RESUMEN
Genetic analysis of selected genome regions of hepatitis A Virus (HAV) suggested that distinct genotype could be defined in different geographic locations. In order to study the degree of genetic variability among HAV isolated during a single epidemic outbreak, sequences from a 148 base pair segment within the VP1 amino terminal region were obtained for eight distinct HAV isolates from an outbreak that occurred in North Bretagne (France). These sequences were compared among themselves and with published sequences from 30 different strains that represented different HAV sub-genotypes that were isolated all over the world. Phylogenetic analysis revealed an extensive genetic heterogeneity among strains belonging to the same outbreak and revealed co-circulation of sub-genotype IA, IB, and the presence of IIIA sub-genotype for the first time in a Mediterranean country.
Asunto(s)
Brotes de Enfermedades , Hepatitis A/epidemiología , Hepatovirus/genética , Adulto , Secuencia de Bases , Clonación Molecular , Femenino , Francia/epidemiología , Variación Genética , Genotipo , Hepatovirus/química , Hepatovirus/clasificación , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Alineación de Secuencia , Análisis de Secuencia de Proteína , Proteínas Estructurales Virales/genéticaRESUMEN
Palmitoyl-VP3(110--121) (PVP3) is a synthetic lipopeptide derivative of a continuous epitope from the VP3 capsid protein of hepatitis A virus, and it is highly immunogenic in vivo. We have investigated the interaction of PVP3 with lipid model membranes of varying surface charge. Binding of PVP3 to anionic vesicles of PC/SM/PE/PS; (PC) 1-palmitoyl-2-oleoyl-phosphatidylcholine, (SM) sphingomyelin, (PE) 1,2-dipalmitoyl-phosphatidylethanolamine and (PS) L-alpha-phosphatidyl-L-serine, a composition that mimics the lipid component of natural membranes, was determined by tryptophan fluorescence and quenching experiments. In addition, and given the anionic net charge of the lipopeptide, binding to zwitterionic (PC/SM/PE) and cationic PC/SM/PE/DOTAP (DOTAP) 1,2-dioleoyl-3-trimethylammonium-propane mixtures was also determined. PVP3 binds to all three types of vesicles, but it adopts different forms depending on the electrical charge of the interface. This conclusion is supported by the insertion of PVP3 into lipid monolayers of the same charges spread at the air-water interface. The bound lipopeptide has membrane-destabilizing effects in all three vesicle compositions, as demonstrated by leakage of vesicle contents, whereas lipid mixing only occurs in cationic liposomes. Our results provide useful information for the design of a liposomal system that promotes a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, potentially increasing the immune response from the host.
Asunto(s)
Cápside/química , Epítopos/farmacología , Hepatovirus/química , Lipoproteínas/química , Fusión de Membrana/efectos de los fármacos , Transferencia de Energía , Espectrometría de Fluorescencia , Termodinámica , Triptófano/químicaRESUMEN
A synthetic peptide with the sequence [Lys113]VP3(110--121): FWRKDLVFDFQV, corresponding to an epitope of the VP3 capsid protein of hepatitis A virus (HAV), was synthesized by solid phase and characterized. To obtain insight into its physicochemical properties and to understand its possible mechanism of action at the membrane level, interaction with DPPC or DPPC/DPPG (95/5) liposomes and lipid monolayers of DPPC, DPPG, SA, PS, PA and SM were studied by fluorescence spectroscopy and Langmuir--Blotgett films technique, respectively. Fluorescence studies showed that the peptide was in a hydrophobic environment when DPPC liposomes were used. The addition of a 5% of a charged lipid, DPPG, to the preparations changed the preference of the peptide towards a polar surrounding. However, the peptide had a high surface activity (nmol/L) and was able to incorporate into lipid monolayers. Interaction was higher with charged phospholipids than with neutral ones. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.
Asunto(s)
Cápside/química , Hepatovirus/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Cinética , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
The present study was undertaken to examine the structural features of two peptide constructs designed on the basis of linear combination of B and T-cell epitopes in different orientations (BT and TB) that may be important to explain the differences in the elicited antihepatitis A virus immune response and in the interaction with biological model membranes. A CD study was carried out and the corresponding quantitative analysis of the experimental data was done using deconvolution computer programs. Moreover, fluorescence experiments were performed to analyze differences in the fluorescence emission spectra of both molecules. The main conformational difference by CD studies was obtained working in aqueous medium. Although the TB sequence adopted a preferably random coil structure, the BT peptide was best fitted with beta-type structures. These results are further supported by fluorescence studies. These findings have relevance for the design of synthetic immunopeptides.
