Sphingolipid and cholesterol dependence of alphavirus membrane fusion. Lack of correlation with lipid raft formation in target liposomes

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped viruses that infect their host cells by receptor-mediated endocytosis and subsequent fusion from within acidic endosomes. Fusion of the viral envelope requires the presence of both cholesterol and sphingolipids in the target membrane. This is suggestive of a possible involvement of sphingolipid-cholesterol microdomains, or "lipid rafts," in the membrane fusion and cell entry process of the virus. In this study, large unilamellar vesicles (LUVs) were prepared from synthetic sphingolipids and sterols that vary with respect to their capacity to promote microdomain formation, as assessed by gradient flotation analysis in the presence of Triton X-100. SFV and SIN fused with LUVs irrespective of the presence or absence of Triton X-100-insoluble microdomains. These results suggest that SFV and SIN do not require the presence of lipid rafts for fusion with target membranes. Furthermore, it is not necessary for sphingolipids to reside in a detergent-insoluble complex with cholesterol to promote SFV or SIN fusion.     

Adaptation of alphaviruses to heparan sulfate: interaction of Sindbis and Semliki forest viruses with liposomes containing lipid-conjugated heparin

Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycosaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 (E2:1, E2:70, and E2:114) have been identified that mutate during adaptation and independently confer on the virus the ability to bind to cell surface HS (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). In this study, we used HS-adapted SIN mutants to evaluate a new model system involving target liposomes containing lipid-conjugated heparin (HepPE) as an HS receptor analog for the virus. HS-adapted SIN, but not nonadapted wild-type SIN TR339, interacted efficiently with HepPE-containing liposomes at neutral pH. Binding was competitively inhibited by soluble heparin. Despite the efficient binding of HS-adapted SIN to HepPE-containing liposomes at neutral pH, there was no fusion under these conditions. Fusion did occur, however, at low pH, consistent with cellular entry of the virus via acidic endosomes. At low pH, wild-type or HS-adapted SIN underwent fusion with liposomes with or without HepPE with similar kinetics, suggesting that interaction with the HS receptor analog at neutral pH has little influence on subsequent fusion of SIN at low pH. Finally, Semliki Forest virus (SFV), passaged frequently on BHK-21 cells, also interacted efficiently with HepPE-containing liposomes, indicating that SFV, like other alphaviruses, readily adapts to cell surface HS. In conclusion, the liposomal model system presented in this paper may serve as a novel tool for the study of receptor interactions and membrane fusion properties of HS-interacting enveloped viruses.     

Vesicular stomatitis virus binds and fuses with phospholipid domain in target cell membranes

Fusion of vesicular stomatitis virus with some cells (HELR 66, KB, and human erythrocytes, both intact and trypsinized) and liposomes made of various natural and synthetic lipids was studied with spin-labeled phospholipid. Binding of virus was assayed separately with radiolabeled and spin-labeled virus. Binding to cells and liposomes was small at neutral pH but enhanced at acidic pHs. Fusion with cells and liposomes was negligibly small at neutral pH but greatly activated at acidic pHs lower than 6.5. Activation of fusion occurred at lower pH values than enhancement of binding. Fusion occurred rapidly and efficiently, reaching a plateau at 50-80% after 3 min at 37 degrees C. Binding and fusion with cells were enhanced by pretreatment of cells with trypsin. Binding to liposomes was dependent on the head group of the phospholipid, stronger to phosphatidylserine than to phosphatidylcholine, but not much dependent on the acyl chain composition. On the other hand, cis-unsaturated acyl chains were required for the efficient fusion, but there was only a small, if any, requirement for the head group. Cholesterol enhanced the fusion further. High fusion efficiency with cis-unsaturated phospholipids cannot be ascribed to the membrane fluidity but may be related to higher tail-to-head volume ratios. Possible mode of interaction of viral G glycoprotein with phospholipid is discussed. The virus cell entry mechanism is suggested as binding to the phospholipid domain in the cell surface membranes, endocytosis, and followed by fusion with the phospholipid domain in endosomes upon acidification.     

Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane.

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pH-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV--liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.     

Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH.

