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1.
PLoS Pathog ; 18(1): e1010210, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35085375

RESUMO

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.


Assuntos
Guanosina/metabolismo , Hepacivirus/metabolismo , Mutação INDEL/fisiologia , Nucleotídeos/metabolismo , Replicação Viral/fisiologia , Linhagem Celular Tumoral , Guanosina/farmacologia , Hepatite C/metabolismo , Humanos , RNA Viral/genética , Replicação Viral/efeitos dos fármacos
2.
Front Microbiol ; 12: 754664, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34745059

RESUMO

Hepatitis C virus (HCV) is a single-stranded RNA virus of positive polarity [ssRNA(+)] that replicates its genome through the activity of one of its proteins, called NS5B. This viral protein is responsible for copying the positive-polarity RNA genome into a negative-polarity RNA strand, which will be the template for new positive-polarity RNA genomes. The NS5B protein is phosphorylated by cellular kinases, including Akt. In this work, we have identified several amino acids of NS5B that are phosphorylated by Akt, with positions S27, T53, T267, and S282 giving the most robust results. Site-directed mutagenesis of these residues to mimic (Glu mutants) or prevent (Ala mutants) their phosphorylation resulted in a reduced NS5B in vitro RNA polymerase activity, except for the T267E mutant, the only non-conserved position of all those that are phosphorylated. In addition, in vitro transcribed RNAs derived from HCV complete infectious clones carrying mutations T53E/A and S282E/A were transfected in Huh-7.5 permissive cells, and supernatant viral titers were measured at 6 and 15 days post-transfection. No virus was rescued from the mutants except for T53A at 15 days post-transfection whose viral titer was statistically lower as compared to the wild type. Therefore, phosphorylation of NS5B by cellular kinases is a mechanism of viral polymerase inactivation. Whether this inactivation is a consequence of interaction with cellular kinases or a way to generate inactive NS5B that may have other functions are questions that need further experimental work.

3.
Viruses ; 13(5)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066055

RESUMO

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


Assuntos
Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Flavivirus/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Flavivirus/efeitos dos fármacos , Genoma Viral , Humanos , Mutação , Fases de Leitura Aberta , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Proteoma , Proteômica/métodos , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/genética , Vírus do Nilo Ocidental/fisiologia , Zika virus/fisiologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia
4.
Pathogens ; 10(2)2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672588

RESUMO

Usutu virus (USUV) is a flavivirus that mainly infects wild birds through the bite of Culex mosquitoes. Recent outbreaks have been associated with an increased number of cases in humans. Despite being a growing source of public health concerns, there is yet insufficient data on the virus or host cell targets for infection control. In this work we have investigated whether the cellular kinase Akt and USUV polymerase NS5 interact and co-localize in a cell. To this aim, we performed co-immunoprecipitation (Co-IP) assays, followed by confocal microscopy analyses. We further tested whether NS5 is a phosphorylation substrate of Akt in vitro. Finally, to examine its role in viral replication, we chemically silenced Akt with three inhibitors (MK-2206, honokiol and ipatasertib). We found that both proteins are localized (confocal) and pulled down (Co-IP) together when expressed in different cell lines, supporting the fact that they are interacting partners. This possibility was further sustained by data showing that NS5 is phosphorylated by Akt. Treatment of USUV-infected cells with Akt-specific inhibitors led to decreases in virus titers (>10-fold). Our results suggest an important role for Akt in virus replication and stimulate further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral target.

5.
FEMS Microbiol Lett ; 366(24)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31995181

RESUMO

Small World Initiative and Tiny Earth are popular citizen science programs that are implemented worldwide in response to the global antibiotic resistance crisis. When starting up the program in Albacete (Spain), we noted that rates of isolated antibiotic-producing bacteria are generally low. To make the activity more stimulating for participating students, we modified the protocol to obtain more positive results by focusing on isolation of actinomycetes, the main producers of most clinically used antibiotics. Adaptations involved redesigning culture media, incubation times and temperatures, and modification of the ESKAPE antibiosis experiment by employing an agar-transplantation step. Of 390 bacterial isolates tested, almost 6% tested positive in antibiosis experiments and DNA sequence analysis confirmed that all positives are actinomycetes, demonstrating that our protocol is efficient toward isolating antibiotic-producing actinomycetes from soil. Evaluation forms filled by participating students indicated that the program was received very positively and that our modifications contribute to make this educational program more stimulating and efficient.


