RESUMEN
The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics.
Asunto(s)
Brotes de Enfermedades/prevención & control , Monitoreo Epidemiológico , Genómica/métodos , Fiebre Amarilla/prevención & control , Fiebre Amarilla/transmisión , Virus de la Fiebre Amarilla/aislamiento & purificación , Aedes/virología , Factores de Edad , Animales , Brasil/epidemiología , Brotes de Enfermedades/estadística & datos numéricos , Evolución Molecular , Humanos , Filogenia , Reacción en Cadena de la Polimerasa , Riesgo , Factores Sexuales , Análisis Espacio-Temporal , Fiebre Amarilla/epidemiología , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/clasificación , Virus de la Fiebre Amarilla/genéticaRESUMEN
Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4(+) and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Proteasa del VIH/inmunología , VIH-1/inmunología , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Mapeo Epitopo/métodos , Epítopos de Linfocito T/metabolismo , Citometría de Flujo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Proteasa del VIH/genética , Proteasa del VIH/metabolismo , VIH-1/metabolismo , Antígenos HLA-DR/inmunología , Antígenos HLA-DR/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Péptidos/inmunología , Péptidos/metabolismo , Unión ProteicaRESUMEN
In this paper, we describe the synthesis of a novel class of pseudo-peptides derived from isomannide and several oxazolones as potential inhibitors of serine proteases as well as preliminary pharmacological assays for hepatitis C. Hepatitis C, dengue and West Nile fever are among the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes and are a primary target in the drug development field. Several pseudo-peptides were obtained in good yields from the reaction of isomannide and oxazolones, and their anti-HCV potential using the HCV replicon-based assay was shown.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/química , Diseño de Fármacos , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Antivirales/síntesis química , Antivirales/química , Antivirales/farmacología , Benzamidas/síntesis química , Benzamidas/química , Benzamidas/farmacología , Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Genes Reporteros , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepatocitos/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Oligopéptidos/química , Oxazoles/síntesis química , Oxazoles/química , Replicón , Inhibidores de Serina Proteinasa/químicaRESUMEN
The synthetic peptide T-20 (enfuvirtide, EFV) represents the first compound approved by the FDA known as entry inhibitors (EIs). The resistance mutations associated with this new class of antiretroviral drug are located in the first heptad repeat (HR1) region of gp41. Amino acid changes in codons G36D/S, I37V, V38A/M/E, Q39H/R, Q40H, N42T, and N43D can confer resistance to EFV. In this work we investigated the presence of resistance mutations that occur in patients never treated with EFV and failing HAART with protease inhibitors (PIs), nucleoside reverse transcriptase (RT) inhibitors (NRTIs), and nonnucleoside RT inhibitors (NNRTIs). This knowledge can reveal whether this salvage therapy can be effective in patients failing HAART. For this, we amplified 65 samples from plasma isolates and than sequenced a fragment of 416 nt encompassing the HR1 and HR2 regions (amino acids 33-170 of gp41). The subtype distribution among the 65 isolates was 45 (69.23%) subtype B, 9 (13.85%) subtype C, 7 (10.77%) subtype F1, and 4 (6.15%) mosaics B/F1, B/C, F1/C, and C/F1/B. We found a high prevalence (7.6%) of EFV-associated mutation G36D in this cohort of patients failing HAART therapy, five isolates from subtype B (11.11% within this group). In contrast, when 1079 sequences from drug-naive patients were analyzed, only one showed the G36D substitution. This finding indicates a strong association between the selected position G36D and HAART therapy (p < 0.0001). The isolates that possess these mutations can develop resistance to EFV more rapidly. Nevertheless, more information about the impact of these mutations in salvage therapy with EFV in patients failing HAART must still be obtained.
