RESUMEN
Three main approaches are used to combat severe viral respiratory infections. The first is preemptive vaccination that blocks infection. Weakened or dead viral particles, as well as genetic constructs carrying viral proteins or information about them, are used as an antigen. However, the viral genome is very evolutionary labile and changes continuously. Second, chemical agents are used during infection and inhibit the function of a number of viral proteins. However, these drugs lose their effectiveness because the virus can rapidly acquire resistance to them. The third is the search for points in the host metabolism the effect on which would suppress the replication of the virus but would not have a significant effect on the metabolism of the host. Here, we consider the possibility of using the copper metabolic system as a target to reduce the severity of influenza infection. This is facilitated by the fact that, in mammals, copper status can be rapidly reduced by silver nanoparticles and restored after their cancellation.
Asunto(s)
Cobre/metabolismo , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Ceruloplasmina/fisiología , Proteínas Transportadoras de Cobre/metabolismo , ATPasas Transportadoras de Cobre/fisiología , Farmacorresistencia Viral , Interacciones Huésped-Patógeno , Humanos , Vacunas contra la Influenza , Gripe Humana/tratamiento farmacológico , Gripe Humana/prevención & control , Gripe Humana/virología , Mamíferos/metabolismo , Nanopartículas del Metal/uso terapéutico , Chaperonas Moleculares/metabolismo , Proteínas PrPC/fisiología , ARN Viral/fisiología , Plata/uso terapéutico , Superóxido Dismutasa-1/fisiología , Proteínas Virales/fisiología , Replicación ViralRESUMEN
Hepatitis C is a contagious liver disease caused by hepacivirus of the Flaviviridae family. It has a RNA genome, a unique highly variable molecule. It encodes ten proteins which are necessary to infect cells and multiply. Replication occurs only in hepatocytes. Because of its wide genomic variability and the absence of symptoms, it is difficult to make an early diagnosis and successful treatment. In this review we analyze the molecular mechanism by which the virus infects the hepatocytes and causes the disease. We focused the analysis on different therapies, with the possibility of improving treatment with the use of new specific vaccines. We highlight the use of new therapies based on nucleic acids, mainly DNA vectors. In the near future, once this treatment is adequately evaluated in clinical trials, and the costs are calculated, it could be a very beneficial alternative to conventional methods.
Asunto(s)
Hepacivirus/efectos de los fármacos , Vacunas contra Hepatitis Viral/uso terapéutico , Animales , Progresión de la Enfermedad , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Genoma Viral , Genotipo , Hepacivirus/genética , Hepacivirus/inmunología , Hepacivirus/fisiología , Hepatitis C/tratamiento farmacológico , Hepatitis C/epidemiología , Hepatitis C/virología , Anticuerpos contra la Hepatitis C/inmunología , Hepatocitos/virología , Humanos , Inmunogenicidad Vacunal , Ratones , ARN Viral/genética , Vacunas de ADN , Vacunas de Subunidad , Proteínas Virales/fisiologíaRESUMEN
We purified ORI2 [3-(3,4-dihydroxyphenyl)acrylic acid 1-(3,4-dihydroxyphenyl)-2-methoxycarbonylethyl ester] from an extract of the plant Isodon excisus. We tested the antiviral effect of ORI2 in a coxsackievirus-induced myocarditis model. Coxsackievirus B3 (CVB3) is a common cause of myocarditis and dilated cardiomyopathy. Activation of extracellular signal-regulated kinase (ERK) and Akt signaling in virus-infected cells is essential for CVB3 replication. Antiviral compounds were screened by HeLa cell survival assay. Several purified natural compounds were added to HeLa cells cultured in 96-well plates for 30 min after 1 multiplicity of infection (m.o.i) CVB3 infection. ORI2 significantly improved HeLa cell survival in a dose-dependent manner. For in vivo studies, BALB/c mice (n=20) were infected with CVB3, then 10 of the mice were treated by daily intraperitoneal injections of ORI2 (100 mM) for 3 consecutive days. ORI2 treatment significantly improved early survival in the treated mice compared to untreated mice (85% vs. 50%, respectively). Organ virus titers and myocardial damage were significantly lower in the ORI2-treated mice than in untreated mice. These results demonstrate that ORI2, delivered by intraperitoneal injection after CVB3 infection, has a significant antiviral effect by markedly inhibiting virus replication, resulting in a decrease in organ virus titer and myocardial damage. ORI2 may be developed as a potential therapeutic agent for the treatment of CVB3 infections.
