RESUMEN
The effect of temperature on fusion of Sendai virus with target membranes and mobility of the viral glycoproteins was studied with fluorescence methods. When intact virus was used, the fusion threshold temperature (20-22 degrees C) was not altered regardless of the different types of target membranes. Viral glycoprotein mobility in the intact virus increased with temperature, particularly sharply at the fusion threshold temperature. This effect was suppressed by the presence of erythrocyte ghosts and/or dextran sulfate in the virus suspension. In these cases also, no change in the fusion threshold temperature was observed. On the other hand, reconstituted viral envelopes (virosomes) bearing viral glycoproteins but lacking matrix proteins were capable of fusing with erythrocyte ghosts even at temperatures lower than the fusion threshold temperature and no fusion threshold temperature was observed over the range of 10-40 degrees C. The mobility of viral glycoproteins on virosomes was much greater and virtually temperature-independent. The intact virus treated with an actin-affector, jasplakinolide, reduced the extent of fusion with erythrocyte ghosts and the mobility of viral glycoproteins, while the treatment of virosomes with the same drug did not affect the extent of fusion of virosomes with erythrocyte ghosts and the mobility of the glycoproteins. These results suggest that viral matrix proteins including actins affect viral glycoprotein mobility and may be responsible for the temperature threshold phenomenon observed in Sendai virus fusion.
Asunto(s)
Membrana Eritrocítica/virología , Liposomas/metabolismo , Fusión de Membrana/fisiología , Virus Sendai/fisiología , Temperatura , Proteínas Virales de Fusión/metabolismo , Células Cultivadas , Citocalasinas/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Depsipéptidos/farmacología , Relación Dosis-Respuesta a Droga , Membrana Eritrocítica/efectos de los fármacos , Humanos , Fusión de Membrana/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Virus Sendai/efectos de los fármacos , Virosomas/efectos de los fármacos , Virosomas/fisiologíaRESUMEN
Effects of various polymers (dextran sulfate, dextran and polyethylene glycol) on binding and fusion of Sendai virus to target cells were studied by use of fluorescence spectroscopy. Direct binding of dextran sulfate but not dextran to Sendai virus was detected. Anionic and nonionic polymers showed definite effects on segmental motions of the viral envelope proteins. Sendai virus binding to human erythrocyte ghost membranes (HEG) was reduced by dextran sulfate and dextran while the fusion temperature dependence remained unaltered at approximately 20 degrees C. Nonionic polymer, polyethylene glycol, caused an increase in extent of fusion of Sendai virus with HEG. Segmental motion of viral envelope proteins, determined in terms of anisotropy of fluorescent probes attached to viral surface proteins, exhibited a temperature dependent transition at 20 degrees C by a sharp change from restricted to less restricted motion. In the presence of each of the polymers, this transition was no longer apparent. Since fusion did occur in the presence of all polymers, the temperature dependent characteristic of Sendai virus target cell fusion can be said not to depend on viral surface protein segmental motion. A reasonable and coherent explanation was given for the apparent disparity between the effects of inhibiting and enhancing polymers on fusion and motion of viral proteins.