Asunto(s)
Terapia Antirretroviral Altamente Activa , Farmacorresistencia Viral , Proteína gp41 de Envoltorio del VIH/farmacología , Inhibidores de Fusión de VIH/farmacología , Infecciones por VIH/virología , VIH-1/genética , Mutación Missense , Fragmentos de Péptidos/farmacología , Adulto , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Brasil , Enfuvirtida , Genotipo , Proteína gp41 de Envoltorio del VIH/genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/clasificación , VIH-1/efectos de los fármacos , VIH-1/aislamiento & purificación , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
The major human immunodeficiency virus type 1 subtype circulating in Brazil is B, followed by F and C. We have genotyped 882 samples from Brazilian patients for whom highly active antiretroviral therapy failed, and we found subtype B and the unique recombinant B/F1 forms circulating. Due to codon usage variation, there is a significantly lower incidence of the substitutions L210W, Q151M, and F116Y in subtype F1 isolates than in the subtype B counterparts.
Asunto(s)
Terapia Antirretroviral Altamente Activa , Codón/genética , Farmacorresistencia Viral/genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Mutación , Brasil , Recuento de Linfocito CD4 , Femenino , Genotipo , Infecciones por VIH/virología , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , Seropositividad para VIH/tratamiento farmacológico , Seropositividad para VIH/virología , VIH-1/clasificación , VIH-1/genética , Humanos , Masculino , ARN Viral/sangre , Insuficiencia del TratamientoRESUMEN
The I50V protease inhibitor (PI) resistance mutation was found in 87.4% of protease gene fragments sequenced from 199 nucleic acid isolates extracted using an NASBA virus load assay, performed between 1997 and 2001 in Brazil. This mutation is an amprenavir-related mutation, and at that particular time this PI was seldom used in Brazil. This mutation was found both in patients with and without therapeutic success. Q calibrators showed the PI resistance mutation I50V when directly amplified and sequenced from the 423-bp PCR product targeting protease gene. The majority of the patients' samples had a mixture of I50I and I50V; however, this artifact was nor seen when a 989-bp PCR product was used. These results show that RNA extracted using virus load kits need to be critically evaluated before being used in home-brew genotypic tests.
Asunto(s)
Reacciones Falso Positivas , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/genética , Juego de Reactivos para Diagnóstico/normas , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral/genética , Infecciones por VIH/genética , Infecciones por VIH/virología , Proteasa del VIH/genética , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/uso terapéutico , VIH-1/efectos de los fármacos , Humanos , Mutación , ARN Viral/análisis , Juego de Reactivos para Diagnóstico/estadística & datos numéricos , Carga ViralRESUMEN
We have investigated the phenotypic and genotypic susceptibility of 14 HIV-1 strains isolated from individuals failing HAART therapy to protease inhibitors (PI). Proviral and plasma viral pol gene fragment were amplified, sequenced and subtyped. Nine samples clustered with protease subtype B reference strains and the remaining samples were classified as non-B subtype corresponding to subtype F (n = 4) and subtype A (n = 1). Although all patients were treated with similar P1 drug regimen, the non-B subtype isolates did not present the L90M and 184V mutations and used mainly G48V and V82A/F to achieve drug resistance. A strong cross-resistance phenotype among all four PI was associated with the mutation L90M in the subtype-B isolates, and with G48V and V82A/F in the non-B counterparts. This observation revealed that the non-B viruses tested had specific genotypic characteristics contrasting with the subtype-B isolates.
