Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.     

Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.     

Functional reconstitution of the integral membrane proteins of influenza virus into phospholipid liposomes

The integral membrane proteins of influenza virus, a hemagglutinin and a neuraminidase, have been incorporated into liposomes composed of either phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylethanolamine (2:1 w/w) using detergent dialysis. The virus spike glycoproteins for reconstitution were selectively solubilized by using cetyltrimethylammonium bromide to leave a "core particle", which lacked a lipid bilayer but possessed quaternary structure as observed by electron microscopy. The viral spike proteins were combined with exogenous phospholipid in excess sodium cholate followed by exhaustive dialysis for 150 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that only the viral glycoproteins were associated with all the complexes formed. The level of sodium cholate remaining after dialysis was shown to be reduced to less than 1 molecule per 80 protein molecules. Viral proteins reconstituted into dimyristoylphosphatidylcholine liposomes were shown to have retained hemagglutination, low-pH-dependent hemolysis, and neuraminidase activities and were associated with a lipid bilayer in two types of complexes with average lipid to protein mole ratios after sucrose density gradient purification of either 590:1 or 970:1. The bilayer vesicles formed were of similar sizes and were shown by negative-stain electron microscopy to be 150-300 nm in diameter with well-defined spikes on their surface. Reconstituted liposomes of dimyristoylphosphatidylcholine were found to be unstable with respect to their trapped volume and therefore were unsuitable for fusion studies, unlike complexes formed with phosphatidylcholine or a mixture of phosphatidylcholine/phosphatidylethanolamine derived from hen eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new method for reconstitution of highly fusogenic sendai virus envelopes

A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.     

Reconstitution of functional influenza virus envelopes and fusion with membranes and liposomes lacking virus receptors.

Reconstituted influenza virus envelopes were obtained following solubilization of intact virions with Triton X-100. Quantitative determination revealed that the hemolytic and fusogenic activities of the envelopes prepared by the present method were close or identical to those expressed by intact virions. Hemolysis as well as virus-membrane fusion occurred only at low pH values, while both activities were negligible at neutral pH values. Fusion of intact virions as well as reconstituted envelopes with erythrocyte membranes--and also with liposomes--was determined by the use of fluorescently labeled viral envelopes and fluorescence dequenching measurements. Fusion with liposomes did not require the presence of specific virus receptors, namely sialoglycolipids. Under hypotonic conditions, influenza virions or their reconstituted envelopes were able to fuse with erythrocyte membranes from which virus receptors had been removed by treatment with neuraminidase and pronase. Inactivated intact virions or reconstituted envelopes, namely, envelopes treated with hydroxylamine or glutaraldehyde or incubated at low pH or 85 degrees C, neither caused hemolysis nor possessed fusogenic activity. Fluorescence dequenching measurements showed that only fusion with liposomes composed of neutral phospholipids and containing cholesterol reflected the viral fusogenic activity needed for infection.     

Functional reconstitution of thymopoiesis after human immunodeficiency virus infection

We have utilized combination antiretroviral therapy following human immunodeficiency virus type 1-induced human CD4(+) thymocyte depletion in the SCID-hu mouse to examine the immune competence of reconstituting thymocytes which appear following administration of combination therapy. These cells express a normal distribution of T-cell receptor variable gene families and are responsive to costimulatory signals. These results suggest that normal thymic function may be restored following antiretroviral treatment.     

Functional reconstitution of influenza A M2(22-62)

Amantadine-sensitive proton uptake by liposomes is currently the preferred method of demonstrating M2 functionality after reconstitution, to validate structural determination with techniques such as solid-state NMR. With strong driving forces (two decades each of both [K(+)] gradient-induced membrane potential and [H(+)] gradient), M2(22-62) showed a transport rate of 78 H(+)/tetramer-s (pH(o) 6.0, pH(i) 8.0, nominal V(m)=-114 mV), higher than previously measured for similar, shorter, and full-length constructs. Amantadine sensitivity of the conductance domain at pH 6.8 was also comparable to other published reports. Proton flux rate was optimal at protein densities of 0.05-1.0% (peptide wt.% in lipid). Rundown of total proton uptake after addition of valinomycin and CCCP, as detected by delayed addition of valinomycin, indicated M2-induced K(+) flux of 0.1K(+)/tetramer-s, and also demonstrated that the K(+) permeability, relative to H(+), was 2.8 × 10(-6). Transport rate, amantadine and cyclooctylamine sensitivity, acid activation, and H(+) selectivity were all consistent with full functionality of the reconstituted conductance domain. Decreased external pH increased proton uptake with an apparent pK(a) of 6.     

Functional interactions of the influenza virus glycoproteins

Hemagglutinin (HA) and neuraminidase (NA) are functionally related coat glycoproteins of the influenza virus (Flu). HA interacts with terminal sialyl residues of oligosaccharides and ensures the binding of the virus particle to the cell surface. NA is a receptor-destroying enzyme that removes sialyl residues from oligosaccharides contained in cell and virus components and thereby prevents aggregation of virus particles. Analysis of reasortants combining low-functional NA of human Flu with HA of avian Flu showed that sialyl residues are not completely removed in some cases. With high HA affinity for sialyl substrates, such virus particles aggregate, aggregates accumulate on the cell surface, and virus yield decreases. Serial passaging of low-yield aggregating reassortants may lead to selection of high-yield variants, which do not aggregate. A loss of aggregation is due to a decrease in HA affinity for high-molecular-weight sialyl substrates. On evidence of sequencing of the HA gene in original reassortants and their nonaggregating variants, HA affinity is reduced and aggregation lost owing to a mechanism common for different HA antigenic subtypes (H2, H3, H4, and H13). This is an increase in the negative charge as a result of an amino acid substitution in the vicinity of the receptor-binding pocket of HA. Taken together, these findings suggest a way of postreassortment adaptation which improves the functional match of HA and NA. The experimental system employed provides a model of natural processes associated with generation of Flu variants having a pandemic potential.     

