Effect of sterol structure on the transfer of sterols and phospholipids from liposomes to erythrocytes in vitro.

Human erythrocytes were incubated for 24 h with liposomes containing [3H]phosphatidylcholine, [14C]cholesterol and one of several other sterols. Of the other sterols, 3-hydroxycholest-3-en-2-one did not appear to be taken up by the cells, sterophenol was taken up at about the same rate as cholesterol, and cholesta-4,6-dien-3-one and 7-oxocholesterol were taken up much more rapidly than cholesterol. Each component of the liposomes was incorporated into the cells independently of the others and the rate of incorporation of the test sterol had little, if any, effect on the rate of incorporation of phospholipid or cholesterol. The results support the proposal that sterol exchange is mediated via the pool of monomers present in the medium rather than by a collision mechanism.

The interaction of synthetic analogs of the N-terminal fusion sequence of influenza virus with a lipid monolayer. Comparison of fusion-active and fusion-defective analogs.

The amino terminus of subunit-2 of influenza virus hemagglutinin (NHA2) plays a crucial role in the induction of fusion between viral and endosomal membranes leading to the infection of a cell. Three synthetic analogs with an amino acid sequence corresponding to NHA2 of variant hemagglutinins were studied in a monolayer set up. Comparison of the interaction of a fusion-active and two fusion-defective analogs with a lipid monolayer revealed a greater surface activity of the fusion-active analog. Pronounced differences were found if the pure peptides were spread at the air/water interface; the fusion-active analog showed a higher collapse pressure and a greater limiting molecular area. Circular dichroism measurements on collected lipid monolayers indicated a high content of alpha-helical structure for the fusion-active and one of the fusion-defective analogs. A simple relation between alpha-helical content and fusogenicity does not seem to exist. Instead, the extent of penetration, a defined tertiary structure or orientation of the alpha-helical peptide may be essential for its membrane perturbing activity.

Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts.

Puromycyl peptides were degraded in MRC5 fibroblasts more rapidly than normal proteins labelled for the corresponding length of time for both long and short labelling periods. The degradation of the puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half-lives of normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal proteins were degraded in the lysosomes. The rapid degradation of the puromycyl peptides was not due to the inhibition of protein synthesis brought about by puromycin but was due to the structure of the substrates themselves. The degree and intracellular site of degradation of puromycyl peptides closely mimic those of abnormal (missense) proteins containing amino acid analogues.

Degradation of peptides and proteins of different sizes by homogenates of human MRC5 lung fibroblasts. Aged cells have a decreased ability to degrade shortened proteins.

The degradation of haemoglobin and haemoglobin-derived peptide fragments by homogenates of MRC5 fibroblasts has been investigated. Results show that the smaller fragments were degraded more rapidly than larger substrates at both pH 5.5 and pH 7.5. Only the smallest of the soluble cyanogen bromide peptides (Mr 3500) was degraded at pH 7.5. Degradation at pH 5.5 proceeded more rapidly than that at pH 7.5 for all substrates tested but was more marked with the larger substrates. Homogenates prepared from aged cells degraded puromycin peptides and, to a lesser extent, cyanogen bromide peptides at a slower rate, at pH 7.5, than those prepared from younger cells. We suggest that cytosolic degradation is less selective and at least one cytosolic proteolytic activity decreases as cells age.

A monoclonal antibody specific to the HA2 glycoprotein of influenza A virus hemagglutinin that inhibits its fusion activity reduces replication of the virus.

Monoclonal antibody (MAb) CF2, which binds to the fusion peptide of influenza A virus hemagglutinin (HA) (amino acids (aa) 1-35 of the N-terminus of the light chain of HA), inhibited the fusion activity of HA. This MAb preferentially bound to pH 5-treated virus (with conformationally altered HA) and bound only weakly to the native wild type (wt) virus. However, a significant binding of MAb CF2 to the amantadine resistant virus mutant Ab4 (with a mutation at aa 17 of HA1 leading to a destabilization of HA trimer) was obtained without pH 5 treatment. Exploiting the fusion-inhibition activity of MAb CF2 the effect of this antibody on the virus replication in vitro was followed using both the wt virus and the amantadine resistant mutant Ab4. No reduction of replication of wt virus and a low reduction of replication of Ab4 mutant (by about 20%) was detected by radioimmunoassay after preincubation of the virus with a high concentration of MAb CF2 at room temperature. An increased reduction of replication of Ab4 mutant (by about 40%) was observed in cell radioimmunoassay (RIA) and in plaque assay when the virus was preincubated with MAb at 37 degrees C. Under these conditions a reduction of the wt virus replication also occurred by about 40%. This is the first report on the capacity of a MAb specific to HA2 gp, the light chain of influenza A virus HA, to reduce replication of the virus. This capacity in relation to (i) the affinity of the antibody to the virus, and (ii) the accessibility of corresponding epitopes on the virus surface as well as the proposed mechanism of inhibition of replication of the virus are discussed.

