RESUMEN
We have determined the sequences of the 5' ends of three strains of Newcastle disease virus, permitting the assembly of the entire genomic sequence, which amounts to 15,186 nucleotides. This length is in agreement with the rule of six, which has been shown to determine replication efficiency in similar viruses. Comparison of the extreme 5' end of the trailer sequence with that of the 3'-terminal leader sequence of the virus reveals a high degree of complementarity. Variation between the 5'-terminal sequences of the different strains reveals the presence of alternative L gene polyadenylation signals, leading to correspondingly different trailer lengths.
Asunto(s)
Genoma Viral , Virus de la Enfermedad de Newcastle/genética , Animales , Secuencia de Bases , Embrión de Pollo , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.
Asunto(s)
Bacillus subtilis/genética , Genoma Bacteriano , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Clonación de Organismos , ADN Bacteriano , Datos de Secuencia MolecularRESUMEN
A recombinant baculovirus expressing the nucleocapsid gene (NP) of Newcastle disease virus (NDV), a member of the genus Rubulavirus, has been generated and shown to express the native protein to high levels in insect cells. In contrast to the NP protein of the rubulavirus human parainfluenza virus 2, the NDV protein has been demonstrated by electron microscopy and caesium chloride gradient analysis to be capable of self-assembly in vivo to form nucleocapsid-like structures in the absence of other NDV proteins. These structures, which contained RNA that was resistant to micrococcal nuclease digestion, were also observed when the protein was expressed in E. coli, a phenomenon which was not inhibited by the presence of a 40 amino acid fusion region at the amino terminus of the protein. Further, the formation of these structures was inhibited by the co-expression of the phosphoprotein (P). Therefore, we conclude that the P protein acts as a chaperone, preventing uncontrolled encapsidation of non-viral RNA by NP protein.
Asunto(s)
Virus de la Enfermedad de Newcastle/fisiología , Nucleocápside/biosíntesis , Fosfoproteínas/fisiología , Proteínas Virales/fisiología , Ensamble de Virus/fisiología , Animales , Baculoviridae/genética , Línea Celular , Escherichia coli/genética , Genes Virales/genética , Humanos , Virus de la Enfermedad de Newcastle/genética , Nucleocápside/genética , Nucleocápside/ultraestructura , Fosfoproteínas/genética , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión , Spodoptera , Proteínas Virales/genéticaRESUMEN
To investigate the role that the individual subunits play in the ATP-dependent helicase activity of the RecBCD protein we have investigated the ability of the RecB, RecC and RecD proteins to displace various 20-mer oligonucleotides annealed to either end or to the centre of an oligonucleotide 60 bases long. The results show that the only subunit which can displace the 20-mers in the absence of the other subunits is the RecB protein. Moreover, the 20-mer is displaced only if it is annealed to the 60-mer at the 5' end or the middle, suggesting that the RecB protein translocates along the 60-mer in the 3' to 5' direction, displacing annealed 20-mers as it proceeds. We have shown that reconstituted RecBC and RecBCD complexes displace the 20-mers but, unlike RecB, they do not require a 3'-ended single-stranded region for helicase action, but can displace the 20-mers from either end of the 60-mer. The level of helicase activity of the RecBC complex is considerably greater than that of RecB alone, and the activity of the RecBCD complex appears to be greater still. This hierarchy of activity is also shown by DNA binding studies, but is not reflected in the ATPase activities of the enzymes. We have also shown that the ability of trypsin to cleave various sites on the RecB molecule is modified by the presence of ATP or ATP-gamma-S, suggesting that conformational changes may be induced in RecB upon ATP binding. We discuss a model for the ATP-driven, unidirectional motion of the RecB translocase along single-stranded DNA. In this model, the RecB molecule binds to single-stranded DNA and then translocates along it, one base at a time, in the 3' to 5' direction, by a 'ratchet' mechanism in which repeated stretching and contraction of the protein is coupled to ATP hydrolysis. The RecC protein in the RecBC complex is proposed to act as a 'sliding clamp' which increases processivity by preventing dissociation.
