RESUMEN
HIV-1 virions are as immature noninfectious particles lacking a central core. Shortly after budding, virions temporally mature and acquire cores and infectious activity. The cause of maturation remains poorly studied. We have revealed that the virions produced early after infection following 24-36 hours, never mature and remain noninfectious, and only virions produced 48-72 hours after infection mature. The mature virions contain 3 times more genomic viral RNA than "early" virus. The "early" virions contain the same proteolytically cleaved Gag proteins as mature virions in contrast to the accepted version. The virus protease inhibitor Indinavir sulfate (IS) fully blocks infectivity when added early after infection. The early proteolysis of Gag precursor in the infected cells and inclusion into the virions of cellularly cleaved matrix protein (cMA) are shown in the IS-treated cells. cMA is associated with genomic viral RNA.
Asunto(s)
Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Precursores de Proteínas/metabolismo , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Inhibidores de la Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/patogenicidad , Humanos , Indinavir/metabolismo , Indinavir/farmacología , Virulencia/efectos de los fármacos , Esparcimiento de VirusRESUMEN
The epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom, its related occurrence of a new type of Creutzfeld-Jacob disease and proven cases of this type of the disease transmitted by blood transfusion initiated intensive studies to develop a inexpensive, prompt, and sensitive method for the early lifetime diagnosis of prion diseases. This would permit initiation of the timely treatment of the patients and prevention of contamination of foodstuffs. However, despite significant progress made in this direction, this objective has not yet been achieved. The present review highlights the currently available methods for the diagnosis of transmissible spongiform encephalopathies, as well as the latest developments in the ultrasensitive detection of these diseases, which is based on the misfolded prion protein complex.
Asunto(s)
Enfermedades por Prión/diagnóstico , Priones/análisis , Animales , Biomarcadores/análisis , Biomarcadores/sangre , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Bovinos , Células Cultivadas , Síndrome de Creutzfeldt-Jakob/diagnóstico , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patología , Humanos , Immunoblotting , Microscopía Fluorescente , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Priones/sangre , Priones/metabolismoRESUMEN
Full-length Bos taurus PrPC protein was obtained in the eu- and prokaryotic expression systems. Immunoblotting and indirect enzyme immunoassay demonstrated high specificity and antigenic activity of full-length proteins in the reactions with monoclonal antibodies (anti-SAF-32 and VRQ-84). Membrane location of recombinant PrPC protein in insect cells was shown by immunofluorescent analysis.
Asunto(s)
Proteínas PrPC/biosíntesis , Proteínas Recombinantes/biosíntesis , Animales , Anticuerpos Monoclonales/inmunología , Antígenos/análisis , Baculoviridae/genética , Bovinos , Membrana Celular/química , Proteínas PrPC/análisis , Proteínas PrPC/inmunología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunologíaRESUMEN
Summarized in the paper are data on the molecular bases of transmissible spongoid encephalopathy (TSE), its etiology, formation mechanism of prion proteins, STE-related strains, specific barriers to TSE, its pathogenesis and treatment.
Asunto(s)
Enfermedades por Prión/etiología , Enfermedades por Prión/veterinaria , Priones/fisiología , Animales , Sistema Nervioso Central/química , Sistema Nervioso Central/metabolismo , Transmisión de Enfermedad Infecciosa , Humanos , Mutación , Enfermedades por Prión/terapia , Priones/genética , Priones/aislamiento & purificaciónAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , Diseño de Fármacos , Infecciones por VIH/tratamiento farmacológico , VIH-1 , VIH-2 , Síndrome de Inmunodeficiencia Adquirida/virología , Quimioterapia Combinada , Infecciones por VIH/virología , VIH-1/clasificación , VIH-2/clasificación , HumanosRESUMEN
L-3,4-Dihydroxyphenylalanine (DOPA) and dopamine encapsulated in liposomes have been obtained. These preparations can be used as drugs for Parkinson's disease. The level of encapsulation of the compounds under study in liposomes and their partition coefficients have been determined. The kinetics of DOPA (or dopamine) exit from the liposomes have been studied.
Asunto(s)
Dihidroxifenilalanina/administración & dosificación , Dopamina/administración & dosificación , Fenómenos Químicos , Química Física , Portadores de Fármacos , LiposomasRESUMEN
The synthesis of gag antigens of human immunodeficiency virus (HIV) by recombinant vaccinia virus strains containing the expressed genes gag and gag-pol and the capacity of these proteins to formation of virus-like particles during infection of various cell cultures were studied. The recombinant strain containing the truncated gag gene (p48gag) was shown to effectively synthesize gag polypeptides and to form immature virus-like particles during infection of all the cell cultures tested (CV-1, Hep-2, HT-29). The morphogenesis of mature virus-like particles was detected by electron microscopy only in infection of Hep-2 cells with a strain containing a complete gag-pol sequence of HIV.