The role of influenza virus haemagglutinin in membrane fusion.

An acid-induced conformational change in influenza virus haemagglutinin triggers the virus to fuse with the endosomal membrane of a host cell during infection. Structural aspects of this change are discussed along with the possible haemagglutinin-mediated processes which occur during membrane fusion.

A comparison of lysosomal involvements in the degradation of normal and abnormal endogenous proteins of differing half-lives in MRC5 cells.

Protein degradation by diploid human-embryo lung fibroblasts (MRC5 cells) in monolayer culture was studied. 1. Varying the labelling period of proteins was found to alter the half-lives of labelled abnormal canavanine-containing proteins to an extent very similar to that obtained with normal proteins. 2. By manipulating the times of labelling it was possible to generate a species of abnormal protein with a greater half-life than that of a species of normal protein. A comparison of the lysosomal involvement in their degradation as determined both by inhibition by methylamine, a lysosomotropic agent, and by the degree of increase in protein degradation in step-down conditions, indicated that the degree of lysosomal involvement was not entirely dependent upon the half-life of the protein, but that abnormal proteins are preferentially degraded non-lysosomally. 3. The microtubule inhibitors colchicine and vinblastine were found to stimulate statistically basal protein degradation of normal long-labelled protein, whereas they had less effect upon the basal degradation of the other species of proteins studied and very little effect upon step-down degradation of all proteins studied. The stimulation in protein degradation found did not seem to involve the acid proteinases of lysosomes.

The suppression of endogenous protein degradation by fractions of foetal calf serum: dialysed serum is less able to suppress degradation in aged cells.

We have studied the effect of various fractions of foetal bovine serum upon the endogenous degradation of long labelled proteins in cultured MRC5 cells, and upon other cellular functions. Only heat-inactivated serum was capable of suppressing protein degradation to a similar extent to complete serum. Acid-treated and delipidized sera were moderately effective. Albumin on its own was able to replace 40 per cent of the effect of serum, indicating the exogenous protein might compete with endogenous protein for degradation in lysosomes. Albumin was not capable of supporting DNA synthesis. Dialysed serum showed an age-related effect suppressing protein degradation to a lesser extent and being less effective in supporting DNA synthesis or cellular proliferation in aged cells. All the effects noted were related to lysosomal protein degradation. Serum diffusate did not suppress protein degradation.

Use of fluorescent probes in the study of phospholipid--sterol bilayers.

1. The transfer of excitation energy between the fluorescent probes 1,6-diphenylhexa-1,3,5-triene and 12-(9-anthroyl)stearic acid and the cholesterol analogue cholesta-4,6-dien-3-one in phosphatidylcholine liposomes has been investigated. 2. The results indicate that probes and steroid are randomly distributed in the bilayer at steroid concentrations up to 35 mol%. 3. The degree of polarization of diphenylhexatriene fluorescence increases with increasing cholesterol content. Other sterols, differing in structure in the region of the polar group or in the side chain at position-17, produce similar but not identical effects. 4. the results are consistent with the proposal that diphenylhexatriene gives a general picture of the state of the bilayer and that there is no segregation of sterols in liquid-crystalline phosphatidylcholine bilayers.

Inhibition of fusion activity of influenza A haemagglutinin mediated by HA2-specific monoclonal antibodies.