Asunto(s)
ADN Bacteriano/genética , ADN de Cadena Simple/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Exodesoxirribonucleasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Helicasas/genética , Exodesoxirribonucleasa V , Datos de Secuencia MolecularRESUMEN
Two major families of peptidylprolyl cis-trans-isomerases, the cyclophilins and the structurally unrelated FK506-binding proteins (FKBPs), have been identified as cellular factors involved in protein folding in vitro. Here we report on the biochemical characterization of a second prolyl isomerase of Bacillus subtilis that was purified from a cyclophilin-negative (ppiB null) mutant and was shown to be the trigger factor (TigBS). N-terminal sequencing of 27 amino acid residues of the purified protein revealed 100% identity to the deduced sequence encoded by the tig gene, sequenced as a part of the B. subtilis genome project. The tigBS gene, located at 246 degrees on the genetic map upstream of the clpX and lonA,B genes, encodes an acidic protein (pI 4.3) of 47.5 kDa. Purified and recombinant TigBS-His proteins share the same substrate specificity and catalytic activity (Kcat/K(m) of 1.5 microM-1 s-1); both are inhibited by the macrolide FK506 with IC50 the range of 500 nM. We also demonstrate that the prolyl isomerase activity of TigBS is mediated by an internal domain of about 13 kDa (homologous to FKPB12) that represents the catalytic core of the trigger factor.
Asunto(s)
Isomerasas de Aminoácido/química , Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Proteínas Portadoras/química , Tacrolimus/química , Isomerasas de Aminoácido/biosíntesis , Isomerasas de Aminoácido/genética , Isomerasas de Aminoácido/aislamiento & purificación , Secuencia de Aminoácidos , Bacillus subtilis/química , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Catálisis , Clonación Molecular , Histidina , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil , Unión Proteica , Tacrolimus/aislamiento & purificaciónRESUMEN
Within the framework of the international programme to sequence the genome of Bacillus subtilis strain 168, we were allocated the region between dnaB (256 degrees) and pheA (240 degrees). The sequencing of this region is now complete and we report our primary analysis of the 114 kb region containing 114 ORFs. In addition to previously characterized genes, we have identified genes involved in the utilization of plant cell wall polysaccharides, stress responses and the metabolism of amino acids, cell walls, DNA and fatty acids. We also discuss various structural and physical features, including the orientation of genes with respect to replication, putative start and stop codons, ribosome binding sites and rho-independent transcription terminators.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Cromosomas Bacterianos/genética , ADN Helicasas , Genes Bacterianos , Proteínas Bacterianas/genética , Secuencia de Bases , Pared Celular/metabolismo , Mapeo Cromosómico , ADN Bacteriano/genética , AdnB Helicasas , Genoma Bacteriano , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plantas/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Regiones Terminadoras GenéticasRESUMEN
The nucleotide sequence of clpX, which is localized between the tig (trigger factor) and the lon (ATP-dependent protease) genes at 245 degrees on the standard Bacillus subtilis (Bs) genetic map, was determined. The putative clpX gene codes for a 46-kDa protein of 421 amino acid (aa) residues. A comparison of the deduced aa sequence with those of the recently described bacterial clpX gene products from Synechocystis sp., Escherichia coli (Ec), Haemophilus influenzae and Azotobacter vinelandii revealed strong similarities. However, in contrast to Ec, clpX and clpP of Bs are located at different loci on the chromosome and are transcribed as monocistronic genes. A heat-inducible sigma A-like promoter was mapped upstream of the clpX structural gene, but no CIRCE element, characteristic of class-I heat-shock genes (e.g., groESL and dnaK), was found between the transcriptional and translational start sites. Although the majority of the heat-inducible general stress genes in Bs are under the control of the alternative sigma factor, sigma B, the heat induction of clpX appears to be sigma B-independent. The latter indicates that clpX belongs to class-III heat-inducible genes.
Asunto(s)
Adenosina Trifosfatasas/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas de Choque Térmico/genética , ATPasas Asociadas con Actividades Celulares Diversas , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , ADN Bacteriano , Endopeptidasa Clp , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Chaperonas Moleculares , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
A recombinant baculovirus containing a cDNA clone encoding the nucleocapsid (NP) protein of Newcastle disease virus (strain Ulster 2C) has been used to infect insect cells (Spodoptera frugiperda). High levels of overexpressed NP protein were observed, comprising up to 40% of total cellular protein, which were subsequently shown to be antigenic. Nucleoprotein derived from the crude soluble lysate of infected insect cells has been used in an indirect ELISA to detect the presence of anti-NDV antibodies in a cohort of chicken sera. Data produced from these tests indicated a good correlation between ELISA titre and haemagglutination inhibition test data. The test was not affected by interference from background cellular proteins nor by cross-reactivity with non-NDV poultry pathogens. Additionally, the test did not generate false-positive readings.
