Inhibition of VSV binding and infectivity by phosphatidylserine: Is phosphatidylserine a VSV-binding site?

Cell, Vol. 32, 639-646,

February

1983,

Copyright

0 1983

by MIT

Inhibition of VSV Binding and Infectivity by Phosphatidylserine: Is Phosphatidylserine a VSV-Binding Richard Schlegel, *+§ Tommie Sue Tralka,* Mark C. Willingham+ and Ira Pastant *Department of Pathology National Cancer Institute + Laboratory of Molecular Biology National Cancer Institute Bethesda, Maryland 20205

Summary Recently we described a saturable, high-affinity binding site for vesicular stomatitis virus (VSV) on the surface of Vero cells that appears to mediate viral infectivity. To isolate this binding site, we have extracted Vero cells with the detergent, octyl+oglucopyranoside. The dialyzed detergent extract specifically inhibits the saturable, high-affinity binding of 35S-methionine-labeled VSV to Vero cells. The inhibitory activity is resistant to protease, neuraminidase and heating to 100°C. It is soluble in chloroform-methanol and inactivated by phospholipase C, suggesting that it is a phospholipid. Of various puriifed lipids tested, only phosphatidylserine was capable of totally inhibiting the high-affinity binding of VSV. The half-maximal inhibitory concentration for phosphatidylserine was 1 PM. Phosphatidylserine also inhibited VSV plaque formation by 80%90%; Herpes simplex virus plaque formation was unaffected. Centrifugation and electron microscopy studies have shown that phosphatidylserine-containing liposomes bind to VSV. The finding that phosphatidylserine directly binds to VSV and inhibits VSV attachment and infectivity suggests that plasma membrane phosphatidylserine could function as a binding site or portion of a binding site for vsv. Introduction Virus attachment to host cells is often mediated by specific plasma membrane binding sites or “receptors” (Lonberg-Holm, 1981). However, few virus receptors have been purified or biochemically identified. Best characterized is glycophorin, a sialoglycoprotein binding site for influenza virus on erythrocytes (Kathan et al., 1961; Jackson et al., 1973). Less well defined is the receptor for Epstein-Barr virus, which appears to be the same as the membrane receptor for the C3 component of the complement system (Yefenof et al., 1976) and the receptor for Semliki Forest virus, which apparently includes the histocompatibility antigens (Helenius et al., 1978) and additional, unrelated binding sites (Oldstone et al., 1980). Other viruses may also use plasma membrane proteins as receptors (Hennache and Boulanger, 1977). In some cases, however, the virus-binding site * To whom

requests

for reprints

should

be addressed.

Site?

appears to be a lipid or complex of lipids (Haywood, 1974; Mooney et al., 1975). The limited evidence available suggests that the binding site for vesicular stomatitis virus (VSV) is not a membrane protein, because proteolytic digestion of the cell surface does not inhibit viral infection (Schloemer and Wagner, 1975). Studies on VSV binding demonstrate that there are about 4000 high-affinity, saturable binding sites for VSV on the surface of Vero cells (Schegel et al., 1982b). Having established that VSV binds to a small number of high-affinity sites and that these sites are apparently involved in VSV entry, it is possible to investigate the biochemical nature of these binding sites. Our approach has been to solubilize membrane constituents from cells and to determine which components are able to bind to VSV and inhibit its binding to cells. Using such an assay we have found one phospholipid, phosphatidylserine (PS), specifically inhibits VSV binding and infectivity. In addition, VSV will bind to liposomes containing PS. These results suggest that PS is the cellular binding site for VSV or an important component of the binding site. Results Solubilization of an Inhibitor of VSV Binding To identify a molecule with the properties of the VSV receptor, we used a competition assay similar to those described for the isolation of Semliki Forest virus and adenovirus receptors (Helenius et al., 1978; Svensson et al., 1981). Confluent monolayers of Vero cells were extracted with 50 mM octyt-fi-p-glucopyranoside (OG) at 4°C. the extract was centrifuged at 100,000 x g for 60 min and the supernatant was then dialyzed against phosphate-buffered saline (PBS) according to the protocol given in the Experimental Procedures. Figure 1 illustrates that the detergent extract totally inhibits the specific binding of 35S-methionine-labeled VSV to Vero cells. Nonsaturable binding of 35S-VSV, which represented 45% to 50% of total binding, was unaffected (data not shown). The protein concentration of the extract was 3 mg/ml, and 10 ~1 of extract represents the amount of material solubilized from one 35 mm culture dish (the size used for the binding assay). Thus the amount of binding activity solubilized from one 35 mm dish is sufficient to reduce VSV saturable binding by approximately 50%. Inhibitor Has phospholipid Properties The detergenpextract was treated with several enzymes and inactivation procedures to obtain an estimate of the chemical nature of the inhibitory activity (Table 1). The inhibitory activity was resistant to both trypsin and pronase digestion as well as to heating to 100°C for 10 min. Neuraminidase also had no effect on its activity. However, the activity was totally soluble in chloroform-methanol and was sensitive to phospholipase C (B. cereus). The resistance to heat and