Asunto(s)
Sulfato de Dextran/farmacología , Membrana Eritrocítica/virología , Fusión de Membrana/efectos de los fármacos , Polietilenglicoles/farmacología , Respirovirus/efectos de los fármacos , Animales , Embrión de Pollo , Humanos , Respirovirus/fisiología , TemperaturaRESUMEN
Several lipoamino acids were synthesized in which palmitic acid was coupled with the alpha-amino group of an amino acid. These lipoamino acids were tested for their inhibitory action against Sendai virus fusion to liposomes composed of egg phosphatidylethanolamine and 5 mol% of the ganglioside GD1a. A commonly employed viral fusion assay based on the dilution of the fluorescent probe octadecylrhodamine (R18) exhibited an additional complication in the presence of Nalpha-palmitoyl tryptophan (palm-Trp). At higher mol fraction of palm-Trp it was observed that there was an increase in R18 quenching. Studies on the dependence of the emission wavelength of palm-Trp on excitation wavelength demonstrated that the presence of R18 alters the environment of the indole. The results illustrate one of the complexities of viral fusion assays using the R18 probe. Despite this complication it was possible to demonstrate that several of the lipoamino acids are effective at inhibiting the fusion of Sendai virus to liposomes as measured by the R18 assay. One of the most effective inhibitors of this process is palm-Trp which, at a concentration of 4 mol% in liposomes, markedly reduces the apparent rate of fusion. At pH 5.0 this amphiphile is also an inhibitor of Sendai virus fusion, indicating that the ionization of the carboxyl group of this amphiphile is not required for its antiviral activity. The inhibitory action of palm-Trp against Sendai virus was confirmed by demonstrating inhibition of Sendai-mediated cytopathic effects studied in tissue culture. A property associated with antiviral activity is the ability of amphiphiles to raise the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine. All of these lipoamino acids were found to possess this property, but a quantitative relationship with inhibition of viral fusion was not found.
Asunto(s)
Aminoácidos/química , Ácidos Grasos/química , Membrana Dobles de Lípidos/química , Aminoácidos/farmacología , Animales , Antivirales/química , Antivirales/farmacología , Rastreo Diferencial de Calorimetría , Embrión de Pollo , Colorantes Fluorescentes , Fusión de Membrana/efectos de los fármacos , Respirovirus/efectos de los fármacos , Respirovirus/fisiologíaRESUMEN
Fusion between Sendai virus (SV) and individual host cells was investigated with confocal laser scanning microscopy (CLSM) and image correlation spectroscopy (ICS). SV was labeled with the fluorescent probe 7-octadecylamino-4-nitrobenz-2-oxa-1,3-diazole (NBD-NH-C18) and was allowed to bind to host cells (HEp-2, BALB-3T3) at 4 degrees C. The effect of lipophosphoglycan (LPG), isolated from Leishmania donovani, on virus fusion was investigated by incorporation of LPG (0, 5, 10 or 20 microM) into the host cell membrane (HEp-2) before addition of SV. LPG did not affect the number of SV bound per cell. After incubation at 37 degrees C for 15 min without LPG, CLSM revealed a redistribution of NBD-NH-C18 from the SV envelope to the host cell membrane and an increase in average fluorescence intensity, indicating dequenching. ICS analysis of images obtained after incubation at 37 degrees C showed an increased mean cluster density to 260% of the value at 4 degrees C, reflecting the disappearance of labeled SV from the cell surface and diffusion of NBD-NH-C18 into the host cell membrane. Preincubation of the cells with LPG inhibited the temperature-induced redistribution and dequenching of NBD-NH-C18 in a concentration-dependent manner, with a total inhibition of fusion at 20 microM LPG. Together, the results demonstrate that CLSM combined with ICS is a powerful tool for studies of fusion of enveloped viruses with individual host cells and that LPG inhibits the fusion process at or before the hemifusion (lipid mixing) stage of SV interaction with cells.
