RESUMEN

Three subtypes of influenza A virus cause human disease: H1N1, H2N2, and H3N2. Although all result in respiratory illness, little is known about how these subtypes infect differentiated airway epithelia. Therefore, we assayed A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and X31 (H3N2) influenza virus strains for binding and infection on fully differentiated primary cultures of airway epithelia isolated from human bronchus, grown on semiporous filters at an air-liquid interface. In this model system, viral infectivity was highest when virus was applied to the apical versus the basolateral surface; Japan was most infectious, followed by PR8. The X31 strain showed very low levels of infectivity. Confocal microscopy and fluorescence-resonance energy transfer studies indicated that Japan virus could enter and fuse with cellular membranes, while infection with X31 virions was greatly inhibited. Japan virus could also productively infect human trachea explant tissues. These data show that influenza viruses with SAalpha2,3Gal binding specificity, like Japan, productively infect differentiated human airway epithelia from the apical surface. These data are important to consider in the development of pseudotyped recombinant viral vectors for gene transfer to human airway epithelia for gene therapy.

Asunto(s)

Bronquios/virología , Subtipo H1N1 del Virus de la Influenza A , Subtipo H2N2 del Virus de la Influenza A , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza A/fisiología , Tráquea/virología , Anticuerpos Antivirales/biosíntesis , Células Cultivadas , Endocitosis , Células Epiteliales/ultraestructura , Células Epiteliales/virología , Hemaglutinación por Virus/inmunología , Humanos , Virus de la Influenza A/inmunología , Fusión de Membrana , Modelos Biológicos , Proteínas Virales/biosíntesis , Replicación Viral

RESUMEN

BACKGROUND: Cell proliferation, vector titer and accessibility of target cells represent hurdles for efficient gene transfer to lung epithelia in vivo using recombinant murine leukemia (MuLV)-based retroviruses. We tested the hypothesis that the pulmonary epithelium is susceptible to retroviral-mediated gene transfer when stimulated to proliferate by a mitogen, keratinocyte growth factor (KGF). METHODS: Rats received keratinocyte growth factor (KGF, 2.5 micrograms/g x 4 doses, two consecutive days) intratracheally followed by high titer amphotropic retrovirus expressing beta-galactosidase. Gene transfer was assessed five days later. RESULTS: KGF stimulated transient proliferation in the bronchiolar and alveolar epithelia (30-40% PCNA positive cells at peak) which decreased to background levels seven days after administration. Gene transfer to epithelia (X-Gal positive cells) occurred more frequently in KGF treated rats, but proliferation exceeded the level of gene transfer. X-gal positive cells were noted in the alveolar epithelium and occasionally in the bronchiolar epithelium. In order to understand the discrepancy between the number of proliferating and transduced cells, primary rat tracheal epithelium cultured at the air-liquid interface was infected from either the apical or basolateral side. Gene transfer was achieved only through basolateral application of vector, suggesting that epithelial polarity represents a barrier to MuLV-based lung gene transfer in vivo. CONCLUSIONS: KGF transiently stimulates epithelial proliferation in vivo, facilitating MuLV-based gene transfer. Retroviral vectors may encounter multiple barriers which have evolved to defend the lung from infections.

Asunto(s)

Factores de Crecimiento de Fibroblastos , Técnicas de Transferencia de Gen , Sustancias de Crecimiento/farmacología , Virus de la Leucemia Murina/genética , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Animales , División Celular/efectos de los fármacos , Polaridad Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Vectores Genéticos , Humanos , Pulmón/citología , Ratones , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Virales/metabolismo , Proteínas Recombinantes/farmacología , beta-Galactosidasa/genética

