Role of membrane phospholipids and glycolipids in cell-to-cell fusion by VSV

Comp. Immun. Microbiol. infect. Dis. Vol. 14, No. 4, pp. 303--313, 1991 Printed in Great Britain. All rights reserved

0147-9571/91 $3.00+0.00 Copyright © 1991 Pergamon Press plc

ROLE OF M E M B R A N E PHOSPHOLIPIDS A N D G L Y C O L I P I D S IN CELL-TO-CELL F U S I O N BY VSV CINZIA CONTI,* PAOLA MASTROMARINO a n d NICOLA ORSI lstituto di Microbiologia, Facolth di Medicina, Universit~ di Roma "La Sapienza", Piazzale A. Moro 5, 00100 Roma, Italia Abstract--To identify membrane components of CER cells interacting with vesicular stomatitis virus (VSV) during fusion at acidic pH (fusion from without, FFWO) two different approaches have been used, i.e. (i) treating the whole cells with enzymes and (ii) testing the ability of isolated membrane molecules to interfere with FFWO. Phospholipase A: and C digestion of cells greatly reduced syncytia formation, pointing towards the involvement of lipid structures as target sites for VSV. Cell susceptibility to F F W O was also reduced after neuraminidase, fl-galactosidase or periodate treatment, suggesting that carbohydrate residues may participate in a complex receptor structure required for virus fusion. When membrane molecules were examined separately for their ability to inhibit viral FFWO, phosphatidylserine, phosphatidylinositol, sphingomyelin, cholesterol and GM3 ganglioside were found to be active, confirming the role of membrane lipid moiety in the cell surface structures involved in the early phases of VSV infection. Key words: Vesicular stomatitis virus, syncytium, fusion from without, lipid, receptor

R f s u m ~ M ) n a 6tudi6 la nature chimique des composants de la membrane cellulaire reconnus par le virus de la stomatite vfsiculaire (VSV) au cours de la fusion ~i pH acide (fusion de l'extfrieur, FFWO) au moyen de deux difffrentes m&hodes, i.e. (i) en traitant les cellules avec des enzymes spfcifiques et (ii) en vfrifiant la capacit6 de plusieurs molfcules de la membrane cellulaire ~ inhiber la FFWO. Le traitement des cellules avec la phospholipase A 2 et C a rfduit la formation des polykaryocytes; ceci indique que les lipides jouent un rfle important au cours de la fusion. En outre, la susceptibilit6 des cellules ~i la F F W O a 6t6 rfduite par la neuraminidase, la fl-galactosidase et le periodate de potassium, dfmontrant que les carbohydrates peuvent aussi 6tre nfcessaires pour la fusion ~t pH acide. Les rfsultats obtenus avec les composants isolfs de la membrane cellulaire ont montr6 que la phosphatidylserine, le phosphatidylinositol, la sphingomyeline, le cholesterol et le ganglioside GM3 sont actifs et ont confirm6 la participation des molfcules lipidiques aux interactions prfcoces virus-cellule h6te. Mots-clefs: Virus de la stomatite vfsiculaire, syncytium, fusion de l'extfrieur, lipides, recepteurs

INTRODUCTION

Fusion between the viral and the cell membranes is a key event in the penetration of the enveloped virus genome into the cytoplasm of host cells. This step can occur either directly with the plasma membrane or with the membrane of a vacuole following endocytosis of virus. Paramyxoviruses belong to the first group, entering cells by fusion with cytoplasmic membrane in the neutral to slightly alkaline pH range encountered at the cell surface [1]. *Author for correspondence. Abbreviations used: vesicular stomatitis virus (VSV), fusion from without (FFWO), chicken embryo related (CER), eagle's minimum essential medium (MEM), fetal calf serum (FCS), bovine serum albumin (BSA), multiplicity of infection (m.o.i.), phosphate buffered saline (PBS), fusion index ( f ) , N-acetyl-neuraminic acid (NANA), plaque forming unit (PFU), potassium metaperiodate (KIO4). 303