Asunto(s)
Membrana Celular/virología , Glicoesfingolípidos/farmacología , Fusión de Membrana/efectos de los fármacos , Microscopía Confocal/métodos , Respirovirus , 4-Cloro-7-nitrobenzofurazano/química , Animales , Línea Celular , Endocitosis , Colorantes Fluorescentes , Humanos , Ratones , Respirovirus/química , Análisis Espectral/métodos , Temperatura , Virión/químicaRESUMEN
An analysis of the R18 fusion assay was made during the fusion of the Sendai virus with erythrocyte ghosts. The increase in R18 fluorescence, reflecting the interaction process, was evaluated in terms of the different processes that in principle may contribute to this increase, that is, monomeric probe transfer, hemifusion, and complete fusion. To this end, the kinetics of the R18-labeled lipid mixing were compared to those obtained with an assay in which the fusion-monitoring probe, eosin-maleimide, was attached to the viral surface proteins. The experiments relied on the use of native and fusion-inactive viruses and studies involving viral and target membranes that were modified by the incorporation of the lysophospholipid. The total dequenching signal detected in the R18 assay consists of components from probe transferred without fusion and from fusion itself. At 37 degrees C, the initial rate of dequenching (within two minutes) was predominately from the probe diluted by fusion with little contribution from transfer. The dequenching signal due to the probe transfer without fusion occurred at temperatures as low as 10 degrees C and increased linearly with time. Complete fusion started at about 20-25 degrees C and increased sharply at 30 degrees C. The extent of hemifusion was deduced from the total R18 dequenching data and those of the eosin-maleimide labeled protein dilution method for the limiting cases; the analysis indicates that hemifusion started at about 15 degrees C and increased over the range 20-25 degrees C. The initial rate of dequenching of the R18 assay measured within 2 min gives an accurate measure of membrane fusion above 30 degrees C.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Fusión de Membrana , Rodaminas/metabolismo , Eosina Amarillenta-(YS)/análogos & derivados , Eosina Amarillenta-(YS)/metabolismo , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/fisiología , Membrana Eritrocítica/virología , Humanos , Liposomas , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Fusión de Membrana/efectos de los fármacos , Fosfatidilserinas/metabolismo , Respirovirus/metabolismo , Respirovirus/fisiología , Espectrometría de Fluorescencia/métodos , Tripsina , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The rate and extent of membrane fusion is markedly sensitive to membrane interfacial properties. Lipopeptides with hydrophilic peptide moieties will insert into membranes, leaving the peptide portion at the membrane-water interface. In this work, we have used a lipopeptide composed of the peptide [Nle15]-gastrin-(2-17)-amide covalently linked to 1,2-diacyl-3-mercaptoglycerol-N(alpha)-maleoyl-beta-alanine to give DM-gastrin or DP-gastrin having 14 or 16 carbon atom acyl chains, respectively. The fluorescence emission from the two Trp residues of these lipopeptides exhibited little or no blue shift upon addition of liposomes of egg-phosphatidylethanolamine containing 5 mol% G(D1a). Iodide quenching of DP-gastrin fluorescence was also independent of lipid. These results indicate that the peptide moiety is exposed to the aqueous environment even though the lipopeptide is firmly anchored to the membrane. Both DM and DP-gastrin markedly raise the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine. However, DM-E5 lowers this phase transition temperature. These lipopeptides have effects on the overall fusion of Sendai virus to liposomes in accord with their opposite effects on lipid curvature. The lipogastrins are potent inhibitors of viral fusion, while DM-E5 slightly promotes this process. Truncated forms of DM-gastrin are also inhibitory to viral fusion, but are less inhibitory than the full lipopeptide. Analysis of the fusion kinetics shows that DP-gastrin causes a reduction in the final extent of fusion and a marked lowering of the fusion rate constant. Binding of Sendai virus to the ganglioside receptor-containing liposomes was not affected. Consideration of the various contributions to the mechanism of inhibition of viral fusion suggests that effects of lipogastrin on membrane intrinsic monolayer curvature is of primary importance.
Asunto(s)
Colecistoquinina/análogos & derivados , Gastrinas/farmacología , Fusión de Membrana/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Respirovirus/fisiología , Proteínas Virales de Fusión/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Rastreo Diferencial de Calorimetría , Embrión de Pollo , Colecistoquinina/farmacología , Datos de Secuencia Molecular , Respirovirus/efectos de los fármacos , Espectrometría de FluorescenciaRESUMEN
Umbilical cord serum samples (380), an average of 10 per month for 3 years (1990 to 1992), were tested by indirect immunofluorescence assay for group C rotavirus immunoglobulin G. Thirty percent were positive, suggesting that approximately one-third of women of childbearing age in western New York have experienced group C rotavirus infection.
Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones por Rotavirus/epidemiología , Rotavirus/inmunología , Femenino , Sangre Fetal/inmunología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoglobulina G/sangre , New York/epidemiología , Rotavirus/clasificación , Estudios SeroepidemiológicosRESUMEN
Glycophorin A was reconstituted into large unilamellar vesicles of egg phosphatidylcholine by detergent dialysis. The observed overall rate of Sendai virus fusion increased approximately 4-fold between 0 and 0.006 mol % glycophorin, roughly proportional to the glycophorin content. However, no further increase in rate was observed at 0.02 mol % glycophorin. Treatment of reassembled glycophorin-liposomes with neuraminidase resulted in a significant decrease in the percent of viral fusion, confirming that the presence of sialic acid residues on glycophorin is essential for its role as a receptor. The sialic acid-containing glycolipid, the ganglioside GD1a, was also incorporated into phosphatidylcholine liposomes, either in addition to or in place of glycophorin A. Comparing, on the basis of sialic acid content, liposomes containing either glycophorin or GD1a, comparable rates and extents of fusion were found. However, on a molar basis glycophorin is much more effective. It was found that the addition of GD1a to glycophorin-containing liposomes only slightly increased the rate of fusion. This was largely due to an increase in the percent of virions capable of fusing.
Asunto(s)
Glicoforinas/metabolismo , Virus de la Parainfluenza 1 Humana/metabolismo , Receptores Virales/metabolismo , Gangliósidos/farmacología , Glicoforinas/farmacología , Liposomas/metabolismo , Fusión de Membrana , Neuraminidasa/metabolismo , Tamaño de la Partícula , Fosfatidilcolinas/metabolismoRESUMEN
Lipophosphoglycan (LPG) is an amphiphile produced by Leishmania. Its chemical structure consists of a hydrophilic flexible polymer of repeating PO4-6Gal beta 1-4Man alpha 1 units (on average 16 units) linked via a hexasaccharide core to a lyso-1-O-alkyl-P1 membrane anchor. In the study of viral fusion we report in this paper, we have introduced LPG into human erythrocyte ghost (HEG) membranes, with the purpose of understanding how the LPG-induced surface-structural changes may modulate the interactions between a viral envelope and the HEG membranes. We have found that LPG, when incorporated at very low concentrations into intact human erythrocyte membranes, strongly inhibits Sendai virus-induced hemolysis. When incorporated into HEGs, it reduces the binding of both Sendai and influenza viruses to HEGs; furthermore, it strongly inhibits the overall viral fusion to HEGs, being among the most potent known inhibitors. We have also shown that LPG stabilizes the bilayer structure of phosphatidylethanolamine against the formation of an inverted-hexagonal structure. We suggest that LPG may give rise to an effective "steric repulsion" between the viral and HEG membranes, thereby modulating some specific modes of interaction between viral-target membranes in the overall fusion process; LPG may also modulate the bending rigidity and the spontaneous curvature of the HEG membrane in the direction of making the destabilization and rearrangement of the underlying lipid bilayer more difficult.
Asunto(s)
Glicoesfingolípidos/farmacología , Virus de la Influenza A/fisiología , Leishmania donovani/química , Fusión de Membrana/efectos de los fármacos , Virus de la Parainfluenza 1 Humana/fisiología , Animales , Secuencia de Carbohidratos , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/virología , Hemólisis , Humanos , Cinética , Liposomas , Datos de Secuencia Molecular , Receptores Virales/efectos de los fármacos , TemperaturaRESUMEN
The ganglioside GD1a, which serves as a receptor for Sendai virus, also affects lipid polymorphism as determined by 31P nuclear magnetic resonance. The ganglioside promotes the formation of isotropic structures in monomethyldioleoylphosphatidylethanolamine. GD1a also raises the bilayer to hexagonal phase transition temperature of this lipid. The effects of GD1a on the kinetics of viral fusion can be understood on the basis of its role in facilitating the binding of Sendai virus to target membranes as well as its effects on membrane physical properties. Fusion of Sendai virus with liposomes composed of egg phosphatidylethanolamine is particularly sensitive to the presence of ganglioside. In the absence of ganglioside no fusion is observed due to the absence of virus binding to the target membrane. Between 2 and 6 mol % GD1a in egg phosphatidylethanolamine liposomes there is a marked increase in the rate constant of binding of the virus to the liposome but a decrease in the fusion rate constant. The latter effect is found to be common to a number of other amphiphiles that raise the bilayer to hexagonal phase transition temperature. The ganglioside enhances virus binding to liposomes of all the compositions studied, but leakage rates and fusion rate constants are either unaffected or reduced. In the systems studied, the enhanced formation of isotropic structures in liposomes containing the ganglioside does not enhance the kinetics of the actual fusion reaction.
