Fusion activity of influenza virus. A comparison between biological and artificial target membrane vesicles

We have investigated the pH-dependent fusion activity of influenza virus toward human erythrocyte ghosts, utilizing a recently developed fluorescence assay, which permits continuous monitoring of the fusion reaction. The rate of fusion is negligible at neutral pH but shows a sharp increase at pH values just below 5.5. This pH dependence profile closely corresponds to that of virus-induced hemolysis. Fusion is rapidly inactivated by a low-pH preincubation of the virus alone either at 37 or at 0 degrees C. The presence of ghosts during this low-pH preincubation, carried out at 0 degree C under which condition there is hardly any fusion, causes a significant protection of the viral fusion activity against inactivation. Fusion initiated at low pH can be arrested instantaneously by readjustment of the pH to neutral. The characteristics of fusion of influenza virus with ghosts deviate from those of fusion with cardiolipin liposomes (Stegmann, T., Hoekstra, D., Scherphof, G., and Wilschut, J. (1985) Biochemistry 24, 3107-3113). Fusion with ghosts is consistent with a requirement of the well-documented pH-dependent conformational change in the viral hemagglutinin, whereas fusion with cardiolipin liposomes does not exhibit a strict dependence on the conformational change. Rather, the negative surface charge on the liposomes plays a critical role, as zwitterionic liposomes containing gangliosides show fusion behavior similar to that of erythrocyte ghosts.     

Kinetic modeling of Sendai virus fusion with PC-12 cells. Effect of pH and temperature on fusion and viral inactivation.

We have studied the fusion activity of Sendai virus, a lipid-enveloped paramyxovirus, towards a line of adherent cells designated PC-12. Fusion was monitored by the dequenching of octadecyl-rhodamine, a fluorescent non-exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus-cell fusion. When the temperature was lowered from 37 degrees C to 25 degrees C, a sharp inhibition of the fusion process was observed, probably reflecting a constraint in the movement of viral glycoproteins at low temperatures. The rate constants of adhesion and fusion were reduced 3.5-fold and 7-fold, respectively, as the temperature was lowered from 37 degrees C to 25 degrees C. The fusion process seemed essentially pH-independent, unlike the case of liposomes and erythrocyte ghosts. Preincubation of the virus in the absence of target cell membranes at neutral and alkaline pH (37 degrees C, 30 min) did not affect the fusion process. However, a similar preincubation of the virus at pH = 5.0 resulted in marked, though slow, inhibition in fusion with the fusion rate constant being reduced 8-fold. Viral preincubation for 5 min in the same acidic conditions yielded a mild inhibition of fusogenic activity, while preincubation in the cold (4 degrees C, 30 min) did not alter viral fusion activity. These acid-induced inhibitory effects could not be fully reversed by further viral preincubation at pH = 7.4 (37 degrees C, 30 min). Changes in internal pH as well as endocytic activity of PC-12 cells had small effect on the fusion process, thus indicating that Sendai virus fuses primarily with the plasma membranes.     

Morphological changes and fusogenic activity of influenza virus hemagglutinin.

The kinetics of low-pH induced fusion of influenza virus with liposomes have been compared to changes in the morphology of influenza hemagglutinin (HA). At pH 4.9 and 30 degrees C, the fusion of influenza A/PR/8/34 virus with ganglioside-bearing liposomes was complete within 6 min. Virus preincubated at pH 4.9 and 30 degrees C in the absence of liposomes for 2 or 10 min retained most of its fusion activity. However, fusion activity was dramatically reduced after 30 min, and virtually abolished after a 60-min preincubation. Cryo-electron microscopy showed that the hemagglutinin spikes of virions exposed to pH 4.9 at 30 degrees C for 10 min underwent no major morphological changes. After 30 min, however, the spike morphology changed dramatically, and further changes occurred for up to 60 min after exposure to low pH. Because the morphological changes occur at a rate corresponding to the loss of fusion activity, and because these changes are much slower than the rate at which fusion occurs, we conclude that the morphologically altered HA is inactive with respect to fusion-promoting activity. Molecular modeling studies indicate that the formation of an extended coiled coil within the HA trimer, as proposed for HA at low pH, requires a major conformational change in HA, and that the morphological changes we observe are consistent with the formation of an extended coiled coil. These results imply that the crystallographically determined low-pH form of HA does occur in the intact virus, but that this form is not a precursor of viral fusion. It is speculated that the motion to the low-pH form may be responsible for the membrane destabilization leading to fusion.     