Asunto(s)
Antígenos Virales/química , Epítopos/química , Hepatovirus/química , Hepatovirus/inmunología , Secuencia de Aminoácidos , Antígenos Virales/genética , Linfocitos B/inmunología , Dicroismo Circular , Epítopos/genética , Hepatovirus/genética , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Linfocitos T/inmunología , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Shorter analogues of a continuous epitope of hepatitis A virus, VP3(110-121) peptide, failed to react with convalescent sera, indicating the importance of the entire peptide in the epitope structure. To better understand the influence of the structural properties of this 12-mer peptide epitope on its biological activity, the interaction of smaller peptide analogues with phospholipid biomembrane models was investigated by a combination of spectroscopic and biophysical techniques. In this article we describe our findings concerning the surface activity and the interaction of peptides with simple mono- and bilayer membranes composed of a zwitterionic phospholipid (dipalmitoyl phosphatidylcholine, DPPC), an anionic phospholipid (dipalmitoyl phosphatidylglicerol, DPPG), or a DPPC/DPPG mixture. The results indicate that the net negative charge of the peptide is in some way responsible of the specific interactions between VP3(110-121) and membrane phospholipids, and necessary to induce beta-type conformations upon vesicle interaction.
Asunto(s)
Proteínas de la Cápside , Cápside/química , Membrana Dobles de Lípidos/metabolismo , Fragmentos de Péptidos/química , Péptidos/química , Cápside/síntesis química , Cápside/metabolismo , Hepatovirus/química , Membrana Dobles de Lípidos/química , Liposomas/química , Liposomas/metabolismo , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Péptidos/síntesis química , Péptidos/metabolismo , Fosfolípidos/metabolismo , Fosfolípidos/farmacología , Unión Proteica , Conformación Proteica/efectos de los fármacos , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
Dense, RNase-sensitive, VP2-containing, non-infectious hepatitis A virus (HAV) particles were found to be formed at early times after the infection of cultured cells. These particles formed with kinetics mirroring those reported for HAV uncoating. The kinetics of the formation of dense HAV particles corresponded to a decrease in detectable, mature input virions, as detected by RNA dot blot hybridization of CsCl density gradient fractions. The dense HAV particles did not appear to have altered sedimentation coefficients, and as the fate of small capsid protein VP4 is not yet known, these particles cannot yet be termed 'A particles' or 'infectosomes', as have the uncoating intermediates in some picornavirus-cell systems.
Asunto(s)
Cápside/metabolismo , Hepatovirus/fisiología , ARN Viral/análisis , Virión/fisiología , Animales , Calcio/farmacología , Línea Celular , Centrifugación por Gradiente de Densidad , Hepatovirus/química , Concentración de Iones de Hidrógeno , Cinética , Hibridación de Ácido Nucleico , Virión/química , Replicación ViralRESUMEN
The interaction of the multiple antigenic peptide MAP4VP3 with lipid membranes has been studied by spectroscopic techniques. MAP4VP3 is a multimeric peptide that corresponds to four units of the sequence 110-121 of the capsid protein VP3 of hepatitis A virus. In order to evaluate the electrostatic and hydrophobic components on the lipid-peptide interaction, small unilamelar vesicles of different compositions, including zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic dipalmitoylphosphatidylcholine/phatidylinositol (DPPC:PI 9:1), and cationic dipalmitoylphosphatidylcholine/stearylamine (DPPC:SA 9.5:0.5), were used as membrane models. Intrinsic tryptophan fluorescence changes and energy transfer experiments show that MAP4VP3 binds to all three types of vesicles with the same stoichiometry, indicating that the electrostatic component of the interaction is not important for binding of this anionic peptide. Steady-state polarization experiments with vesicles labeled with 1,6-diphenyl-1,3,5-hexatriene or with 1-anilino-8-naphtalene sulphonic acid indicate that MAP4VP3 induces a change in the packing of the bilayers, with a decrease in the fluidity of the lipids and an increase in the temperature of phase transition in all the vesicles. The percentage of lipid exposed to the bulk aqueous phase is around 60% in intact vesicles, and it does not change upon binding of MAP4VP3 to DPPC vesicles, indicating that the peptide does not alter the permeability of the membrane. An increase in the amount of lipid exposed to the aqueous phase in cationic vesicles indicates either lipid flip-flop or disruption of the vesicles. Binding to DPPC vesicles occurs without leakage of entrapped carboxyfluorescein, even at high mol fractions of peptide. However, a time-dependent leakage is seen with cationic DPPC/SA and anionic DPPC/PI vesicles, indicating that the peptide induces membrane destabilization and not lipid flip-flop. Resonance energy transfer experiments show that MAP4VP3 leakage from cationic vesicles is due to membrane fusion, whereas leakage from anionic vesicles is not accompanied by lipid mixing. Results show that MAP4VP3 interacts strongly with the lipid components of the membrane, and although binding is not of electrostatic nature, the bound form of the peptide has different activity depending on the membrane net charge; thus, it is membrane disruptive in cationic and anionic vesicles, whereas no destabilizing effect is seen in DPPC vesicles.
