Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing.

We have monitored the mixing of both aqueous intracellular and membrane-bound fluorescent dyes during the fusion of human red blood cells to influenza hemagglutinin-expressing fibroblasts using fluorescence spectroscopy and low light, image-enhanced video microscopy. The water-soluble fluorescent dye, N-(7-nitrobenzofurazan-4-yl)taurine, was incorporated into intact human red blood cells. The fluorescence of the dye in the intact red blood cell was partially quenched by hemoglobin. The lipid fluorophore, octadecylrhodamine, was incorporated into the membrane of the same red blood cell at self-quenching concentrations (Morris, S. J., D. P. Sarkar, J. M. White, and R. Blumenthal. 1989. J. Biol. Chem. 264: 3972-3978). Fusion, which allowed movement of the water-soluble dye from the cytoplasm of the red blood cell into the hemagglutinin-expressing fibroblasts, and movement of octadecylrhodamine from membranes of red blood cell to the plasma membrane of the fibroblasts, was observed by fluorescence microscopy as a spatial relocation of dyes, and monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, fluorescence increased after a delay of about 30 s at 37 degrees C, reaching a maximum within 3 min. The kinetics, pH profile, and temperature dependence were similar for both fluorescent events measured simultaneously, indicating that influenza hemagglutinin-induced fusion rapidly establishes bilayer continuity and exchange of cytoplasmic contents.

The MCP-4/MCP-1 ratio in plasma is a candidate circadian biomarker for chronic post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is psychiatric disease, which can occur following exposure to traumatic events. PTSD may be acute or chronic, and can have a waxing and waning course of symptoms. It has been hypothesized that proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) or plasma might be mediators of the psychophysiological mechanisms relating a history of trauma exposure to changes in behavior and mental health disorders, and medical morbidity. Here we test the cytokine/chemokine hypothesis for PTSD by examining levels of 17 classical cytokines and chemokines in CSF, sampled at 0900 hours, and in plasma sampled hourly for 24 h. The PTSD and healthy control patients are from the NIMH Chronic PTSD and healthy control cohort, initially described by Bonne et al. (2011), in which the PTSD patients have relatively low comorbidity for major depressive disorder (MDD), drug or alcohol use. We find that in plasma, but not CSF, the bivariate MCP4 (CCL13)/ MCP1(CCL2) ratio is ca. twofold elevated in PTSD patients compared with healthy controls. The MCP-4/MCP-1 ratio is invariant over circadian time, and is independent of gender, body mass index or the age at which the trauma was suffered. By contrast, MIP-1ß is a candidate biomarker for PTSD only in females, whereas TARC is a candidate biomarker for PTSD only in males. It remains to be discovered whether these disease-specific differences in circadian expression for these specific immune signaling molecules are biomarkers, surrogates, or drivers for PTSD, or whether any of these analytes could contribute to therapy.

Continuous monitoring of transport by fluorescence on cells and vesicles.

Fluorescence techniques are gaining wider applicability in the field of membrane transport due to their high temporal resolution, modest demand for biological material and the kinetic information which is made available by fluorescence tracings. The development of novel fluorescent substrates for particular transport systems and of novel fluorescent indicators for permeant ions, have opened the way for studying transport kinetics and regulation of transport in a variety of cellular and vesicular systems. The various methods of continuous monitoring of transport by fluorescence (CMTF) which are presently in use, are reviewed with emphasis on both analytical and applicative properties.

New permeability pathways induced in membranes of Plasmodium falciparum infected erythrocytes.

The permeability properties of the membrane of human erythrocytes infected with malaria parasites (Plasmodium falciparum) were studied by the method of osmotic hemolysis. At the trophozoite stage, the host membrane becomes permeable to substrates such as sorbitol and glucose. The new permeability pathway is insensitive to most inhibitors of the glucose carrier, but is highly susceptible to the membrane dipole modifier phloretin. It is blocked by disaccharides and oligosaccharides, both of which are impermeant to non-infected and infected cells. It has an enthalpy of activation of solute penetration of 10 +/- 1 kcal mol-1 (range of 5-37 degrees C). It appears that new permeability pathways with pore-like properties are induced in parasitized cells. The pore(s) admit(s) neutral and anionic substances of a discrete molecular volume, but exclude(s) cations. Apparently they play an essential role in parasite development.