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Like most enveloped viruses, vesicular stomatitis virus (VSV), a rhabdovirus, is internalized by adsorptive endocytosis and is subsequently delivered into the cytoplasm by fusion of the viral membrane with that of prelysosomal endocytic vacuoles [2, 3]. Under physiological conditions, no fusion between VSV and the cytoplasmic membrane seems to take place [2]. Fusion with plasma membranes of tissue culture cells can be artificially triggered by a short treatment of the membrane-bound virus with a slightly acidic buffer (fusion from without, FFWO) [4]. The virions form bridges between the membranes promoting the complete fusion of cells and the formation of giant multinucleated cells (syncytium). The sharply defined pH threshold of viral FFWO at 5.5~5 [4] is similar to that required for hemolysis of erythrocytes [5], a process that may be related to fusion, and for fusion with liposomes [6]. The fusogenic activity of VSV is mediated by the G envelope glycoprotein [7, 8]. This protein, in the absence of other viral proteins, produces a low-pH-induced fusion of cells [9, 10] and hemolysis of erythrocytes when incorporated into liposomes [11]. The activation of the fusion function of G protein seems to be due to a conformational change triggered by a brief exposure to a low pH environment (5.5-6) [12, 13]. This treatment probably exposes a previously buried hydrophobic domain [14]. Furthermore, a synthetic peptide corresponding to the first six amino acids of the amino-terminal region of the G protein has been demonstrated to possess a pH-dependent hemolytic activity [15]. However, the chemical nature of the cellular counterpart involved in FFWO has not as yet been characterized. We have studied the nature of CER cell membrane components required for syncytia formation by VSV using two different approaches: by enzyme treatment of the whole cells and by testing the ability of isolated membrane molecules to compete with cells for virus fusion. MATERIALS AND METHODS Virus

VSV, Indiana strain, was grown in Chicken Embryo Related cells (CER) or HeLa $3 cells using Eagle's minimum essential medium (MEM) containing 2% fetal calf serum (FCS). Cell monolayers were infected with virus at a multiplicity of infection (m.o.i.) of 0.01 and incubated at 37°C for about 24 h. The medium was collected and centrifuged (30 min, 10,000g, 4°C) to remove cellular debris. The virus was pelleted by ultracentrifugation at 80,000g for 2 h, resuspended in a small volume of TN buffer (50 mM Tris-base, 0.1 M NaC1, pH 7.4) to yield about 100-fold concentration and stored at -80°C. The virus titer was determined by plaque assay as previously described [16]. Cells

CER and HeLa $3 cells were grown in MEM supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100/~g/ml streptomycin. For fusion experiments, the cells (2.4 × 104 CER cells/well; 3 × 104 HeLa $3 cells/well) were seeded in 96-well plates and grown for 24 h at 37°C in a 5% CO2 incubator. Cell fusion and cell fusion inhibition assay

The fusion assay was an adaptation of the method described by White et al. [4]. Briefly, plates cooled to 4°C and kept on crushed ice were washed three times with ice-cold binding

Membrane receptors for VSV fusion at acidic pH

305

medium [RPMI 1640 without bicarbonate containing 0.2% bovine serum albumin (BSA), 10 mM morpholinoethanesulfonic acid and 10 mM Hepes, pH 6.8] and incubated with 10 #1 of virus dilution in binding medium at pH 6.8. The virus was grown in the same cell line in which the titration was performed. After 1 h at 4°C, 100/~1 of pH 5.5 binding medium prewarmed to 37°C was added and the plates transferred in a water bath at 37°C for 60 s. The medium was then removed, replaced with binding medium at pH 7.2 and the plates kept at 37°C. After 1 h, the cells were washed with phosphate buffered saline (PBS), fixed with cold methanol for 15 min and stained with Giemsa stain for 1 h at room temperature. After washing extensively with PBS, the plates were dried and examined under light microscopy. The fusion index (f) was calculated according to the following formula [1-(number of cells/number of nuclei)]. When virtually all of the nuclei in a microscope field were present in a single polykaryon (100% of fusion) the value o f f was considered 1 whereas f = 0.1 corresponded to the background value (0 point). At a magnification of x 200, approx. 250-300 nuclei per field were counted, and the average fusion index of six fields in duplicate wells was adopted.