RESUMEN

Gene transfer with recombinant murine leukemia viruses (MuLV) provides the potential to permanently correct inherited lung diseases, such as cystic fibrosis (CF). Several problems prevent the application of MuLV-based recombinant retroviruses to lung gene therapy: (i) the lack of cell proliferation in mature pulmonary epithelia, (ii) inefficient gene transfer with a vector applied to the apical surface, and (iii) low titers of many retroviral preparations. We found that keratinocyte growth factor (KGF) stimulated proliferation of differentiated human tracheal and bronchial epithelia. Approximately 50% of epithelia divided in response to KGF as assessed by bromodeoxyuridine histochemistry. In airway epithelia stimulated to divide with KGF, high-titer ampho- and xenotropic enveloped vectors preferentially infected cells from the basal side. However, treatment with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells expressed the transgene after gene transfer. By using this approach, cells throughout the epithelial sheet, including basal cells, were targeted. Moreover, the Cl- transport defect in differentiated CF airway epithelia was corrected. These findings suggest that barriers to apical infection with MuLV can be overcome.

Asunto(s)

Factores de Crecimiento de Fibroblastos , Técnicas de Transferencia de Gen , Virus de la Leucemia Murina/genética , Proteínas de Transporte de Fosfato , Simportadores , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/virología , Diferenciación Celular , División Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular , Polaridad Celular , Células Cultivadas , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Expresión Génica , Sustancias de Crecimiento/administración & dosificación , Humanos , Receptores Virales/genética , Receptores Virales/fisiología , Proteínas Cotransportadoras de Sodio-Fosfato , Tráquea/citología , Tráquea/efectos de los fármacos , Tráquea/virología

RESUMEN

Cationic liposome-mediated intracellular delivery of a fluorescein-labeled chimeric DNA-RNA ribozyme targeted to the HIV-1 5' LTR was investigated, using THP-1, THP-1/HIV-1IIIB or HeLa/LAV cells. Different fluorescence patterns were observed when the cells were exposed to Lipofectamine, Lipofectin or DMRIE:DOPE (1:1) complexed to the ribozyme. With Lipofectamine intense cell-associated fluorescence was found. Incubation with Lipofectin resulted in less intense diffuse fluorescence, while with DMRIE an intense but sporadic fluorescence was observed. Differentiated THP-1/HIV-1IIIB cells were more susceptible to killing by liposome-ribozyme complexes than THP-1 cells. Under non-cytotoxic conditions (a 4-h treatment) complexes of 5, 10 or 15 microM Lipofectin or DOTAP:DOPE (1:1) and ribozyme, at lipid:ribozyme ratios of 8:1 or 4:1, did not affect p24 production in THP-1/HIV-1IIIB cells in spite of the intracellular accumulation of the ribozyme. A 24-h exposure of THP-1/HIV-1IIIB cells to 5 microM Lipofectin or DOTAP:DOPE (1:1) complexed with either the functional or a modified control ribozyme reduced virus production by approximately 30%. Thus, the antiviral effect of the liposome-complexed ribozyme was not sequence-specific. In contrast, the free ribozyme at a relatively high concentration inhibited virus production by 30%, while the control ribozyme was ineffective, indicating a sequence-specific effect. Both Lipofectin and DOTAP complexed with ribozyme were toxic at 10 and 15 microM after a 24-h treatment. A 4-h treatment of HeLa/LAV cells with Lipofectin at 5, 10 or 15 microM was not toxic to the cells, but also did not inhibit p24 production. In contrast, treatment of HeLa CD4+ cells immediately after infection with HIV-1IIIB at the same lipid concentrations and lipid:ribozyme ratios was cytotoxic. Our results indicate that the delivery of functional ribozyme into cells by cationic liposomes is an inefficient process and needs extensive improvement before it can be used in ex vivo and in vivo applications.