Asunto(s)
Infecciones por Coxsackievirus/tratamiento farmacológico , Miocarditis/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Proteínas Virales/fisiología , Replicación Viral/efectos de los fármacos , Animales , Infecciones por Coxsackievirus/patología , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Miocarditis/patología , Miocarditis/virología , Extractos Vegetales/farmacología , Replicación Viral/fisiologíaRESUMEN
The Triple Gene Block 1 (TGBp1) protein encoded by the Potato virus X is a multifunctional protein that acts as a suppressor of RNA silencing or facilitates the passage of virus from cell to cell by promoting the plasmodesmata opening. We previously showed that the membrane raft protein StRemorin1.3 is able to impair PVX infection. Here, we show that overexpressed StRemorin1.3 does not impair the silencing suppressor activity of TGBp1, but affects its ability to increase plasmodesmata permeability. A similar effect on plasmodesmata permeability was observed with other movement proteins, suggesting that REM is a general regulator of plasmodesmal size exclusion limit. These results add to our knowledge of the mechanisms underlying the StREM1.3 role in virus infection.
Asunto(s)
Proteínas Portadoras/fisiología , Fosfoproteínas/fisiología , Proteínas de Plantas/fisiología , Plasmodesmos/metabolismo , Potexvirus/fisiología , Solanum tuberosum/virología , Proteínas Virales/fisiología , Agrobacterium/genética , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno , Microscopía Fluorescente , Permeabilidad , Plasmodesmos/virología , Isoformas de Proteínas/fisiología , Transporte de Proteínas , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Solanum tuberosum/metabolismo , Nicotiana/metabolismoRESUMEN
INTRODUCTION: Ebolaviruses and marburgviruses cause severe and often lethal human hemorrhagic fevers. As no FDA-approved therapeutics are available for these infections, efforts to discover new therapeutics are important, especially because these pathogens are considered biothreats and emerging infectious diseases. All methods for discovering new therapeutics should be considered, including compound library screening in vitro against virus and in silico structure-based drug design, where possible, if sufficient biochemical and structural information is available. AREAS COVERED: This review covers the structure and function of filovirus proteins, as they have been reported to date, as well as some of the current antiviral screening approaches. The authors discuss key studies mapping small-molecule modulators that were found through library and in silico screens to potential sites on viral proteins or host proteins involved in virus trafficking and pathogenesis. A description of ebolavirus and marburgvirus diseases and available animal models is also presented. EXPERT OPINION: To discover novel therapeutics with potent efficacy using sophisticated computational methods, more high-resolution crystal structures of filovirus proteins and more details about the protein functions and host interaction will be required. Current compound screening efforts are finding active antiviral compounds, but an emphasis on discovery research to investigate protein structures and functions enabling in silico drug design would provide another avenue for finding antiviral molecules. Additionally, targeting of protein-protein interactions may be a future avenue for drug discovery since disrupting catalytic sites may not be possible for all proteins.
Asunto(s)
Infecciones por Filoviridae/tratamiento farmacológico , Proteínas Virales/fisiología , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Simulación por Computador , Diseño de Fármacos , Filoviridae , Infecciones por Filoviridae/fisiopatología , Humanos , Proteínas Virales/químicaRESUMEN
Post-transcriptional gene silencing (PTGS), or RNA silencing, is one of the key mechanisms of antiviral defence used by plants. To counter this defence response, viruses produce suppressor proteins that are able to inhibit the PTGS pathway or to interfere with some of its function. The aim of this study was to evaluate the RNA silencing suppressor (RSS) activity of P0 proteins from selected European isolates of the beet-infecting poleroviruses beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV) using two different experimental systems: (i) agro-infiltration of Nicotiana benthamiana green fluorescent protein-positive plants and (ii) mechanical inoculation of Chenopodium quinoa using a beet necrotic yellow vein virus (BNYVV, genus Benyvirus) RNA3-based replicon. The results demonstrated that P0 of most BMYV isolates exhibited RSS activity, although at various efficiencies among isolates. Conversely, P0 of BChV isolates displayed no RSS activity in either of the two systems under the experimental conditions used. These results are the first reported evidence that P0 proteins of two closely related beet poleroviruses show strain-specific differences in their effects on RNA silencing.