Kinetics of pH-dependent fusion between influenza virus and liposomes

The pH-dependent fusion between influenza virus and liposomes (large unilamellar vesicles) has been investigated as a model for the fusion step in the infectious entry of the virus into cells. Fusion was monitored continuously, with a fluorescence assay based on resonance energy transfer (RET) [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099], which allows an accurate quantitation of the fusion process. Evidence is presented indicating that the dilution of the RET probes from the liposomal bilayer into the viral membrane is not due to transfer of individual lipid molecules. The initial rate and final extent of the fusion reaction increase dramatically with decreasing pH, fusion being virtually complete within 1 min at pH 4.5-5.0. From experiments in which the ratio of virus to liposomes was varied, it is concluded that virus-liposome fusion products continue to fuse with liposomes, but not with virus. Fusion is most efficient with liposomes consisting of negatively charged phospholipids, while phosphatidylcholine and sphingomyelin are inhibitory. The reaction is completely blocked by an antiserum against the virus and inhibited by pretreatment of the virus with trypsin. The effect of proteolytic pretreatment at pH 7.4 is enhanced after preincubation of the virus at pH 5.0, consistent with the occurrence of a low pH induced, irreversible, conformational change in the viral fusion protein, the hemagglutinin (HA), exposing trypsin cleavage sites. When, after initiation of the fusion reaction at pH 5.0, the pH is readjusted to neutral, the process is arrested instantaneously, indicating that the low pH induced conformational change in the HA protein, in itself, is not sufficient to trigger fusion activity.     

Intermediates in influenza induced membrane fusion.

Our results show that the mechanism by which influenza virus fuses with target membranes involves sequential complex changes in the hemagglutinin (HA, the viral fusion protein) and in the contact site between virus and target membrane. To render individual steps amenable to study, we worked at 0 degree C which decreased the rate of fusion and increased the efficiency. The mechanism of fusion at 0 degree C and 37 degrees C was similar. The process began with a conformational change in HA which exposed the fusion peptides but did not lead to dissociation of the tops of the ectodomain of the trimer. The change in the protein led to immediate hydrophobic attachment of the virus to the target liposomes. Attachment was followed by a lag period (4-8 min at 0 degree C, 0.6-2 s at 37 degrees C) during which rearrangements occurred in the site of membrane contact between the virus and liposome. After a further series of changes the final bilayer merger took place. This final fusion event was not pH dependent. At 0 degree C efficient fusion occurred without dissociation of the top domains of the HA trimer, suggesting that a transient conformation of HA is responsible for fusion at physiological temperatures. The observations lead to a revised model for HA mediated fusion.     

Conformational changes and fusion activity of influenza virus hemagglutinin of the H2 and H3 subtypes: effects of acid pretreatment.

Marked differences were observed between the H2 and H3 strains of influenza virus in their sensitivity to pretreatment at low pH. Whereas viral fusion and hemolysis mediated by influenza virus X:31 (H3 subtype) were inactivated by pretreatment of the virus at low pH, influenza virus A/Japan/305/57 (H2 subtype) retained those activities even after a 15-min incubation at pH 5.0 and 37 degrees C. Fusion with erythrocytes was measured by using the octadecylrhodamine-dequenching assay with both intact virions and CV-1 monkey kidney cells expressing hemagglutinin (HA) on the plasma membrane. To study the nature of the differences between the two strains, we examined the effects of low-pH treatment on the conformational change of HA by its susceptibility to protease digestion, exposure of the fusion peptide, and electron microscopy of unstained, frozen, hydrated virus. We found that the respective HA molecules from the two strains assumed different conformational states after exposure to low pH. The relationship between the conformation of HA and its fusogenic activity is discussed in the context of these experiments.     