Asunto(s)
Antígenos Virales/química , Cápside/química , Hepatovirus/química , Lípidos/química , Antígenos Virales/inmunología , Cápside/inmunología , Proteínas de la Cápside , Hepatovirus/inmunología , Membranas Artificiales , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunologíaRESUMEN
To establish correlation between structural properties (charge, composition, and conformation) and membrane penetration capability, the interaction of epitope peptide-carrier constructs with phospholipid model membranes was studied. For this we have conjugated a linear epitope peptide, (110)FWRGDLVFDFQV(121) (110-121), from VP3 capside protein of the Hepatitis A virus with polylysine-based branched polypeptides with different chemical characteristics. The epitope peptide elongated by one Cys residue at the N-terminal [C(110-121)] was attached to poly[Lys-(DL-Ala(m)()-X(i)())] (i < 1, m approximately 3), where x = ø(AK), Ser (SAK), or Glu (EAK) by the amide-thiol heterobifunctional reagent, 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester. The interaction of these polymer-[C(110-121)] conjugates with phospholipid monolayers and bilayers was studied using DPPC and DPPC/PG (95/5 mol/mol) mixture. Changes in the fluidity of liposomes induced by these conjugates were detected by using two fluorescent probes 1,6-diphenyl-1,3, 5-hexatriene (DPH) and sodium anilino naphthalene sulfonate (ANS). The binding of conjugates to the model membranes was compared and the contribution of the polymer component to these interactions were evaluated. We found that conjugates with polyanionic/EAK-[C(110-121)] or polycationic/SAK-[C(110-121)], AK-[C(110-121)]/character were capable to form monomolecular layers at the air/water interface with structure dependent stability in the following order: EAK-[C(110-121)] > SAK-[C(110-121)] > AK-[C(110-121)]. Data obtained from penetration studies into phospholipid monolayers indicated that conjugate insertion is more pronounced for EAK-[C(110-121)] than for AK-[C(110-121)] or SAK-[C(110-121)]. Changes in the fluorescence intensity and in polarization of fluorescent probes either at the polar surface (ANS) or within the hydrophobic core (DPH) of the DPPC/PG liposomes suggested that all three conjugates interact with the outer surface of the bilayer. Marked penetration was documented by a significant increase of the transition temperature only with the polyanionic compound/EAK-[C(110-121)]. Taken together, we found that the binding/penetration of conjugates to phospholipid model membranes is dependent on the charge properties of the constructs. Considering that the orientation and number of VP3 epitope peptides attached to branched polypeptides were almost identical, we can conclude that the structural characteristics (amino acid composition, charge, and surface activity) of the carrier have a pronounced effect on the conjugate-phospholipid membrane interaction. These observations suggest that the selection of polymer carrier for epitope attachment might significantly influence the membrane activity of the conjugate and provide guidelines for adequate presentation of immunogenic peptides to the cells.