Cell 640

proteases, the solubility in chloroform-methanol and the inactivation by phospholipase C suggest that the activity is due to a phospholipid. In an analogous fashion, saturable VSV binding to Vero monolayers is insensitive to trypsin and neuraminidase (Schloemer and Wagner, 1975; Schlegel et al., 1982b). Pretreatment of Vero cells with pronase or papain also has no effect on VSV binding (Table 2). In contrast, incubation of Vero monolayers with phospholipase C (6. cereus) inhibits specific VSV binding. These combined results suggest that the soluble activity has properties similar to the cell surface binding sites. Only Phosphatidylserine Completely Inhibits Saturable VSV Binding When the soluble inhibitor was extracted with chloroform:methanol (2:1), dried and separated by preparative thin-layer chromatography (see Experimental Procedures), >80% of the inhibitory activity was found in the region where phosphatidylserine and phosphatidylinositol migrate (Table 3). A mixture of all of the other chromatographed lipids produced only a 17% inhibitory effect. To document further that the binding inhibitor was a phospholipid, the major plasma membrane lipids were assayed for their ability to inhibit saturable VSV binding. All purified lipids (except cholesterol) were solubilized in OG at 1 mg/ml, and then the solution was dialyzed or diluted below the detergent’s critical micelle concentration as described in the Experimental Procedures. Cholesterol was dissolved in 100% ethanol at 1 mg/ml. Samples of the lipid preparations were then tested for inhibition of 35S-VSV binding (Figure 2). PS was the most potent inhibitor of 35S-VSV binding to Vero cells. PS at 1 PM inhibited saturable VSV binding by 60% and was more effective than any other lipid at 10 PM concentrations. Although there were slight variations between experiments, 10 PM PS could usually completely inhibit saturable VSV binding. Phosphatidylethanolamine (PE) was the only other phospholipid to have some inhibitory effect on VSV binding. However, when PE was assayed over a wider concentration range, it was not possible to increase this inhibitory effect beyond 30% (Figure 3). The inhibitory effect of PS appears to account for all or most of the activity detected in detergent extracts. Using estimates that the content of PS in fibroblasts is 20 nmoles/mg protein (Pastan and Friedman, 1968), we have calculated that 400 ~1 of OG extract should produce a final PS concentration of 8 PM. Since the inhibitory effects of 400 ~1 OG extract (Figure 1) and 8 PM PS (Figure 3) are virtually identical (90% inhibition), it appears that PS is the major inhibitory factor present in OG extracts. Domains of Phosphatidylserine Necessary for Inhibitory Activity Sensitivity to phospholipase C (Table 1) as well as the specificity for PS (Figure 2) suggested that the serine

0

100

200

300

400

500

600

700

800

~1 OG EXTRACT Figure Cells

1. Inhibition

of VSV Binding

by a Detergent

Extract

of Vero

35S-VSV (50 ng) was added to confluent Vero monolayers which had been equilibrated at 4°C. At the same time, different amounts of dialyzed OG extracts of Vero cells were added. Cultures were kept at 4OC for 18 hr and the amount of saturable VSV binding was quantitated as described in the Experimental Procedures. The protein concentration of the OG extract was 3 mg/ml. The OG extract completely inhibited saturable VSV binding. Half-maximal inhibition was obtained with approximately 10 ~1 OG extract.