The flavivirus envelope protein E undergoes irreversible conformational changes at a mildly acidic pH which are believed to be necessary for membrane fusion in endosomes. In this study we used a combination of chemical cross-linking and sedimentation analysis to show that the envelope proteins of the flavivirus tick-borne encephalitis virus also change their oligomeric structure when exposed to a mildly acidic environment. Under neutral or slightly alkaline conditions, protein E on the surface of native virions exists as a homodimer which can be isolated by solubilization with the nonionic detergent Triton X-100. Solubilization with the same detergent after pretreatment at an acidic pH, however, yielded homotrimers rather than homodimers, suggesting that exposure to an acidic pH had induced a simultaneous weakening of dimeric contacts and a strengthening of trimeric ones. The pH threshold for the dimer-to-trimer transition was found to be 6.5. Because the pH dependence of this transition parallels that of previously observed changes in the conformation and hydrophobicity of protein E and that of virus-induced membrane fusion, it appears likely that the mechanism of fusion with endosomal membranes involves a specific rearrangement of the proteins in the viral envelope. Immature virions in which protein E is associated with the uncleaved precursor (prM) of the membrane protein M did not undergo a low-pH-induced rearrangement. This is consistent with a protective role of protein prM for protein E during intracellular transport of immature virions through acidic compartments of the trans-Golgi network.     

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.     

Evidence that rabies virus forms different kinds of fusion machines with different pH thresholds for fusion

Fusion of rabies virus with membranes is triggered at a low pH and is mediated by a viral glycoprotein (G). Fusion of rabies virus with liposomes was monitored by using a lipid mixing assay based on fluorescence resonance energy transfer. Fusion was detected below pH 6.4, and its extent increased with H(+) concentrations to be maximal around pH 6.15. The origin of the partial fusion activity of rabies virus under suboptimal pH conditions (i.e., between pH 6.15 and 6.4) was investigated. We demonstrate unambiguously that fusion at a suboptimal pH is distinct from the phenomenon of low-pH-induced inactivation and that it is not due to heterogeneity of the virus population. We also show that viruses that do not fuse under suboptimal pH conditions are indeed bound to the target liposomes and that the fusion complexes they have formed are blocked at an early stage of the fusion pathway. Our conclusion is that along the fusion reaction, different kinds of fusion machines with different pH thresholds for fusion can be formed. Possible explanations of this difference of pH sensitivity are discussed.     

Fusion of Semliki Forest virus with cholesterol-containing liposomes at low pH: a specific requirement for sphingolipids

Semliki Forest virus (SFV) utilizes a membrane fusion strategy to introduce its genome into the host cell. After binding to cell-surface receptors, virus particles are internalized through receptor-mediated endocytosis and directed to the endosomal cell compartment. Subsequently, triggered by the acid pH in the lumen of the endosomes, the viral envelope fuses with the endosomal membrane. As a result of this fusion reaction the viral RNA gains access to the cell cytosol. Low-pH-induced fusion of SFV, in model systems as well as in cells, has been demonstrated previously to be strictly dependent on the presence of cholesterol in the target membrane. In this paper, we show that fusion of SFV with cholesterol-containing liposomes depends on sphingomyelin (SM) or other sphingolipids in the target membrane, ceramide representing the sphingolipid minimally required for mediating the process. The action of the sphingolipid is confined to the actual fusion event, cholesterol being necessary and sufficient tor low-pH-dependent binding of the virus to target membranes. The 3-hydroxyl group on the sphingosine backbone plays a key role in the SFV fusion reaction, since 3-deoxy-sphingomyelin does not support the process. This, and the remarkably low levels of sphingolipid required for half-maximal fusion (1–2 mol%), suggest that the sphingolipid does not play a structural role in SFV fusion, but rather acts as a co-factor, possibly through activation of the viral fusion protein. Domain formation between cholesterol and sphingolipid, although it may facilitate SFV fusion, is unlikely to play a crucial role in the process.     

Role of metastability and acidic pH in membrane fusion by tick-borne encephalitis virus.