Asunto(s)
Terapia Antirretroviral Altamente Activa , Farmacorresistencia Microbiana/genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Mutación , Secuencia de Aminoácidos , Genotipo , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Fenotipo , Homología de Secuencia de AminoácidoRESUMEN
In Nigeria, the most populous country in Africa, the characterization of HIV-1 strains has been limited. In this study we evaluated the genetic diversity of the protease coding region, one of the anti-retroviral therapy target, and investigated the presence of mutations related to resistance to HIV protease inhibitors. We analyzed samples collected during 1996 and all patients were anti-retroviral drug naïves. Ten samples were evaluated by sequencing of the protease gene. The majority, 80%, were classified as subtype A and the two others were unclassified-divergent strains, something in between A and G subtypes. The gag region from these outliners were sequenced and the phylogenetic analysis classified them as subtype G. The protease amino acid consensus sequence of the Nigerian subtype A are in complete agreement with the consensus A differing from the USA subtype B consensus in 10 positions (L10V, I13V, K14R, I15V, K20I, M36I, R41K, P63L, H69K and L89M). The secondary substitutions associated with protease inhibitor resistance were observed in all Nigerian sequences at the positions L10V, M36I and L89M. The majority of sequence variation was concentrated in the interval between aminoacids 70-90 where the protease substrate binding region is located.
Asunto(s)
Variación Genética , Infecciones por VIH/virología , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/genética , VIH-1/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral , Farmacorresistencia Microbiana , VIH-1/clasificación , VIH-1/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , NigeriaRESUMEN
Development of drug resistance is the inevitable consequence of incomplete suppression of virus plasma levels in HIV-1-infected patients treated with highly active antiretroviral therapy. Resistance mutations previously characterized have been found in B subtype viruses of developed countries. Moreover, mutation profiles for non-B and more divergent B subtype viruses found in developing countries shall be analyzed together with their ex vivo phenotyping in order to establish an exact correlation between the genotyping data and the clinical management counseling for those uncommon virus subtypes. In the present study, we evaluated the mutation profile for individuals infected with B subtype and non-B subtype viruses. Viral DNA fragments corresponding to the RT gene were amplified, sequenced, and subtyped. Phenotyping analysis for reverse transcriptase nucleoside (NRTI) and nonnucleoside inhibitor susceptibility was performed using the recombinant virus assay technology. Brazilian non-B subtypes (subtype F, n = 4, and subtype A, n = 1) isolates showed essentially the same B subtype mutation profile, presenting an NRTI drug resistance with similar MIC50% and MIC90% values for all drugs analyzed regardless of their subtypes. A strong cross-resistance phenotype among AZT, 3TC, and abacavir could be seen in all isolates analyzed. A novel result was that some RT sequences not only revealed the presence of G333D/E mutations but also correlated to the presence of mutation T386I that could abrogate the M184V-surpassing effect of L210W or L210W plus G333D/E. These findings suggest that Brazilian non-B subtype HIV-1 strains use an identical RT drug resistance mutation pattern when compared to B isolates and will contribute to the validation of the genotypic and phenotypic tests in these predominant worldwide-spread viral variants.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , Resistencia a Múltiples Medicamentos/genética , Transcriptasa Inversa del VIH/genética , VIH-1/clasificación , VIH-1/enzimología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/epidemiología , Síndrome de Inmunodeficiencia Adquirida/virología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacología , Brasil/epidemiología , Análisis Mutacional de ADN , Farmacorresistencia Microbiana , Quimioterapia Combinada , Femenino , Variación Genética/genética , Genotipo , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Masculino , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Filogenia , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Inhibidores de la Transcriptasa Inversa/farmacología , Factores de Riesgo , Alineación de Secuencia , Factores de Tiempo , Insuficiencia del TratamientoRESUMEN
We have developed a replication-competent human immunodeficiency virus (HIV) carrying a selective marker that can be used in vivo. This recombinant virus (Z6 Delta nef gpt) was generated by replacing the 5' half of the HIV nef gene with the Escherichia coli guanine phosphoribosyl transferase gene (gpt). This new vector can express the gpt product on infection and works as a positive selective marker for mycophenolic acid (MPA) resistance, a potent immunosuppressive drug used in organ rejection therapy. Conversely, gpt expression also served as a negative selectable marker, since its intracellular expression induces host-cell susceptibility to 6-thioxantine (6-TX), a nucleotide analog that is toxic to the infected cell under these conditions. In this manner, we could suppress the recombinant virus replication through 6-TX selection in both transformed cells and primary human peripheral blood mononuclear cells (PBMCs), suggesting the vector's potential as a model for a new live-attenuated vaccine approach against HIV.