Asunto(s)
Productos del Gen gag/genética , VIH-1/genética , Virus Vaccinia/genética , Virión , Animales , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Proteínas de Fusión gag-pol/genética , VIH-1/inmunología , Humanos , Microscopía Electrónica , Recombinación Genética , Células Tumorales CultivadasRESUMEN
Influenza viruses A, B, and C belonging to Orthomyxoviridae comprise an internal ribonucleoprotein (RNP) and an outer lipoprotein envelope with surface spike glycoproteins and the M1 protein matrix. The lipoprotein envelope and spike glycoproteins are solubilized by nonionic detergent treatment in a pH-independent manner. In contrast, disassembly of the M1 protein matrix appears to depend on pH. Treatment of influenza C viruses with nonionic detergent in neutral or alkaline medium (pH 9.0-7.2) results in disintegration of the virion M1 matrix and leads to a significant release of RNP free of the M1 protein. In acidic medium (pH 6.0-5.0) the M1 matrix fails to be removed and the viral core-like complex of RNP along with the M1 matrix cover is released. Since influenza A and B viruses were characterised by acid-dependent disassembly of the virion M1 matrix, influenza C viruses seem to be more resemble the paramyxoviruses, which also show a neutral-alkaline pH dependence of the matrix disintegration. These observations suggest that uncoating of influenza C viruses and paramyxoviruses in target cells may have similar events.
Asunto(s)
Gammainfluenzavirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Concentración de Iones de Hidrógeno , Gammainfluenzavirus/ultraestructura , Microscopía Electrónica , Ribonucleoproteínas/metabolismoRESUMEN
The relation of the initial products of the HIV-1 gag gene to the final products was determined in virus samples and cell fractions of infected H9 and Jurkat-tat cell cultures. The proteins were identified by immunoblotting with pooled sera from AIDS patients or monoclonal antibodies. The proportion in the virions of gag precursor proteins and the products of their proteolytic cleavage varied according to the maturity of the virus particles as determined by electron microscopy. The distribution of viral gag proteins in the cell fractions was determined 2, 4, and 24 h after infection. Treatment of cells with cycloheximide to block de novo protein synthesis did not significantly affect the results. Gag proteins containing the N terminus of the precursor p55 (including p55, the intermediate precursors p41(45) and p39, and mature protein p17) were found in the cell nuclei up to 24 h after infection. The major core protein p24 was located in the cytoplasmic fraction. These data strongly suggest that gag precursors from the p55 N terminus and the matrix protein p17 enter the infected cell separately from the major core protein p24, or become separated from it in the cytoplasm.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/metabolismo , Productos del Gen gag/análisis , VIH-1/química , Proteínas Virales , Virión/química , Células Cultivadas , Productos del Gen gag/inmunología , Antígenos VIH/análisis , Antígenos VIH/inmunología , Proteína p24 del Núcleo del VIH/química , VIH-1/crecimiento & desarrollo , VIH-1/ultraestructura , Humanos , Fracciones Subcelulares/química , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
Monkey kidney cells CV-1 were infected with recombinant vaccinia virus carrying HIV-1 gag gene with a deletion of 230 nucleotide pairs from the 3'-terminus. The main gene product detected in the lysates of infected cells was the gag precursor rp50. The protein was accumulated on the cell membranes suggesting that it had a myristylated N-terminus, and was cleaved by a recombinant virus specific protease with the formation of two proteins, p17 and p24 corresponding in molecular masses to mature gag proteins. Virus-like particles similar to immature HIV virions were budding from the surface of infected cells. They look like the ring of optically dense material covered with a lipid bilayer, of the same size (100-120 nm) and of the same density in a sucrose gradient (1.16-1.18 g/ml) as HIV-1 virions. The particles contained rp50 and cellular heterogeneous RNA. Thus, the unprocessed gag precursor with deleted 77 amino acid residues from the C-terminus is able to form virus-like particles in the absence of env proteins and virus-specific RNA, and these particles are budding from the cell surface. The question about the use of extracellular Gag-particles for AIDS diagnostic work and construction of vaccines is discussed.