Cystic fibrosis and the use of pharmacogenomics to determine surrogate endpoints for drug discovery.

Cystic fibrosis (CF) is caused by a mutation in the CFTR gene, encoding a chloride channel. For the most common mutation, Delta F508, the basis of the deficit is the failure of the mutant CFTR channel protein to traffic properly to the apical plasma membrane of the affected epithelial cell. The trafficking failure results in loss of the cyclic adenosine monophosphate (cAMP)-activated chloride channel function of the CFTR protein in the plasma membrane. The lung is the principal site affecting patient morbidity and mortality in CF. The main reason is that the CF airway epithelial cells also secrete high levels of the proinflammatory cytokine interleukin (IL)-8, resulting in massive cellular inflammation, infection, tissue damage and lung destruction. The relationship between the trafficking defect, the loss of chloride channel activity, and inflammation is not known. However, gene therapy of CF lung epithelial cells with the wild-type CFTR gene can repair the chloride channel defect, as well as suppress the intrinsic hypersecretion of IL-8. Repair of both defective channels and high IL-8 secretion can also be effected by treatment with the candidate CF drug CPX, which is in clinical trials in CF patients. CPX acts by binding to the mutant CFTR protein, and helps the protein to mature and gain access to the plasma membrane. CPX also suppresses the synthesis and secretion of IL-8 from CF epithelial cells, presumably by virtue of its repair of the trafficking defect of mutant CFTR. To guide pharmacogenomic experiments we have therefore hypothesized that the genomic signature of CF epithelial cells treated with CPX should resemble the signature of the same cells repaired by gene therapy. We have developed two algorithms for identifying genes modified by repair of CFTR defects. The GRASP algorithm uses a statistical test to identify the most profoundly changing genes. The GENESAVER algorithm allows us to identify those genes whose pattern of expression changes in-phase or out-of-phase with IL-8 secretion by CF cells. For the latter algorithm we modified IL-8 secretion from CF cells by treatment with wild-type CFTR, with CPX, or by exposure to bacteria. The results have supported the hypothesis, and have provided a basis for considering the common pharmacogenomic expression signature as a surrogate endpoint for CF drug discovery. Significantly, the nature of the hypothesis, as well as the algorithm developed for this study, can be easily applied to pharmacogenomic studies with other goals.

ANX7 as a bio-marker in prostate and breast cancer progression.

The ANX7 gene codes for a Ca2+-activated GTPase, which has been implicated in both exocytotic secretion in cells and control of growth. In this review, we summarize information regarding increased tumor frequency in the Anx7 knockout mice, ANX7 growth suppression of human cancer cell lines, and ANX7 expression in human tumor tissue micro-arrays. The loss of ANX7 is significant in metastatic and hormone refractory prostate cancer compared to benign prostatic hyperplasia. In addition, ANX7 expression has prognostic value for predicting survival of breast cancer patients.

The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: I. Kinetic properties of NBD-taurine transfer in symmetric conditions.