Asunto(s)
Beta vulgaris/virología , Luteoviridae/fisiología , Interferencia de ARN , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Proteínas Fluorescentes Verdes , Luteoviridae/genética , Datos de Secuencia Molecular , Nicotiana/genéticaRESUMEN
Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are important human pathogens that cause a variety of diseases from mild skin diseases such as herpes labialis and herpes genitalis to life-threatening diseases such as herpes encephalitis and neonatal herpes. A number of studies have elucidated the roles of this virus in viral replication and pathogenicity, the regulation of gene expression, interaction with the host cell and immune evasion from the host system. This research has allowed the development of potential therapeutic agents and vectors for human diseases. This review focuses on the basic functions and roles of HSV gene products and reviews the current knowledge of medical applications of genetically engineered HSV mutants using different strategies. These major HSV-derived vectors include: (i) amplicons for gene delivery vectors; (ii) replication-defective HSV recombinants for vaccine vectors; (iii) replication-attenuated HSV recombinants for oncolytic virotherapy.
Asunto(s)
Terapia Biológica , Simplexvirus , Animales , Técnicas de Transferencia de Gen , Ingeniería Genética , Vectores Genéticos , Humanos , Mutación , Viroterapia Oncolítica , Simplexvirus/genética , Simplexvirus/fisiología , Vacunas , Proteínas Virales/fisiología , Replicación ViralRESUMEN
Beet necrotic yellow vein virus (BNYVV) induces the most important disease threatening sugar beet. The growth of partially resistant hybrids carrying monogenic dominant resistance genes stabilize yield but are unable to entirely prevent virus infection and replication. P25 is responsible for symptom development and previous studies have shown that recently occurring resistance-breaking isolates possess increased P25 variability. To better understand the viral pathogenicity factor's interplay with plant proteins and to possibly unravel the molecular basis of sugar beet antivirus resistance, P25 was applied in a yeast two-hybrid screen of a resistant sugar beet cDNA library. This screen identified candidate proteins recognized as orthologues from other plant species which are known to be expressed following pathogen infection and involved in plant defense response. Most of the candidates potentially related to host-pathogen interactions were involved in the ubiquitylation process and plants response to stress, and were part of cell and metabolism components. The interaction of several candidate genes with P25 was confirmed in Nicotiana benthamiana leaf cells by transient agrobacterium-mediated expression applying bimolecular fluorescence complementation assay. The putative functions of several of the candidates identified support previous findings and present first targets for understanding the BNYVV pathogenicity and antivirus resistance mechanism.
Asunto(s)
Beta vulgaris/virología , Proteínas de Plantas/metabolismo , Virus de Plantas/metabolismo , Proteínas Virales/fisiología , Factores de Virulencia/fisiología , Biblioteca de Genes , Inmunidad Innata/genética , Microscopía Fluorescente , Enfermedades de las Plantas/virología , Proteínas de Plantas/genética , Virus de Plantas/patogenicidad , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismoAsunto(s)
Aminobutiratos/farmacología , Antivirales/farmacología , Orthomyxoviridae/efectos de los fármacos , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Modelos Biológicos , Modelos Moleculares , Orthomyxoviridae/fisiología , Fenilbutiratos , Conformación Proteica , Estructura Terciaria de Proteína , Caperuzas de ARN/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/fisiología , Proteínas Virales/química , Proteínas Virales/fisiologíaAsunto(s)
Apoptosis , Fragmentación del ADN , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Cromatina/metabolismo , Efecto Citopatogénico Viral , ADN Complementario , Proteínas Fluorescentes Verdes , Humanos , Etiquetado Corte-Fin in Situ , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/crecimiento & desarrollo , Síndrome Respiratorio Agudo Grave/metabolismo , Coloración y Etiquetado , Células Vero , Proteínas del Envoltorio Viral , Proteínas Virales/fisiología , Proteínas ViroporinasRESUMEN
The RNA3-encoded p25 protein of beet necrotic yellow vein virus (BNYVV) is responsible for the production of rhizomania symptoms of sugar beet roots (Beta vulgaris subsp. vulgaris). Here, it was found that the presence of the p25 protein is also associated with the resistance response in rub-inoculated leaves of sugar beet and wild beet (Beta vulgaris subsp. maritima) plants. The resistance phenotype displayed a range of symptoms from no visible lesions to necrotic or greyish lesions at the inoculation site, and only very low levels of virus and viral RNA accumulated. The susceptible phenotype showed large, bright yellow lesions and developed high levels of virus accumulation. In roots after Polymyxa betae vector inoculation, however, no drastic differences in virus and viral RNA accumulation levels were found between plants with susceptible and resistant phenotypes, except at an early stage of infection. There was a genotype-specific interaction between BNYVV strains and two selected wild beet lines (MR1 and MR2) and sugar beet cultivars. Sequence analysis of natural BNYVV isolates and site-directed mutagenesis of the p25 protein revealed that 3 aa residues at positions 68, 70 and 179 are important in determining the resistance phenotype, and that host-genotype specificity is controlled by single amino acid changes at position 68. The mechanism of the occurrence of resistance-breaking BNYVV strains is discussed.