Enzymatic treatments of cells Enzymatic treatments were performed on CER cells, grown in 96-well plates. After washing three times in PBS, the cells were incubated for 30 min at 37°C in the presence or absence of enzymes (50#l/well) dissolved in PBS (papain, trypsin, pronase E, phospholipase C, sphingomyelinase, ~-mannosidase, ~-fucosidase, 13-N-acetyl-oglucosaminidase), PBS containing 2 mM MgC12 (/~-galactosidase) or PBS, 1 mM CaC12, 1 mM MgC12 (phospholipase A2, neuraminidase). After phospholipase A2 digestion, the cells were incubated for 5 min at room temperature with 50 mg/ml BSA to extract the reaction products [17]. The monolayers were then washed three times with PBS and incubated with 10 #1 of virus dilution giving approximately anfvalue of 0.7. Cell viability after enzymatic treatments was estimated by the trypan blue exclusion test in parallel treated cultures.

Potassium metaperiodate (KI04) treatment of cells Cell monolayers were treated with 50/~l/well of KIO4 solutions in PBS for 30min at 37°C in the dark. An equal volume of 0.22% glycerol (w/v) in PBS was added to stop the reaction [18]. After washing three times with PBS, the cells were tested in fusion assay as described above.

Enzymes and chemicals Neuraminidase (from Vibrio cholerae) was obtained from Behring. Phospholipase A 2 (from bee venom) and C (from Bacillus cereus), sphingomyelinase (from human placenta), trypsin (from bovine pancreas), pronase E (from Streptomyces griseus), papain (from Papaya latex) were obtained from Sigma. /~-galactosidase (from Escherichia coli), ~-mannosidase (from Canavalia ensiformis), ~-fucosidase (from beef kidney), fl-N-acetylD-glucosaminidase (from beef kidney) were purchased from Boehringer Mannheim. KIO4 was from Merck. N-Acetyl-D-glucosamine, ~-methyl-D-mannoside, D-glucose, D-galactose, N-acetyl-neuraminic acid (NANA), phosphatidylserine, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, cholesterol, GM 3 ganglioside, galactocerebroside type I and II, cerebroside sulfate, glucocerebroside, glucopsychosine, glucosylceramide, lactosylceramide were purchased from Sigma. Stock

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solutions of compounds were made up in PBS. Before use, lipids were dispersed by sonication at 4°C and diluted at the appropriate concentration in binding medium. RESULTS

Relationship between m.o.i, and fusion index of VSV The ability of VSV to fuse from without cultured cells has been studied in CER cells of avian origin and in HeLa cells of human origin. Figure I(A) and (B) shows that cell-to-cell fusion in CER cells exposed to VSV can only be demonstrated in monolayers treated with low pH binding medium, and not in those treated at neutral pH. Furthermore, no cell fusion could be observed in uninfected cells when treated with binding medium at low or neutral pH (data not shown). The viability of the polykaryons was checked in parallel cultures by evaluating their ability to incorporate the vital dye neutral red [Fig. I(C)]. In the same experimental conditions, only few polykaryons could be observed in HeLa $3 cells as compared to CER cells (data not shown). Plot of the fusion index ( f ) of VSV in CER cells vs the m.o.i, is reported in Fig. 2. A large polykaryocyte formation, involving almost the complete monolayer in a microscopic field, occurred at a m.o.i, of at least 1.6-3.2 x 104 plaque forming units (PFU)/cell. The range of maximum sensitivity was between 0.2-0.8 x 104 PFU/cell corresponding to a fusion index of 0.3-0.7. For this reason in the fusion studies we used dilutions of virus giving approximately an f value of 0.7, corresponding to about 3 4 nuclei per cell.