Table 1. Sensitivity of Inhibitory Inactivation Procedures

Activity

to Various Saturable

Enzymes

35S-VSV Binding

VSV per Plate (w)”

Control Binding

Control

9.0

100

OG extract (Vera cells) + Trypsin (100 pg/ml) + Pronase E (100 fig/ml) + Neuraminidase (1 U/ml) + 1 OO’C, 10 min + Freeze-thaw + Phospholipase C (0.1 U/ml) + Phospholipase C (1 .O U/ml)

0 0 0 0 1.4 0.6 0.5 6.0

Chloroform-methanol OG extract

o-o.5

a Values

represent

fraction

the average

and

(%)

0 0 0 0 15 7 5 67

of

of duplicate

O-6 samples.

head group of PS was essential for its interaction with VSV. To evaluate the activity of a mol.ecule containing an intact serine head group but lacking the fatty acid chains, we subjected PE and PS to methanolysis and separated the ethanolamine and serine phosphoglycerol from the free fatty acids (see Experimental Pro-

KS;

Binds to Phosphatidylserine

Table Cells

2. Enzyme

Sensitivity

of Saturable

VSV-Binding

1

Sites on Vero

Saturable 35S-VSV Binding (96 of controlY Control

100

Pronase

E, 10 bg/ml

112

Pronase

E, 100 pg/ml

103

Papain.

50 &ml

97

Phospholipase

C, 5 U/ml

Phospholipase

C, 50 U/ml

23 7

a Saturable VSV binding represents 55% of the total VSV bound (6 ng) to a confluent 35 mm culture plate of Vero cells. Experimental values represent the average of triplicate samples. The maximum standard deviation was f 1.5%.

Table 3. Inhibition of 35S-VSV Binding Octylglucoside Detergent Extract

by Lipid Fractions

35S-VSV Binding

Total Control 10 pl PS/PI 10 ~1 all other combined

fraction lipid fractions

Binding

from the

(cpm/plate) Saturable Binding

13.210 13,900

6,710 7,200

6,940 7,430

440 930

11,670 12,900

5,170 6,400

CE

Figure

2. Inhibition

VSV Binding

by Purified

Lipids

35S-VSV (50 ng) plus concentrations of 1 PM (hatched bars) or 10 gM (open bars) of purified lipids were added to confluent Vero monolayers at 4°C. All lipids (except cholesterol) were dissolved in 50 mM OG at 1 mg/ml and then the solution was diluted to below the detergent’s critical micelle concentration. Cholesterol was dissolved in ethanol. The small amount of OG or ethanol did not interfere with the VSV binding assay. Saturable VSV binding was quantitated as in Figure 1. PI: phosphatidylinositol. PC: phosphatidylcholine. PG: phosphatidylglycerol. PE: phosphatidylethanolamine. PA: phosphatidic acid. CE: cerebroside. SP: sphingomyelin. PS: phosphatidylserine. GM,: GM, ganglioside. GD,: GD, ganglioside. CH: cholesterol. PIP/P: phosphatidylinositol monophosphate and diphosphate

cedures). Figure 3 shows that the serine phosphoglycerol molecule did not inhibit VSV binding. The inhibitory effect of PE was also reduced by methanolysis. Thus, despite retention of the serine head group, the serine phosphoglycerol was inactive in the VSV binding assay. Apparently both the hydrophobic fatty acid chains and the polar serine groups are necessary for the specific interaction with VSV. Phosphatidylcholine/Phosphatidylserine Liposomes Duplicate the Inhibitory Effects of Purified Phosphatidylserine To demonstrate that the inhibitory activity of PS is not due to a unqiue property of pure PS liposomes, we also used mixed phosphatidylcholine (PC) and PS liposomes. PC liposomes did not inhibit the specific binding of VSV to Vero cells (Table 4), consistent with ou previous data (Figure 2). However, PC/PS liposomes did compete for saturable VSV binding. PC/ PS liposomes at 5 PM showed the same ability to inhibit saturable VSV binding to confluent Vero monolayers as was previously observed for pure PS preparations at 5 IJM (-15% of control). These results illustrate that PC/PS liposomes inhibit VSV-cell binding and that the inhibition is dependent upon PS. To determine whether PS could directly interact with VSV, a liposome-VSV binding assay was utilized.