Fusion of reconstituted influenza virus envelopes with liposomes mediated by streptavidin/biotin interactions

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) represent an efficient fusogenic cellular delivery system. By interaction of HA with its natural receptors, sialylated lipids (gangliosides) or proteins, virosomes bind to cells and, following endocytic uptake, deliver their contents to the cytosol through fusion from within acidic endosomes. Here, we show that binding to sialic acid is not necessary for fusion. In the presence of streptavidin, virosomes containing a biotinylated lipid fused with liposomes lacking sialic acid if these liposomes also had a biotinylated lipid in their membranes. Moreover, fusion characteristics corresponded well with fusion of virosomes with ganglioside-containing liposomes.     

Functional motions of influenza virus hemagglutinin: a structure-based analytical approach.

Influenza virus hemagglutinin (HA), a homotrimeric integral membrane glycoprotein essential for viral infection, is engaged in two biological functions: recognition of target cells' receptor proteins and fusion of viral and endosomal membranes, both requiring substantial conformational flexibility from the part of the glycoprotein. The different modes of collective motions underlying the functional mobility/adaptability of the protein are determined in the present study using an extension of the Gaussian network model (GNM) to treat concerted anisotropic motions. We determine the molecular mechanisms that may underlie HA function, along with the structural regions or residues whose mutations are expected to impede function. Good agreement between theoretically predicted fluctuations of individual residues and corresponding x-ray crystallographic temperature factors is found, which lends support to the GNM elucidation of the conformational dynamics of HA by focusing upon a subset of dominant modes. The lowest frequency mode indicates a global torsion of the HA trimer about its longitudinal axis, accompanied by a substantial mobility at the viral membrane connection. This mode is proposed to constitute the dominant molecular mechanism for the translocation and aggregation of HAs, and for the opening and dilation of the fusion pore. The second and third collective modes indicate a global bending, allowing for a large lateral surface exposure, which is likely to facilitate the close association of the viral and endosomal membranes before pore opening. The analysis of kinetically hot residues, in contrast, reveals a localization of energy centered around the HA2 residue Asp112, which apparently triggers the solvent exposure of the fusion peptide.     

Functional reconstitution of halorhodopsin. Properties of halorhodopsin-containing proteoliposomes

A one-step purification method for halorhodopsin was developed. Functional proteoliposomes were prepared from this preparation using cholate, which is removed by dialysis in the presence of asolectin or the polar halobacterial lipids. Light-induced outward directed transport of chloride by halorhodopsin was followed by measuring passive proton efflux in the presence of uncoupler; initial rates and extents amounted to significant fractions of values obtained for halorhodopsin-containing cell envelope vesicles. The transport activity was much higher when cholate rather than octyl glucoside was used in the reconstitution. Since CD spectra in cholate but not in octyl glucoside showed band-splitting in the visible region, suggestive of exciton interaction between halorhodopsin monomers, the reconstitution may depend on an aggregate state of the halorhodopsin. The rate constants for three thermal steps in the halorhodopsin photocycle were greatly reduced in the detergent-solubilized samples, but they increased in the proteoliposomes to values similar to those for halorhodopsin in cell envelope vesicles. Thus, the reconstitution yields halorhodopsin with both photochemical and transport activities restored. Freeze-fracture electron micrographs of the proteoliposomes showed unilammellar liposomes with numerous particles of 100-150 A diameter at the fracture faces. These should correspond to halorhodopsin aggregates, formed in the bilayer in an apparently concentration-dependent manner.     

Functional reconstitution of the glycine receptor

The functional reconstitution of the chloride channel coupled glycine receptor is described. Glycine receptors were purified from the cholate extract of rat spinal cord membranes by affinity chromatography and incorporated into phospholipid vesicles by the addition of phosphatidylcholine and removal of detergent by gel filtration. The reconstituted vesicles showed the same polypeptide composition as the purified receptor (proteins of Mr 48,000 and 58,000). The pharmacological characteristics of the glycine receptor were also preserved in the proteoliposomes, as demonstrated by the displacement of [3H]strychnine binding by several glycinergic ligands and by photoaffinity labeling experiments. In order to observe functional responses (i.e., specific agonist-induced anion translocation), we have developed an assay based on the fluorescence quenching of an anion-sensitive entrapped probe, SPQ [6-methoxy-N-(3-sulfopropyl)quinolinium]. Reconstituted vesicles were loaded with the fluorescent probe during a freeze-thaw-sonication cycle in the presence of added liposomes containing cholesterol. In such a reconstituted system, glycine receptor agonists are able to increase the rate of anion influx into the vesicles. The action of agonists is blocked by the simultaneous presence of strychnine or other glycine antagonists. Our results show that the purified 48,000- and 58,000-dalton polypeptides reconstituted into phospholipid vesicles can bind ligands and promote specific ion translocation in a way similar to the glycine receptor in its native environment.     

Infectious particles derived from Semliki Forest virus vectors encoding murine leukemia virus envelopes.

Semliki Forest virus vectors encoding murine leukemia virus (MLV) envelope protein with a truncated cytoplasmic tail generate submicrometer, cell-associated, membranous particles that transmit replication-competent vector RNA specifically to cells bearing the MLV receptor. Such "minimal" viruses could have applications as retroviral vaccines or in the study of virus evolution.     

Functional reconstitution of a chloride channel protein from bovine trachea.

We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.