Fusion of membrane vesicles bearing only the influenza hemagglutinin with erythrocytes, living cultured cells, and liposomes

Membrane vesicles, bearing only the influenza viral hemagglutinin glycoprotein, were reconstituted following solubilization of intact virions with Triton X-100. The viral hemagglutinin glycoprotein was separated from the neuraminidase glycoprotein by agarose sulfanilic acid column. The hemagglutinin glycoprotein obtained was homogenous in gel electrophoresis and devoid of any neuraminidase activity. A quantitative determination revealed that the hemolytic activity of the hemagglutinin vesicles was comparable to that of intact virions. Incubation of fluorescently labeled hemagglutinin vesicles with human erythrocyte ghosts (HEG) or with liposomes composed of phosphatidylcholine/cholesterol or phosphatidylcholine/cholesterol/gangliosides, at pH 5.0 but not at pH 7.4, resulted in fluorescence dequenching. Very little, if any, fluorescence dequenching was observed upon incubation of fluorescently labeled HA vesicles with neuraminidase or glutaraldehyde-treated HEG or with liposomes composed only of phosphatidylcholine. Hemagglutinin vesicles were rendered non-hemolytic by treatment with NH2OH or glutaraldehyde or by incubation at 85 degrees C or low pH. No fluorescence dequenching was observed following incubation of non-hemolytic hemagglutinin vesicles with HEG or liposomes. These results clearly suggest that the fluorescence dequenching observed is due to fusion between the hemagglutinin vesicles and the recipient membranes. Incubation of hemagglutinin vesicles with living cultured cells, i.e. mouse lymphoma S-49 cells, at pH 5.0 as well as at pH 7.4, also resulted in fluorescence dequenching. The fluorescence dequenching observed at pH 7.4 was inhibited by lysosomotropic agents (methylamine and ammonium chloride) as well as by EDTA and NaN3, indicating that it is due to fusion of hemagglutinin vesicles taken into the cells by endocytosis.     

Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes

Fusion of influenza virus with liposomes composed of negatively charged phospholipids differs from fusion with biological membranes or zwitterionic liposomes with ganglioside receptors [Stegmann, T., Hoekstra, D., Scherphof, G., & Wilschut, J. (1986) J. Biol. Chem. 261, 10966-10969]. In this study, we investigated how the kinetics and extent of fusion of influenza virus, monitored with a fluorescence resonance energy-transfer assay, are influenced by the surface charge and the presence of receptors on liposomal membranes. The results were analyzed in terms of mass action kinetic model, providing separate rate constants for the initial virus-liposome adhesion, or aggregation, and for the actual fusion reaction. Incorporation of increasing amounts of cardiolipin (CL) or phosphatidylserine (PS) into otherwise zwitterionic phosphatidylcholine (PC)/phosphatidylethanolamine (PE) vesicles results in a gradual shift of the pH threshold of fusion to neutral, relative to the pH threshold obtained with PC/PE vesicles containing the ganglioside GD1a, while also the rate of fusion increases. This indicates the emergence of a fusion mechanism not involving the well-documented conformational change in the viral hemagglutinin (HA). However, only with pure CL liposomes this nonphysiological fusion reaction dominates the overall fusion process; with pure PS or with zwitterionic vesicles containing CL or PS, the contribution of the nonphysiological fusion reaction is small. Accordingly, preincubation of the virus alone at low pH results in a rapid inactivation of the viral fusion capacity toward all liposome compositions studied, except pure CL liposomes. The results of the kinetic analyses show that with pure CL liposomes the rates of both virus-liposome adhesion and fusion are considerably higher than with all other liposome compositions studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusion of Sendai virus with negatively charged liposomes as studied by pyrene-labelled phospholipid liposomes

Sendai virus particles fuse with negatively charged liposomes but not with vesicles made of zwitterionic phospholipids. The liposome-virus fusion process was studied by dilution of the concentration-dependent excimer-forming fluorophore 2-pyrenyldodecanoylphosphatidylcholine contained in the liposomes by the viral lipids. The data were analyzed in the framework of a mass action kinetic model. This provided analytical solutions for the final levels of probe dilution and numerical solutions for the kinetics of the overall fusion process, in terms of rate constants for the liposome-virus adhesion, deadhesion and fusion. This analysis led to the following conclusions: At neutral pH and 37 degrees C, only 15% of the virus particles can fuse with the phospholipid vesicles, although all the virions may aggregate with the liposomes. The rate constants for aggregation, fusion and deadhesion are of the orders of magnitude of 10(7) M-1 X s-1, 10(-3) s-1 and 10(-2), s-1, respectively. The fraction of active virus increases with temperature. At acidic pH, both the fraction of 'fusable' virus and the rate of fusion increase markedly. The optimal pH for fusion is 3-4, where most of the virus particles are active. At higher pH values, an increasing fraction of the virus particles become inactive, probably due to ionization of viral glycoproteins, whereas at pH values below 3.0 the fusion is markedly reduced, most likely due to protonation of the negatively charged vesicles. While only 15% of the virions fuse with the liposomes at pH 7.4 and 37 degrees C, all the liposomes lose their content (Amselem, S., Loyter, A. Lichtenberg, D. and Barenholz, Y. (1985) Biochim. Biophys. Acta 820, 1-10). We therefore propose that release of entrapped solutes is due to liposome-virus aggregation, and not to fusion. Both trypsinization and heat inactivation of the virus particles inhibit not only the fusion process but also the release of carboxyfluorescein. This demonstrates the obligatory role of viral membrane proteins in liposome-virus aggregation. Reconstituted vesicles made of the viral lipid and the hemagglutinin/neuraminidase (HN) glycoprotein fuse with negatively charged liposomes similar to the intact virions. This suggests that the fusion of virions with negatively charged vesicles, unlike the fusion of the virus with biological membranes, requires only the HN and not the fusion glycoprotein.     