Asunto(s)
Anticuerpos Antivirales/análisis , Cápside/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Virus de la Enfermedad de Newcastle/aislamiento & purificación , Proteínas del Núcleo Viral/inmunología , Animales , Anticuerpos Antivirales/inmunología , Cápside/genética , Línea Celular , Pollos , Estudios de Evaluación como Asunto , Vectores Genéticos/genética , Pruebas de Inhibición de Hemaglutinación , Inmunoglobulina G/inmunología , Enfermedad de Newcastle/diagnóstico , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/inmunología , Nucleopoliedrovirus/genética , Enfermedades de las Aves de Corral/diagnóstico , Enfermedades de las Aves de Corral/virología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Spodoptera/citología , Proteínas del Núcleo Viral/genéticaRESUMEN
The V protein of Newcastle disease virus (NDV) is produced by the insertion of a single nontemplated G residue at a specific point during transcription of the phosphoprotein (P) gene, accessing a new reading frame upon translation. The V protein, in common with its counterpart in other paramyxoviruses contains a highly cysteine rich motif near the carboxyl terminus, suggestive of a zinc-binding domain. By constructing E. coli overexpression plasmids for the NDV P and V proteins, and monitoring the binding of 65ZnCl2 to proteins electroblotted onto nitrocellulose membranes, we have demonstrated that the V protein strongly binds zinc.
Asunto(s)
Proteínas Virales/metabolismo , Zinc/metabolismo , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
The co-transcriptional editing of the Newcastle disease virus (NDV) P gene has been studied by sequence analysis of cloned viral genomic RNA and mRNA. Evidence has been obtained for the specific insertion of non-templated G nucleotides, the consequence of which is the generation of three populations of P gene-derived mRNAs. The three populations encode proteins (P, V and W) which have a common N-terminal region, but which utilize three different reading frames at their C termini. Paradoxically, NDV edits its P gene mRNA by the insertion of non-templated G residues in a manner similar to Sendai and measles viruses (P-->V editing) despite its apparent closer evolutionary relationship to the simian virus type 5, mumps and related group of viruses which edit a V genomic sequence to generate an mRNA to encode a functional P protein (V-->P editing).
Asunto(s)
Genes Virales/genética , Virus de la Enfermedad de Newcastle/genética , Fosfoproteínas/genética , Edición de ARN , Transcripción Genética/genética , Proteínas Virales/genética , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Viral/genética , Sistemas de Lectura/genética , Respirovirus/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas no Estructurales Virales/genética , Proteínas Estructurales Virales/clasificaciónRESUMEN
In attempt to increase the induction of peptide-specific cytolytic T cells (CTL) we investigated the effect of the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene product on the activation of peptide-specific CTL. Spleen cells of CH3 mice immunized against the influenza nucleoprotein peptide 50-63 (NP 50-63) were restimulated in vitro (i) with peptide-pulsed syngeneic fibroblast cells (Ltk-) as antigen-presenting cells, which were in addition (ii) infected with NDV or (iii) stably transfected with the HN cDNA of NDV. A greater than sixfold increase in peptide-specific CTL responses was observed in cultures restimulated with peptide-pulsed Ltk- cells which co-expressed viral hemagglutinin due to either infection or transfection. A similar augmentation was seen in CTL responses against other types of antigen (major histocompatibility complex alloantigens, minor histocompatibility antigens or tumor antigens) when suboptimal cultures were stimulated with the respective antigen-presenting cells modified by NDV infection. These findings suggest that NDV or viral HN expressed on antigen-presenting cells or tumor cells can exert a T cell co-stimulatory function.