Asunto(s)
Escherichia coli/genética , Genes nef , VIH-1/enzimología , VIH-1/genética , Hipoxantina Fosforribosiltransferasa/genética , Vacunas contra el SIDA , Línea Celular , Escherichia coli/enzimología , Productos del Gen nef/genética , Vectores Genéticos , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Leucocitos Mononucleares/virología , Ácido Micofenólico/farmacología , Vacunas Atenuadas , Replicación Viral/efectos de los fármacos , Xantinas/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
The presence of human immunodeficiency virus type 1 (HIV-1) bearing mutations resistant to nucleosidic inhibitors of the viral reverse transcriptase (RT) derived from HIV-seropositive asymptomatic and untreated volunteer blood donors was examined. The RT amplicons of 32 specimens were analyzed by using a reverse hybridization line probe assay technique that detects resistance against zidovudine (3'-azido-3'-deoxythymidine [AZT], didanosine (2',3'-dideoxyinosine [ddI], zalcitabine (2',3'-dideoxycytidine [ddC]), and lamivudine ((-)-beta-L-2',3'-dideoxy-3'-thiacytidine [3TC]) at amino acid positions 41, 69, 70, 74, 184, and 215 of the HIV RT. One sample (brp004, subtype B) showed an AZT resistance secondary mutation at position K70R. Fifteen specimens revealed one or more sites of nonreactivity to both wild-type- and mutant-specific probes (dual nonreactivity). Samples were also submitted to RT direct sequencing and phylogenetic analysis. Nine of 32 specimens belonged to non-B subtypes (C, D, F, and F/B or B/F mosaics). Three of these non-B isolates, named brp004, brp063, and brp069, revealed three other relevant AZT resistance mutations-a T215F mutation and two M41L mutations, respectively-hidden by the nonreactivity to line probe assay strips on the respective codon regions. The isolate brp004 also carried a D67N AZT resistance mutation revealed by direct sequencing. No nonnucleosidic RT inhibitor-resistant mutation was found. The analysis revealed a frequency of 2.26 x 10(-4) mutations per nucleotide for independent samples related to RT resistance. These findings emphasize the magnitude of naturally occurring reservoirs of drug-resistant virus among untreated HIV-1-positive individuals in Brazil.
Asunto(s)
Transcriptasa Inversa del VIH/química , Seropositividad para VIH/virología , Secuencia de Bases , Codón , Farmacorresistencia Microbiana , Genotipo , VIH-1/clasificación , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Filogenia , Zidovudina/farmacologíaRESUMEN
The prevalence of HIV infection in Brazil is one of the highest in the world. In addition, transfusion-transmitted HIV accounts for 2.3% of all AIDS cases in Brazil. The objective of this study was to evaluate genetic diversity and distribution of HIV-1 strains circulating in the blood-donor population. We characterized 43 seropositive blood units collected from volunteer blood donors residing throughout Rio de Janeiro, Brazil. Viral RNA was extracted from plasma, reverse transcribed, and amplified by nested polymerase chain reaction (PCR) using HIV group M degenerate primers. Genetic heterogeneity was evaluated by direct automated cycle sequencing of the following gene fragments: gag p24 (399 bp), env C2V3 (345 bp), and env gp41 (369 bp). Phylogenetic analysis reflected the complexity of the Brazilian HIV epidemic: the majority of specimens, 33 of 43 (76.7%) were subtype B, and 6 of 43 (14%) were subtype F. The remaining 4 samples (9.3%) involved potential mosaic viruses of subtypes B and F or B and D. This survey is the first to document HIV-1 genetic variation in the Brazilian blood-donor population.