Kinetics of fusion and lipid transfer between virus receptor containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay

Octadecylrhodamine B chloride (R18) and ganglioside GD1a (virus receptor) were incorporated into small unilamellar liposomes [Hoekstra et al. (1984) Biochemistry 23, 5675-5681]. Upon interaction of these liposomes with PR8 influenza viruses without prebinding, two types of dequenching were observed at 37 degrees C, both second-order processes: a fast reaction at pH 5.3, 2k = 17.53 x 10(-3) (Q.s)-1, and a slow reaction at pH 7.4, 2k = 0.335 x 10(-3) (Q.s)-1. The maximal level of dequenching was the same for both. Upon prebinding of liposomes to PR8 viruses (30 min, 0 degrees C, pH 7.4) at high concentrations, a very fast dequenching occurred when the prebinding mixture was diluted into prewarmed (37 degrees C) 10 mM PBS, pH 5.3. For the initial phase, a first-order rate constant of 0.5 s-1 could be extrapolated. After a quick drop in velocity during the first 30 s, the reaction was kinetically indistinguishable from the one found without prebinding. A second-order process with 2k = 16.52 x 10(-3) (Q.s)-1 became rate-limiting. The fast reactions at pH 5.3 can be abolished by inactivation or removal of the virus hemagglutinin. We conclude that the reaction at pH 5.3 reflects the hemagglutinin-dependent fusion process known to occur between influenza viruses and partner membranes at low pH; however, second-order kinetics indicate that specific binding rather than fusion is the rate-limiting step. For the slow dequenching, which is not affected by prebinding, the rate constant is 20 times lower than for the fast reaction, and the process is independent of viral hemagglutinin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature-dependent kinetics of the activities of influenza virus

The temperature dependence of membrane interactions between PR8 influenza virus and virus receptor (GD1a)-containing liposomes was studied. For quantitation, the octadecylrhodamine B chloride (R18) membrane marker was incorporated into liposomes at quenched concentrations. Upon interaction with target membranes, the marker gets diluted, and dequenching can be measured in a fluorescence spectrophotometer. Rate constants were calculated from the dequenching curves under low pH conditions, which allow for fusion, and at neutral pH, where no specific fusion occurs. Activation energies were determined from Arrhenius plots. The results were compared with the temperature dependence of other viral activities like infectivity, hemolysis, and fusion with erythrocytes. For the slow reaction at pH 7.4, where only non-specific lipid transfer takes place, the activation energy was about 24 kcal/mole between 15 degrees C and 45 degrees C. For the fast, hemagglutinin (HA)-specific fusion reaction (pH 5.3), a very low activation energy (approximately 7 kcal/mole) was found between 25 degrees C and 37 degrees C, whereas below 25 degrees C it was much higher (approximately 34 kcal/mole). The temperature range with low activation energy coincides with the one for optimal infectivity, hemolysis, and fusion with erythrocytes. Furthermore, it is the same range in which the conformational change of HA takes place, which in the absence of a partner membrane leads to an irreversible inactivation of the fusion protein.     

Influenza virus-model membrane interaction. A morphological approach using modern cryotechniques

The membrane fusion activity of influenza virus was characterized morphologically using a model system composed of a highly purified influenza B virus suspension and ganglioside-containing zwitterionic liposomes. Electron microscopical analysis was performed after a combination of fast-freezing with either freeze-fracture or freeze-substitution-thin sectioning, ensuring maximal time resolution and avoiding preparation artifacts. In a parallel fluorescence 'lipid mixing' fusion assay, influenza virus-membrane fusion was characterized biochemically. Biochemical and morphological data are in full agreement, indicating negligible membrane fusion activity at neutral pH and high fusion activity at low pH. The freeze-fracture morphology strongly suggests a local point contact between viral and liposomal membrane at neutral pH, and a local point fusion mechanism for influenza virus-membrane fusion upon lowering of the pH. Fusion is followed by lipid mixing, lateral diffusion of viral spike proteins and exposure of viral contents at the inner liposomal surface.     

Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8

The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37 degrees C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37 degrees C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4 degrees C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37 degrees C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37 degrees C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S(A59)H716D virions with trypsin at 4 degrees C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37 degrees C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.     

Conformational intermediates and fusion activity of influenza virus hemagglutinin

Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824-3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37 degrees C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20 degrees C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37-43, 1994).     

Fusion activity of influenza virus PR8/34 correlates with a temperature-induced conformational change within the hemagglutinin ectodomain detected by photochemical labeling

Fusion of influenza viruses with membranes is catalyzed by the viral spike protein hemagglutinin (HA). Under mildly acidic conditions (approximately pH 5) this protein undergoes a conformational change that triggers the exposure of the "fusion peptide", the hydrophobic N-terminal segment of the HA2 polypeptide chain. Insertion of this segment into the target membrane (or viral membrane?) is likely to represent a key step along the fusion pathway, but the details are far from being clear. The photoreactive phospholipid 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine ([3H]PTPC/11), inserted into the bilayer of large unilamellar vesicles (LUVs), allowed us to investigate both the interaction of viruses with the vesicles under "prefusion" conditions (pH 5; 0 degrees C) and the fusion process itself occurring at elevated temperatures (greater than 15-20 degrees C) only. Despite the observed binding of viruses to LUVs at pH 5 and 0 degrees C, labeling of HA2 was very weak (less than 0.002% of the radioactivity originally present). In contrast, fusion could be readily monitored by the covalent labeling of that polypeptide chain. We have studied also the effect of temperature on the acid-induced (pH 5) interaction of bromelain-solubilized HA (BHA) with vesicles. Labeling of the BHA2 polypeptide chain was found to show a remarkable correlation with the temperature dependence of the fusion activity of whole viruses. A temperature-induced structural change appears to be critical for both the interaction of BHA with membranes and the expression of fusion activity of intact viruses.     

Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration

The conditions that optimize Sendai virus membrane fusion with liposomes have been studied. No fusion occurs in the absence of ganglioside receptors. Maximum fusion occurs when the molar ratio of ganglioside GD1a to phospholipid is 0.02 or greater. The amount of fusion at 37 degrees C increases with time up to at least 6.5 h. The rate of fusion increases from the lowest temperature tested, 10 degrees C, to 40 degrees C. Above 43 degrees C the amount of fusion decreases because of thermal inactivation of the viral proteins. There is a broad pH maximum between pH 7.5 and pH 9.0. At both ends of the pH range the amount of fusion increases and exceeds that found in the physiologic pH range. Neither ethylenediaminetetraacetic acid nor Ca2+ changes the amount of membrane fusion. The optimal conditions for membrane fusion of Sendai virus membranes with liposomes are the same as the optimal conditions for fusion with host cells and with red blood cells. Since the liposomes contain no proteins, the optimal conditions for Sendai virus membrane fusion must be determined by the viral proteins and be mostly independent of the nature or presence of the host proteins.     

Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence

Fusion between membranes of 3T3 fibroblasts expressing hemagglutinin (HA) from the Japan strain of influenza virus and human red blood cells (RBC) was measured using an assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecylrhodamine (R18). The probe was incorporated into the membrane of intact RBC at self-quenching concentrations, and the RBCs were bound to the 3T3 cells. Fusion, which allowed movement of R18 into 3T3 cell membranes, was monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, the fluorescence increased after a delay of about 30 s at 37 degrees C, and leveled off within 2 min. In control experiments where R18 RBCs bound to 3T3 cells expressing the uncleaved precursor hemagglutinin (HA0) were incubated at 37 degrees C and low pH, no fluorescence increase was observed. This indicated that the R18 dequenching occurred as a result of HA-induced fusion of plasma membranes. Fusion showed a very steep pH dependence with a threshold at pH 5.4 and a maximum at pH 5.0, similar to HA-induced fusion seen previously using cell biological techniques. The fusion rate increased and the delay for the onset of fusion decreased as the temperature was raised above 20 degrees C. Low pH activation of the fusion process at 37 degrees C could be partially arrested by raising the pH after 2-10 s, but not after 15 s, indicating that the irreversible pH-activated conformational change of HA necessary for fusion was complete within about 15 s. Analysis of the data indicates that the pH-induced membrane fusion activity of HA is a highly cooperative event.     

