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The influenza virus enters target cells via the action of hemagglutinin proteins (HA) inserted into the viral envelope. HA promotes membrane fusion between the viral envelope and endosomal membrane at low pH, following viral binding to sialic acid-containing receptors on target cells, and internalization by endocytosis. The effect of target membrane sialic acid residues on the fusion activity of the influenza virus towards model membranes was evaluated by both reduction, (i.e. treating somatic cells with neuraminidase- (NA-) prior to virus-cell interactions), and by supplementing liposomes with the gangliosides GD1a and GT1b. The harshness of the neuraminidase pretreatment of target cells required to affect virus-induced membrane merging was found to greatly depend on the assay conditions, i.e. whether a virus-cell prebinding step at neutral pH was included prior to acidification. Minor concentrations of neuraminidase were found to greatly reduce virus fusion, but only in the absence of a prebinding step; they had no effect if this step was included. Although membrane merging was greatly reduced following cell neuraminidase pretreatment, virus-cell association at low pH was not disturbed proportionately. This probably reflects unspecific virus-cell binding under these conditions, probably of inactivated or aggregated virus particles, which does not translate into membrane merging. This seems to suggest both that target membrane sialic acid can protect the virus from losing its activity before triggering membrane merging, and that the importance of this interaction is not merely to ensure virus-target proximity. With liposomes, we found that both types of ganglioside supported efficient fusion, with GD1a promoting a slightly faster initial rate. However, in this case, virus-target proximity closely mirrored fusion activity, thus pointing to differential specificity between targets routinely used to assay influenza virus fusion activity.

Membrane fusion is important to reproduction because it occurs in several steps during the process of fertilization. Many events of intracellular trafficking occur during both spermiogenesis and oogenesis. The acrosome reaction, a key feature during mammalian fertilization, is a secretory event involving the specific fusion of the outer acrosomal membrane and the sperm plasma membrane overlaying the principal piece of the acrosome. Once the sperm has crossed the zona pellucida, the gametes fuse, but in the case of the sperm this process takes place through a specific membrane domain in the head, the equatorial segment. The cortical reaction, a process that prevents polyspermy, involves the exocytosis of the cortical granules to the extracellular milieu. In lower vertebrates, the formation of the zygotic nucleus involves the fusion (syngamia) of the male pronucleus with the female pronucleus. Other undiscovered membrane trafficking processes may also be relevant for the formation of the zygotic centrosome or other zygotic structures. In this review, we focus on the recent discovery of molecular machinery components involved in intracellular trafficking during mammalian spermiogenesis, notably related to acrosome biogenesis. We also extend our discussion to the molecular mechanism of membrane fusion during the acrosome reaction. The data available so far suggest that proteins participating in the intracellular trafficking events leading to the formation of the acrosome during mammalian spermiogenesis are also involved in controlling the acrosome reaction during fertilization.

Fluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), or rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B), inserted into the virus membrane. Experimental conditions were designed to allow a clear distinction between membrane mixing and non-specific probe transfer. Fluorescence dequenching observed with Rh-PE was much slower than with R18, unless a particular experimental procedure was used. Using liposomes as a target membrane, the kinetics and extent of the decrease in resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and Rh-PE, initially embedded in the liposome membrane, were matched by that of the dequenching of viral R18, but not of viral Rh-PE. DORh-B was found not to be appropriate to follow membrane merging. Our results indicate that on a time scale of several minutes R18 more accurately reflects the kinetics of membrane fusion. Nevertheless, control experiments should be performed to evaluate non-specific probe transfer of R18 molecules, whose contribution to fluorescence dequenching can become significant after long incubation times.

We have directly compared the effect of two types of dextran sulfate with distinct molecular weights (500 kDa and 5 kDa) on the fusion activity and infectivity of both Sendai and influenza viruses, two lipid-enveloped viruses that differ in their routes of entry into target cells. To correlate membrane merging and infectivity MDCK cells were used as targets for the viruses in both approaches. In either case pronounced inhibition of virus-cell interactions by dextran sulfate was only observed at low pH, even though Sendai virus fuses maximally at pH 7.4. Although membrane merging could not be fully abolished, the inhibitory effect was always greater when the higher molecular weight dextran sulfate was used. The presence of this residual fusion activity, that could not be reduced even with high concentrations of agent, suggests that a limited number of binding sites for dextran sulfate may exist on the viral envelopes. The compounds also inhibited fusion of bound virions, and all results could be reproduced using erythrocyte ghosts as target membranes in the fusion assay, instead of MDCK cells. In agreement with these observations only the infectivity of influenza virus (which requires a low pH-dependent step to enter target cells) was affected by dextran sulfate, again the higher molecular weight compound showing a more pronounced inhibitory effect.