Effect of enzyme digestion and periodate oxidation of CER cells on VSV-mediated cell-to -cell fusion To study the role of the main components of the plasma membrane in cell-to-cell fusion by VSV, CER cell monolayers were digested with several enzymes and the susceptibility of modified cells to viral F F W O was studied. Results reported in Table 1 demonstrated that after phospholipase A 2 and C treatment the capacity of cells to form syncytia was strongly reduced (from 83 to 100%). The effect was dose dependent. Sphingomyelinase digestion produced only a slight inhibition of F F W O (about 30%). Enzymatic removal of surface carbohydrates with glycosidases produced different effects. The cells treated with ~-mannosidase, ~-fucosidase and/%N-acetyl-n-glucosaminidase appeared normal in their ability to be fused, whereas digestion with neuraminidase or /~-galactosidase caused a reduction in the extent of fusion (60-85%). Gentle periodate oxidation of cell membrane oligosaccharides also produced a dose-dependent reduction of syncytia. Membrane protein degradation by papain, trypsin and pronase E had no effect on VSV FFWO.

Inhibiting activity of isolated membrane components on VSV-mediated cell-to-cell fusion The results from the enzyme digestion and periodate oxidation of the cell surface suggested that lipids and carbohydrates are required for membrane fusion by VSV at acidic pH. To confirm the role of these molecules, a number of individual phospholipids, glycolipids, neutral lipids and carbohydrates were tested for their ability to inhibit syncytia formation. Molecules were added either during the 1 h viral binding step at 4°C or during the polykaryocyte formation at 37°C (1 h). The dose-response effect of compounds is reported in Table 2. Phosphatidylserine, phosphatidylinositol, cholesterol and GM3 ganglioside were the membrane lipid components active on both binding and fusion.

Fig. I. VSV-induced polykaryocytosys in C E R cells. The virus was bound to cells for 1 h in the cold. Prewarmed medium, pH 5.5 (A) or 7.4 (B), was added for 6 0 s and the cells were postincubated with pH 7.2 medium for I h at 37°C and then fixed and stained (magnification x 200). Unfixed polykaryons (C) and control untreated cells (D) stained with the vital dye neutral red (magnification × 100).

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Membrane receptors for VSV fusion at acidic pH

309

1.0

/

0.8

/

x 0.6

-~ 0 . 4 U.

//.

0.2

0

I I I 0.2 0.4 0.8

I I I I I 1.6 :3.2 6.4 12.8 25.6

m.o.i. ( x 10 4 )

Fig. 2. Relationship between multiplicity of infection and fusion index of VSV in CER cells. Serial ten-fold dilutions (10/al) of virus in binding medium at pH 6.8 were added to the cells and allowed to adsorb for 1 h at 4°C. The inoculum was then removed and the cells processed for fusion assay as described in Materials and Methods. Sphingomyelin, and to a lesser extent glucocerebroside, were able to compete with cells for virus attachment only. Phosphatidylcholine, phosphatidylethanolamine, galactocerebroside type I and II, cerebroside sulfate, glucopsychosine, glucosylceramide, lactosylceramide and simple monosaccharides (NANA, N-acetyl-D-glucosamine, c(-methyl-Dmannoside, D-glucose, o-galactose) were unable to affect neither viral binding nor fusion. In these experiments controls were included which consisted of C E R cell monolayers incubated with compounds for 30 min at 4°C and washed three times before the addition of virus. Controls gave results similar to untreated cells (data not shown). DISCUSSION Attachment and fusion with cell membranes represent two fundamental steps of virus life cycle. Although extensive studies have been performed to identify membrane molecules to which VSV binds to successfully infect cells [16, 19, 20], as yet only limited data are available on the chemical nature of membrane components interacting with virus during fusion at acidic p H [21]. In this report we have attempted to investigate this point by means of two different approaches, i.e. by enzyme treatment of cells and by testing the capacity of isolated membrane molecules to interfere with virus fusion. The fusion function of VSV was monitored as formation of polykaryocytes in two kinds of cultured cell monolayers (CER and HeLa). It is generally accepted that virus-induced cell-to-cell fusion (syncytia formation) is caused by fusion of viral envelope with cellular membranes [22]. In our experimental conditions, cells of human origin (HeLa) were found to be less sensitive than cells of avian origin (CER) to VSV-mediated fusion. A difference in the ability of various cells to undergo fusion from within after infection with VSV has already been described [23, 24]. Cell-to-cell fusion caused by VSV was observed at a very high multiplicity of infection. The amount of virus used by us to approach an f = 1 (at least 30,000 PFU/cell) was larger than that reported for Semliki Forest virus (about 2000 viruses bound per cell) [4] and La Crosse Bunyavirus (about 2600 PFU/celI) [25]. However, taking into account