Asunto(s)
Gangliósidos/metabolismo , Virus de la Parainfluenza 1 Humana/metabolismo , Receptores Virales/química , Proteína HN/metabolismo , Técnicas In Vitro , Liposomas , Fusión de Membrana , Lípidos de la Membrana/química , Fosfatidiletanolaminas/química , Proteínas Virales de Fusión/metabolismoRESUMEN
The ability of lysophosphatidylcholine to inhibit membrane fusion at subsolubilizing concentrations (between 1 and 9 mol % with respect to the membrane lipids) was examined. Fusion between N-methyldioleoylphosphatidylethanolamine (DOPE) large unilamellar vesicles (LUV) and fusion between Sendai virus and N-methyl-DOPE LUV were measured. A contents mixing fusion assay was used for LUV fusion (ANTS/DPX), and a lipid mixing assay (octadecylrhodamine B) was used for the virus fusion experiments. Lysophosphatidylcholine was effective at inhibiting both LUV fusion and Sendai virus/LUV fusion. Lysophosphatidylcholine also inhibited leakage from N-methyl-DOPE LUV, 31P nuclear magnetic resonance data were obtained of N-methyl-DOPE in the presence of lysophosphatidylcholine. Lysophosphatidylcholine stabilized the lamellar phase and reduced the incidence of nonlamellar structures at all temperatures. The destabilization of nonlamellar structures with a negative radius of curvature may be a mechanism for inhibition of fusion by lysophosphatidylcholine in these systems.
Asunto(s)
Lisofosfatidilcolinas/farmacología , Fusión de Membrana/efectos de los fármacos , Concentración de Iones de Hidrógeno , Cinética , Liposomas , Espectroscopía de Resonancia Magnética , Virus de la Parainfluenza 1 Humana/fisiología , Fosfatidiletanolaminas , SolubilidadRESUMEN
A number of amphiphiles which raise the bilayer to hexagonal phase transition temperature (TH) of phosphatidylethanolamine (PE) have been shown to inhibit viral fusion. In this study we have further evaluated the mechanism of this inhibition. Several anionic amphiphiles, including cholesterol sulfate, a component of mammalian plasma membranes, lower the final extent of Sendai virus fusion with both human erythrocyte ghosts and liposomes composed of PE and 5% of the ganglioside, GD1a. A cationic amphiphile slightly increased the final extent of fusion. The fusion rate constant is not greatly affected by the presence of as much as 20% cholesterol sulfate or other charged amphiphiles. The zwitterionic amphiphile, cholesterol phosphorylcholine has no effect on the final extent of fusion but it lowers the fusion rate constant. This amphiphile is potent in raising TH. The amphiphile cholesterol hemisuccinate (CHEMS) stabilizes the bilayer relative to the hexagonal phase at neutral pH, while at acidic pH the formation of the hexagonal phase is promoted. When CHEMS is added to vesicles of egg PE containing 5% GD1a, the rate of Sendai virus fusion is little affected at neutral pH but the rate is significantly enhanced at pH 5.0. These results demonstrate that viral fusion can be modulated, in part, by the tendency of the membrane to convert to the hexagonal phase.
Asunto(s)
Membrana Eritrocítica/fisiología , Liposomas , Fusión de Membrana , Virus de la Parainfluenza 1 Humana/fisiología , Animales , Rastreo Diferencial de Calorimetría , Embrión de Pollo , Colesterol/análogos & derivados , Colesterol/química , Membrana Eritrocítica/ultraestructura , Gangliósidos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Membrana Dobles de Lípidos , Virus de la Parainfluenza 1 Humana/ultraestructura , Fosfatidiletanolaminas , Relación Estructura-ActividadRESUMEN
The effect of anionic polymers (dextran sulfate, heparin and chondroitin sulfate) on fusion of Sendai virus with erythrocyte ghosts was studied. The effect of pH on the activity of these anionic polymers was also investigated. In order to examine the interaction of such polymers with the Sendai virion and erythrocyte ghost surfaces, the binding of virions to erythrocyte ghosts and the aggregation of virions and/or erythrocyte ghosts were also measured with respect to the same parameters. It was found that the anionic polymers suppressed the fusion of Sendai virus with erythrocyte ghosts. The order of effectiveness of the polymers in suppression was dextran sulfate greater than heparin greater than chondroitin sulfate, for the application of a same quantity (weight/ml) of the polymers. The lower the pH of the suspending medium, the more effective were the polymers in suppressing virion-erythrocyte ghost aggregation and fusion. The suppression of fusion was dependent on the concentration of the polymers applied: the higher the concentration of the polymer applied, the more the suppression was observed. Evidence from binding studies, turbidity measurements and electrophoretic mobility measurements indicates that the anionic polymers interact preferentially with the virion surface.
Asunto(s)
Membrana Eritrocítica/microbiología , Virus de la Parainfluenza 1 Humana/efectos de los fármacos , Polímeros/farmacología , Adsorción , Sulfatos de Condroitina/farmacología , Sulfato de Dextran/farmacología , Electroforesis , Agregación Eritrocitaria/efectos de los fármacos , Membrana Eritrocítica/química , Membrana Eritrocítica/efectos de los fármacos , Heparina/farmacología , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Nefelometría y Turbidimetría , Virus de la Parainfluenza 1 Humana/química , Espectrometría de FluorescenciaRESUMEN
The characteristics of fusion of respiratory syncytial virus (RSV) with HEp-2 cells were studied by the R18 fluorescence dequenching assay of membrane fusion. A gradual increase in fluorescence intensity indicative of virion-cell fusion was observed when R18-labeled RSV was incubated with HEp-2 cells. Approximately 35% dequenching of the probe fluorescence was observed in 1 h at 37 degrees C. Fusion showed a temperature dependence, with significant dequenching occurring above 18 degrees C. The dequenching was also dependent on the relative concentration of target membrane. Thus, increasing the concentration of target membrane resulted in increased levels of dequenching. In addition, viral glycoproteins were shown to be involved in this interaction, since dequenching was significantly reduced by pretreatment of labeled virus at 70 degrees C for 5 min or by trypsinization of R18-labeled virions prior to incubation with HEp-2 cells at 37 degrees C. The fusion of RSV with HEp-2 cells was unaffected over a pH range of 5.5 to 8.5, with some increase seen at lower pH values. Treatment of HEp-2 cells with ammonium chloride (20 and 10 mM), a lysosomotropic agent, during early stages of infection did not inhibit syncytium formation or progeny virion production by RSV. At the same concentrations of ammonium chloride, the production of vesicular stomatitis virus was reduced approximately 4 log10 units. These results suggest that fusion of the virus with the cell surface plasma membrane is the principal route of entry.
Asunto(s)
Membrana Celular/microbiología , Virus Sincitiales Respiratorios/fisiología , Cloruro de Amonio/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Glutaral/farmacología , Glicoproteínas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Virus Sincitiales Respiratorios/efectos de los fármacos , Temperatura , Tripsina/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18), a lipid mixing assay for fusion. This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. 31P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not. One possible interpretation of these 31P NMR results was that the inhibitory peptide stabilized a membrane structure with a large radius of curvature, when the fusion pathway demanded a membrane defect with a small radius of curvature. This hypothesis was tested by determining the influence of an inhibitory and a noninhibitory peptide on the formation of membraneous structures with small radii of curvature, through ultrasonic irradiation of phospholipid dispersions. The inhibitory peptide prevented the formation of membrane structures with small radii of curvature, while the noninhibitory peptide did not prevent the formation of such structures.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Fusión de Membrana , Virus de la Parainfluenza 1 Humana/ultraestructura , Péptidos/química , Membrana Eritrocítica/ultraestructura , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Virus de la Parainfluenza 1 Humana/crecimiento & desarrollo , Péptidos/síntesis química , Fosfolípidos , Dispersión de Radiación , Solubilidad , Relación Estructura-ActividadRESUMEN
31P nuclear magnetic resonance spectroscopy (31P-NMR) was used to study phospholipid organization in hydrated preparations of N-methyl dioleoylphosphatidylethanolamine and a 'fusion peptide' with the sequence: FAGV-VLAGAALGVAAAAQI, which corresponds to the amino terminus of the F1 subunit of the membrane fusion protein of measles virus. These amino acids are believed to mediate syncytia formation, host-cell penetration and hemolysis by infectious virus. The presence of the peptide at 0.5 mole percent significantly facilitated the formation of isotropic 31P resonances. The effects at 1 mole percent peptide were substantially enhanced over the effects observed at 0.5 mole percent, leading to a decrease in the onset temperature of the formation of the isotropic 31P-NMR resonances by about 30 degrees C. The formation of such isotropic 31P-NMR resonances has been previously associated with an increased rate of fusion of large unilamellar vesicles composed of N-methyl dioleoylphosphatidylethanolamine. Enhanced fusion of octadecyl rhodamine-labelled Sendai virus with N-methyl dioleoylphosphatidylethanolamine large unilamellar vesicles was observed when the 'fusion peptide' was incorporated into the large unilamellar vesicles.
Asunto(s)
Liposomas , Virus del Sarampión/fisiología , Fusión de Membrana/efectos de los fármacos , Virus de la Parainfluenza 1 Humana/fisiología , Fosfatidiletanolaminas/química , Proteínas Virales de Fusión/farmacología , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Espectroscopía de Resonancia Magnética/métodos , Datos de Secuencia Molecular , Virus de la Parainfluenza 1 Humana/efectos de los fármacos , Termodinámica , Proteínas Virales de Fusión/síntesis químicaRESUMEN
The effect of dextran sulfate on the fusion of Sendai virus and erythrocyte ghosts was studied as a function of dextran sulfate concentration and pH of cell suspension solutions. In order to examine the interaction of dextran sulfate with Sendai virion and erythrocyte ghost surfaces, the turbidity of cell suspensions was also measured with respect to the same parameters as above. It was found that dextran sulfate inhibited the fusion of Sendai virus with erythrocyte ghosts. The lower the pH of the suspension solution was, the more effective was dextran sulfate in suppressing virion erythrocyte ghost fusion. Dextran sulfate of a higher molecular weight was slightly more effective in suppressing fusion than that of a lower molecular weight. From turbidity measurements of Sendai virus and erythrocyte ghost suspensions, it is likely that dextran sulfate interacts preferentially with Sendai virion surfaces and inhibits interaction between Sendai virions and erythrocyte ghosts.