Asunto(s)
Beta vulgaris/inmunología , Enfermedades de las Plantas/inmunología , Hojas de la Planta/inmunología , Virus ARN/fisiología , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Beta vulgaris/virología , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Enfermedades de las Plantas/virología , Hojas de la Planta/virología , ARN Viral/genética , Análisis de Secuencia de ADN , Proteínas Virales/genéticaRESUMEN
Being highly pathogenic for human and nonhuman primates and the subject of former weapon programmes makes Ebola virus one of the most feared pathogens worldwide today. Due to a lack of licensed pre- and postexposure intervention, the current response depends on rapid diagnostics, proper isolation procedures and supportive care of case patients. Consequently, the development of more specific countermeasures is of high priority for the preparedness of many nations. Over the past years, enhanced research efforts directed to better understand virus replication and pathogenesis have identified potential new targets for intervention strategies. The authors discuss the most promising therapeutic approaches for Ebola haemorrhagic fever as judged by their efficacy in animal models. The current development in this field encourages discussions on how to move some of the experimental approaches towards clinical application.
Asunto(s)
Antivirales/uso terapéutico , Productos Biológicos/uso terapéutico , Fiebre Hemorrágica Ebola/terapia , Animales , Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Vacunas contra el Virus del Ébola , Ebolavirus/clasificación , Ebolavirus/efectos de los fármacos , Ebolavirus/patogenicidad , Ebolavirus/fisiología , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/fisiopatología , Fiebre Hemorrágica Ebola/virología , Humanos , Sueros Inmunes , Inmunización Pasiva , Primates , Roedores , Proteínas Virales/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
beta protein from bacteriophage lambda promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. beta protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of beta protein in the presence and absence of DNA using limited proteolysis. Residues 1-130 form an N-terminal "core" domain that is resistant to proteases in the absence of DNA, residues 131-177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178-261 of beta protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of beta protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of beta protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of beta protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1-177 of beta protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA.
Asunto(s)
Bacteriófago lambda/metabolismo , Proteínas de Unión al ADN/química , ADN/química , Proteínas Virales/química , Alanina/química , Secuencia de Aminoácidos , Biotinilación , Proteínas de Unión al ADN/fisiología , Relación Dosis-Respuesta a Droga , Lisina/química , Espectrometría de Masas , Datos de Secuencia Molecular , Oligonucleótidos/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Virales/fisiologíaRESUMEN
Monopartite geminiviruses of the genus Begomovirus have two virion-sense genes, V1 and V2. V2 encodes the viral coat protein, but the function of V1 is largely unknown, although some studies suggest that it may play a role in cell-to-cell movement. Yeast two-hybrid technology was used to identify possible host binding partners of V1 from Tomato leaf curl virus (TLCV) to better understand its function. A protein closely related to a family of plant reversibly glycosylated peptides, designated SlUPTG1, was found to interact with V1 in yeast and in vitro. SlUPTG1 may function endogenously in the synthesis of cell wall polysaccharides, since a bacterially expressed form of the protein acted as an autocatalytic glycosyltransferase in vitro, a SlUPTG1:GFP fusion protein localized to the cell wall, and expression of SlUPTG1 appeared to be highest in actively dividing tissues. However, expression of SlUPTG1 in a transient TLCV replication assay increased the accumulation of viral DNA, suggesting that this host factor also plays a role in viral infection. Together, these data provide new insight into the role of V1 in TLCV infection and reveal another host pathway which geminiviruses may manipulate to achieve an efficient infection.