Asunto(s)
Regulación Viral de la Expresión Génica , Productos del Gen gag/genética , VIH-1/genética , Virus Vaccinia/genética , Virión , Northern Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Microscopía Electrónica , Microscopía Fluorescente , Hibridación de Ácido Nucleico , ARN Viral/análisis , Recombinación GenéticaRESUMEN
The results of a comparative study of the features of antigenic determinants of avid and non-avid variants of influenza virus hemagglutinin H3 and of the influence of the mode of antigen presentation on the degree of its avidity are presented. The avidity of influenza virus hemagglutinin was shown to be determined by the capacity of individual antigenic determinants to interaction with antibodies. The antigenic determinants of avid hemagglutinin possessing a high functional activity in interaction with antibodies may have the spatial configuration which does not change in different modes of the antigen presentation. Isolation of hemagglutinin from virions of non-avid virus variants may lead to increased functional activity of individual antigenic determinants (and the antigen molecule as a whole) probably due to an increased degree of exposure and/or complementarity of the determinants for active centres of antibody.
Asunto(s)
Afinidad de Anticuerpos , Hemaglutininas Virales/inmunología , Virus de la Influenza A/inmunología , Animales , Anticuerpos Monoclonales , Embrión de Pollo , Pruebas de Inhibición de Hemaglutinación , Pruebas de NeutralizaciónRESUMEN
A post-embedding technique for immunocytochemical analysis at the ultrastructural level was used to detect and localize HIV antigens on ultrathin sections of Lowicryl-embedded HIV-infected cells. A genomic probe containing ribosomal sequences and labeled with biotin was used to hybridize rRNA molecules in sections of animal cells embedded in Lowicryl. The method presently described offers the possibility to detect rapidly and precisely ribosomal gene expression and viral proteins at the ultrastructural level.
Asunto(s)
Antígenos VIH/análisis , VIH-1/inmunología , VIH-2/inmunología , ARN Ribosómico/análisis , Resinas Acrílicas , Animales , Sondas de ADN , Regulación de la Expresión Génica/genética , Humanos , Métodos , Hibridación de Ácido Nucleico , ARN Ribosómico/genética , TemperaturaRESUMEN
The principal possibility of using antibodies conjugated with colloid gold for better visualization of viruses, viral antigens, and their immune complexes in electron microscope was studied on the experimental model of HBs-antigen and Venezuelan equine encephalomyelitis virus. The same method was used for the study of the thin structure of influenza A (H1N1) virus hemagglutinins using monoreceptor antisera.
Asunto(s)
Antígenos Virales/análisis , Oro , Animales , Complejo Antígeno-Anticuerpo/análisis , Reacciones Antígeno-Anticuerpo , Coloides , Virus de la Encefalitis Equina Venezolana/inmunología , Antígenos de Superficie de la Hepatitis B/análisis , Humanos , Inmunohistoquímica , Virus de la Influenza A/inmunología , Ratones , Microscopía Electrónica , Proteínas Recombinantes/análisisRESUMEN
Specific multimer complexes of glycoproteins of enveloped viruses were prepared by treatment of suspensions of purified concentrated virus with a non-ionic detergent MECK mixed with 0.2% glycoside Quil A. Mixed complexes of glycoproteins with glycoside Quil A were formed upon removal of the detergent from the mixture of solubilized glycoproteins and glycoside Quil A. According to the results of electron microscopy, the formed complexes differed morphologically from the conventional micelles of glycoproteins and presented spherical reticular structures 20-30 nm in diameter. The immunogenic potency of the complexes was much higher than that of virus particles and micelles of purified glycoproteins and was comparable to the immunogenic potency of glycoproteins mixed with complete Freund adjuvant. The protective activity of the complex of protein G of rabies virus with glycoside Quil A was higher than that of the subunit rabies vaccine adsorbed on aluminium hydroxide.
Asunto(s)
Adyuvantes Inmunológicos , Glicoproteínas/aislamiento & purificación , Glicósidos/farmacología , Polímeros/aislamiento & purificación , Saponinas/farmacología , Proteínas del Envoltorio Viral/aislamiento & purificación , Animales , Anticuerpos Antivirales/análisis , Relación Dosis-Respuesta Inmunológica , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/análisis , Glicoproteínas/inmunología , Glicósidos/inmunología , Inmunización , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/inmunología , Virus de la Influenza A/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Micelas , Virus de la Parainfluenza 1 Humana/efectos de los fármacos , Virus de la Parainfluenza 1 Humana/inmunología , Virus de la Parainfluenza 1 Humana/aislamiento & purificación , Polímeros/análisis , Polímeros/inmunología , Saponinas de Quillaja , Virus de la Rabia/efectos de los fármacos , Virus de la Rabia/inmunología , Virus de la Rabia/aislamiento & purificación , Árboles , Proteínas del Envoltorio Viral/análisis , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The paper presents the results of generating influenza virus recombinants by hybridization of the laboratory A/PR8/34 strain with epidemic A/Philippines 2/82 virus and studies of a number of their biological properties. A highly temperature-sensitive recombinant with mutational damages in the hemagglutinin gene was detected.