The molecular mechanism of anion exchange across the human red blood cell membrane was assessed with the fluorescent substrate analog NBD-taurine and the method of continuous monitoring of transport by fluorescence. The efflux of NBD-taurine was studied under a variety of experimental conditions such as temperature, pH and anion composition of cells and media. The temperature profile of NBD-taurine transfer from Cl-loaded cells into Cl media resembled that of Cl self-exchange, whereas that of NBD-taurine transfer from sulfate-loaded cells into sulfate media resembled that of sulfate self-exchange. Although the pH profiles of NBD-taurine transfer from Cl-loaded cells into Cl media and that of Cl self-exchange resembled each other, the analogous transfer with sulfate replacing Cl was markedly different. These and other data were analyzed and found to be consistent with a model which comprises the following: (a) a H+-titratable group in the carrier mechanism; (b) alteration of transport sites between the two sides of the membrane (i.e., ping-pong kinetics); and (c) transmembrane distribution of transport sites which is modulated by pH. It is shown that NBD-taurine transfer represents a tracer flux of a fluorescent substrate which gives a measure for the presence of monovalent transport sites at the inner surface of the membrane. The latter is markedly affected by the relative concentrations of anions and H+ on both sides of the red blood cell membrane.

The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: II. Kinetic properties of NBD-taurine transfer in asymmetric conditions.

The transport of inorganic anions across human red blood cell membranes is accomplished by a carrier-like mechanism which involves an electroneutral and obligatory one-for-one anion exchange. The transport kinetics were described by models that involve alternation of single transport sites between the two membrane surfaces. These models predict that each carrier shows either an inward-facing Ei or an outward-facing Eo, conformation, each capable of binding either a monovalent anion or a divalent anion + a proton, to yield an electroneutral translocating complex. Unidirectional transport rates provide, therefore, a measure for the relative concentration of carriers at a given membrane surface. In the present work we assessed how modulation of the transmembrane distribution of carriers by the anion composition of cells and media, and by pH, affect the anion transport system. We have set the system in asymmetric conditions with respect to anions, so that a fast transportable anion (e.g., chloride) was present in one side of the membrane and slow transportable anions (e.g., sulfate, phosphate, oxalate, isethionate, gluconate, HEPES) were present on the other side of the membrane. The skewed distribution of carriers induced in these conditions were assessed by two methods: 1) NBD-taurine transfer which provided a measure for [Ei], the monovalent inward-facing form of the carrier, and 2) inhibition of NBD-taurine transfer by the specific impermeant and competitive inhibitor 4,4'-dinitro-2,2'-stilbene disulfonic acid (DNDS), which provided a measure for the availability of the carrier at the outer membrane surface. In the various symmetric and asymmetric conditions, we found marked differences in transport rates and transport profiles as well as in the susceptibility of the system to inhibition by DNDS. Direct binding studies of DNDS to cells in the various asymmetric conditions supported the conclusion derived from transport studies that transport sites can be recruited towards the membrane surface facing the slow transportable anions.

Composition of octyl glucoside-phosphatidylcholine mixed micelles.

The composition of mixed micelles of egg phosphatidylcholine (PC) and octyl glucoside was studied by a novel technique based on measuring resonance energy-transfer efficiency between two fluorescent lipid probes present in trace amounts. Equations were derived for calculating the stoichiometry of the composition of mixed micelles from the energy-transfer measurements. These were applied to determining the average number of lipid molecules in the octyl glucoside-egg PC mixed micelle as a function of detergent concentration. The average number of detergent molecules in these mixed micelles was independent of lipid concentration in the range studied (0-500 microM). The dependence of mixed micelle stoichiometry on the concentration of aqueous (monomeric) octyl glucoside is consistent with the assumptions of ideal mixing of the two amphiphiles in the mixed micelles and that mixed micelles can be treated as a distinct phase.

Micelle-vesicle transition of egg phosphatidylcholine and octyl glucoside.