We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R 18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already bound to the erythrocyte/erythrocyte ghost in the cold, since with virus prebinding fusion can be triggered more rapidly. Although virus binding to both erythrocytes and erythrocyte ghosts was similar, fusion activity was much more pronounced when erythrocyte ghosts were used as target membranes. These observations indicate that intact erythrocytes and erythrocyte ghosts are not equivalent as target membranes for the study of Sendai virus fusion activity. Fusion of Sendai virus with both target membranes was inhibited when erythrocytes or erythrocyte ghosts were pretreated with proteinase K, suggesting a role of target membrane proteins in this process. Treatment of both target membranes with neuraminidase, which removes sialic acid residues (the biological receptors for Sendai virus) greatly reduced viral binding. Interestingly, this treatment had no significant effect on the fusion reaction itself.

Following reports suggesting that membrane fusion mediated by the influenza virus hemagglutinin might be dependent on a pH gradient across a putative target membrane, we have designed experiments in which this issue could be addressed directly. Accordingly, we have prepared two populations of liposomes, both simulating the plasma membrane of target cells, but with the pH of the internal aqueous medium buffered either at pH 7.4 (physiological cytosol pH) or pH 5.0 (endosomal pH at which influenza virus displays maximal fusion activity). Monitoring fusion as the relief in self-quenching of the fluorescent probe octadecylrhodamine B chloride we have found that the internal pH of the target liposomes did not influence membrane merging as mediated by the influenza virus hemagglutinin, thus demonstrating that a transmembrane pH gradient is not required for the fusion process to take place.

We have developed a model system, consisting of rat brain synaptic vesicles and rat brain synaptic plasma membrane vesicles, to study the fusion process associated with the exocytotic release of neurotransmitters. Our results show a significant increase in the extent of fusion when the reaction takes place in cytosol compared to that obtained when fusion is carried out in buffer. This effect is mediated by cytosolic proteins, although N-ethylmaleimide-sensitive factor does not play a role in fusion. We also registered an almost complete inhibition of fusion when synaptic vesicles were pre-incubated with botulinum toxin B, indicating that synaptobrevin plays an important role in the coalescence of membrane lipids of the interacting membranes.

Final extents of fusion of influenza virus (A/PR/8/34 strain) with neutral and partially acidic liposomes were monitored with (i) a fluorescence resonance energy-transfer assay in which the liposomes were labeled and (ii) by the dequenching of octadecylrhodamine, initially incorporated in the viral membrane. The latter assay was also employed in the fusion of influenza virus and Sendai virus with erythrocyte ghosts. In all cases, a phenomenon of partial fusion activity of the virus was observed, which is distinct from low pH inactivation. The unfused influenza or Sendai virions, which were separated by sucrose gradient centrifugation from liposomes or erythrocyte ghosts exhibited again partial fusion activity toward freshly added liposomes or ghosts, respectively. The conclusion is that the fraction of initially bound and unfused virions does not consist of defective particles, but rather of particles bound to the target membranes via inactive sites on the virus (or on cellular membranes), or else, partial fusion activity is a manifestation of a certain probability of production of fusion inactive sites by irreversible association of viral glycoproteins or peptides in the target membrane.

Fusion of African swine fever virus (ASFV) with model membranes was monitored by a fluorescence dequenching assay. ASFV was able to fuse with liposomes of various compositions. Fusion was more extensive with liposomes made of negatively charged phospholipids, and reduced in the presence of the neutral phospholipid phosphatidylcholine (PC). Fusion activity of ASFV was pH-dependent, the extent of fusion increasing with decreasing pH for all target membranes. These results are consistent with the in vivo characteristics of fusion of ASFV with the endosome membrane. An increase in fusion activity was obtained with dehydrating agents, similarly to what occurs with other lipid-enveloped viruses. Dehydrating agents such as dimethylsulfoxide and dimethylsulfone greatly enhanced fusion, the effect of dimethylsulfone being more pronounced than that of dimethylsulfoxide. Poly(ethylene glycol) also potentiated ASFV fusion activity, and the effect of this polymer was found to be dependent on its molecular weight. These results stress the importance of dehydration and hydrophobic interactions on the early events of viral penetration into target cells.