Asunto(s)

Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Liposomas/metabolismo , ARN Catalítico/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Cationes , Diferenciación Celular/efectos de los fármacos , Línea Celular , Portadores de Fármacos , Infecciones por VIH/genética , Infecciones por VIH/patología , VIH-1/metabolismo , Células HeLa , Humanos , Liposomas/farmacología , Sustancias Macromoleculares , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/virología , ARN Catalítico/farmacología

RESUMEN

Liposomes that destabilize at mildly acidic pH are efficient tools for delivering water-soluble drugs into the cell cytoplasm. However, their use in vivo is limited because of their rapid uptake from circulation by the reticuloendothelial system. Lipid-anchored polyethylene glycol (PEG-PE) prolongs the circulation time of liposomes by steric stabilization. We have found that addition of PEG-PE to the membrane of pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) confers steric stability to these vesicles. This modification significantly decreases the pH-dependent release of a charged water-soluble fluorophore, calcein, from liposomes suspended in buffer or cell culture medium. However, the ability of such liposomes to release calcein intracellularly, measured by a novel flow cytometry technique involving dual fluorescence labeling, remains unaltered. As expected, the release of calcein from liposomes endocytosed by cells is inhibited upon pretreatment of the cells with NH4Cl, an inhibitor of endosome acidification. The unique properties of these liposomes were also demonstrated in vivo. The distribution kinetics of 111In-containing CHEMS/DOPE/PEG-PE liposomes injected intravenously into rats has pharmacokinetic parameters similar to control, non-pH-sensitive, sterically stabilized CHEMS/distearoylphosphatidylcholine/PEG-PE liposomes. In contrast, regular pH-sensitive liposomes lacking the PEG-PE component are cleared rapidly. Sterically stabilized pH-sensitive liposomes may therefore be useful for the intracellular delivery in vivo of highly negatively charged molecules such as genes, antisense oligonucleotides, and ribozymes for the treatment of various diseases.

Asunto(s)

Sistemas de Liberación de Medicamentos/métodos , Liposomas/síntesis química , Animales , Tampones (Química) , Células Cultivadas , Citometría de Flujo , Fluoresceínas/farmacocinética , Concentración de Iones de Hidrógeno , Fosfatidiletanolaminas , Ratas , Rodaminas/farmacocinética

RESUMEN

Liposomes can be targeted to HIV-infected cells by either reconstituting transmembrane CD4 in the membrane or covalently coupling soluble CD4 to modified lipids. We investigated whether synthetic peptides could be used as ligands for targeting liposomes. A synthetic peptide from the complementarity determining region 2 (CDR-2)-like domain of CD4 could bind specifically to HIV-infected cells and mediate the binding of peptide-coupled liposomes to these cells. A peptide from the CDR-3-like domain of CD4 inhibited HIV-induced syncytia formation, but failed to target liposomes to infected cells. This apparent discrepancy may be due to the requirement for a conformational change in the CD4 receptor for the CDR-3 region to interact with the HIV envelope protein. Our results demonstrate the feasibility of using synthetic peptides to target liposomes containing antiviral drugs to HIV-infected cells.

Asunto(s)

Antígenos CD4/metabolismo , VIH-1 , Liposomas , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Antígenos CD4/química , Línea Celular , Región Variable de Inmunoglobulina , Datos de Secuencia Molecular

RESUMEN

Influenza virus binds to cell surface sialic acid receptors, and following endocytosis fuses with the endosome membrane at low pH. Whether sialic acid plays a role in the virus-cell membrane fusion step is not known. We investigated the effect of the removal of cell membrane sialic acid on the fusion activity of influenza virus (A/PR/8/34 strain) toward human T lymphocytic leukemia (CEM) cells at low pH. Fusion was monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Removal of sialic acid by neuraminidase resulted in a drastic reduction in both viral binding and fusion. The association of the virus with neuraminidase-treated cells was enhanced at pH 5, compared to that at neutral pH, probably due to the unfolding of the hemagglutinin and the resulting increase in viral surface hydrophobicity, but the fusion capacity of the virus was reduced significantly. The results were analysed with a mass-action kinetic model which could explain and predict the kinetics of fusion. Our results indicate that binding of influenza virus to sialic acid residues on the cell surface leads to rapid and extensive fusion and partially inhibits the low pH-induced viral inactivation.