The dissolution and formation of egg phosphatidylcholine (PC) vesicles by the detergent octyl glucoside were examined systematically by using resonance energy transfer between fluorescent lipid probes, turbidity, and gel filtration chromatography. Resonance energy transfer was exquisitely sensitive to the intermolecular distance when the lipids were in the lamellar phase and to the transitions leading to mixed micelles. Turbidity measurements provided information about the aggregation of lipid and detergent. Several reversible discrete transitions between states of the PC-octyl glucoside system were observed by both methods during dissolution and vesicle formation. These states could be described as a series of equilibrium structures that took the forms of vesicles, open lamellar sheets, and mixed micelles. As detergent was added to an aqueous suspension of vesicles, the octyl glucoside partitioned into the vesicles with a partition coefficient of 63. This was accompanied by leakage of small molecules and vesicle swelling until the mole fraction of detergent in the vesicles was just under 50% (detergent:lipid ratio of 1:1). Near this point, a transition was observed by an increase in turbidity and release of large molecules like inulin, consistent with the opening of vesicles. Both a turbidity maximum and a sharp increase in fluorescence were observed at a detergent to lipid mole ratio of 2.1:1. This was interpreted as the lower boundary of a region where both lamellar sheets and micelles are at equilibrium. At a detergent:lipid ratio of 3.0:1, another sharp change in resonance energy transfer and clarification of the suspension were observed, demarcating the upper boundary of this two-phase region. This latter transition is commonly referred to as solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)

pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles.

Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.

Inhibition of immune reactions in vivo by liposome associated transforming growth factor (TGF) type beta 1.

In view of its potent inhibitory capacity on immune cells in culture, we wished to determine the ability of transforming growth factor (TGF) beta 1 to down-regulate immune responses in vivo. Preliminary experiments suggested that, at the doses used, systemic injection of soluble TGF beta 1 could not affect bacterial-induced spleen enlargement in mice. Therefore, we sought to utilize a physiochemical property of this molecule, namely its high pI, to determine possible association between the ligand and preformed liposomes possessing an opposite charge. TGF beta 1 was preferentially associated with negatively charged, but not with neutral, liposomes. These TGF beta 1 associated liposomes were able to deliver a suppressive signal to indicator cells in vitro. Intravenous injection of TGF beta 1, associated with liposomes possessing an opposite charge, into mice immunized with heat-killed Corynebacterium parvum significantly reduced the size of the spleen as well as the number of splenocytes. Systemically administered TGF beta 1 associated liposomes could also inhibit delayed type hypersensitivity reactions to Listeria monocytogenes. These data suggest that appropriately administered, TGF beta 1 can inhibit immune responses in vivo.

Pharmacogenomics of the cystic fibrosis transmembrane conductance regulator (CFTR) and the cystic fibrosis drug CPX using genome microarray analysis.

BACKGROUND: Cystic fibrosis (CF) is the most common lethal recessive disease affecting children in the U.S. and Europe. For this reason, a number of ongoing attempts are being made to treat the disease either by gene therapy or pharmacotherapy. Several phase 1 gene therapy trials have been completed, and a phase 2 clinical trial with the xanthine drug CPX is in progress. The protein coded by the principal CFTR mutation, DeltaF508-CFTR, fails to traffic efficiently from the endoplasmic reticulum to the plasma membrane, and is the pathogenic basis for the missing cAMP-activated plasma membrane chloride channel. CPX acts by binding to the mutant DeltaF508-CFTR and correcting the trafficking deficit. CPX also activates mutant CFTR channels. The comparative genomics of wild-type and mutant CFTR has not previously been studied. However, we have hypothesized that the gene expression patterns of human cells expressing mutant or wild-type CFTR might differ, and that a drug such as CPX might convert the mutant gene expression pattern into one more characteristic of wild-type CFTR. To the extent that this is true, a pharmacogenomic profile for such corrective drugs might be deduced that could simplify the process of drug discovery for CF. MATERIALS AND METHODS: To test this hypothesis we used cDNA microarrays to study global gene expression in human cells permanently transfected with either wild-type or mutant CFTR. We also tested the effects of CPX on global gene expression when incubated with cells expressing either mutant or wild-type CFTR. RESULTS: Wild-type and mutant DeltaF508-CFTR induce distinct and differential changes in cDNA microarrays, significantly affecting up to 5% of the total genes in the array. CPX also induces substantial mutation-dependent and -independent changes in gene expression. Some of these changes involve movement of gene expression in mutant cells in a direction resembling expression in wild-type cells. CONCLUSIONS: These data clearly demonstrate that cDNA array analysis of cystic fibrosis cells can yield useful pharmacogenomic information with significant relevance to both gene and pharmacological therapy. We suggest that this approach may provide a paradigm for genome-based surrogate endpoint testing of CF therapeutics prior to human administration.