The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus is, like the alphavirus E1 protein, a class II viral fusion protein that differs structurally and probably mechanistically from class I viral fusion proteins. The surface of the native TBE virion is covered by an icosahedrally symmetrical network of E homodimers, which mediate low-pH-induced fusion in endosomes. At the pH of fusion, the E homodimers are irreversibly converted to a homotrimeric form, which we have found by intrinsic fluorescence measurements to be more stable than the native dimers. Thus, the TBE virus E protein is analogous to the prototypical class I fusion protein, the influenza virus hemagglutinin (HA), in that it is initially synthesized in a metastable state that is energetically poised to be converted to the fusogenic state by exposure to low pH. However, in contrast to what has been observed with influenza virus HA, this transition could not be triggered by input of heat energy alone and membrane fusion could be induced only when the virus was exposed to an acidic pH. In a previous study we showed that the dimer-to-trimer transition appears to be a two-step process involving a reversible dissociation of the dimer followed by an irreversible trimerization of the dissociated monomeric subunits. Because the dimer-monomer equilibrium in the first step apparently depends on the protonation state of E, the lack of availability of monomers for the trimerization step at neutral pH could explain why low pH is essential for fusion in spite of the metastability of the native E dimer.     

Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes

It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion.     

Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.     

Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.     

Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein.

This paper presents a kinetic analysis of low-pH-induced fusion of Semliki Forest virus (SFV) with cholesterol-containing unilamellar lipid vesicles (liposomes), consisting otherwise of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. Fusion is monitored continuously with a lipid mixing assay, involving virus bio-synthetically labeled with the fluorophore pyrene. At pH 5.55, 37 degrees C, SFV-liposome fusion occurs on the time scale of seconds. Extensive fusion (up to 60% of the virus) requires an excess of liposomes, while a low-pH preincubation of the virus alone results in inactivation of its fusion capacity. The onset of fusion after acidification of virus-liposome mixtures is preceded by a pH- and temperature-dependent lag phase. Early in this lag phase, a conformational change in the E2E1 spike glycoprotein occurs, involving formation of a trypsin-resistant E1 homotrimer, exposing a conformation-specific epitope (E1"). These changes are followed by a rapid, cholesterol-dependent binding of the virus to the liposomes (as assessed by sucrose density gradient analysis), subsequent fusion starting only after an additional delay. This sequence of events strongly suggests that the E1 homotrimeric structure represents the fusion-active conformation of the SFV spike, the actual fusion complex possibly involving a higher order oligomer of E1 trimers.     

Hepatitis C virus is primed by CD81 protein for low pH-dependent fusion

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.     

Formation and characterization of the trimeric form of the fusion protein of Semliki Forest Virus

Enveloped animal viruses infect cells via fusion of the viral membrane with a host cell membrane. Fusion is mediated by a viral envelope glycoprotein, which for a number of enveloped animal viruses rearranges itself during fusion to form a trimeric alpha-helical coiled-coil structure. This conformational change from the metastable, nonfusogenic form of the spike protein to the highly stable form involved in fusion can be induced by physiological activators of virus fusion and also by a variety of destabilizing conditions. The E1 spike protein subunit of Semliki Forest virus (SFV) triggers membrane fusion upon exposure to mildly acidic pH and forms a homotrimer that appears necessary for fusion. We have here demonstrated that formation of the E1 homotrimer was efficiently triggered under low-pH conditions but not by perturbants such as heat or urea, despite their induction of generalized conformational changes in the E1 and E2 subunits and partial exposure of an acid-specific E1 epitope. We used a sensitive fluorescence assay to show that neither heat nor urea treatment triggered SFV-liposome fusion at neutral pH, although either treatment inactivated subsequent low-pH-triggered fusion activity. Once formed, the low-pH-induced E1 homotrimer was very stable and was only dissociated under harsh conditions such as heating in sodium dodecyl sulfate. Taken together, these data, as well as protein structure predictions, suggest a model in which the less stable native E1 subunit specifically responds to low pH to form the more stable E1 homotrimer via conformational changes different from those of the coiled-coil type of fusion proteins.     