of Saturable

SP P! PM

!\

PE

0

0

\

0

Figure urable

I 5

10

15

PS I 20

I 25

3. Effect of PS and PE (pre and post VSV Binding

I 30

Methanolysis)

I 35

0 40

on Sat-

Several concentrations of PS and PE were tested for their ability to inhibit saturable VSV binding as described in Figure 2. VSV binding was completely inhibited by 10 pM PS, whereas PE maximally produced a 30% inhibition. When each of these lipids was subjected to methanolysis, the resultant serine and ethanolamine phosphoglycerols showed greatly reduced inhibitory activities,

Cdl 642

Table 4. Inhibition

of Saturable

VSV Binding

Saturable vsv (w)

by PC/PS

35S-VSV Binding

per Plate

Control W)

Control

6.1

100

10 gM PC liposomes

5.6

92

100 gM PC liposomes

4.6

75

5 pM PC/5 somes

0.92

15

0.19

3

50 pM PC/50 liposomes a Values

Liposomes

Binding

gM PS lipopM PS

represent

the average

of duplicate

samples.

PC/PS Liposomes Bind to VSV ‘?-PC liposomes with and without PS were used to demonstrate the direct interaction betweeen PS and VSV. Purified VSV (100 pg) was added to 50 pg of “C-PC/PS liposomes and incubated at room temperature for varying time periods. The liposome-VSV mixture was then layered over 2.0 ml of 50% glycerol and centrifuged at 100,000 x g for 45 min. Unbound liposomes did not pellet under these conditions, but VSV did. The amount of 14C in the viral pellet was measured in a liquid scintillation counter and indicates the amount of phospholipid bound to VSV. Figure 4 shows that the VSV-liposome interaction is nearly complete in 2 hr at room temperature. To investigate the dose dependence and PS specificity of this liposome-VSV interaction, increasing amounts of VSV were added to 100 pg of either 14CPC or “C-PC/PS liposomes. The mixtures were then incubated at room temperature for 3 hr, and the amount of VSV-associated liposomes determined as described above. Virion-associated 14C-PC was proportional to the amount of VSV added (Figure 5). One hundred micrograms of purified VSV bound 14,000 cpm “C-PC/PS liposomes, which represented 54% of the total amount of liposomes added. 14C-PC liposomes, on the other hand, showed a much reduced binding to the VSV particles (3000 cpm bound per 1.50 pg VSV). Thus PS was responsible for most of the binding of PC/PS liposomes to VSV. Liposome Density Is Altered after Binding to VSV Evidence for the direct interaction of PS and VSV was also obtained by using Percoll gradients to separate free liposomes from VSV-liposome complexes. VSV (100 pg) was added to “C-PC/PS liposomes (100 pg) and kept at room temperature for 3 hr. The mixtures were then applied to 40% Percoll gradients, centrifuged and analyzed as described in the Experimental Procedures. The density of free VSV was 1.050. The density of PC/PS liposomes was 1.030. After the incubation between VSV and PC/PS liposomes, approximately 70% of the liposomes shifted to an intermediate density of 1.044. The other 30% of liposomes

HOURS Figure

4. Time Dependence

of Liposome

Binding

to VSV

Purified VSV (100 pg) and 14C-PC/PS liposomes (50 pg) were incubated at 23°C for different time periods. Free and bound liposomes were separated by centrifugation through a 50% glycerol-PBS solution. Only liposomes bound to VSV were Delleted under these conditions (see Experimental Procedures)

P9 VW Figure 5. Dependence of Liposome-VSV Concentration and Phospholipid Content