Asunto(s)
Hemaglutininas Virales/inmunología , Nucleoproteínas/inmunología , Péptidos/inmunología , Proteínas de Unión al ARN , Linfocitos T Citotóxicos/inmunología , Proteínas del Núcleo Viral/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Línea Celular , Citotoxicidad Inmunológica , Hemaglutininas Virales/genética , Virus de la Influenza A/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neuraminidasa/genética , Neuraminidasa/inmunología , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/inmunología , Proteínas de la Nucleocápside , TransfecciónRESUMEN
The Escherichia coli RecBCD holoenzyme and the individual constituent subunits have been purified from overproducing strains. The purified RecBCD holoenzyme has a native molecular mass of approximately 330 kDa, indicative of a heterotrimer subunit assembly. The RecB, RecC, and RecD subunits can associate in vitro to give nuclease, helicase, ATPase, and Chi-specific endonuclease activities which are indistinguishable from those of the RecBCD holoenzyme. At concentrations at which the reconstituted RecB + C + D enzyme is very active, none of the individual RecB, RecC, or RecD subunits have readily detectable activities of the holoenzyme, except RecB protein which had previously been shown to exhibit DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). At higher concentrations and with shorter DNA substrates reconstituted RecBC protein exhibits low levels of helicase and exonuclease activity.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Exodesoxirribonucleasas/metabolismo , Adenosina Trifosfato/metabolismo , Cromatografía Liquida , ADN Bacteriano/metabolismo , Electroforesis en Gel de Poliacrilamida , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Mutagénesis Sitio-DirigidaRESUMEN
The RecB subunit of the Escherichia coli RecBCD enzyme has previously been reported to possess DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). Here we demonstrate that a specific interaction between RecB protein and ATP can also be shown by photoaffinity labeling with the ATP analogue 8-azido-ATP. Furthermore, the capacity of the RecB protein to support ATP hydrolysis varies with the structure and length of the DNA cofactor. Single-stranded linear and circular DNA are markedly better in promoting ATP hydrolysis than duplex DNA. The purified RecB protein can function as a DNA helicase, displacing oligonucleotides annealed to viral M13 DNA in an ATP-dependent and orientation-specific manner.
Asunto(s)
Adenosina Trifosfato/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Exodesoxirribonucleasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Marcadores de Afinidad , Azidas/química , Secuencia de Bases , Catálisis , Cromatografía en Gel , ADN Helicasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Exodesoxirribonucleasa V , Hidrólisis , Cinética , Datos de Secuencia Molecular , FotoquímicaRESUMEN
In vitro, Escherichia coli RecA protein acts upon gapped and partially homologous linear duplex DNA to generate recombination products linked by Holliday junctions. When strand exchange reactions are supplemented with purified RecBCD enzyme, we observe the formation of products that resemble "patch" recombinants. The formation of "splice" recombinant products was not observed. The individual subunits, RecB, RecC, or RecD, had no effect on RecA protein-mediated strand exchange nor on the Holliday junctions formed in the reaction. Analysis of the way in which patch products arise indicates exonucleolytic digestion of the linear arms of the recombination intermediates (alpha-structures) by RecBCD enzyme. We find no evidence for specific resolution events at the site of the Holliday junction by RecBCD enzyme using these DNA substrates.
Asunto(s)
Intercambio Genético , ADN/metabolismo , Proteínas de Escherichia coli , Exodesoxirribonucleasas/metabolismo , Rec A Recombinasas/metabolismo , ADN/química , Escherichia coli/metabolismo , Exodesoxirribonucleasa V , Estructura Molecular , Rec A Recombinasas/química , Especificidad por SustratoRESUMEN
The intracellular levels of the Escherichia coli RecBCD proteins have been amplified by fusing the recBCD genes to the strong tac promoter/operator in the expression vector, pKK223-3. The overproduced proteins occur at levels amounting to approx. 10% of total cellular protein. Strains harbouring these overexpression plasmids have been used to purify the RecB. RecC and RecD protein subunits, as well as the RecBCD holoenzyme. The individually purified protein subunits can be used to reconstitute the ATP-dependent DNase activity of the RecBCD enzyme.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Exodesoxirribonucleasas/genética , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/biosíntesis , Exodesoxirribonucleasas/aislamiento & purificación , Expresión Génica/genética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/biosíntesisAsunto(s)
Proteína HN/genética , Virus de la Enfermedad de Newcastle/genética , Secuencia de Aminoácidos , Codón , Análisis Mutacional de ADN , ADN Viral , Datos de Secuencia Molecular , Neuraminidasa/farmacología , Virus de la Enfermedad de Newcastle/efectos de los fármacos , Virus de la Enfermedad de Newcastle/fisiología , Conformación Proteica , TemperaturaRESUMEN
The hemagglutinin-neuraminidase (HN) gene from the Beaudette C strain of Newcastle disease virus (NDV) has been expressed in a recombinant fowlpox virus vector. The HN gene, under the control of the vaccinia p7.5 promoter, was inserted into a nonessential gene in the terminal inverted repeats of fowlpox virus. Expression was demonstrated in tissue culture, a protein of the correct size for fully glycosylated HN protein being recognized by an HN-specific monoclonal antibody on Western blots. When the recombinant fowlpox virus was inoculated into chickens by intravenous or wing-web routes, antibody which recognizes HN from purified NDV virions was produced. Protective immunity to NDV was generated in the chickens; at the highest dose of vaccine 100% of the chickens tested were protected against challenge with a virulent strain of NDV.