Assuntos
Actinobacteria/isolamento & purificação , Actinobacteria/metabolismo , Antibacterianos/metabolismo , Resistência Microbiana a Medicamentos , Testes de Sensibilidade Microbiana , Microbiologia do Solo
6.
Nat Microbiol ; 1(7): 16045, 2016 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-27572964

RESUMO

Spontaneous mutations are the ultimate source of genetic variation and have a prominent role in evolution. RNA viruses such as hepatitis C virus (HCV) have extremely high mutation rates, but these rates have been inferred from a minute fraction of genome sites, limiting our view of how RNA viruses create diversity. Here, by applying high-fidelity ultradeep sequencing to a modified replicon system, we scored >15,000 spontaneous mutations, encompassing more than 90% of the HCV genome. This revealed >1,000-fold differences in mutability across genome sites, with extreme variations even between adjacent nucleotides. We identify base composition, the presence of high- and low-mutation clusters and transition/transversion biases as the main factors driving this heterogeneity. Furthermore, we find that mutability correlates with the ability of HCV to diversify in patients. These data provide a site-wise baseline for interrogating natural selection, genetic load and evolvability in HCV, as well as for evaluating drug resistance and immune evasion risks.


Assuntos
Genoma Viral , Hepacivirus/genética , Hepatite C/virologia , Taxa de Mutação , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Nucleotídeos , RNA Viral , Replicon , Replicação Viral/genética
7.
Antimicrob Agents Chemother ; 60(6): 3540-50, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27021315

RESUMO

Hepatitis C virus (HCV) interacts with cellular components and modulates their activities for its own benefit. These interactions have been postulated as a target for antiviral treatment, and some candidate molecules are currently in clinical trials. The multifunctional cellular kinase Akt/protein kinase B (PKB) must be activated to increase the efficacy of HCV entry but is rapidly inactivated as the viral replication cycle progresses. Viral components have been postulated to be responsible for Akt/PKB inactivation, but the underlying mechanism remained elusive. In this study, we show that HCV polymerase NS5B interacts with Akt/PKB. In the presence of transiently expressed NS5B or in replicon- or virus-infected cells, NS5B changes the cellular localization of Akt/PKB from the cytoplasm to the perinuclear region. Sequestration of Akt/PKB by NS5B could explain its exclusion from its participation in early Akt/PKB inactivation. The NS5B-Akt/PKB interaction represents a new regulatory step in the HCV infection cycle, opening possibilities for new therapeutic options.


Assuntos
Hepacivirus/enzimologia , Hepatite C/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular Tumoral , Hepatite C/virologia , Humanos , Fosforilação , Replicação Viral/fisiologia
8.
Antiviral Res ; 108: 14-24, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24815023

RESUMO

The hepatitis C virus (HCV) replicates its genome in replication complexes located in micro-vesicles derived from endoplasmic reticulum. The composition of these replication complexes indicates that proteins, both viral and cellular in origin, are at high concentrations. Under these conditions, protein-protein interactions must occur although their role in the replication pathways is unknown. HCV RNA-dependent RNA-polymerase (NS5B) initiates RNA synthesis in these vesicles by a de novo (DN) mechanism. After initiation, newly synthesized dsRNA could induce conformational changes that direct the transition from an initiating complex into a processive elongation complex. In this report, we analyze the role played by NS5B-NS5B intermolecular interactions controlling these conformational rearrangements. Based on NS5B protein-protein docking and molecular dynamics simulations, we constructed mutants of residues predicted to be involved in protein-protein interactions. Changes at these positions induced severe defects in both the activity of the enzyme and the replication of a subgenomic replicon. Thus, mutations at the interaction surface decreased both DN synthesis initiation and processive elongation activities. Based on this analysis, we define at an atomic level an NS5B homomeric interaction model that connects the T-helix in the thumb subdomain of one monomer, with the F-helix of the fingers subdomain in other monomer. Knowing the molecular determinants involved in viral replication could be helpful to delineate new and powerful antiviral strategies.


Assuntos
Antivirais/metabolismo , Hepacivirus/enzimologia , Hepacivirus/fisiologia , Multimerização Proteica , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Antivirais/isolamento & purificação , Desenho de Fármacos , Hepacivirus/química , Hepacivirus/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
9.
Antimicrob Agents Chemother ; 53(2): 728-34, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19064890

RESUMO

The NS3/4A protease from hepatitis C virus (HCV) plays a key role in viral replication. We report a system for monitoring the activity of this enzyme in single living mammalian cells. We constructed a fluorescence resonance energy transfer (FRET) probe that consists of an enhanced cyan fluorescent protein-citrine fusion, with a cleavage site for HCV NS3/4A protease embedded within the linker between them. Expression of the biosensor in mammalian cells resulted in a FRET signal, and cotransfection with the NS3/4A expression vector produced a significant reduction in FRET, indicating that the cleavage site was processed. Western blot and spectrofluorimetry analysis confirmed the physical cleavage of the fusion probe by the NS3/4A protease. As the level of FRET decay was a function of the protease activity, the system allowed testing of NS3/4A protease variants with different catalytic efficiencies. This FRET probe could be adapted for high-throughput screening of new HCV NS3/4 protease inhibitors.


Assuntos
Hepacivirus/enzimologia , Proteínas não Estruturais Virais/genética , Western Blotting , Catálise , Linhagem Celular , Clonagem Molecular , Primers do DNA , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Plasmídeos/genética , Espectrometria de Fluorescência
10.
Microsc Res Tech ; 70(12): 1010-21, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17722057

RESUMO

Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/química , Fluorescência , Células HeLa , Humanos
11.
J Virol ; 80(2): 571-7, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16378959

RESUMO

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5' end or the 3' end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5' and 3' ends are partially tolerated.


Assuntos
Inativação Gênica , HIV-1/fisiologia , RNA Interferente Pequeno/genética , Substituição de Aminoácidos , Infecções por HIV/terapia , Transcriptase Reversa do HIV/genética , HIV-1/genética , Mutação , Replicação Viral
12.
Curr Pharm Des ; 10(30): 3741-56, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15579068

RESUMO

The concept of using RNA molecules as therapeutic agents is receiving increasing attention by basic science and pharmaceutical research. Over the past five years, a number of clinical trials have been initiated to evaluate the efficacy and safety of several RNA agents for the treatment of a range of conditions from cancer to infectious disease. From a molecular biology perspective, two main factors are implicated in RNA therapeutics against pathogenic RNAs: i/ The activity, stability and delivery of the inactivating agent (ribozyme, RNase P, "decoy" RNA, aptamer, small interfering-RNA) and its co-localisation with the target; and ii/ The properties of the RNA substrate, which, in the case of an RNA virus, most likely limit the effectiveness of the inactivating agent. The main reasons are the limited size of the viral genome and the restrictions imposed by the RNA structure and variations at the target. In the first section of this article we review three properties of the HCV RNA genome, from primary sequence to tertiary structure, which imply restrictions and opportunities for RNA-based treatment. In the second section, we briefly describe several of the RNA-based therapeutic strategies against HCV now under development.


Assuntos
Genoma Viral , Hepacivirus/genética , Mimetismo Molecular , Interferência de RNA , RNA de Transferência/genética , RNA Viral/genética , Regiões 5' não Traduzidas/genética , Animais , Marcação de Genes , Hepacivirus/fisiologia , Hepatite C/terapia , Humanos , Conformação Proteica , RNA Catalítico/genética , RNA de Transferência/química , RNA Viral/química , Replicação Viral/genética
13.
FEBS Lett ; 577(3): 517-22, 2004 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-15556639

RESUMO

Previously, we described two RNA structural motifs in the hepatitis C viral (HCV) genome that can be processed in vitro by human ribonuclease P (RNase P) enzyme [J. Biol. Chem. 277 (2002) 30606]. One of these structures is located in the internal ribosome entry site and is conserved in the related animal pestiviruses [J. Biol. Chem. 278 (2003) 26844]. Here, we tested two prokaryotic RNase P ribozymes (P RNA) against this conserved structural motif. In vitro experiments indicated that P RNA from Synechocystis sp. can specifically process the viral transcript preparations in a position close to the human RNase P cleavage site. This provides additional support for the presence of an RNA structure similar to tRNA near the AUG start codon and suggests that Synechocystis P RNA may be an active agent for HCV antigenomic interventions.


Assuntos
Códon de Iniciação/química , Hepacivirus/química , Hepacivirus/metabolismo , RNA Viral/metabolismo , Ribonuclease P/metabolismo , Synechocystis/enzimologia , Autorradiografia , Sequência de Bases , Sítios de Ligação , Catálise , Escherichia coli/enzimologia , Variação Genética , Hepacivirus/genética , Mutação , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Catalítico/metabolismo , RNA Viral/química , RNA Viral/genética
14.
J Biol Chem ; 277(15): 13184-91, 2002 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-11821417

RESUMO

DNA polymerase lambda (pol lambda) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in pol lambda supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol lambda to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol lambda inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5'-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol lambda is a novel beta-like DNA polymerase. However, the high affinity of pol lambda for dNTPs (37-fold over pol beta) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol beta and pol lambda have nonredundant in vivo functions.


Assuntos
DNA Polimerase beta/metabolismo , Reparo do DNA , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Polimerase beta/química , DNA Polimerase beta/genética , Primers do DNA , DNA Complementar , Humanos , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
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