Binding

upon

Liposome

14C-PC or “C-PC/PS liposomes (100 pg) were bound to increasing amounts of VSV at 23°C for 3 hr. Free and bound liposomes were separated according to the methods outlined in Figure 4. Liposomes containing PS exhibited a much greater degree of VSV binding.

remained at a density of 1.030. The altered liposome density probably reflects binding to VSV particles. Electron Microscopic Visualization of VSVLiposome Binding An amount of 100 pg of PC or PC/PS liposomes was mixed with VSV (100 pg viral protein) in 1 .O ml PBS at

K;

Binds to Phosphatidylserine

Table 5. Effect

of PS on VSV and HSV Plaque Formation

Virus

pfu x 10-S

vsv

98 f 5.7

VSV + 20 pM PS

21 f 4.0

HSV

119 + 20

HSV + 20 PM PS Values

represent

127 * 18 mean

-C SD.

5). Other experiments indicate that the degree of inhibition varies from 60% to 90%. Liposomes derived from total cell lipid (OG extracts) were also able to inhibit VSV plaque formation (data not shown). When this same procedure was applied to a Herpes simplex virus plaque assay, there was no evidence of an inhibitory effect. Thus the inhibition by PS appears specific for VSV. Discussion

Figure

6. Micrographs

of VSV-Liposome

Binding

Purified VSV (a) was mixed with either PC or PC/PS liposomes in PBS for 60 min at 23°C. The mixture was then negatively stained with phosphotungstic acid and examined by electron microscopy as described in the Experimental Procedures. VSV plus PC liposomes (b); VSV plus PC/PS liposomes (c, d). Bar: 100 nm.

22°C for 60 min. The VSV-liposome mixture was then applied to Formvar-coated grids, stained with phosphotungstic acid and examined by electron microscope as described in the Experimental Procedures. Figure 6 illustrates the morphology of VSV after its interaction with either PC or PC/PS liposomes. Although these studies are not quantitative, PC/PS liposomes appear to have a greater affinity for VSV than PC liposomes. Also, it is apparent that the VSV virions remain morphologically intact after binding to PC/PS liposomes. There was no evidence of liposome-virus membrane fusion under these ionic and pH conditions. PS Inhibits VSV but Not Herpes Simplex Virus Plaque Formation If inhibition of VSV binding by PS represents interference with a functional route of VSV uptake, one would predict that PS would also be able to inhibit VSV plaque formation. VSV stock was diluted (~10~) in Dulbecco’s medium containing 10% calf serum and then divided into two aliquots, one of which was supplemented with 20 PM PS. The VSV dilutions were then used in a plaque assay. PS (20 PM) inhibited VSV plaque formation by approximately 80% (Table

At low virus multiplicities of infection and at 4”C, VSV binds predominantly to a protease-resistant saturable site on the surface of Vero cells. By developing a VSV binding assay that is specific for this saturable interaction, we have detected in detergent extracts of Vero cells a phospholipid component which inhibits this specific host cell-VSV binding. PS was the most potent lipid tested and was capable of totally inhibiting this saturable site interaction. The finding that VSV bound to PS suggests that this lipid functions as a component of the cell binding site for VSV. It is generally believed that lipids have little role in the binding of animal viruses. Apparent exceptions to this generalization are seen in the role of gangliosides in Sendai virus infection (Markwell et al., 1981; Haywood, 1974) and of PE and cholesterol in the binding of Sindbis virus by liposomes (Mooney et al., 1975). Consequent to binding, VSV is internalized via an endocytic pathway morphologically identical with that of receptor-bound ligands (Dickson et al., 1981; Schlegel et al., 1982a). Many ligands that utilize this route of internalization have protein receptors (King and Cuatrecasas, 1981; Pastan and Willingham, 1982). In contrast, VSV apparently attaches to PS molecules. Both the serine head group and the hydrophobic fatty acid portions of PS are essential for its binding activity (Table 1, Figure 3). VSV presumably interacts with the hydrophilic, polar head region of PS, since it is that domain which faces the external environment. Deacylation of PS by methanolysis, however, produces a serine phosphoglycerol molecule that exhibits little or no interaction with VSV at micromolar concentrations. This could result from the removal of unique fatty acids characteristic of PS that might participate in the specific binding of VSV, or from the conversion of PS from a hydrophobic, multivalent, liposomal arrangement to a hydrophilic, uni-

Cell 644

valent, soluble form. The latter form would be expected to have a greatly reduced affinity for VSV. The predominant segregation of PS to the inner leaflet of plasma membranes may explain the limited number of binding sites (4000) observed on the external membrane surface of Vero cells. Using estimates that 3% of the total cell lipid is PS and that 80%-90% of plasma membrane PS faces the cytoplasmic compartment (Op den Kamp, 1979; Rothman and Lenard, 19771, it was calculated that the ratio of PS molecules to VSV particles at saturating concentrations of VSV would be approximately lo4 to 1 on the cell surface. It must be emphasized, however, that many of these cell surface PS molecules may be tightly associated with integral membrane proteins and, therefore, unavailable for interactions with VSV. It is not possible at present to determine whether the 4000 binding sites for VSV reflects the availability of a limited quantity of PS molecules. In addition, we coGId not study the stoichiometry of the VSV-PS interaction using PS/PC liposomes because we are uncertain of the proportion of PS in the outer leaflet of these liposomes. The specific binding of VSV to PS is probably mediated by the VSV G protein since trypinization of VSV results in a marked decrease in PS binding (Schlegel, unpublished results). Since the amino acid sequence of VSV G protein has been predicted from the cloned VSV genome (Rose and Gallione, 1981), it should be possible to define the domain of G protein that is responsible for the PS binding. It is interesting that there are several regions of G protein that exhibit a clustering of positively charged amino acids and that might facilitate an interaction with the negatively charged head groups of PS. Such an interaction, however, would not explain the specificity of G protein for PS. Other anionic phospholipids such as phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol monophosphate and diphosphate do not inhibit saturable VSV binding (Figure 2). Evidently the G protein-PS binding involves more than simple ionic interactions. One might expect that the specific association of multivalent particles (such as VSV) with PS would result in an increased amount of PS in the membrane region where VSV binds. Whether VSV actually promotes such PS sequestration or whether it binds to preexisting PS-rich domains is unknown. Either possibility, however, might be important to VSV infection. The internalization of VSV into host cells involves an early association with plasma membrane coated pits and subsequent appearance in receptosomes (Simpson et al., 1969; Dahlberg, 1974; Dickson et al., 1981; Matlin et al., 1982). Following the acidification of receptosomes (Tycko and Maxfield, 1982), VSV probably fuses with the vesicle membrane and releases its nucleocapsid into the cell cytoplasm. It is possible that PS has a role in promoting such mem-

brane fusion events (Portis et al., 1979) within acidic vesicles. Although an acidic environment may be an important factor for VSV infection, it is clear that VSV G protein can mediate membrane fusion even at neutral pH (Hughes et al., 1979). Our findings indicate that PS markedly increases the interaction of VSV with PC liposomes. A previous study has indicated that G protein aggregates can partition and apparently fuse with PC liposomes in the absence of PS (Petri and Wagner, 1980). However, the interaction between G protein aggregates and liposomes may be very different than that observed between intact viruses and liposomes. Despite the apparent role of PS in VSV binding and infection, it is also possible that PS constitutes only a portion of a larger protein-lipid complex which functions in VSV uptake. Definitive evidence for the role of PS in VSV infection will depend upon the experimental manipulation of available plasma membrane PS. There are indications that other enveloped viruses may also bind to plasma membrane phospholipids. For example, the treatment of host cells with phospholipase C inhibits infectivity by Semliki Forest virus (Friedman and Pastan, 1968). In addition, arboviruses appear to interact specifically with phosphatidylinositol and polyphosphoinsitides (Frisch-Niggemeyer, 1967, 1971). The broad host range of some of these viruses, as well as VSV, may relate to their binding to membrane lipids. Experimental