PIP: Brazil has the highest prevalence of HIV infection in Latin America and one of the highest such prevalences in the world. By 1996, 110,000 AIDS cases had been cumulatively reported by the Brazil National AIDS Program. HIV-1 subtypes B and F have previously been described in Brazil, accounting for 85% and 15% of infections, respectively. Findings are presented from a study conducted to evaluate the genetic diversity and distribution of HIV-1 strains circulating in the blood donor population. The authors characterized 43 HIV-seropositive blood units collected from volunteer blood donors living throughout Rio de Janeiro. Viral RNA was extracted from plasma, reverse transcribed, and amplified by nested polymerase chain reaction using HIV group M degenerate primers. Genetic heterogeneity was assessed through the direct automated cycle sequencing of gene fragments gag p24 (399 bp), env C2V3 (345 bp), and env gp41 (369 bp). 33 of the 43 (76.7%) specimens were of subtype B and 6 (14%) of subtype F, while the remaining 4 (9.3%) involved potential mosaic viruses of subtypes B and F or B and D.
Asunto(s)
Donantes de Sangre , Seropositividad para VIH/virología , VIH-1/clasificación , Secuencia de Bases , Brasil , VIH-1/genética , Humanos , Datos de Secuencia MolecularRESUMEN
1. Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. 2. Two constructions were developed to express either a full-length or truncated enzyme lacking the 20 amino acids at the N-terminal end. Bacterial extracts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. 3. The biological activity of these two molecules was tested using a poly(A)-dependent oligo(U)-primed poly(U)-polymerase assay. The full-length replicase is active. The aminoterminal truncated enzyme had 0.02% activity of the intact one. 4. This result indicates the importance of the twenty N-terminal amino acids for the activity of FMDV RNA-dependent RNA polymerase.
Asunto(s)
Aphthovirus/enzimología , Replicación del ADN , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Replicación Viral , Secuencia de Aminoácidos , Aphthovirus/fisiología , Secuencia de Bases , ADN Polimerasa Dirigida por ADN/análisis , Escherichia coli/genética , Datos de Secuencia Molecular , Plásmidos , Relación Estructura-ActividadRESUMEN
1. The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. 2. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10(5). The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha 32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. 3. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. 4. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other picornaviruses.
Asunto(s)
Aphthovirus/genética , Replicación del ADN , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Regulación Viral de la Expresión Génica/genética , Replicación Viral , Aphthovirus/enzimología , Aphthovirus/fisiología , ADN Viral/biosíntesis , ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Viral de la Expresión Génica/fisiología , Plásmidos/genética , Regiones Promotoras Genéticas/genética , ARN Viral/genética , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Moldes GenéticosRESUMEN
Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. Two constructions were developed to express either a full-lenghtt or truncated enzyme lacking the 20 aminoacids at the N-terminal en. Bacterial extr5acts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. The biological activity of these two molecules was tested using a poly(A)-dep[endent oligo(U)-primed poly(U)-polymer4ase assay. The full-lenght replicase is active. The aminoterminal truncated wnzyme had 0.02% activity o9f the intact5 one. This result indicates the importaqnce of the twenty N-terminal amino acids for the activity of FMDV RNA dependent RNMA polymerase
Asunto(s)
Secuencia de Aminoácidos , Fiebre Aftosa , Péptidos/análisis , ARN Polimerasa Dependiente del ARN , Replicación ViralRESUMEN
The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10 5. The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other piconaviruses
Asunto(s)
Aphthovirus , ARN Polimerasas Dirigidas por ADN , Enzimas/aislamiento & purificación , Escherichia coli , Técnicas In Vitro , Picornaviridae , Recombinación GenéticaRESUMEN
The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to rebuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid.
Asunto(s)
Aphthovirus/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Secuencia de Aminoácidos , Aphthovirus/enzimología , Secuencia de Bases , Clonación Molecular , Codón/genética , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Electroforesis en Gel de Agar , Datos de Secuencia Molecular , Oligonucleótidos/química , Plásmidos , TransfecciónRESUMEN
The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to renuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid