Adenovirus-dependent release of choline from plasma membrane vesicles at an acidic pH is mediated by the penton base protein.

It has been suggested that during receptor-mediated endocytosis of human adenovirus (Ad) type 2 into cells, Ad disrupts the membrane of endocytic vesicles to enter the cytosol. To understand the mechanism of Ad-mediated disruption of the endocytic vesicles, I exposed Ad to plasma membrane vesicles derived from KB cells. Ad caused release of choline from the plasma membrane vesicles preloaded with [3H]choline. The efflux of choline was dependent on (i) the concentration of Ad, with a half-maximal effect at 0.5 microgram/ml; (ii) the pH of the buffer, with the optimum pH of the reaction ranging from 5.5 to 6.0; (iii) the length of the incubation, with a half-maximal release at 2 min; and (iv) the temperature of the incubation, with the optimum temperature being 37 degrees C. The Ad-dependent release of choline was inhibited by anti-penton base, while antihexon did not block the effect. These results suggest roles for a low-pH environment and the penton base protein in the Ad-dependent efflux of choline from plasma membrane vesicles.     

Interaction of rotavirus particles with liposomes.

We have studied the interactions of purified viral particles with liposomes as a model to understand the mechanism of entry of rotavirus into the cell. Liposomes, made from pure as well as mixed lipids, that contained encapsulated self-quenching concentrations of the fluorophore carboxyfluorescein (CF) were used. Rotavirus-liposome interactions were studied from the fluorescence dequenching of CF resulting from its release to the bulk solution. Purified infectious double-shelled virus particles induced a concentration- and temperature-dependent release of CF. The rate and extent of CF release was maximum between pH 7.3 and 7.6. The removal of outer structural proteins VP4 and VP7 from virus, which results in the formation of single-shelled particles, prevented virus interaction with liposomes. Rotavirus particles with uncleaved VP4 did not interact with liposomes, but treatment in situ of these particles with trypsin restored the interaction with the liposomes and resulted in CF dequenching. Our data support the view that rotavirus enters the cell through direct penetration of the plasma membrane. In contrast, adenovirus, the only other nonenveloped virus studied by this method, shows the optimum rate of marker release from liposomes at around pH 6 (R. Blumenthal, P. S. Seth, M. C. Willingham, and I. Pastan, Biochemistry 25:2231-2237, 1986). The interaction between rotavirus and liposomes is sensitive to specific divalent metal ions, unlike the adenovirus-liposome interaction, which is independent of them.     

β2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of β₂-microglobulin (β₂m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which β₂m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of β₂m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that β₂m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between β₂m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of β₂m amyloid-associated osteoarticular tissue destruction in DRA.     

Optimal transfection with the HK polymer depends on its degree of branching and the pH of endocytic vesicles

We have recently reported that liposomes in combination with histidine (HK)-containing polymers enhanced the expression of luciferase in transfected cells. In transformed or malignant cell lines, branched HK polymers (combined with liposome carriers) were significantly more effective than the linear HK polymer in stimulating gene expression. In the current study, we found that the linear HK polymer enhanced gene expression in primary cell lines more effectively than the branched polymers. The differences in the optimal carrier (linear versus branched) were not due to initial cellular uptake, size of the complexes or level of gene expression. There was, however, a strong association between the optimal type of HK polymer and the pH of endocytic vesicles (P = 0.0058). By altering the percentage of histidines carrying a positive charge, the endosomal pH of a cell may determine the amount of DNA released from the linear or branched HK polymer. In the two cell lines in which the linear HK was the optimal polymer, the endocytic vesicles were strongly acidic with a pH of <5.0. Conversely, in the four cell lines in which the branched polymers were optimal transfection agents, the pH of endocytic vesicles was >6.0. Furthermore, binding data support the relationship between DNA release from the optimal HK polymer and endosomal pH. The interplay between optimal HK polymers and the endosomal pH may lead to improved gene-delivery polymers tailored to a particular cell.     

Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay

The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS)     

Vincristine-sulphate–loaded liposome-templated calcium phosphate nanoshell as potential tumor-targeting delivery system

Vincristine-sulfate–loaded liposomes were prepared with an aim to improve stability, reduce drug leakage during systemic circulation, and increase intracellular uptake. Liposomes were prepared by the thin-film hydration method, followed by coating with calcium phosphate, using the sequential addition approach. Prepared formulations were characterized for size, zeta potential, drug-entrapment efficiency, morphology by transmission electron microscopy (TEM), in vitro drug-release profile, and in vitro cell cytotoxicity study. Effect of formulation variables, such as drug:lipid ratio as well as nature and volume of hydration media, were found to affect drug entrapment, and the concentration of calcium chloride in coating was found to affect size and coating efficiency. Size, zeta potential, and TEM images confirmed that the liposomes were effectively coated with calcium phosphate. The calcium phosphate nanoshell exhibited pH-dependent drug release, showing significantly lower release at pH 7.4, compared to the release at pH 4.5, which is the pH of the tumor interstitium. The in vitro cytotoxicity study done on the lung cancer cell line indicated that coated liposomes are more cytotoxic than plain liposomes and drug solution, indicating their potential for intracellular drug delivery. The cell-uptake study done on the lung cancer cell line indicated that calcium-phosphate–coated liposomes show higher cell uptake than uncoated liposomes.     

Vincristine-sulphate-loaded liposome-templated calcium phosphate nanoshell as potential tumor-targeting delivery system

Vincristine-sulfate-loaded liposomes were prepared with an aim to improve stability, reduce drug leakage during systemic circulation, and increase intracellular uptake. Liposomes were prepared by the thin-film hydration method, followed by coating with calcium phosphate, using the sequential addition approach. Prepared formulations were characterized for size, zeta potential, drug-entrapment efficiency, morphology by transmission electron microscopy (TEM), in vitro drug-release profile, and in vitro cell cytotoxicity study. Effect of formulation variables, such as drug:lipid ratio as well as nature and volume of hydration media, were found to affect drug entrapment, and the concentration of calcium chloride in coating was found to affect size and coating efficiency. Size, zeta potential, and TEM images confirmed that the liposomes were effectively coated with calcium phosphate. The calcium phosphate nanoshell exhibited pH-dependent drug release, showing significantly lower release at pH 7.4, compared to the release at pH 4.5, which is the pH of the tumor interstitium. The in vitro cytotoxicity study done on the lung cancer cell line indicated that coated liposomes are more cytotoxic than plain liposomes and drug solution, indicating their potential for intracellular drug delivery. The cell-uptake study done on the lung cancer cell line indicated that calcium-phosphate-coated liposomes show higher cell uptake than uncoated liposomes.     

Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein.

Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.     

A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, a novel thermolysis method was used to disrupt E. coli cells to release a recombinant thermostable esterase. We found that heat treatment of E. coli was highly effective to destroy the integrity of bacterial cell walls and release the recombinant hyperthermophilic esterase at temperatures above 60 degrees C. The effects of temperature, pH and cell concentration on the efficiency of cell disruption were examined. The most effective temperature for cell disruption was at 80 degrees C. The pH and cell concentration had only minor effect on the release of the hyperthermophilic esterase. In addition, we found that the hyperthermophilic esterase could be purified at the early stage of the thermolysis, which is a major advantage of the thermolysis method. Finally, a comparison between thermolysis and traditional methods for the disruption of cells and the release of the thermostable enzyme was made.     

Enhanced Neutrophil Stimulation by Phorbol Ester Encapsulated in pH-Sensitive Liposomes

Abstract

Phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA) are both hydrophobic stimulators for superoxide release by guinea pig neutrophils. However AA incorporated into liposomes is no longer an effective stimulator. In contrast, PMA incorporated into liposomes is more effective in neutrophil stimulation than free PMA. the ED₅₀ of superoxide release was 3.1 × 10^?8M, and 4.0 × 10^?10 M for free PMA and liposomes composed of egg phosphatidylethanolamine (PE) /AA/ PMA (molar ratio 7:2:1), respectively. PMA incorporated into PE/AA liposomes could also shorten the lag period of superoxide release in a concentration-dependent fashion. the enhanced stimulation activity of PMA in liposomes was correlated with the enhanced liposome uptake by neutrophils, probably via phagocytosis. Weak bases and a proton ionophore inhibited superoxide release by cells stimulated with either free or liposomal PMA. these results suggested that free PMA attached to cell membranes might be endocytosed and stimulate the superoxide-generating systems via an endocytic compartment(s). Since liposomes effectively deliver the contents into the compartments, liposomal PMA may thus be a potent stimulator for neutrophils. This hypothesis is further supported by the observation that pH-sensitive liposomes, which are active in the acidic endocytic compartments, are more effective carriers for PMA than the conventional pH-insensitive liposomes.     

Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.     

Role of vesicles during adenovirus 2 internalization into HeLa cells.

In this investigation, the early period of adenovirus type 2 (Ad2)-HeLa cell interaction was analyzed by electron microscopy and biochemical techniques. Events observed in this period ranged from the disappearance of virions from the cell surface to their subsequent association with the cell nucleus. Destabilization of the virions attached to the intact cell was necessary for virions to escape from intracellular vesicles. Strong temperature dependence and rapid escape from a vesicular compartment were shown in temporal kinetic experiments. These vesicles appeared to be acidic, since lysosomotropic agents partly inhibited the release of virions from vesicles. Studies of Ad2 binding to cells in buffers of different pH values suggested that adenovirus binds to cells by two different mechanisms. At low pH the binding was most probably mediated by the penton base and at neutral pH by the fiber protein. The number of receptor sites per cell was 25,000 and 6,000 at low and neutral pH, respectively. This study suggests that the low-pH affinity between the penton base and a vesicular membrane is important inside acid vesicles when Ad2 quickly enters the cytoplasm. However, a significant fraction of the virions was possibly internalized by a pathway not requiring a passage through such vesicles.     

Effect of steroidal glycoalkaloids of the potato on the permeability of liposome membranes

At pH 7.2, the potato glycoalkaloid α-chaconine caused release of entrapped peroxidase from phosphatidylcholine liposomes containing different free sterols but was ineffective against sterol-free liposomes. The alkaloid was able to complex with all the tested sterols in vitro although there was no close correlation between the extent of sterol binding and liposome disruption. α-Solanine also complexed with sterols in vitro but had no effects on sterol-containing liposomes under these conditions. Both sterol concentration and alkaloid concentration were limiting factors in the action of chaconine but did not markedly affect that of solanine. Solanine destabilized liposome membranes only at pH values of 8 and above but was less effective than chaconine. The importance of the carbohydrate moiety of glycoalkaloids was further demonstrated by the inability of β₂-chaconine to complex with sterols or disrupt liposomes.     

Membrane fusion mutants of Semliki Forest virus

Previous reports have indicated that the entry of Semliki Forest virus (SFV) into cells depends on a membrane fusion reaction catalyzed by the viral spike glycoproteins and triggered by the low pH prevailing in the endosomal compartment. In this study the in vitro pH-dependent fusion of SFV with nuclease-filled liposomes has been used to select for a new class of virus mutants that have a pH-conditional defect. The mutants obtained had a threshold for fusion of pH 5.5 as compared with the wild- type threshold of 6.2, when assayed by polykaryon formation, fusion with liposomes, or fusion at the plasma membrane. They were fully capable of infecting cells under standard infection conditions but were more sensitive to lysosomotropic agents that increase the pH in acidic vacuoles of the endocytic pathway. The mutants were, moreover, able to penetrate and infect baby hamster kidney-21 cells at 20 degrees C, indicating that the endosomes have a pH below 5.5. The results confirm the involvement of pH-triggered fusion in SFV entry, emphasize the central role played by acidic endosomal vacuoles in this reaction, shed further light on the mechanism of SFV inhibition by lysosomotropic weak bases, and demonstrate the usefulness of mutant viruses as biological pH probes of the endocytic pathway.     

Octaarginine- and octalysine-modified nanoparticles have different modes of endosomal escape

The present study examines the role of surface modification with an octaarginine peptide (R8) in liposomal escape from endocytic vesicles, using octalysine (K8) as a control cationic peptide; the mechanism of endosomal escape of liposomes was also investigated. Gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than 1 order of magnitude higher than that of K8-modified nanoparticles, and 2 orders of magnitude higher than gene expression using unmodified nanoparticles. The difference in gene expression could not be attributed to differences in uptake, as R8- and K8-modified liposomes were taken up primarily via macropinocytosis with comparable efficiency. The extent of R8-nanoparticle escape to the cytosol was double that of K8-nanoparticles. Suppression of endosome acidification inhibited R8-nanoparticle endosomal escape, but enhanced that of K8-nanoparticles. Using spectral imaging in live cells, we showed that R8- and K8-liposomes escaped from endocytic vesicles via fusion between the liposomes and the endosomal membrane. R8-liposomes fused efficiently at both acidic and neutral pH, whereas K8-liposomes fused only at neutral pH. Similar behavior was observed during in vitro lipid mixing and calcein-release experiments. Co-incubation of cells with distinctly labeled K8- and R8-modified nanoparticles confirmed a common uptake pathway and different rates of endosomal escape particularly at longer time intervals. Therefore, it was concluded that R8 on the liposome surface stimulates efficient escape from endocytic vesicles via a fusion mechanism that works at both neutral and acidic pH; in contrast, K8 mediates escape mainly at neutral pH.     

Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides

Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system. The extent of such damage depends in part on the antioxidant defenses of the target cell. To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system. The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid. Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density. Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr. Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase. Reagent H2O2 could be replaced by glucose plus glucose oxidase. Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min. The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions. Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0. In the absence of halides, no lysis was observed. Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride. Fluoride was inactive. Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride. Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury. These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system. They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells. By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.     

Adenovirus-induced leakage of co-endocytosed macromolecules into the cytosol

The early interaction between KB cells and adenovirus was studied by examining the uptake of an extracellular fluorescent macromolecule, FITC (fluorescein isothiocyanate)-labeled dextran (FD). When cells in suspension were incubated with both adenovirus and FD, cell-associated FD increased 2- to 3-fold the value obtained without adenovirus. Under fluorescence microscopy, cells incubated with adenovirus showed bright, whole-cellular fluorescence; whereas, those incubated without adenovirus, or with heat-inactivated virus, showed weaker fluorescence, mainly of the pinocytic vesicles. The increased uptake of the FD by adenovirus was inhibited by treating KB cells with the drugs chloroquine, ammonium chloride and monensin that raise the pH of the acidic compartment. Entry of adenovirus into the KB cell's nucleus also was inhibited by these drugs. The conclusion is that entry of adenovirus into the cell involves its passage of an acidic compartment (probably the endocytic vesicle) and that co-endocytosed macromolecules are released into the cytosol on entry.     

pH-dependent fusion of vesicular stomatitis virus with Vero cells. Measurement by dequenching of octadecyl rhodamine fluorescence

We have studied fusion between membranes of vesicular stomatitis virus (VSV) and Vero cells using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We could identify the two pathways of fusion by the kinetics of R18 dequenching, effects of inhibitors, temperature dependence, and dependence on osmotic pressure. Fusion at the plasma membrane began immediately after lowering the pH below 6 and showed an approximately exponential time course, whereas fusion via the endocytic pathway (pH 7.4) became apparent after a time delay of about 2 min. Fusion via the endocytic pathway was attenuated by treating cells with metabolic inhibitors and agents that raise the pH of the endocytic vesicle. A 10-fold excess of unlabeled virus arrested R18VSV entry via the endocytic pathway, whereas R18 dequenching below pH 6 (fusion at the plasma membrane) was not affected by the presence of unlabeled virus. The temperature dependence for fusion at pH 7.4 (in the endosome) was much steeper than that for fusion at pH 5.9 (with the plasma membrane). Fusion via the endocytic pathway was attenuated at hypo-osmotic pressures, whereas fusion at the plasma membrane was not affected by this treatment. The pH profile of Vero-VSV fusion at the plasma membrane, as measured by the dequenching method, paralleled that observed for VSV-induced cell-cell fusion. Fusion was blocked by adding neutralizing antibody to the Vero-VSV complexes. Activation of the fusion process by lowering the pH was reversible, in that the rate of fusion was arrested by raising the pH back to 7.4. The observation that pH-dependent fusion occurred at similar rates with fragments and with intact cells indicates that pH, voltage, or osmotic gradients are not required for viral fusion.     

Structural studies of adeno-associated virus serotype 8 capsid transitions associated with endosomal trafficking

The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.     

The pH-dependent interaction of cinnamomin with lipid membranes investigated by fluorescence methods

Cinnamomin, a new type II ribosome-inactivating protein (RIP), was found to be able to induce the release of calcein loaded in lecithin small unilamellar vesicles and the fusion or aggregation of the lecithin liposomes. Such induction could be promoted severalfold by a pH 5.0 environment, a condition similar to that in endocytic vesicles. Lowering the pH from 7.5 to 5.0 evoked conformational changes of cinnamomin and unmasked its hydrophobic areas, including the exposure of 1-anilino-8-naphthalenesulfonate (1,8-ANS) binding sites of the molecule. Some tryptophan residues with affinity to acrylamide were demonstrated to participate in the lipid-protein interaction. The pH dependent fusogenicity of type II RIP might suggest its in vivo function as a fusogen to exert its cytotoxicity.