Asunto(s)
Proteínas de la Cápside , Cápside/química , Proteínas Portadoras/química , Epítopos/química , Hepatovirus/química , Inmunoconjugados/química , Fragmentos de Péptidos/química , Péptidos/química , Fosfolípidos/química , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Secuencia de Aminoácidos , Naftalenosulfonatos de Anilina , Cápside/síntesis química , Cápside/inmunología , Cápside/metabolismo , Proteínas Portadoras/síntesis química , Proteínas Portadoras/metabolismo , Reactivos de Enlaces Cruzados/química , Disulfuros/química , Epítopos/metabolismo , Colorantes Fluorescentes , Hepatovirus/inmunología , Inmunoconjugados/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Liposomas , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Fosfolípidos/metabolismo , Propiedades de SuperficieRESUMEN
The RNA genome of hepatitis A virus (HAV) shares common characteristics of the picornavirus family. However, the nucleotide or amino acid sequences are distantly related with other members of the family. Like other picornaviruses, HAV proteins are cleaved from a large polyprotein (PO), but the processing and some products are quite different. The 3C protein is the sole processing enzyme, and the primary cleavage takes place at the 2A/2B site. Several VP1-2A sites are proposed. In some strains, the intermediate VP1-2A polypeptides are assembled in the virion. The VP4 is very small and not detected in the mature virion. Some mutations in 2B, 2C and 3A proteins are identified to enhance viral replication or to induce cytopathogenic effects in the viruses adapted to cell cultures.
Asunto(s)
Hepatovirus/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Genoma Viral , Hepatovirus/genética , Humanos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Proteínas Virales/químicaRESUMEN
Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1.
Asunto(s)
Cápside/química , Hepatovirus/química , Proteínas Virales , Proteínas Estructurales Virales/química , Proteasas Virales 3C , Secuencia de Aminoácidos , Animales , Cápside/genética , Cápside/metabolismo , Línea Celular , Chlorocebus aethiops , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Hepatovirus/genética , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Péptidos , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Conejos , Análisis de Secuencia , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismoRESUMEN
Low pH values encountered during uptake of viruses by receptor-mediated endocytosis have been shown to expose hydrophobic residues of many viruses and result in viral conformational changes leading to uncoating of the viral genome. An assay for hydrophobicity utilising the non-ionic detergent Triton X-114 was established, making use of metabolically-labelled hepatitis A virus (HAV). In this assay, hydrophilic proteins interact with the aqueous (buffer) phase, while hydrophobic proteins interact with the Triton (detergent) phase. HAV particles interact with the aqueous phase at neutral pH, whereas, under acidic conditions, HAV was found predominantly in the detergent phase. This indicates that the capsid of HAV undergoes conformational changes rendering the particle more hydrophobic under acidic conditions. A further two conformational changes were found in HAV on exposure to low pH, as detected by changes in buoyant density in CsCl gradients. These were maturation of provirions to virions and the formation of dense particles. These results may have implications for uncoating of the HAV RNA genome, and these conformational changes could represent intermediates in the viral uncoating process.
Asunto(s)
Ácidos/farmacología , Cápside/química , Hepatovirus/química , Cápside/efectos de los fármacos , Endocitosis , Hepatovirus/efectos de los fármacos , Hepatovirus/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Modelos Biológicos , Conformación ProteicaRESUMEN
The complete RNA genome of avian encephalomyelitis virus (AEV) has been molecularly cloned and sequenced. This revealed AEV to be a member of the Picornaviridae and consequently it is the first avian picornavirus for which the genome has been sequenced. Excluding the poly(A) tail the genome comprises 7032 nucleotides, which is shorter than that of any mammalian picornavirus sequenced to date. An open reading frame commencing at nucleotide 495 and terminating at position 6896 (6402 nucleotides) potentially encodes a polyprotein of 2134 amino acids. The polyprotein sequence has 39% overall amino acid identity with hepatitis A virus (HAV; genus Hepatovirus), compared to 19 to 21% for viruses from the other five picornavirus genera. Eleven cleavage products were predicted. The highest identity (49%) with HAV was in the P1 region, encoding the capsid proteins. The 5' and 3' untranslated regions (UTRs) comprise 494 and 136 nucleotides, respectively. The 5' UTR is the shortest of any picornavirus sequenced to date and, unlike HAV, it does not contain a long polypyrimidine tract.