Asunto(s)
Geminiviridae/patogenicidad , Proteínas de Plantas/metabolismo , Solanum lycopersicum/virología , Proteínas Virales/metabolismo , ADN Viral/metabolismo , Geminiviridae/genética , Geminiviridae/metabolismo , Biblioteca de Genes , Glicosilación , Proteínas Fluorescentes Verdes/análisis , Solanum lycopersicum/metabolismo , Cebollas/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes de Fusión/análisis , Análisis de Secuencia de Proteína , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/fisiologíaRESUMEN
Here we report recovery of infectious Marburg virus (MARV) from a full-length cDNA clone. Compared to the wild-type virus, recombinant MARV showed no difference in terms of morphology of virus particles, intracellular distribution in infected cells, and growth kinetics. The nucleocapsid protein VP30 of MARV and Ebola virus (EBOV) contains a Zn-binding motif which is important for the function of VP30 as a transcriptional activator in EBOV, whereas its role for MARV is unclear. It has been reported previously that MARV VP30 is able to support transcription in an EBOV-specific minigenome system. When the Zn-binding motif was destroyed, MARV VP30 was shown to be inactive in the EBOV system. While it was not possible to rescue recombinant MARV when the VP30 plasmid was omitted from transfection, MARV VP30 with a destroyed Zn-binding motif and EBOV VP30 were able to mediate virus recovery. In contrast, rescue of recombinant EBOV was not supported by EBOV VP30 containing a mutated Zn-binding domain.
Asunto(s)
ADN Complementario/metabolismo , Marburgvirus/fisiología , Factores de Transcripción/fisiología , Proteínas Virales/fisiología , Ebolavirus/química , Ebolavirus/crecimiento & desarrollo , Genoma Viral , Enfermedad del Virus de Marburg/virología , Marburgvirus/química , Marburgvirus/crecimiento & desarrollo , Datos de Secuencia Molecular , Recombinación Genética , Factores de Transcripción/química , Proteínas Virales/química , Zinc/fisiologíaRESUMEN
Potato mop-top virus (PMTV) RNA3 contains a triple gene block (TGB) encoding viral movement proteins and an open reading frame for a putative 8 kDa cysteine-rich protein (CRP). In this study, PMTV CRP was shown to be expressed in the course of virus infection, and a PMTV CRP-specific subgenomic RNA was mapped. CRP has previously been shown to be dispensable for infection of PMTV in Nicotiana benthamiana. In this study, PMTV CRP was found to increase the severity of disease symptoms when expressed from Potato virus X or Tobacco mosaic virus in N. benthamiana and Nicotiana tabacum, suggesting that the protein affects virulence of the virus or might suppress a host defence mechanism. However, PMTV CRP did not show RNA silencing suppression activity in three assays. Host responses to the PMTV CRP expression from different viral genomes ranged from an absence of response to extreme resistance at a single cell level and were dependent on the viral genome. These findings emphasized involvement of viral proteins and/or virus-induced cell components in the plant reaction to CRP. PMTV CRP was predicted to possess a transmembrane segment. CRP fused to the green fluorescent protein was associated with endoplasmic reticulum-derived membranes and induced dramatic rearrangements of the endoplasmic reticulum structure, which might account for protein functions as a virulence factor of the virus.
Asunto(s)
Cisteína/metabolismo , Enfermedades de las Plantas/virología , Virus de Plantas/metabolismo , Virus de Plantas/patogenicidad , Solanum tuberosum/virología , Proteínas Virales/fisiología , Peso Molecular , Virus de Plantas/genética , Interferencia de ARN , Transcripción Genética , Proteínas Virales/genética , VirulenciaRESUMEN
The Triple Gene Block proteins TGBp1, TGBp2, and TGBp3 of Beet necrotic yellow vein virus (BNYVV) are required for efficient cell-to-cell spread of the infection. The TGB proteins can drive cell-to-cell movement of BNYVV in trans when expressed from a co-inoculated BNYVV RNA 3-based 'replicon'. TGBp2 and TGBp3 expressed from the replicon were nonfunctional in this assay if they were fused to the green fluorescent protein (GFP), but addition of a hemagglutinin (HA) tag to their C-termini did not incapacitate movement. Immunogold labeling of ultrathin sections treated with HA-specific antibodies localized TGBp2-HA and TGBp3-HA to what are probably structurally modified plasmodesmata (Pd) in infected cells. A similar subcellular localization was observed for TGBp1. Large gold-decorated membrane-rich bodies containing what appear to be short fragments of endoplasmic reticulum were observed near the cell periphery. The modified gold-decorated Pd and the membrane-rich bodies were not observed when the TGB proteins were produced individually in infections using the Tobacco mosaic virus P30 protein to drive cell-to-cell movement, indicating that these modifications are specific for TGB-mediated movement.