The effect of seven monoclonal antibodies (MAbs) specific to the light chain (HA2) of influenza A haemagglutinin (HA) on its fusion activity was investigated. These MAbs, which are non-virus neutralizing, defined four distinct antigenic sites on HA2 glycopolypeptide and the corresponding epitopes were attributed to the sequence stretches on HA2. The accessibility of all seven HA2 epitopes significantly increased after trypsin cleavage and pH 5 treatment of the HA (X-31). The influence of anti-HA2 MAbs on the fusion process was followed by cell-cell fusion of CHO cells expressing precursor HA, virus-liposome fusion assay, and haemolysis mediated by virus. MAb CF2, which bound directly to the fusion peptide 1-35 of HA2, was positive in all three fusion-inhibition assays and was the only one inhibiting the polykaryon formation of CHO-X-31 cells. Two other MAbs belonging to the same antigenic site but not binding directly to the fusion peptide inhibited virus to liposome fusion (EB12) or inhibited haemolysis (BB8). Moreover, MAb IIF4 binding to distinct antigenic site within 125-175 HA2 inhibited haemolysis, too. Thus, fusion activity of HA may be inhibited by anti-HA2 MAbs, mainly those binding to or near the fusion peptide. These antibodies represent useful probes for studies of influenza virus to cell membrane fusion.

Studies of influenza haemagglutinin-mediated membrane fusion.

A resonance energy transfer assay of membrane fusion developed by P. S. Uster and D. W. Deamer (Biochemistry 24, 1-8 (1985)) was used in a study of influenza haemagglutinin-mediated fusion. The characteristics of fusion and haemolysis by X-31 (H3N2) virus, a number of mutants of X-31 which fuse membranes at higher pH, and purified haemagglutinins released from virus particles either by detergent dissociation or by bromelain digestion were compared with particular regard to pH and temperature dependence. The finding that membrane fusion activity, haemolysis, and changes in haemagglutinin conformation covary with pH and temperature provide support for the role of haemagglutinin in fusion and are discussed in relation to the stability of its structure.

Temperature dependence of fusion by sendai virus.

Studies of the temperature dependence of liposome fusion by Sendai virus indicate that fusion occurs maximally at 55 degrees C. The fusion capacity of the virus is also inactivated maximally by preincubation at this temperature and, under the same conditions, the F glycoprotein becomes resistant to proteolysis. By analogy with the activation at elevated temperatures of fusion by influenza virus our results suggest that temperature is also a variable in the activation of fusion by paramyxoviruses and possibly in the activation of other members of the group of viruses that includes myxo-, paramyxo-, retro-, and filoviruses, which all contain cleaved, trimeric fusion glycoproteins.

Electron microscopy of the influenza virus submembranal structure.

Electron micrographs of stain-penetrated influenza virus particles show a variety of internal structures that all consist of units of 4 X 4 nm. Isolated influenza virus M-protein, that is reassociated with lipid, forms very similar structures on liposomes. We therefore conclude that the 4 X 4 nm unit is M-protein. We further argue that the M-protein is not arranged with icosahedral symmetry.

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers.

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.

The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil.

The ectodomain of the Ebola virus Gp2 glycoprotein was solubilized with a trimeric, isoleucine zipper derived from GCN4 (pIIGCN4) in place of the hydrophobic fusion peptide at the N terminus. This chimeric molecule forms a trimeric, highly alpha-helical, and very thermostable molecule, as determined by chemical crosslinking and circular dichroism. Electron microscopy indicates that Gp2 folds into a rod-like structure like influenza HA2 and HIV-1 gp41, providing further evidence that viral fusion proteins from diverse families such as Orthomyxoviridae (Influenza), Retroviridae (HIV-1), and Filoviridae (Ebola) share common structural features, and suggesting a common membrane fusion mechanism.

Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin.

Influenza virus hemagglutinin (HA) fuses membranes at endosomal pH by a process which involves extrusion of the NH2-terminal region of HA2, the fusion peptide, from its buried location in the native trimer. We have examined the amino acid sequence requirements for a functional fusion peptide by determining the fusion capacities of site-specific mutant HAs expressed by using vaccinia virus recombinants and of synthetic peptide analogs of the mutant fusion peptides. The results indicate that for efficient fusion, alanine can to some extent substitute for the NH2-terminal glycine of the wild-type fusion peptide but that serine, histidine, leucine, isoleucine, or phenylalanine cannot. In addition, mutants containing shorter fusion peptides as a result of single amino acid deletions are inactive, as is a mutant containing an alanine instead of a glycine at HA2 residue 8. Substitution of the glycine at HA2 residue 4 with an alanine increases the pH of fusion, and valine-for-glutamate substitutions at HA2 residues 11 and 15 are without effect. We confirm previous reports on the need for specific HAo cleavage to generate functional HAs, and we show that both inappropriately cleaved HA and mutant HAs, irrespective of their fusion capacities, upon incubation at low pH undergo the structural transition required for fusion.