Asunto(s)

Membrana Celular/virología , Orthomyxoviridae/fisiología , Ácidos Siálicos/fisiología , Línea Celular , Membrana Celular/química , Concentración de Iones de Hidrógeno , Ácido N-Acetilneuramínico , Neuraminidasa/farmacología , Receptores de Superficie Celular/fisiología

RESUMEN

Antiviral activity of vegetan, a new natural immunostimulator, in herpetic meningoencephalitis of mice, genital herpes of guinea pigs, and parvoviral enteritis of dogs was compared to that of acylguanosine and phosphonoformic acid (PFA). Vegetan incorporated in liposomes or pure was the most active if used for prevention 5 days before intracerebral infection of mice with herpes simplex type 1 virus, whatever the dose of infective agent (10 to 100 LD50). Protective efficacy of vegetan was 57-63%, whereas for ara-A, acylguanosine, and PFA these values were, respectively, 20, 25, and 33%. Use of liposomal vegetan preparation ensured a 80% protective effect. Vegetan had a manifest antiviral effect in guinea pigs with genital herpes and in dogs with parvoviral enteritis.

Asunto(s)

Adyuvantes Inmunológicos/uso terapéutico , Antivirales/uso terapéutico , Infecciones por Herpesviridae/tratamiento farmacológico , Infecciones por Parvoviridae/tratamiento farmacológico , Animales , Chlorocebus aethiops , Perros , Enteritis/tratamiento farmacológico , Cobayas , Meningoencefalitis/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Compuestos Orgánicos , Células Vero

RESUMEN

We have investigated the potential of the conserved transmembrane M2 protein of influenza A/Ann Arbor/6/60 virus, expressed by a baculovirus recombinant, to induce protective immunity in BALB/c mice. Vaccination of mice with M2 shortened the duration of virus shedding and protected mice from a lethal infection with A/Ann Arbor/6/60 virus but not B/Ann Arbor/1/55 virus, suggesting that the protection was mediated by an M2-specific mechanism. Serum antibodies were detected which reacted with synthetic peptides defining three antigenic determinants located on both the external N- and internal C-termini of the M2 protein. Furthermore, vaccination with M2 protected mice from death following a lethal challenge with the heterologous A/Hong Kong/68 (H3N2) virus. These results demonstrate the potential to elicit heterosubtypic immunity to type A influenza viruses through vaccination with a conserved transmembrane protein.

Asunto(s)

Baculoviridae/genética , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/biosíntesis , Especificidad de Anticuerpos , Femenino , Vectores Genéticos/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Infecciones por Orthomyxoviridae/virología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/inmunología

RESUMEN

To investigate the mechanism of action of the 22-amino-acid HIV fusion peptide on HIV infection, we studied its influence on virus adsorption and HIV-induced syncytium formation. The effect of the peptide preparations on the synthesis of viral antigens in HIV-infected cell cultures was determined by antigen capture assay, and the inhibition of proviral DNA synthesis was detected by hybridization with a HIV-specific oligonucleotide probe after PCR amplification. Fusion peptides inhibited HIV-induced syncytium formation and antigen production in lytic infected cells, and this effect was increased in conjugation with bovine serum albumin or with synthetic net-charged polymer by its C-terminus. The association of peptide with carrier by N-terminus, or with positive-charged polymer or gelatin completely abolished its effect on HIV infection. No peptide preparations influenced HIV-1 chronically infected cells. Because peptide preparations blocked the HIV-specific DNA synthesis 2 hr after infection without influencing virus adsorption and reverse transcription, we concluded that the block of infection occurred during the penetration of virions through the cell membrane. On the basis of results obtained we propose that our peptide preparations could be used for anti-HIV chemotherapy. The possibility of the existence of receptors for gp41 N-terminal region on target cell membrane is discussed.

