of effective vaccine prophylaxis, measles outbreaks continue to occur worldwide. We demonstrate that prophylactic and post-exposure therapeutic treatment with an orally bioavailable small-molecule polymerase inhibitor, ERDRP-0519, prevents measles disease in squirrel monkeys (Saimiri sciureus). Treatment initiation at the onset of clinical signs reduced virus shedding, which may support outbreak control. Results show that this clinical candidate has the potential to alleviate clinical measles and augment measles virus eradication.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Sarampo/prevenção & controle , Morfolinas/uso terapêutico , Piperidinas/uso terapêutico , Pirazóis/uso terapêutico , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacocinética , Tolerância Imunológica/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Vírus do Sarampo/efeitos dos fármacos , Morfolinas/farmacocinética , Piperidinas/farmacocinética , Pirazóis/farmacocinética , Saimiri , Replicação Viral/efeitos dos fármacos , Eliminação de Partículas Virais/efeitos dos fármacos
17.
Sci Rep ; 11(1): 17878, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504255

RESUMO

As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/economia , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Viroporinas/genética
18.
Arch Virol ; 166(11): 3211-3216, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34495411

RESUMO

Fusarium oxysporum is a cosmopolitan plant pathogen that causes fusarium wilt and fusarium root rot in many economically important crops. There is still limited information about mycoviruses that infect F. oxysporum. Here, a novel mitovirus tentatively named "Fusarium oxysporum mitovirus 1" (FoMV1) was identified in F. oxysporum strain B2-10. The genome of FoMV1 is 2,453 nt in length with a predicted AU content of 71.6% and contains one large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF putatively encodes an RNA-dependent RNA polymerase (RdRp) of 723 aa with a molecular mass of 84.98 kDa. The RdRp domain of FoMV1 shares 29.01% to 68.43% sequence identity with the members of the family Mitoviridae. Phylogenetic analysis further suggested that FoMV1 is a new member of a distinct species in the genus Mitovirus.


Assuntos
Micovírus/genética , Fusarium/virologia , Genoma Viral , Filogenia , Vírus de RNA/genética , Micovírus/isolamento & purificação , Fusarium/patogenicidade , Fases de Leitura Aberta , Doenças das Plantas/microbiologia , Vírus de RNA/isolamento & purificação , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Sequenciamento Completo do Genoma
19.
Arch Virol ; 166(11): 3233-3237, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34535823

RESUMO

The entomopathogenic fungus Beauveria bassiana is used worldwide for biological control of insects. Seven dsRNA segments were detected in a single B. bassiana strain, RCEF1446. High-throughput sequencing indicated the presence of three mycoviruses in RCEF1446. Two were identified as the known mycoviruses Beauveria bassiana victorivirus 1 and Beauveria bassiana polymycovirus 1, and the novel mycovirus was designated as "Beauveria bassiana bipartite mycovirus 1" (BbBV1). The complete sequence of the BbBV1 is described here. The mycovirus contains two dsRNA segments. The RNA 1 (dsRNA 4) of BbBV1 is 2,026 bp in length, encoding a RNA-dependent RNA polymerase (RdRp) (68.54 kDa), while the RNA 2 (dsRNA 6) is 1,810 bp in length, encoding a hypothetical protein (35.55 kDa) with unknown function. Moreover, the amino acid sequence of RdRp showed the highest sequence identity of 62.31% to Botryosphaeria dothidea bipartite mycovirus 1. Phylogenetic analysis based on RdRp sequences revealed that BbBV1 represents a distinct lineage of unassigned dsRNA mycoviruses infecting fungi.


Assuntos
Beauveria/virologia , Vírus de RNA de Cadeia Dupla/genética , Micovírus/genética , Genoma Viral , Filogenia , Beauveria/patogenicidade , RNA de Cadeia Dupla , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética
20.
J Gen Virol ; 102(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34524075

RESUMO

Viruses require host factors to support their replication, and genetic variation in such factors can affect susceptibility to infectious disease. Influenza virus replication in human cells relies on ANP32 proteins, which are involved in assembly of replication-competent dimeric influenza virus polymerase (FluPol) complexes. Here, we investigate naturally occurring single nucleotide variants (SNV) in the human Anp32A and Anp32B genes. We note that variant rs182096718 in Anp32B is found at a higher frequency than other variants in either gene. This SNV results in a D130A substitution in ANP32B, which is less able to support FluPol activity than wild-type ANP32B and binds FluPol with lower affinity. Interestingly, ANP32B-D130A exerts a dominant negative effect over wild-type ANP32B and interferes with the functionally redundant paralogue ANP32A. FluPol activity and virus replication are attenuated in CRISPR-edited cells expressing wild-type ANP32A and mutant ANP32B-D130A. We propose a model in which the D130A mutation impairs FluPol dimer formation, thus resulting in compromised replication. We suggest that both homozygous and heterozygous carriers of rs182096718 may have some genetic protection against influenza viruses.


Assuntos
Vírus da Influenza A Subtipo H3N2/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Polimerase Dependente de RNA/metabolismo , Linhagem Celular , Humanos , Vírus da Influenza A Subtipo H3N2/enzimologia , Modelos Moleculares , Proteínas Nucleares/química , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , Replicação Viral
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