Fusion characteristics of influenza C viruses.

A number of different influenza C virus strains were tested for their fusion properties using a resonance energy assay which allows direct monitoring of fusion between virus membranes and artificial lipid vesicles. The fusion pH of various strains was found to range between 5.6 and 6.1. Haemolytic activity of the different strains with chicken erythrocytes was observed at slightly lower pH values and varied between 5.1 and 5.7. Studies of the kinetics of influenza C virus fusion showed distinct characteristics in fusion activity. A lag before onset of fusion was found with influenza C virus which was not observed for influenza A or B viruses. In addition, studies on the rate of conformational change of the influenza C virus glycoprotein, as determined by morphological changes and endogenous tryptophan fluorescence, suggest that the conformational change is rate-limiting in the fusion process, whereas for influenza A viruses the glycoprotein conformational change is fast and a later step in the fusion process is rate-limiting. Monitoring the conformational change of influenza C virus glycoprotein by the onset of trypsin susceptibility showed, however, that membrane fusion occurred in some cases without onset of trypsin susceptibility, indicating that the trypsin-susceptible conformation is a post-fusogenic conformation.

Deacylation of the hemagglutinin of influenza A/Aichi/2/68 has no effect on membrane fusion properties.

The effects of deacylating the H3 influenza hemagglutinin (HA) on its membrane fusion activity were investigated. Chemical deacylation caused no change in the ability of HA to fuse liposomes in vitro. Site-specific mutagenesis of the three cysteine residues in the cytoplasmic tail singly, or in combination, showed that all three were palmitoylated. Substitution of one, two, or all three cysteines with serine and subsequent lack of palmitoylation at mutated sites had no effect on the pH of the conformational change in HA required for fusion activity or the extent of fusion activity.

Interaction of influenza virus hemagglutinin with a lipid monolayer. A comparison of the surface activities of intact virions, isolated hemagglutinins, and a synthetic fusion peptide.

In the infectious entry pathway of influenza virus, the low pH of the endosomal compartment induces an irreversible conformational change in influenza virus hemagglutinin, leading to fusion of viral and endosomal membranes. In the current report, we characterized the low-pH-induced activation of hemagglutinin of influenza strain X31 by studying its interaction with a lipid monolayer. The surface activities of virions, of isolated hemagglutinins and its proteolytic fragments, and of a synthetic peptide mimicking the amino terminus of subunit 2 of hemagglutinin are compared. The data indicate that the surface activity of both virions and isolated hemagglutinin develop as a result of the low-pH-induced conformational change in hemagglutinin. The surface activity of isolated hemagglutinin is mainly caused by penetration into the lipid monolayer of protein domains other than the amino terminus of subunit 2 of hemagglutinin; domains in subunit 1 may be involved. The surface activity of virions appears to be a secondary effect of the conformational change and is explained by assuming a net transfer of viral lipids to the lipid monolayer.

The strong positive correlation between effective affinity and infectivity neutralization of highly cross-reactive monoclonal antibody IIB4, which recognizes antigenic site B on influenza A virus haemagglutinin.

Monoclonal antibody (MAb) IIB4 displays a rare combination of virus neutralization (VN) activity and broad cross-reactivity with influenza A virus strains of the H3 subtype isolated in a period from 1973 to 1988. The epitope of this antibody has been identified as around HA1 residues 198, 199 and 201. Here we report that residues 155, 159, 188, 189 and 193 also influence the binding of this antibody. We have used this antibody to study the relationship between antibody affinity and VN activity. Using one MAb and a single epitope on the haemagglutinin (HA) of different influenza viruses we found a strong positive correlation between effective affinity and VN activity of MAb IIB4. A 10-fold increase in effective affinity corresponded to the 2000-fold increase in VN titre. It follows from the law of mass action that for an effective affinity K=9x10(8) l/mol, 50% VN was achieved at approx. 10% occupation of HA spikes with antibody. In contrast, for an effective affinity K=6x10(7) l/mol, to achieve 50% VN, occupation of up to 98% of HA spikes was required. An effective affinity about K=6x10(7) l/mol thus represents the limiting value for VN because a further decrease in the affinity cannot be compensated by a higher concentration of antibody.