Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor

A variety of ligands and macromolecules enter cells by receptor-mediated endocytosis. Ligands bind to their receptors on the cell surface and ligand-receptor complexes are localized in specialized regions of the plasma membrane called coated pits. Coated pits invaginate and give rise to intracellular coated vesicles containing ligand-receptor complexes which are thus internalized. Transferrin, a major serum glycoprotein which transports iron into cells, enters cells by this pathway. It binds to its receptor on the cell surface, transferrin-receptor complexes cluster in coated pits and are internalized in coated vesicles. Coated vesicles then lose their clathrin coat and fuse with endosomes, an organelle with an internal pH of about 5-5.5. Most ligands dissociate from their receptors in endosomes and they finally end up in lysosomes where they are degraded, while their receptors remain bound to membrane structures and recycle to the cell surface. Transferrin has a different fate: in endosomes iron dissociates from transferrin but apotransferrin remains bound to its receptor because of its high affinity for the receptor at acid pH. Apotransferrin thus recycles back to the plasma membrane still bound to its receptor. When the ligand-receptor complex reaches the plasma membrane or a compartment at neutral pH, apotransferrin dissociates from its receptor with a half-life of 18 s because of its low affinity for its receptor at neutral pH. The receptor is then ready for a new cycle of internalization, while apotransferrin enters the circulation, reloads iron in the appropriate organs and is ready for a new cycle of iron transport.     

Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.     

pH-dependent lysis of liposomes by adenovirus

Purified adenovirus induced a dose-dependent release of the water-soluble markers calcein and carboxyfluorescein from liposomes. Marker release was strongly dependent on pH, and at temperatures below 5 degrees C, the rate of release showed an optimum at a pH of about 6. This pH dependence parallels disruption of endocytic vesicles by adenovirus and the permeabilization that adenovirus induces on the cell surface. There did not seem to be a striking dependence on the lipid composition of the liposomes. Electron microscopy using a negative stain shows liposomes bound to adenovirus. In some cases, the liposomes were still intact, but many liposomes, which were attached to the vertices of the virus, appeared lysed. These data support the notion that adenovirus, which enters the host cell by receptor-mediated endocytosis, gains access to the cytoplasm by a subsequent pH-dependent disruption of the membrane of the endocytic vesicle.     

Comparison of folic acid uptake characteristics by human placental choriocarcinoma cells at acidic and physiological pH

The aim of this work was to characterize the placental uptake of folic acid from the maternal circulation. Using 2 human trophoblast cell lines (BeWo and JAR), we verified that uptake of 3H-folic acid was pH-dependent, increasing significantly with decreasing extracellular pH. In BeWo cells, uptake of 3H-folic acid at pH 5.5 was (i) Na+-independent; (ii) inhibited by folic acid, 5-methyltetrahydrofolate (5-MTHF), and methotrexate (MTX); (iii) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS); (iv) inhibited by the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP); (v) not inhibited by blockers of receptor-mediated endocytosis (cytochalasin D and monensin); (vi) trans-inhibited by MTX and folic acid; and (vii) not affected by an anti-reduced folate transporter-1 (RFC) antibody. At pH 7.5, uptake of 3H-folic acid was (i) Na+-independent; (ii) inhibited by folic acid and MTX, but not by 5-MTHF; (iii) inhibited by SITS, but not by DIDS; (iv) not affected by FCCP; (v) inhibited by monensin (but not by cytochalasin D); (vi) trans-inhibited by folic acid (but not by MTX); and (vii) inhibited by an anti-RFC antibody. In conclusion, in BeWo cells, both RFC and receptor-mediated endocytosis seem to be involved in 3H-folic acid uptake at pH 7.5, whereas at pH 5.5, RFC and (or) a low pH-operating transporter distinct from RFC are involved.     

Prostaglandin synthesis by fetal rat bone in vitro: evidence for a role of prostacyclin.

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE2, PGF2 alpha and 6-keto-PGF1 alpha, the metabolite of prostacyclin (PGI2). Since PGI2 had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI2 stimulated release of previously incorporated 45Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10(-5) to 10(-9)M. Because of the short half life of PGI2 in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI2) which was found to be a stimulator of bone resorption at concentrations of 10(-5) to 10(-6)M. These studies suggest that endogenous PGI2 production may play a role in bone metabolism. Since vessels produce PGI2 it is possible that PGI2 release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.     