Pharmacogenomics of cystic fibrosis.

Pharmacogenomics is becoming a frontline instrument of drug discovery, where the drug-dependent patterns of global gene expression are employed as biologically relevant end points. In the case of cystic fibrosis (CF), cells and tissues from CF patients provide the starting points of genomic analysis. The end points for drug discovery are proposed to reside in gene expression patterns of CF cells that have been corrected by gene therapy. A case is made here that successful drug therapy and gene therapy should, hypothetically, converge at a common end point. In response to a virtual tidal wave of genomic data, bioinformatics algorithms are needed to identify those genes that truly reveal drug efficacy. As examples, we describe the hierarchical clustering, GRASP, and GENESAVER algorithms, particularly within a hypothesis-driven context that focuses on data for a CF candidate drug. Pharmacogenomic approaches to CF, and other similar diseases, may eventually give us the opportunity to create drugs that work in a patient- or mutation-specific manner.

Temperature dependence of the vesicle-micelle transition of egg phosphatidylcholine and octyl glucoside.

The temperature dependence of octyl glucoside micellization was determined and compared to the phase behavior of the octyl glucoside--egg phosphatidylcholine (PC) mixed system in excess water to help elucidate the process of vesicle formation from mixed surfactant-phospholipid micelles. The critical micelle concentration of octyl glucoside (OG) was determined from the sharp increase of ANS fluorescence at micellization in an NaCl buffer at temperatures ranging from 5 to 40 degrees C. The cmc decreased with increasing temperature from 31 mM at 5 degrees C to 16 mM at 40 degrees C. A similar but less steep temperature dependence is observed for the solubilization of egg PC vesicles by OG as monitored by the surfactant-dependent changes in (1) solution turbidity and (2) the resonance energy transfer between NBD-PE and Rho-PE incorporated in the vesicles. These assays identify two breakpoints, most likely the boundaries of the cylindrical micelle and spheroidal micelle coexistence region. The [OG]aq values at these two breakpoints have similar temperature dependencies. However, the cylindrical mixed micelles at the boundary have nearly identical OG:PC ratios over the temperature range studied, whereas the spheroidal mixed micelles have relatively more OG at the higher temperatures (OG:PC ratio increases from 2.92 to 3.72 between 5 and 35 degrees C). Estimation of the acyl volume to surface area ratio for the compositions observed suggests that this parameter remains constant over temperature. The spheroidal mixed micelles, but not the cylindrical PC-OG micelles, exhibit ideal mixing between the two components at all temperatures (5-35 degrees C). This temperature sensitivity may be utilized to improve the efficacy of membrane protein reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)

The anion-transfer system of erythrocyte membranes. N-(7-Nitrobenzofurazan-4-yl)taurine, a fluorescent substrate-analogue of the system.

The fluorescent probe Nbd-Tau [N-(7-nitrobenzofurazan-4-yl)taurine] was synthesized and evaluated as a potential substrate of the anion-transport system of human erythrocyte membrane. The probe inhibited Cl- exchange in a competitive manner from either surface of the membrane, displaying Ki values in the mM range at the inner surface and in the microM range at the outer surface. Inhibition from within cells was via interaction with Cl--transport sites, whereas from it was via interaction with sites of unidentified nature. Nbd-Tau efflux from cells was monitored fluorimetrically in a continuous mode by a novel method that circumvents separation of the cells from the medium. Using this method, it is shown that Nbd-Tau efflux fulfils the following criteria of a substrate of the anion transport system: (a) susceptibility to classical and specific inhibitors of the system; (b) competitive inhibition with Cl- for anion-transport sites; and (c) temperature coefficient comparable with that of Cl- exchange. The fluorometric method is highly sensitive, versatile, and kinetically informative. With minor modifications it can be used for measuring anion transport across "ghost" and isolated membrane vesicles.

pH-dependent fusion of vesicular stomatitis virus with Vero cells. Measurement by dequenching of octadecyl rhodamine fluorescence.