Asunto(s)
Genes Virales , Luteovirus/fisiología , Beta vulgaris/virología , Luteovirus/clasificación , Luteovirus/genética , Luteovirus/ultraestructura , Movimiento , Filogenia , Enfermedades de las Plantas/virología , Proteínas Virales/fisiologíaRESUMEN
The NIa proteinase from pepper vein banding virus (PVBV) is a sequence-specific proteinase required for processing of viral polyprotein in the cytoplasm. It accumulates in the nucleus of the infected plant cell and forms inclusion bodies. The function of this protein in the nucleus is not clear. The purified recombinant NIa proteinase was active, and the mutation of the catalytic residues His-46, Asp-81, and Cys-151 resulted in complete loss of activity. Most interesting, the PVBV NIa proteinase exhibited previously unidentified activity, namely nonspecific double-stranded DNA degradation. This DNase activity of the NIa proteinase showed an absolute requirement for Mg(2+). Site-specific mutational analysis showed that of the three catalytic residues, Asp-81 was the crucial residue for DNase activity. Mutation of His-46 and Cys-151 had no effect on the DNase activity, whereas mutant D81N was partially active, and D81G was completely inactive. Based on kinetic analysis and molecular modeling, a metal ion-dependent catalysis similar to that observed in other nonspecific DNases is proposed. Similar results were obtained with glutathione S-transferase-fused PVBV NIa proteinase and tobacco etch virus NIa proteinase, confirming that the DNase function is an intrinsic property of potyviral NIa proteinase. The NIa protein present in the infected plant nuclear extract also showed the proteinase and the DNase activities, suggesting that the PVBV NIa protein that accumulates in the nucleus late in the infection cycle might serve to degrade the host DNA. Thus the dual function of the NIa proteinase could play an important role in the life cycle of the virus.
Asunto(s)
Potyvirus/enzimología , Proteínas Virales/química , Proteínas Virales/fisiología , Ácido Aspártico/química , Núcleo Celular/metabolismo , Cisteína/química , Citoplasma/metabolismo , ADN/química , Análisis Mutacional de ADN , Desoxirribonucleasas/química , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Endopeptidasas , Escherichia coli/metabolismo , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Histidina/química , Concentración de Iones de Hidrógeno , Iones , Cinética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Magnesio/química , Modelos Moleculares , Mutación , Plásmidos/metabolismo , Mutación Puntual , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Cloruro de Sodio/farmacología , Solanum tuberosum/metabolismo , Factores de TiempoRESUMEN
A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of the Replication-associated protein translation start codon, was tested for promoter activity in solanaceous plants. The promoter activity of this fragment (pRep(459::Rep)) was demonstrated when it was introduced upstream the uidA reporter gene into tobacco, potato and tomato plants by genetic transformation. It became active in 7-day-old transgenic tobacco seedlings as revealed by a vascular-specific pattern of gene expression which was maintained during the continued growth of the plant. Transformed potato and tomato plants also showed a vascular-specific pattern of expression. In comparative assays, pRep(459::Rep) showed an expression activity 10-40-fold less than the 35S promoter from Cauliflower mosaic virus. To delimit the minimal cis-acting elements necessary for vascular specificity of this promoter, a set of PCR deletion mutants of pRep(459::Rep) (pRep(459), pRep(324), pRep(203), pRep(145), pRep(132) and pRep(115)), were generated and used to transform tobacco plants. Transgenic tobacco plants belonging to all the pRep versions were blue stained in the vascular system except those from the pRep(115) version. The results described in this report demonstrate that the minimal sequences necessary for the pRep promoter activity are confined in a segment of 132 nts (located between the nts 2454 and 2585 of the ToMoTV DNA A) and that this promoter harbors those elements sufficient for vascular-specific expression.
Asunto(s)
Geminiviridae/genética , Geminiviridae/fisiología , Regiones Promotoras Genéticas , Proteínas Virales/genética , Región de Flanqueo 5' , Fusión Artificial Génica , Caulimovirus/genética , Regulación Viral de la Expresión Génica , Genes Reporteros , Glucuronidasa/genética , Glucuronidasa/metabolismo , Plantas Modificadas Genéticamente/virología , Eliminación de Secuencia , Solanum tuberosum/virología , Nicotiana/virología , Transformación Genética , Proteínas Virales/fisiología , Replicación ViralRESUMEN
Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EGdelta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(delta4AB)SEAP. EG(delta4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, delta4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(delta4AB)SEAP cells. In conclusion, Ava5-EG(delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.