Asunto(s)

Antivirales/farmacología , Proteína gp41 de Envoltorio del VIH/farmacología , VIH-1/fisiología , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/microbiología , Fusión Celular/efectos de los fármacos , ADN Viral/análisis , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/ultraestructura , Transcriptasa Inversa del VIH , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Reacción en Cadena de la Polimerasa , ADN Polimerasa Dirigida por ARN/análisis , Replicación Viral/efectos de los fármacos

RESUMEN

The N-terminal region of the human immunodeficiency virus type 1 (HIV-1) gp41 appears to be involved in virus-cell membrane fusion. To study the influence of fusion domain structure on gp41 interaction with artificial lipid membranes, two families of peptides were synthesized. The peptides of the first family starting from the C-terminal Gly-532 of gp160 (BRU isolate) were assembled in a stepwise manner to N-terminus of gp41(Ala-517). These hydrophobic peptides, containing 10-16 amino acid residues (a.a.), were able to form channel-like current fluctuation through planar lipid membranes, and the longest 15-16 a.a. peptides lysed the liposomes. Peptides of the second family beginning from the C-terminal Arg-538 and continuing to Val-510 contained several hydrophilic amino acid residues. These 15-22 a.a. peptides also increased the conductance of planar lipid bilayers and lysed liposomes. The degree of liposome lysis depended upon peptide length and concentration. The attachment of gp120 C-terminal amino acid or peptides to N-terminus of 517-538 peptide resulted in complete loss of activity. The effects of the second family of peptides on membranes were reduced to a great extent at acidic pH. The conjugation of 22 a.a. Lys peptide with bovine serum albumin decreased its lytic activity. The circular dichroism study of these peptides revealed alpha-helix configuration in hydrophobic and aqueous media only for deca- and longer peptides. The electron microscopy of 22 a.a. peptide performed in the aqueous medium showed large spherical aggregates about 0.5-0.7 micron in diameter consisting of long filaments approximately 5 nm in diameter. Other tested peptides could generate only short strings. Thus, the effects of fusion peptides on lipid membranes depends on their sequence and length, secondary and tertiary structures, and freedom of their N-terminus.

Asunto(s)

Proteína gp41 de Envoltorio del VIH/química , Fusión de Membrana , Péptidos/química , Secuencia de Aminoácidos , Dicroismo Circular , Proteína gp41 de Envoltorio del VIH/fisiología , Proteína gp41 de Envoltorio del VIH/ultraestructura , Membrana Dobles de Lípidos/química , Liposomas/química , Lípidos de la Membrana/química , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/fisiología , Relación Estructura-Actividad

RESUMEN

Using a panel of sera from HIV-infected persons and donors, the authors showed that radioimmunoprecipitation assays compare favourably with immunoblotting assays. With radioimmunoprecipitation, cross reactions were observed between HIV-2 antigens and HIV-2 antibodies, and that the nature of cross reactivity differs from that observed with immunoblotting. Potentials of radioimmunoprecipitation assays as a confirmatory test for use with sera that have given indeterminate results in immunoblotting assays and contradictory results in enzyme immunoassays are examined.

Asunto(s)

Serodiagnóstico del SIDA/métodos , Infecciones por VIH/diagnóstico , VIH-1 , VIH-2 , Ensayo de Radioinmunoprecipitación/métodos , Síndrome de Inmunodeficiencia Adquirida/diagnóstico , Reacciones Cruzadas , Diagnóstico Diferencial , Estudios de Evaluación como Asunto , Técnica del Anticuerpo Fluorescente , Anticuerpos Anti-VIH/sangre , Antígenos VIH/sangre , VIH-1/inmunología , VIH-2/inmunología , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Radioinmunoensayo , Sensibilidad y Especificidad