Asunto(s)
Aves/virología , Virus de la Encefalomielitis Aviar/genética , Hepatovirus/genética , Picornaviridae/genética , Secuencia de Aminoácidos , Animales , Cápside/química , Cápside/genética , Clonación Molecular , Virus de la Encefalomielitis Aviar/química , Virus de la Encefalomielitis Aviar/clasificación , Genoma Viral , Hepatovirus/química , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Filogenia , Picornaviridae/química , Picornaviridae/clasificación , Procesamiento Proteico-Postraduccional , Proteínas/química , Proteínas/genética , ARN Viral/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Regiones no Traducidas/genéticaRESUMEN
The present study was undertaken to examine the structural features that may be important to explain the immunogenicity of the (110-121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus. A conformational analysis of the preferred conformations by CD and molecular mechanics was carried out. Present results suggest that the interaction with liposomes as biomembrane model induces and stabilizes the amphipathic beta-structure of the peptide. To study the contribution of amino acid replacements at the RGD tripeptide as well as the influence of the peptide chain length on peptide conformation, solid-phase peptide synthesis of several peptide analogs was carried out and the peptide conformation was studied using CD spectroscopy. The results show that the RGD sequence is necessary to induce the beta-structure in the presence of liposomes.
Asunto(s)
Proteínas de la Cápside , Cápside/química , Hepatovirus/química , Fragmentos de Péptidos/química , Dicroismo Circular , Computadores , Liposomas/metabolismo , Modelos Moleculares , Conformación Molecular , Oligopéptidos/química , Estructura Secundaria de Proteína , Dodecil Sulfato de Sodio/farmacologíaRESUMEN
Although detection of hepatitis A virus (HAV) has been greatly aided by the development of polymerase chain reaction (PCR) technology, identification of genetic variants requires sequencing PCR products, which necessarily limits the length of the HAV genome (typically 2%) that can be analyzed. From a regulatory standpoint, identification of the specific strain detected by PCR is a prerequisite not only to overrule contamination of test samples in the diagnostic laboratory, but also to possibly locate the origin of the virus detected by PCR. We explored alternatives to sequencing PCR products to achieve these goals. The findings indicate that restriction fragment length polymorphism (RFLP) analysis of PCR products from two noncontiguous regions of the HAV genome encompassing 765 nucleotides (approximately 10% of the genome) by the restriction endonucleases HinfI and AluI, which cut frequently within the HAV genome, can distinguish the common tissue culture adapted strains of HAV from stool isolates. The resolution can be greatly enhanced by combining single strand conformation polymorphism (SSCP) analysis with restriction enzyme digestion, when most of the seventeen strains analyzed could be identified.
Asunto(s)
Hepatovirus/genética , ADN Viral/química , ADN Viral/aislamiento & purificación , Variación Genética , Hepatovirus/química , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Auto immune haemolytic anaemia has been described in association with a variety of hepatotropic viruses, in particular cytomegalovirus, Epstein-Barr virus and hepatitis B. There is a well-recognized association between chronic active hepatitis and auto immune haemolytic anaemia. We present the first reported case of acute hepatitis A which resulted in a fall in haemoglobin concentration from 14.6 to 4.5 g/dl due to an acute haemolytic anaemia with an associated rise in bilirubin from 149 to 960 mumol/l.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Anemia Hemolítica Autoinmune/virología , Hepatitis A/complicaciones , Hepatitis A/inmunología , Enfermedad Aguda , Anemia Hemolítica Autoinmune/sangre , Hepatitis A/sangre , Hepatovirus/química , Hepatovirus/inmunología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Hepatitis A virus (HAV) is composed mainly of three structural capsid proteins: VP1, VP2 and VP3. Our group has reported the synthesis and the immunogenic evaluation of VP3 (110-121) peptide sequence. In the present work, in order to stimulate a T-cell immune response, we have selected the HAV-VP3 (102-121) peptide which has maximum amphipathicity. Its synthesis was carried out manually in the solid phase and semipreparative HPLC was used for purification of the crude peptide. Finally the purified peptide was characterized by analytical HPLC, amino acid analysis and MS. A palmitoyl derivative of VP3 (102-121) was synthesized to modify the hydrophobicity of the peptide. Both free and lipophilically derivatized peptides were incorporated into multilamelar liposomes. Physicochemical studies of the HAV-related peptides described above were carried out using monolayers as membrane models. Compression isotherms, surface activity and penetration kinetics into dipalmitoylphosphatidylcholine monolayers were determined. Moreover, changes in the fluidity of bilayers induced by these peptides were determined by means of polarizable probes such as 8-anilino-1-naphthalenesulfonic acid and 1,6-diphenyl-1,3,5-hexatriene. The integrity of the membranes has also been ascertained with the carboxyfluorescein.