Fluid-phase endocytosis by intrahepatic bile duct epithelial cells isolated from normal rat liver

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.     

The low-density lipoprotein receptor: ligands, debates and lore

Like pieces belonging to a large mosaic, the structures of low-density lipoprotein receptor (LDL-R) modules have been elucidated one by one in recent years. LDL-Rs localized on hepatocytes play an important role in removing cholesterol-transporting LDL particles from the plasma by receptor-mediated endocytosis. Key steps in this process involve the LDL-R binding LDL at neutral pH at the cell surface and, after internalization, releasing it again at acidic pH in the endosomes. How the modules of the LDL-R might interact within the intact receptor to carry out ligand binding and release has been revealed by the recent crystal structure of the extracellular domain of the LDL-R.     

Nonrandom distribution of sialic acid over the cell surface of bristle- coated endocytic vesicles of the sinusoidal endothelium cells

Previous studies with protein tracers have shown that the luminal surface of the vascular endothelium of the bone marrow is endocytic. The endocytosis occurs through the formation of large bristle-coated vesicles (LCV). The anionic charge distribution in this process was examined at the luminal surface of the endothelial cell, At pH 1.8, colloidal iron (CI), native ferritin, and polycationic ferritin (PCF) are bound by the luminal surface of the endothelial cell, but not at the sites of LCV formation. PCF used over a pH range of 1.8--7.2 (CI is unstable at higher pH levels) revealed LCV binding of this agent in increasing manner from pH 3.5 upwards. PCF binding at low pH (1.8) at the endothelial cell surface was markedly reduced by neuraminidase. Neuraminidase did not reduce PCF binding by the endothelial cell surface nor by the LCV at higher pH levels. It is concluded that the luminal surface of the endothelial cell has exposed sialic acid groups which are absent or significantly diminished at endocytic sites. The free surface of the endothelial cells as well as the sites of endocytosis have, in addition, anionic material with a pKa higher than that of sialic acid (pKa 2.6). These anionic materials may be different at the sites of endocytosis as compared to those present at the free cell surface.     

Self-organized nanogels responding to tumor extracellular pH: pH-dependent drug release and in vitro cytotoxicity against MCF-7 cells

The principal objective of this study was to fabricate doxorubicin-loaded self-organized nanogels composed of hydrophobized pullulan (PUL)-Nalpha-Boc-L-histidine (bHis) conjugates. Their responses to tumor extracellular pH (pHe) were determined, and they were also evaluated with regard to their anticancer efficacy against breast cancer cell lines (MCF-7). bHis was grafted to a PUL-deoxycholic acid (DO) conjugate (PUL-DO) via an ester linkage. PUL-DO/bHis conjugates with two different degrees of bHis substitutions (PUL-DO/bHis36 and PUL-DO/bHis78) were synthesized. PUL-DO/bHis nanogels formed via dialysis at a pH of 8.5 evidenced larger particle sizes (<140 nm) and lower critical aggregation concentrations (CACs) than did the PUL-DO nanogels (90 nm). The pH-dependent CAC of PUL-DO/bHis78 changed dramatically, from 1.2 microg/mL at pH 8.5, to 10 at 7.0, and to 660 at 6.2. A similar tendency in pH-dependent size was also noted. The ionization of the imidazole ring in bHis is principally responsible for pH dependency. The bHis moieties function as a switching tool responding to external pH. Doxorubicin (DOX)-loaded nanogels were assessed for pH-dependent releasing kinetics. The release rate of DOX from the PUL-DO/bHis78 nanogels increased significantly with reductions in pH. This resulted in increased cytotoxicity (30% cell viability at a dose of 10 microg/mL DOX equivalent) against sensitive MCF-7 cells at a pH of 6.8 and low cytotoxicity at pH 7.4 (65% cell viability at an identical dose). The results show that PUL-DO/bHis nanogels may potentially be employed as anti-tumor drug carriers.     