Asunto(s)
Variación Genética , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza A/genética , Recombinación Genética , Animales , Antígenos Virales/análisis , Antígenos Virales/inmunología , Hemaglutininas Virales/análisis , Hemaglutininas Virales/inmunología , Virus de la Influenza A/inmunología , Virus de la Influenza A/ultraestructura , Ratones , Ratones Endogámicos CBA , Microscopía Electrónica , Mutación , Filipinas , Temperatura , Virión/ultraestructura , Replicación ViralRESUMEN
Endocytic vacuoles (receptosomes) containing influenza virus were isolated from the cytoplasm of Ehrlich ascitic carcinoma cells and characterized. In the sucrose density gradient, the virus-containing material was detected in two peaks with a buoyant density of 1.175-1.16 and 1.155-1.135 g/cm3 with which the activity of marker enzymes of cell plasma membranes was associated. The virus was present in receptosomes in morphologically and electrophoretically intact condition. Examinations for the lipid composition of endocytic vacuoles showed the presence in their membranes of large amounts of cholesterol and glycolipids, particularly asialo-GM1 which, according to some authors may enhance the fusion of viral and cell membranes.
Asunto(s)
Endocitosis , Virus de la Influenza A/patogenicidad , Organoides/microbiología , Vacuolas/microbiología , Animales , Carcinoma de Ehrlich/enzimología , Carcinoma de Ehrlich/microbiología , Carcinoma de Ehrlich/ultraestructura , Fraccionamiento Celular , Membrana Celular/análisis , Membrana Celular/enzimología , Membrana Celular/microbiología , Embrión de Pollo , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Endosomas/análisis , Endosomas/enzimología , Endosomas/microbiología , Glucolípidos/análisis , Lípidos/análisis , Microscopía Electrónica , Vacuolas/análisis , Vacuolas/enzimología , Cultivo de VirusRESUMEN
A method for incorporation of influenza virus surface proteins into monolayer liposomes has been developed on the basis of removal of dialysed non-ionic detergent (beta-octyl glucoside) from a mixture of viral proteins and lipids. The resulting lipid-protein complexes (virosomes) are similar to intact virus particles by their morphological parameters. The immunogenicity of hemagglutinin incorporated into virosomes was significantly higher than that in the preparation of surface proteins isolated from virions which was inoculated into mice with or without complete Freund's adjuvant. The high immunogenicity of hemagglutinin incorporated into liposomes correlated with their high hemagglutinating activity. The possibility of using virosomes as subunit influenza vaccines is discussed.
Asunto(s)
Virus de la Influenza A/inmunología , Liposomas/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Matriz Viral , Proteínas Virales/inmunología , Animales , Glicoproteínas/inmunología , Glicoproteínas/aislamiento & purificación , Hemaglutinación por Virus , Inmunización , Proteínas de la Membrana/aislamiento & purificación , Ratones , Microscopía Electrónica , Proteolípidos/inmunología , Factores de Tiempo , Proteínas Virales/aislamiento & purificación , Virión/inmunologíaRESUMEN
DNA from bacteriophage lambda or monkey adenovirus type 7 have been condensed with spermine and included into three types of liposomes: ethereal, obtained by inverted phases technique and by Ca++ fusion. Compact DNA form presents a tor with 100 nm external diameter and 430 nm width. It can be included into 100 nm or larger liposomes. Total preparation contains 15-18% of the required liposomes. Liposomal fractions with included DNA were separated from empty liposomes by step gradient of ficoll 400. Liposomal fraction having included DNA contains 15-18% of common lipid. Liposomal interaction with monkey cell cultures has been studied.
Asunto(s)
ADN Viral/efectos de los fármacos , Liposomas/análisis , Espermina/farmacología , Animales , Bacteriófago lambda/análisis , Línea Celular , Chlorocebus aethiops , ADN Viral/análisis , Conformación de Ácido Nucleico , Virus 40 de los Simios/análisisRESUMEN
Dissociation of influenza virus RNP under the effect of salt was studied. Separation of RNA and protein components of influenza virus RNP was shown to occur in a linear 15-30% sucrose concentration gradient containing 1.1 M NaCl. Upon RNP dissociation, protein-protein interactions between individual molecules of the structural protein were retained. The sedimentation coefficient of the protein component was 52S. The possibility of reassociation of the RNA-protein complex was studied. More complete reassociation was observed to occur in the presence of 0.1 M NaCl. The resulting RNA-protein complex morphologically is similar to the native RNP of influenza virus.