RESUMEN

The interaction of 11 overlapping synthetic peptides corresponding to N-terminal segment of HIV transmembrane glycoprotein gp41 (fusion domain) with artificial lipid membranes has been studied. For this purpose the increase of a bilayer lipid membrane (BLM) conductivity and the changes in ESR spectra of spin-labelled liposomes were registrated. Peptide fragment 523-532 gp160 (BRU strain) had the critical length with regard to channel-forming activity on BLM. The degree of such membranotropic action increased simultaneously with the growth of peptide length and the temperature in the cell. Peptides 518-532 and 517-532 lysed TEMPOcholine-containing liposomes at 37 degrees C. The significance of observed effects for explanation of the mechanism of HIV-induced membrane fusion is discussed.

Asunto(s)

Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas Virales de Fusión/metabolismo , Secuencia de Aminoácidos , Espectroscopía de Resonancia por Spin del Electrón , Potenciales de la Membrana , Datos de Secuencia Molecular , Péptidos/síntesis química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo

RESUMEN

Experiments in two strains of mice; CBA, susceptible to influenza, and CBAXC57Bl/cXFl, resistant to it, demonstrated stimulation of influenza infection caused by gangliosides. The stimulating effect of gangliosides (GMl, GDla, GTlb) seems to be explained by their insertion into plasma membranes of epithelial cells of the respiratory tract and by an increase, due to it, of the number of superficial virus-specific receptors.

Asunto(s)

Gangliósidos/farmacología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Animales , Membrana Celular/efectos de los fármacos , Gangliósidos/uso terapéutico , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/inmunología , Virus de la Influenza A/fisiología , Ratones , Ratones Endogámicos CBA , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/microbiología , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/microbiología

RESUMEN

A comparative study of hemolytic activity of influenza type A, B, and C viruses, human parainfluenza type 3, and Sendai virus showed the pattern of pH-dependence and the nature of the curve to differ not only for different viruses under study but also for different erythrocyte species. Studies of virus-induced hemolysis of influenza C virus demonstrated that, depending on the erythrocyte species used, it had common properties both with influenza types A and B viruses and with paramyxoviruses.

Asunto(s)

Hemólisis , Orthomyxoviridae/patogenicidad , Paramyxoviridae/patogenicidad , Animales , Pollos , Cobayas , Hemaglutinación por Virus , Humanos , Concentración de Iones de Hidrógeno , Virus de la Influenza A/patogenicidad , Virus de la Influenza B/patogenicidad , Gammainfluenzavirus/patogenicidad , Virus de la Parainfluenza 1 Humana/patogenicidad , Virus de la Parainfluenza 3 Humana/patogenicidad

RESUMEN

The ESR data on the influence of membrane potential of the fusion of Sendai virus envelope with erythrocyte membrane are presented. The hyperpolarization of cell membrane takes place at low concentration of KCl (1-5 mM) in extracellular medium in the presence of valinomycin, while at high concentration of KCl (125-150 mM) its depolarization occurs. The hyperpolarization of erythrocyte plasma membrane is accompanied by the increase of its fusion with viral envelope and virus-induced hemolysis. At the same time depolarization of erythrocyte membrane leads to the decrease of virus fusion activity. This evidence together with previously obtained by patch-clamp method data on potential-dependence of virus-induced increase of cell membrane conductivity provide us an opportunity to make a proposal that the electric field membrane damage may be the initial stage of the virus-induced membrane fusion.

Asunto(s)

Membrana Eritrocítica/microbiología , Hemólisis , Virus de la Parainfluenza 1 Humana/patogenicidad , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/genética , Animales , Fusión Celular , Embrión de Pollo , Efecto Citopatogénico Viral , Espectroscopía de Resonancia por Spin del Electrón , Membrana Eritrocítica/fisiología , Potenciales de la Membrana , Marcadores de Spin

RESUMEN

Intranasal inoculation of liposomes from egg phosphatidylcholine to white mice infected with influenza A virus resulted in the death of practically all animals whereas the death rate in the control group was 30%. A possible mechanism of the effect of liposomes on the virus infection is discussed.