Cell-to-cell spread of Borna disease virus proceeds in the absence of the virus primary receptor and furin-mediated processing of the virus surface glycoprotein

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of Mononegavirales. BDV cell entry follows a receptor-mediated endocytosis pathway, which is initiated by the recognition of an as-yet-unidentified receptor at the cell surface by the virus glycoprotein G. BDV G is synthesized as a precursor (GPC) that is cleaved by the cellular protease furin to produce the mature glycoproteins GP1 and GP2, which have been implicated in receptor recognition and pH-dependent fusion events, respectively. BDV is highly neurotropic and its spread in cultured cells proceeds in the absence of detectable extracellular virus or syncytium formation. BDV spread has been proposed to be strictly dependent on the expression and correct processing of BDV G. Here we present evidence that cell-to-cell spread of BDV required neither the expression of cellular receptors involved in virus primary infection, nor the furin-mediated processing of BDV G. We also show that in furin-deficient cells, the release of BDV particles induced by the treatment of BDV-infected cells with hypertonic buffer was not significantly affected, while virion infectivity was dramatically impaired, correlating with the decreased incorporation of BDV G species into viral particles. These findings support the view that the propagation of BDV within the central nervous systems of infected hosts involves both a primary infection that follows a receptor-mediated endocytosis pathway and a subsequent cell-to-cell spread that is independent of the expression of the primary receptor and does not require the processing of BDV G into GP1 and GP2.     

Effect of Glutaraldehyde on Cell Viability, Triphenyltetrazolium Reduction, Oxygen Uptake, and β-Galactosidase Activity in Escherichia coli

Acid (pH 5) and alkaline (pH 8.5) glutaraldehyde solutions were compared for their effects on cell viability, oxygen uptake, and beta-galactosidase activities in Escherichia coli. The action of glutaraldehyde at pH 7 on dehydrogenase activity was also studied. Dehydrogenase activity was inhibited at aldehyde concentrations which had little effect on cell viability. In contrast, oxygen uptake and beta-galactosidase activity took place in cells killed by acid or alkaline glutaraldehyde. The effect of glutaraldehyde on dehydrogenase activity and beta-galactosidase activity of disrupted suspensions was also investigated. The dialdehyde was considerably less inhibitory to these enzyme systems than to those of whole cells, and it is thus feasible that the results with whole cells are a consequence of its interaction with, and strengthening of, the outer cell surface, thereby preventing ready access of substrate to enzyme.     

In silico modeling of pH-optimum of protein-protein binding

Protein-protein association is a pH-dependent process and thus the binding affinity depends on the local pH. In vivo the association occurs in a particular cellular compartment, where the individual monomers are supposed to meet and form a complex. Since the monomers and the complex exist in the same micro environment, it is plausible that they coevolved toward its properties, in particular, toward the characteristic subcellular pH. Here we show that the pH at which the monomers are most stable (pH-optimum) or the pH at which stability is almost pH-independent (pH-flat) of monomers are correlated with the pH-optimum of maximal affinity (pH-optimum of binding) or pH interval at which affinity is almost pH-independent (pH-flat of binding) of the complexes made of the corresponding monomers. The analysis of interfacial properties of protein complexes demonstrates that pH-dependent properties can be roughly estimated using the interface charge alone. In addition, we introduce a parameter beta, proportional to the square root of the absolute product of the net charges of monomers, and show that protein complexes characterized with small or very large beta tend to have neutral pH-optimum. Further more, protein complexes made of monomers carrying the same polarity net charge at neutral pH have either very low or very high pH-optimum of binding. These findings are used to propose empirical rule for predicting pH-optimum of binding provided that the amino acid compositions of the corresponding monomers are available.     

Effects of prolonged exposure to ammonia on fluid-phase,receptor-mediated,and adsorptive (non specific) endocytosis in cultured neuroblastoma cells

Summary The effect of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma (Neuro-2a) cells were studied using fluorescein-labeled dextran, concanavalin A conjugated with fluorescein isothiocyanate, and cationized ferritin as tracers. Ammonia treatment increased the rate of endocytosis of cationized ferritin as well as the number of cell elements involved in the process. Moreover, the number of cytoplasmic components containing acid phosphatase activity was also found to increase following ammonia treatment. In contrast, flow-cytometric analyses showed that, under experimental conditions, exposure to ammonia did not alter the intralysosomal pH and had little effect on the fluid-phase and receptor-mediated endocytosis of fluorescein-labeled dextran and concanavalin-A fluorocrome, respectively.     