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Table 1. Effect of enzyme treatment and periodate oxidation of CER cells on VSVmediated cell-to-cell fusion

Papain Trypsin Pronase E Phospholipase A 2

Phospholipase C

Sphingomyelinase

Neuraminidase

c~-Mannosidase -L-Fucosidase fl-Galactosidase

fl-N -acetyl-D-glucosaminidase KIO4

Enzyme concentration*

Reduction of syncytia formation ( % )

1 0.001 0.001 10 5 1 0,5 20 10 5 1 0.5 1 O.5 0.05 0.5 0.1 0.05 1 0. I 30 15 1.5 0.15 0.1 2.5 I 0.5 0.1

0 0 0 83 66 15 0 100 80 5O 10 0 30 10 0 60 15 0 0 0 85 60 10 0 0 58 43 5 0

*Protease concentrations are in mg/ml. Phospholipase and glycosidase concentrations are in units/ml. The units of enzymes correspond to the activity defined by the manufacturers in standard conditions. KIO 4 concentration is in raM. Cells grown in 96-well plates were incubated in the presence and absence of enzymes or metaperiodate. After 30min of incubation at 3T'C, cell monolayers were washed three times with PBS and tested in fusion assay by VSV as described in Materials and Methods. The f of virus in untreated cells was approx. 0.7.

that at the optimal pH (6.3) <10% of the added virus became cell-associated after incubation for 1-2.5 h at 0°C [4], we can hypothesize that in our conditions about 3000 viruses were bound per cell. Removal of fatty acids or polar head groups from phospholipids by phospholipase A2 and C respectively and of sialic acid or galactose residues by specific glycosidases strongly reduced the susceptibility of cells to form syncytia. The involvement of carbohydrates in VSV FFWO was further stressed by the inhibiting effect produced by gentle periodate oxidation of cell membrane oligosaccharides. At similar concentrations, periodate treatment is known to alter the adsorption of influenza virus to erythrocyte receptor by acting upon the sialic acid residues contained in them [26] and the hemagglutinating capacity of certain enteroviruses and reoviruses [27]. Periodate treatment has been found to affect also VSV-induced hemolysis of goose erythrocytes (Conti C., unpublished data). Proteins and glycoproteins did not appear to have a role in VSV-mediated cell fusion, since protease digestion did not modify the susceptibility of cells to form syncytia. Active carbohydrate residues are therefore probably part of surface glycolipid moiety. The involvement of individual lipid components of membrane in virus-cell fusion was ascertained by competition experiments performed during the time of virus-promoted polykaryocyte formation and, as a control, during the viral attachment step. Phos-

M e m b r a n e receptors for V S V fusion at acidic p H

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Table 2. Dose-response effect of individual membrane components on cell-to-cell fusion by VSV Reduction of syncytia formation (%) Concentration (mg/ml)