Asunto(s)

Liposomas/uso terapéutico , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Animales , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/genética , Virus de la Influenza A/patogenicidad , Ratones , Infecciones por Orthomyxoviridae/mortalidad , Recombinación Genética , Estimulación Química

RESUMEN

It has already been shown that influenza virus binds unspecifically to liposomes containing ganglioside GM1 wheras with gangliosides GD1b and GT1b binding occurs in a specific and saturable manner [Slepushkin et al. (1986) Biol. Membr. 3, 229-235]. In the present study the mode of interaction between influenza virus and various gangliosides or phospholipid liposomes containing cholesterol and gangliosides has been investigated. The influence of exogenous gangliosides on the structure of the viral envelope was studied using fluorescent and photoactivatable phospholipids incorporated into the viral membrane. With both types of probes maximal effects of gangliosides were caused by GT1b. Addition of that ganglioside resulted in a marked decrease in the fluorescence polarization (P) of fluorescent labeled virus as well as in substantial changes in the binding of photoactivatable analogues of sphingomyelin and phosphatidylcholine to virus proteins, mainly hemagglutinin. The effects of GT1b and GD1b on P value were comparable, whereas gangliosides with other oligosaccharide chains caused much smaller changes in P. Furthermore GT1b but not GM1 influenced phospholipid-hemagglutinin cross-linking. Interaction of the virus with large unilamellar liposomes was monitored by two fluorescence assays based on resonance-energy transfer from the tryptophans and tyrosines of viral proteins to vesicles labeled with a triacylglycerol (anthrylvinyldioleoylglycerol) or from these labeled vesicles to virions labeled with a perylenoyl derivative of galactosylcerebroside (PGalSph). A third fluorescence assay was based on relief of self-quenching in PGalSph-labeled virions, upon low-pH-induced virus-liposome fusion. With all three fusion assays the changes of fluorescence caused by GT1b were more pronounced than those induced by GM1. On the other hand, virus-induced release of [14C]glucose from multilamellar liposomes was enhanced by GM1 but not by GT1b or GD1b. It is concluded that the interaction of GT1b or GD1b with virus hemagglutinin induces a rearrangement of the viral lipids rendering lipid bilayer areas of the viral envelope significantly fluid, which in turn promotes fusion of the virus with target membranes. Probably virus-liposome fusion and virus-induced liposome leakage are brought about by different mechanisms depending on specific or unspecific binding of the virions to the target.

Asunto(s)

Gangliósidos/metabolismo , Liposomas/metabolismo , Orthomyxoviridae/metabolismo , Sitios de Unión , Transferencia de Energía , Gangliósido G(M1)/metabolismo , Pruebas de Hemaglutinación , Concentración de Iones de Hidrógeno , Espectrometría de Fluorescencia

RESUMEN

The method of transfection of eukaryotic cells by plasmid DNA and production of recombinant vaccinia virus is described. The method is based on DNA encapsulation into reverse evaporation vesicles containing gangliosides, with a subsequent incubation of liposomes with vaccinia-infected cells in the presence of UV-inactivated Sendai virus. The recombinant DNA of vaccinia virus containing part of env gene of HTLV-3/LAV virus was obtained.

Asunto(s)

ADN Recombinante , ADN Viral/genética , Liposomas/administración & dosificación , Plásmidos , Transfección , Virus Vaccinia/genética , Animales , Clonación Molecular/métodos , Gangliósidos/genética , Genes Virales , Vectores Genéticos , VIH/genética , Virus de la Parainfluenza 1 Humana/genética , Virus de la Parainfluenza 1 Humana/efectos de la radiación , Rayos Ultravioleta , Células Vero , Cultivo de Virus

RESUMEN

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 Virus