We investigated the effect of several parameters, such as temperature, pH and proteins, on the fusion between synaptosomes, freshly isolated from rat brain cortex, and large unilamellar phosphatidylserine liposomes. These studies were carried out in both peroxidized and nonperoxidized synaptosomes. Mixing of membrane lipids was monitored using a fluorescence resonance energy transfer assay. Ascorbate (0.8 mM)/Fe2+ (2.5 microM)-induced peroxidation of synaptosomes enhanced the fusion process (twofold) which may reflect an increase in synaptosomal protein hydrophobicity and hence a facilitation of intermembrane aggregation. The fusion process was shown to be temperature sensitive, a reduction in the extent being observed (twofold) as the temperature was lowered from 37 to 25 degrees C. This effect may be due to changes in membrane fluidity. The fusion process is pH dependent, an increase in both kinetics and extent being observed when the pH was lowered from 7.4 to 5.5. A significant inhibition (92% at pH 7.4; 35% at pH 5.5) of the interaction between synaptosomes and liposomes by trypsin pretreatment of synaptosomes was found, thus indicating that the fusion reaction is a protein-mediated process. The inhibitory effect of trypsin at pH 5.5 is not so strong as that at physiological pH. These results suggest that, in addition to the involvement of proteins, nonspecific interactions between the synaptosomal and liposomal membranes under acidic conditions may also play a role in the fusion process. The investigation of binding of synaptosomes to liposomes under several experimental conditions provided evidence for the participation of proteins in membrane aggregation, as well as for the role of electrostatic forces in this process, at mild acidic pH.

We have studied the importance of hydrophobic interactions in the fusion activity of Sendai virus. Viral fusion activity towards model lipid membranes was monitored using a fluorescence dequenching assay. Fusion was dependent on the composition of the target membrane and was more rapid and extensive at low pH. Dehydrating agents such as dimethylsulfoxide and dimethylsulfone greatly enhanced the initial rate and extent of the fusion process, the effect of dimethylsulfone doubling that of dimethylsulfoxide. However, dehydrating agents had a less pronounced stimulatory effect at low pH. Using the fluorescent probe ANS (1-aminonaphthalene-8-sulfonate) we have shown that low pH by itself involves a marked increase in the viral envelope hydrophobicity, probably resulting in the observed lower stimulation of viral fusion activity by dehydrating agents. Therefore, there seems to be a strong correlation between viral envelope hydrophobicity and viral fusion activity.

We have studied the role of hydrophobic interactions in the fusion activity of two lipid enveloped viruses, influenza and Sendai. Using the fluorescent probe ANS (1-aminonaphtalene-8-sulfonate) we have shown that low-pH-dependent influenza virus activation involves a marked increase in the viral envelope hydrophobicity. The effect of dehydrating agents on the fusion activity of both viruses towards model lipid membranes was studied using a fluorescence dequenching assay. Dehydrating agents such as dimethylsulfoxide and dimethylsulfone greatly enhanced the initial rate of the fusion process, the effect of dimethylsulfone doubling that of dimethylsulfoxide. The effect of poly(ethylene glycol) on the fusion process was found to be dependent on the polymer concentration and molecular weight. In general, similar observations were made for both viruses. These results stress the importance of dehydration and hydrophobic interactions in the fusion activity of influenza and Sendai viruses, and show that these factors may be generally involved in membrane fusion events mediated by many other lipid enveloped viruses.

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.

Membrane fusion is a very important process in gametes. The mechanism of membrane fusion during the AR has been proposed to involve SNAREs. Our aim is to quantify patterns of localization of Caveolin 1, SNAREs (Syntaxin 1A, Syntaxin 2 and VAMP 1) and NSF on human sperm, to determine how the differential distribution of these proteins might be interdependent and to evaluate if this distribution is related with seminal parameters. These proteins are present in different regions of the head of human sperm: anterior, equatorial and posterior regions and that Syntaxin 2 and Syntaxin 1A had a slightly different pattern of labelling. The presence and localization of SNAREs, NSF and Caveolin 1 do not correlate with seminal parameters. There is significant correlation between NSF and SNAREs, which may indicate a cooperation of these proteins in membrane fusion mechanisms of human sperm. [ABSTRACT FROM AUTHOR]