Effects of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma cells. A flow-cytometry and cytochemical study

The effect of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma (Neuro-2a) cells were studied using fluorescein-labeled dextran, concanavalin A conjugated with fluorescein isothiocyanate, and cationized ferritin as tracers. Ammonia treatment increased the rate of endocytosis of cationized ferritin as well as the number of cell elements involved in the process. Moreover, the number of cytoplasmic components containing acid phosphatase activity was also found to increase following ammonia treatment. In contrast, flow-cytometric analyses showed that, under experimental conditions, exposure to ammonia did not alter the intralysosomal pH and had little effect on the fluid-phase and receptor-mediated endocytosis of fluorescein-labeled dextran and concanavalin-A fluorocrome, respectively.     

Transferrin-modified liposomes equipped with a pH-sensitive fusogenic peptide: an artificial viral-like delivery system

Liposomes are one of the most promising systems for selective cellular targeting via introduction of specific ligands for cell-surface receptors. After being taken up by the cells, these liposomes usually follow intracellular pathways of receptor-mediated endocytosis. Control of intracellular trafficking is required for optimized drug delivery. In this study, we elucidated the intracellular fate of transferrin-modified liposomes and succeeded in altering it by introducing the pH-sensitive fusogenic peptide, GALA (WEAALAEALAEALAEHLAEALAEALEALAA). Transferrins that are chemically attached to a liposomal surface (Tf-L) were internalized via receptor-mediated endocytosis more slowly than unmodified transferrins. In contrast to the recyclable nature of transferrin, liposome-attached transferrins together with encapsulated rhodamines were retained in vesicular compartments. When GALA was introduced into liposomal membranes using a cholesteryl moiety for anchoring (Chol-GALA), rhodamines were efficiently released and diffused into the cytosol. The addition of GALA to the Tf-L-containing medium or the encapsulation of GALA in Tf-L did not induce similar effects. These results clearly indicate that GALA must be present on the surface of liposomes to exert its function. In vitro energy transfer and dynamic light scattering experiments suggested that the endosomal escape of the encapsulates in Tf-L equipped with Chol-GALA can be attributed to pH-dependent membrane fusion. With GALA present on the surface, intracellular trafficking of liposomes after receptor-mediated endocytosis could be successfully controlled.     

Effect of Glutaraldehyde on the Outer Layers of Escherichia coli

S ummary : Sodium lauryl sulphate (SLS) at pH 3 and 8 lysed cell walls of Escherichia coli. Pretreatment with glutaraldehyde at pH 3 and at pH 8 prevented this lysis. SLS induced maximum lysis of E. coli cells at 40°; pretreatment of cells with glutaraldehyde prevented this lysis also. Electrophoretic studies indicated that glutaraldehyde accumu lated on the surface of E. coli cells more rapidly in acid than in alkaline conditions, and that it blocked amino groups on the surface layer of Bacillus subtilis spores. The relationship of these findings to the bactericidal efficiency of glutaraldehyde in acid and alkaline solution is discussed.     

Infectious entry of murine retroviruses into mouse cells: evidence of a postadsorption step inhibited by acidic pH.