We have studied fusion between membranes of vesicular stomatitis virus (VSV) and Vero cells using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We could identify the two pathways of fusion by the kinetics of R18 dequenching, effects of inhibitors, temperature dependence, and dependence on osmotic pressure. Fusion at the plasma membrane began immediately after lowering the pH below 6 and showed an approximately exponential time course, whereas fusion via the endocytic pathway (pH 7.4) became apparent after a time delay of about 2 min. Fusion via the endocytic pathway was attenuated by treating cells with metabolic inhibitors and agents that raise the pH of the endocytic vesicle. A 10-fold excess of unlabeled virus arrested R18VSV entry via the endocytic pathway, whereas R18 dequenching below pH 6 (fusion at the plasma membrane) was not affected by the presence of unlabeled virus. The temperature dependence for fusion at pH 7.4 (in the endosome) was much steeper than that for fusion at pH 5.9 (with the plasma membrane). Fusion via the endocytic pathway was attenuated at hypo-osmotic pressures, whereas fusion at the plasma membrane was not affected by this treatment. The pH profile of Vero-VSV fusion at the plasma membrane, as measured by the dequenching method, paralleled that observed for VSV-induced cell-cell fusion. Fusion was blocked by adding neutralizing antibody to the Vero-VSV complexes. Activation of the fusion process by lowering the pH was reversible, in that the rate of fusion was arrested by raising the pH back to 7.4. The observation that pH-dependent fusion occurred at similar rates with fragments and with intact cells indicates that pH, voltage, or osmotic gradients are not required for viral fusion.

Identification of isolate-specific proteins on sorbitol-enriched Plasmodium falciparum infected erythrocytes from Gambian patients.

We have compared the surface radio-iodinated proteins of uninfected and Plasmodium falciparum-infected erythrocytes from natural infections of human patients. Cryopreserved infected blood from Gambian children with falciparum malaria was thawed, cultured to the middle trophozoite stage, and surface radio-iodinated. Trophozoite-infected cells were enriched about 10-fold on a Percoll gradient newly designed to separate cells based on their differential permeability to sorbitol. Infected blood was radio-iodinated and erythrocytes from the fraction enriched in parasitized cells and uninfected erythrocytes from the same sample obtained from the gradient and compared by SDS-PAGE and autoradiography. In each sample, parasitized erythrocytes contained one or more polypeptides of very high molecular weight (Mr 250 000-300 000) that were not found on uninfected erythrocytes from the same patient. These proteins were isolate-specific in size and number, suggesting that natural isolates contain a variable number of different P. falciparum phenotypes for this surface protein. In addition, these radio-iodinated surface proteins could not be extracted from the host cell membrane by the non-ionic detergent Triton X-100, but were extracted by SDS. The properties of these proteins suggest they are the equivalent for natural infections of the strain-dependent antigen previously described (Leech, Barnwell, Miller & Howard, 1984) on the surface of P. falciparum-infected Aotus erythrocytes. In addition, we observed a second parasite-dependent modification of labelled proteins on infected erythrocytes with the appearance of a new band of Mr 30 000. There were also variations in the pattern of radio-isotope labelled proteins on uninfected erythrocytes from different patients.

Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH.

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus to low pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodamine was used to monitor fusion. Incubation of the virus at pH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusion as nontreated virus, and it was blocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, when VSV was first bound to target cells and subsequently exposed at 4 degrees C to the low pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation and only the effects of the supernatant, which consist of single virions and/or microaggregates, were considered. The data were analyzed in the framework of an allosteric model according to which viral spike glycoproteins undergo a pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis we conclude that the conformational transition to the active state is rate-limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.