pH-dependent membrane fusion activity of a synthetic twenty amino acid peptide with the same sequence as that of the hydrophobic segment of influenza virus hemagglutinin

A twenty amino acid hydrophobic peptide with the same sequence as that of the HA2 N-terminal segment of influenza virus hemagglutinin was synthesized and studied as to its fusion activity. The peptide caused rapid and efficient fusion of egg yolk phosphatidylcholine sonicated vesicles at acidic pH but not at neutral pH. The threshold pH was ca. 6.2 and the maximum fusion occurred at pH 4.8, the half-maximal pH for fusion being 5.6. The pH dependence was similar to that of the parent virus. The fusion efficiency was dependent on the ration of lipid to peptide, increasing with decreasing ratio. The fusion can be rapidly switched on and off by adjusting the pH, to the acidic side and neutral, respectively. The peptide with an acetylated or succinylated N-terminus also showed low pH-induced fusion activity but the pH range was shifted by ca. 1 unit to the acidic side. The results indicate that the HA2 hydrophobic segment in the virus fusion protein is directly involved in the fusion reaction and protonation of the acidic residues in the segment is required for the activity.     

Active function of membrane receptors for enveloped viruses : I. Specific requirement for liposome-associated sialoglycolipids, but not sialoglycoproteins, to allow lysis of phospholipid vesicles by reconstituted Sendai viral envelopes

Phospholipid liposomes composed of phosphatidylcholine (PC) and cholesterol (chol), bearing the sialoglycoprotein glycophorin (GP), are able to effectively bind Sendai virus particles, but not to be lysed by them. Incorporation of gangliosides (gangl) into the above phospholipid vesicles (yielding liposomes composed of PC/chol/gangl/GP), although not increasing their ability to interact with Sendai virions, rendered them susceptible to the viral lytic activity. This was inferred from the ability of the virus to induce release of carboxyfluorescein (CF) upon interaction at 37 degrees C with liposomes composed of PC/chol/gangl/GP. Lysis of liposomes required the presence of the two viral envelope glycoproteins, namely the hemagglutinin/neuraminidase (HN) and the fusion (F) polypeptides, and was inhibited by phenylmethyl sulfonylfluoride (PMSF), dithiothreitol (DTT) and trypsin, showing that virus-induced lysis of PC/chol/gangl/GP liposomes reflects the fusogenic activity of the virus. Incubation of Sendai virus particles with liposomes containing the acidic phospholipid dicetylphosphate (DCP) but lacking sialic acid containing receptors, also resulted in release of the liposome content. Lysis of these liposomes was due to the activity of the viral HN glycoprotein, therefore not reflecting the natural viral fusogenic activity. Fluorescence dequenching studies, using fluorescently labeled reconstituted Sendai virus envelopes (RSVE), have shown that the viral envelopes are able to fuse with neutral, almost to the same extent, as with negatively charged liposomes. However, fusion with negatively charged liposomes, as opposed to fusion with neutral liposomes, was mediated by the viral HN glycoprotein and not by the viral fusion polypeptide.     

Influence of calcium on lipid mixing mediated by influenza hemagglutinin

We studied the influence of calcium on lipid mixing mediated by influenza hemagglutinin (HA). Lipid mixing between HA-expressing cells and liposomes containing disialoganglioside, influenza virus receptor, was studied at 37 degrees C and neutral pH after a low-pH pulse at 4 degrees C. With DSPC/cholesterol liposomes, calcium present after raising the temperature significantly promoted lipid mixing only when it was triggered by a short low-pH application. In case of DOPC/cholesterol liposomes, calcium promotion was observed regardless of the duration of the low-pH pulse. Calcium present during a short, but not long, low-pH application to HA-expressing cells with bound DSPC/cholesterol liposomes at 4 degrees C inhibited subsequent lipid mixing. We hypothesize that calcium influences lipid mixing because it binds to a vestigial esterase domain of hemagglutinin or causes expulsion of the fusion peptide from an electronegative cavity. We suggest that calcium promotes the transition from early and reversible conformation(s) of low pH-activated HA towards an irreversible conformation that underlies both HA-mediated lipid mixing and HA inactivation.