The entry into cells by many enveloped RNA viruses is accomplished by endocytosis and subsequent penetration of the endosomal membrane by an acidic pH-dependent fusion event. In the current study, we examined early events in the infectious entry of mouse retroviruses, using as a framework the observation that infection of a mouse tail skin cell line by the ecotropic virus Friend murine leukemia virus was inhibited at mildly acidic pH (pH 6). This inhibition operated on a postadsorption step, since binding of virus was unaffected at this pH. The rate of penetration of preadsorbed virus, which displayed first-order kinetics, was markedly affected by changes in the pH of the medium. The half-time for disappearance of infectious cell surface virus at 37 degrees C was approximately 10 min at pH 7.6. At pH 6.0, however, greater than 98% of the adsorbed infectivity remained at the cell surface after 45 min. This cell surface virus, though not infecting the cell at pH 6.0, retained its capacity to enter and infect the cell when the pH of the medium was raised. Acidic pH had little effect on the rate of fluid uptake by the cells, as measured by internalization of [3H]sucrose, indicating that global inhibition of endocytosis had not occurred. In contrast, cell fusion induced by Friend murine leukemia virus was optimal at pH 7.6 but markedly inhibited at a pH of less than 6.4. This inhibitory effect of acidic pH on membrane fusion is unique among the enveloped viruses which have been studied and would preclude entry of Friend murine leukemia virus from within acidified endocytic vesicles. Entry of other members of the ecotropic, mink cell focus-forming, and xenotropic host range groups displayed similar pH sensitivity. However, one xenotropic virus was relatively resistant to the effect of acidic pH, suggesting that differences might exist in the requirements for entry of different retroviruses.     

pH-independent endocytic cycling of the chemokine receptor CCR5

Following agonist activation, the chemokine receptor CCR5 is internalised through clathrin-coated pits and delivered to recycling endosomes. Subsequently, ligand- free and resensitised receptors are recycled to the cell surface. Currently little is known of the mechanisms regulating resensitisation and recycling of this G-protein coupled receptor. Here we show that raising the pH of endocytic compartments, using bafilomycin A, monensin or NH(4)Cl, does not significantly affect CCR5 endocytosis, recycling or dephosphorylation. By contrast, these reagents inhibited recycling of another well-characterised G protein coupled receptor, the beta(2)-adrenergic receptor, following agonist-induced internalisation. CCR5-bound RANTES (CCL5) and MIP-1beta (CCL4) only exhibit pH-dependent dissociation at pH < 4.0, below the values normally found in endocytic organelles. Although receptor-agonist dissociation is not dependent on low pH, the subsequent degradation of released chemokine is inhibited in the presence of reagents that raise endosomal pH. Our data show that exposure to low pH is not required for RANTES or MIP-1beta dissociation from CCR5, or for recycling of internalised CCR5 to the cell surface.     

Expression of chloride channel, ClC-5, and its role in receptor-mediated endocytosis of albumin in OK cells

By using Western blot and RT-PCR analyses, the expression of ClC-5, a member of the ClC family of voltage-gated chloride channels, and its mRNA was detected in OK cells. The effect of chloride channel inhibitors on receptor-mediated endocytosis of albumin was examined in OK cells and compared to that of vacuolar H(+)-ATPase inhibitors. Accumulation of fluorescein-isothiocyanate (FITC)-albumin, a receptor-mediated endocytosis marker, was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a chloride channel inhibitor, in a concentration-dependent fashion. In contrast, uptake of FITC-inulin, a fluid-phase endocytosis marker, was not affected by NPPB. Other chloride channel inhibitors, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid and diphenylamine-2-carboxylic acid, also inhibited FITC-albumin uptake. NPPB, as well as a vacuolar H(+)-ATPase inhibitor bafilomycin A(1), caused a decrease in the affinity and in the maximal velocity of FITC-albumin uptake. These results suggest that chloride channel, most likely ClC-5, plays an important role in the receptor-mediated endocytosis of albumin in OK cells.     

Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion.

The baculovirus gp64 envelope glycoprotein is a major component of the envelope of the budded virus (BV) and is involved in BV entry into the host cell by endocytosis. To determine whether gp64 alone was sufficient to mediate membrane fusion, the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus gp64 protein was transiently expressed in uninfected insect cells. Cells expressing the baculovirus gp64 protein were examined for membrane fusion activity by using a syncytium formation assay under various conditions of exposure to low pH. Cells expressing the gp64 protein mediated membrane fusion and syncytium formation in a pH-dependent manner. A pH of 5.5 or lower was required to induce membrane fusion. In addition, exposure of gp64-expressing cells to low pH for as little as 5 s was sufficient to induce gp64-mediated syncytium formation. These studies provide direct evidence that gp64 is a pH-dependent membrane fusion protein and suggest that gp64 is the protein responsible for fusion of the virion envelope with the endosome membrane during BV entry into the host cell by endocytosis.