Asunto(s)
Hepatovirus/química , Proteínas Virales/química , Secuencia de Aminoácidos , Cápside/química , Polarización de Fluorescencia , Liposomas , Fluidez de la Membrana , Datos de Secuencia Molecular , Solubilidad , TermodinámicaRESUMEN
The RNA genome of hepatitis A virus (HAV) encodes a giant polyprotein that is putatively cleaved proteolytically into four structural and seven non-structural proteins. So far, most of the proposed non-structural proteins and their respective cleavage sites have not been identified. A vaccinia virus recombinant (vRGORF) containing the complete HAV ORF under the control of the bacteriophage T7 promoter was used to express HAV in recombinant animal cells (BT7-H) that constitutively expressed T7 DNA-dependent RNA polymerase. A HAV-specific 27.5 kDa expression product was identified as peptide 2B. The 27.5 kDa 2B antigen was also found in HAV-infected MRC-5 cells. The N-terminal amino acid residues of the new peptide 2B are Ala-Lys-Ile-Ser-Leu-Phe and polyprotein cleavage between 2A and 2B occurred at amino acids 836-837 (Gln-Ala). Furthermore, heterologous expression in the same system of regions P1-P2 and of the protease 3C (3Cpro) gene, showed that P1-P2 polyprotein is not cleaved autocatalytically but by 3Cpro. Hence, 3Cpro is effective in cleaving the polyprotein 2A-2B junction.
Asunto(s)
Cisteína Endopeptidasas/farmacología , Hepatovirus/química , Proteínas no Estructurales Virales/análisis , Proteínas Virales , Proteasas Virales 3C , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sueros Inmunes/inmunología , Datos de Secuencia Molecular , Conejos , Virus Vaccinia/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/inmunologíaRESUMEN
The purification and protein characterization of one of the Cuban isolated strains of hepatitis A virus was carried out. For this, it was necessary to separate the virus from the infected cell by extraction steps with detergents, concentration by ultrafiltration and finally, ultracentrifugation in saccharosoglycerol discontinuous gradient. Protein concentration, as well as the antigenic activity in the different fractions of the gradient were determined. For the protein characterization of the microorganism, those fractions with the greatest specific activity were analyzed by polyacrylamide gel electrophoresis and by Western blotting. It was shown that the viral material was purified and concentrated in the last fractions of the gradient. Bands corresponding to the structural proteins of hepatitis A were observed through electrophoresis and Western blotting.
Asunto(s)
Hepatovirus/aislamiento & purificación , Proteínas Virales/aislamiento & purificación , Western Blotting , Electroforesis en Gel de Poliacrilamida , Hepatovirus/química , HumanosRESUMEN
The junction between 2A and 2B proteins of the hepatitis A virus (HAV) polyprotein is processed by the virus-encoded 3C protease to liberate the precursor for capsid proteins, but details of this cleavage remain poorly defined. We identified the location of this primary cleavage by a novel approach involving expression of HAV polypeptides in eukaryotic cells via recombinant vaccinia viruses. A substrate polyprotein spanning the putative HAV 2A/2B site was fused at its C-terminus to a poliovirus VP1 reporter sequence. This substrate was cleaved efficiently in trans by protease 3C derived from another recombinant vaccinia virus expressing a 3C precursor protein. N-terminal sequencing of the 2B-poliovirus VP1 fusion product identified the site of cleavage as the Gln836/Ala837 dipeptide, 144 residues upstream of the originally predicted site. Two mutations were introduced at the P1 position of the 2A/2B site. Gln836-->Asn, and Gln836-->Arg. Asn substitution at the P1 residue reduced the efficiency of cleavage in the vaccinia expression system and resulted in a small replication focus phenotype of virus rescued from infectious HAV RNA transcripts. Arg substitution abolished cleavage and was lethal to HAV replication. In addition to identifying the site of the primary HAV polyprotein cleavage, these results shed light on the in vivo specificities of the HAV 3C protease.