Detection of cell-cell fusion mediated by Ebola virus glycoproteins

Ebola viruses (EboV) are enveloped RNA viruses infecting cells by a pH-dependent process mediated by viral glycoproteins (GP) involving endocytosis of virions and their routing into acidic endosomes. As with well-characterized pH-dependent viral entry proteins, in particular influenza virus hemagglutinin, it is thought that EboV GP require activation by low pH in order to mediate fusion of the viral envelope with the membrane of endosomes. However, it has not yet been possible to confirm the direct role of EboV GP in membrane fusion and the requirement for low-pH activation. It was in particular not possible to induce formation of syncytia by exposing cells expressing EboV GP to acidic medium. Here, we have used an assay based on the induction of a beta-galactosidase (lacZ) reporter gene in target cells to detect cytoplasmic exchanges, indicating membrane fusion, with cells expressing EboV GP (Zaire species). Acidic activation of GP-expressing cells was required for efficient fusion with target cells. The direct role of EboV GP in this process is indicated by its inhibition by anti-GP antibodies and by the lack of activity of mutant GP normally expressed at the cell surface but defective for virus entry. Fusion was not observed when target cells underwent acidic treatment, for example, when they were placed in coculture with GP-expressing cells before the activation step. This unexpected feature, possibly related to the nature of the EboV receptor, could explain the impossibility of inducing formation of syncytia among GP-expressing cells.     

Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane.

The membrane fusion activities of the isolated single-envelope intracellular form of vaccinia virus (INV) and the double-envelope extracellular (EEV) form were studied by using a lipid-mixing assay based on the dilution of a fluorescent probe. Fluorescently labeled INV and EEV from both the IHD-J and WR strains of vaccinia virus fused with HeLa cells at neutral pH, suggesting that fusion occurs with the plasma membrane during virus entry. EEV fused more efficiently and with faster kinetics than INV: approximately 50% of bound EEV particles fused over the course of 1 h, compared with only 25% of the INV particles. Fusion of INV and EEV was strongly temperature dependent, being decreased by 50% at 34 degrees C and by 90% at 28 degrees C. A monoclonal antibody to a 14-kilodalton envelope protein of INV that has been implicated in the fusion reaction (J. F. Rodriguez, E. Paez, and M. Esteban, J. Virol. 61:395-404, 1987) completely suppressed the initial rate of fusion of INV but had no effect on the fusion activity of EEV, suggesting that vaccinia virus encodes two or more membrane fusion proteins. Finally, cells infected with the WR strain of vaccinia virus formed syncytia when briefly incubated at pH 6.4 or below, indicating that an acid-activated viral fusion protein is expressed on the cell surface. However, WR INV and EEV did not display increased fusion activity at acid pH, suggesting that the acid-dependent fusion factor is not incorporated into virions or that its activity there is masked.     

The fusion peptide of Semliki Forest virus associates with sterol-rich membrane domains

Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl beta-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.     

Fusion of Sendai virions or reconstituted Sendai virus envelopes with liposomes or erythrocyte membranes lacking virus receptors

Incubation of intact Sendai virions or reconstituted Sendai virus envelopes with phosphatidylcholine/cholesterol liposomes at 37 degrees C results in virus-liposome fusion. Neither the liposome nor the virus content was released from the fusion product, indicating a nonleaky fusion process. Only liposomes possessing virus receptors, namely sialoglycolipids or sialoglycoproteins, became leaky upon interaction with Sendai virions. Fusion between the virus envelopes and phosphatidylcholine/cholesterol liposomes was absolutely dependent upon the presence of intact and active hemagglutinin/neuraminidase and fusion viral envelope glycoproteins. Fusion between Sendai virus envelopes and phosphatidylcholine/cholesterol liposomes lacking virus receptors was evident from the following results. Anti-Sendai virus antibody precipitated radiolabeled liposomes only after they had been incubated with fusogenic Sendai virions. Incubation of N-4-nitrobenzo-2-oxa-1,3-diazole-labeled fusogenic reconstituted Sendai virus particles with phosphatidylcholine/cholesterol liposomes resulted in fluorescence dequenching. Incubation of Tb3+-containing virus envelopes with phosphatidylcholine/cholesterol liposomes loaded with sodium dipicolinate resulted in the formation of the chelation complex Tb3+-dipicolinic acid, as was evident from fluorescence studies. Virus envelopes fuse efficiently also with neuraminidase/Pronase-treated erythrocyte membranes, i.e. virus receptor-depleted erythrocyte membranes, although fusion occurred only under hypotonic conditions.     

Membrane fusion mutants of Semliki Forest virus

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