Asunto(s)
Pollos/inmunología , Virus de la Viruela de las Aves de Corral/genética , Genes Virales , Proteína HN/genética , Virus de la Enfermedad de Newcastle/genética , Recombinación Genética , Animales , Anticuerpos Monoclonales/inmunología , Pollos/genética , Pollos/microbiología , Clonación Molecular , Técnicas de Cultivo , Vectores Genéticos , Proteína HN/biosíntesis , Virus de la Enfermedad de Newcastle/enzimología , Regiones Promotoras Genéticas , Vacunas ViralesRESUMEN
In this paper we report the development and testing of a fowlpox virus vector system. Insertion sites in non-essential regions within the terminal inverted repeats of the virus have been characterised. Foreign genes inserted into these sites are shown to be present in two copies in the resultant recombinant virus. To test the potential use of this vector as a live vaccine the fusion gene of Newcastle disease virus (NDV) has been inserted into a vaccine strain of fowlpox virus, and inoculated into chickens. The experiments demonstrate the ability of the recombinant to protect chickens against challenge by a virulent strain of NDV and to elicit the formation of anti-fusion protein antibody.
Asunto(s)
Pollos , Virus de la Viruela de las Aves de Corral/genética , Vectores Genéticos , Enfermedad de Newcastle/prevención & control , Virus de la Enfermedad de Newcastle/genética , Vacunas Virales , Animales , Anticuerpos Antivirales/análisis , Western Blotting , ADN Viral/análisis , Regulación Viral de la Expresión Génica , Virus de la Enfermedad de Newcastle/inmunología , Plásmidos , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Vacunación/veterinaria , Vacunas SintéticasRESUMEN
In this paper we report on the identification of non-essential genes in the terminal repeats of the avipox-virus fowlpox virus and the use of these as insertion sites in a vector system. Foreign genes inserted into these sites are shown to be present in two copies in the resultant recombinant virus. To test the potential use of this vector as a live vaccine the fusion gene of Newcastle disease virus has been inserted into a vaccine strain of fowlpox virus and inoculated into chickens. The experiments demonstrate the ability of the recombinant to protect chickens against challenge by a virulent strain of Newcastle disease virus and to elicit the formation of an anti-fusion protein antibody.
Asunto(s)
Clonación Molecular , Elementos Transponibles de ADN , Virus de la Viruela de las Aves de Corral/genética , Genes Virales , Inmunidad , Virus de la Enfermedad de Newcastle/genética , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Western Blotting , Línea Celular , Pollos , Ensayo de Inmunoadsorción Enzimática , Virus de la Viruela de las Aves de Corral/inmunología , Vectores Genéticos , Pruebas de Inhibición de Hemaglutinación , Virus de la Enfermedad de Newcastle/inmunología , Plásmidos , Mapeo RestrictivoRESUMEN
A panel of eight neutralizing monoclonal antibodies (MAbs) against the fusion (F) protein of Newcastle disease virus (NDV) has been shown to locate a major antigenic site on the basis of competitive binding assay and additivity index studies. Five epitopes (A1 to A5) have been located within this site on the F protein of the Beaudette C strain of NDV on the basis of cross-resistance plaque assays of MAb-resistant mutants raised against these MAbs. Epitopes A1, A4 and A5 are distinct; epitope A2 partially overlaps epitope A3. Nucleotide sequence analysis of the F genes of MAb-resistant mutants showed that each predicted single amino acid substitutions ranging from amino acid residues 157 to 171 for epitope A4 and at residues 72, 78, 79 and 343 for epitopes A1, A2, A3 and A5 respectively. These locations indicate that both the F1 and F2 fragments are involved in the formation of a single antigenic site and suggest the involvement of extensive protein folding in the active form of this F protein.