During attachment

During fusion

Cholesterol

0.5 0.1 0.5 0.1 0.05 0.01 0.005 0.5 0.1 0.05 0.01 0.5 0.5 0.1 0.05 0.5

GM3 ganglioside

0.05 2

5 0 60 50 25 0 0 20 10 0 0 0 35 25 0 30 20 5 40

0 0 50 40 30 10 0 50 40 15 0 0 0 0 0 35 20 0 90

1

40

90

0.5 0.1 0.5 0.5 0.5 0.5 0.1 0.05 1 1 20 20 20 20 20

5 0 0 0 0 15 0 0 0 0 0 0 0 0 0

50 5 0 0 5 0 0 0 0 0 0 0 0 0 0

Phosphatidylcholine Phosphatidylserine

Phosphatidylinositol

Phosphatidylethanolamine Sphingomyelin

0. I

Galactocerebroside type I Galactocerebroside type n Cerebroside sulfate Glucocerebroside Glucopsychosine Glucosylceramide Lactosylceramide N-Acetyl-D-glucosamine ct-MethyI-D-mannoside D-Glucose D-Galactose NANA

Molecules were added to cells during the I h attachment step of virus at 4°C or during syncytia formation at 37°C (1 h). Compounds were tested at the highest non toxic concentration for cell morphology and viability after a 24 h exposure period. The f o f virus was approx. 0.7.

phatidylserine, phosphatidylinositol, sphingomyelin and the neutral lipid cholesterol were able to affect virus-cell interaction. Simple monosaccharides (NANA, D-glucose, ~-methylD-mannoside, D-galactose, N-acetyl-D-glucosamine), monohexosides (glucosylceramide, glucocerebroside, lactosylceramide, glucopsychosine, galactocerebroside) and sulfatides, were poorly active or completely ineffective. GM3, one of the main gangliosides of CER cell membranes [28], was a very potent inhibitor of VSV FFWO. The finding that among the galactose-containing glycolipids only gangliosides caused a reduction in the extent of fusion indicates that specific carbohydrate sequences are required for fusion. The importance of glycolipid moieties in membrane fusion has been proved also for myxoviruses [29]. The analysis of results obtained, expressed by the ratio between the % reduction of FFWO during polykaryocyte formation and the attachment step, allowed us to divide the active lipids into three groups. A first one including molecules effective only during the attachment step (sphingomyelin, glucocerebroside; ratio = 0); a second one comprising compounds affecting attachment and fusion with about the same efficacy

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( p h o s p h a t i d y l s e r i n e , c h o l e s t e r o l ; r a t i o ~ 1) a n d a t h i r d o n e w i t h lipids m o r e a c t i v e w h e n p r e s e n t d u r i n g t h e p o s t - a d s o r p t i o n step ( p h o s p h a t i d y l i n o s i t o l , GM3 ganglioside; r a t i o > 1). T h e r e f o r e , the first class o f m o l e c u l e s is a b l e to p r e v e n t the b i n d i n g o f virus to t h e cell s u r f a c e w i t h o u t affecting a n y o t h e r p o s t - a d s o r p t i o n e v e n t w h e r e a s c o m p o n e n t s b e l o n g i n g to the s e c o n d g r o u p s e e m to be e q u a l l y r e q u i r e d f o r V S V a t t a c h m e n t a n d fusion. I n s t e a d , p h o s p h a t i d y l i n o s i t o l a n d G M 3 g a n g l i o s i d e , w h i c h affect m a i n l y s y n c i t y u m f o r m a t i o n step, s e e m to be the m e m b r a n e c o m p o n e n t s f u r t h e r o n e n g a g e d by the virus for f u s i o n to o c c u r . A c c o r d i n g l y , t h e y w e r e f o u n d to be the lipid m o l e c u l e s m o s t active in the i n h i b i t i o n o f V S V h e m o l y s i s , a p h e n o m e n o n r e l a t e d to f u s i o n [21]. I n c o n c l u s i o n , the m e t h o d o f the i n h i b i t i o n o f p o l y k a r y o c y t e f o r m a t i o n in cell c u l t u r e s p r o v i d e s a s i m p l e a n d useful t o o l to s t u d y the c h e m i c a l n a t u r e o f m e m b r a n e c o m p o n e n t s r e q u i r e d for v i r u s f u s i o n at acidic p H . Acknowledgements--This research was supported by grants from lstituto Pasteur-Fondazione Cenci Bolognetti and from the Italian Ministry of Public Instruction.

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CIMID 14/4~-B