Asunto(s)
Sulfato de Dextran/farmacología , Membrana Eritrocítica/efectos de los fármacos , Fusión de Membrana/efectos de los fármacos , Virus de la Parainfluenza 1 Humana/efectos de los fármacos , Depresión Química , Humanos , Concentración de Iones de Hidrógeno , Peso Molecular , Nefelometría y Turbidimetría , Propiedades de Superficie , Proteínas del Envoltorio Viral/efectos de los fármacos , Proteínas del Envoltorio Viral/metabolismo , Virión/efectos de los fármacosRESUMEN
Small hydrophobic peptides that are capable of inhibiting Sendai virus infection of cells (Richardson, C. D., Scheid, A., and Choppin, P. W. (1980) Virology 105, 205-222) are also capable of inhibiting membrane fusion in a pure lipid vesicle system. Large unilamellar vesicles of N-methyl dioleoylphosphatidylethanolamine containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid and/or p-xylene bis (pyridinium bromide) were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) fluorescence assay. Sendai virus fusion with lipid vesicles was measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride, a lipid mixing assay for fusion. The efficiency with which the peptides carbobenzoxy-D-Phe-L-PheGly, carbobenzoxy-L-Phe-L-Tyr, and carbobenz-oxy-Gly-L-Phe inhibit fusion of N-methyl dioleoyl-phosphatidylethanolamine large unilamellar vesicles directly paralleled their previously known effectiveness in blocking virus infectivity of cultured cells. In addition, above a certain concentration threshold, the inhibitory peptides decreased the initial rate of leakage from lipid vesicles. The inhibition by these peptides of virus-vesicle fusion followed the same order of potency as for vesicle-vesicle fusion. The observation of the same relative potency of these peptides toward inhibition of virus-cell infection, and virus-vesicle and vesicle-vesicle membrane fusion suggested that these peptides inhibited virus-cell infection by inhibiting the ability of the virus to fuse with the cell. Furthermore, these results suggest that the mechanism of inhibition of all three fusion events may have steps in common.
Asunto(s)
Fusión de Membrana , Péptidos/farmacología , Proteínas Virales de Fusión/fisiología , Secuencia de Aminoácidos , Técnicas In Vitro , Fusión de Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Virus de la Parainfluenza 1 Humana , Solubilidad , Relación Estructura-ActividadRESUMEN
Cholesterol sulfate is a component of several biological membranes. In erythrocytes, cholesterol sulfate inhibits hypotonic hemolysis, while in sperm, it can decrease fertilization efficiency. We have found cholesterol sulfate to be a potent inhibitor of Sendai virus fusion to both human erythrocyte and liposomal membranes. Cholesterol sulfate also raises the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine as demonstrated by differential scanning calorimetry and 31P nuclear magnetic resonance spectrometry. Although hexagonal phase structures are not readily found in biological membranes, there is a correlation between the effects of membrane additives on bilayer/non-bilayer equilibria and membrane stabilization. It is proposed that the ability of cholesterol sulfate to alter the physical properties of membranes contributes to its stabilization of biological membranes and the inhibition of membrane fusion.
Asunto(s)
Ésteres del Colesterol/farmacología , Fusión de Membrana , Virus de la Parainfluenza 1 Humana/fisiología , Rastreo Diferencial de Calorimetría , Membrana Eritrocítica , Gangliósidos/fisiología , Geles , Hemaglutinación/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Técnicas In Vitro , Temperatura , Proteínas Virales de Fusión/fisiologíaRESUMEN
The authors evaluated the reliability of the proposal review process used by a faculty-student committee to choose medical student researchers for summer fellowships. A total of 82 proposals were reviewed by the committee over a two-year period (43 in 1987 and 39 in 1988); the proposals were assigned ratings in the spring of each year. The 39 students whose proposals received the highest ratings over the two-year period received fellowships and carried out their projects during ten weeks in the summer of each year. In December of both years, the research fellows, along with a total of 12 students whose proposals had been rejected by the program over the two-year period but who had received support from other programs, presented their findings. These research presentations were also judged and rated, and the ratings of all the presentations were compared with the student committee's original ratings of the research proposals. A significant correlation was found between both years' sets of ratings for the proposals and the research presentations. The "accepted" students generally received higher ratings on their presentations than did the "rejected" students; however, the mean of the ratings received by the "accepted" students for their presentations was not significantly higher than that received by the "rejected" students. Even so, the significant correlation between each year's set of ratings for all the students involved demonstrates the soundness of the review process.