Procedures

Ceils Vero monkey cells were grown in Dulbecco-Vogt’s modified Eagle’s medium (DMEM) that was supplemented with 10% calf serum and 50 pg/ml gentamicin. Stock cultures were maintained in 150 cm* flasks and transferred to 35 mm Costar plastic culture dishes for virus binding studies. Virus Strain Indiana VSV was added to 850 cm2 roller bottle cultures of Vero cells at l-5 VSV pfu/cell. After overnight incubation at 37”C, culture supernatants were centrifuged at 10,000 x g for 10 min and the pellet was discarded. Supernatant fluids were then centrifuged on a 5.0 ml cushion of 50% glycerol at 55,000 x g for 90 min, and the pelleted virus was purified by sequential sucrose rate-velocity and density gradlents (Schlegel et al., 1981). Purified virus was dialyzed against Ca*+ -Mg’+ free Dulbecco’s PBS (Gibco), and stored at 4°C. 35S-methionine-labeled VSV was prepared by an identical purification procedure except that the Vero cells were changed to methionine-free Eagle’s minimal essential medium number 2 immediately after viral infection. Two hours later, the cultures were supplemented with 333 &i/ml ?S-methionine (Amersham; 954 mCi/ml; 1385 Ci/ mmole). Virus was stored as aliquots in Ca*+-Mg2+ free Dulbecco’s PBS at -70°C. Virus preparations were never refrozen. 35!Z-VSV Binding to Vero Monolayers and Detection of a Soluble Inhibitor The conditions for quantification of “S-VSV bound to saturable binding sites on the surface of Vero cells have been described (Schlegel et al., 1982b). Binding experiments were performed at 4°C in 35 mm plastic dishes containing 2-4 x 10’ cells per plate. One milliliter of Dulbecco’s medium containing 10% calf serum and buffered with

;z;

Binds to Phosphatidylserine

100 mM HEPES buffer was added to each plate. Then, 10 @I of purified ?S-VSV (-50 ng) was added, with or without various cell extracts or lipid preparations. The cultures were then kept at 4°C for 18 hr, after which they were rinsed gently three times with ice-cold PBS and solubilized in 1% Triton X-100. The entire sample was assayed for radioactivity. Duplicate samples varied by 5 10%. Under the stated conditions, 2 50% of bound 3*S-VSV was competable by excess unlabeled VSV. When a soluble “receptor-like’ activity was present during the binding conditions described above, ?S-VSV binding to the Vero cell monolayer was blocked. Binding studies have also been performed in the presence of 5% bovine serum albumin rather than 10% calf serum. In such experiments, total VSV binding is increased by 15%, although the proportions of saturable and nonsaturable binding remained the same. Solubilization of Binding Activity from Vero Cells VSV binding activity was solubilized from confluent monolayers of confluent Vero cells grown in 850 cm2 roller bottles. Roller bottles were rinsed three times with 4°C PBS, and the cells were extracted for 2 min on ice with 1.0 ml PBS containing 50 mM octyl-P-Dglucopyranoside (Cal Biochem). Extracts from ten roller bottles were then centrifuged at 100,000 X g for 60 min at 4’C. The supernatant was dialyzed against 2.5 x 1 O3 volume equivalents of PBS at 4°C for 24 hr and stored at 4°C or -70°C. Solubilization of Lipids Chloroform-methanol solutions of purified lipids (Supelco and Sigma) were dried under nitrogen gas at 4°C and solubilized in 50 mM octyl,&o-glucopyranoside (in PBS) at a concentration of 1 mg/ml. Lipid solutions were then dialyzed against 1 O3 volume equivalents of PBS and stored at 4°C. Small samples of these preparations were then used in ?+-VSV binding inhibition assays. Preparation of Liposomes Small, unilamellar liposomes were prepared by a modification of the cholate-dilution technique (Enoch and Strittmatter, 1979). Lipids were dissolved in 100 mM sodium cholate, 0.1 mM EDTA, at a concentration of 10 mM. The solutions were then dialyzed at room temperature against lo6 volume equivalents of PBS and stored at 4OC. When negative stained with 1% phosphotungstic acid and examined by electron microscopy, liposome diameter varied from 40 to 100 nm. Liposomes were composed of either PC or PC: PS (1 :I) and were radioactively labeled with 14C-PC (O.i5,&i/pg; New England Nuclear) to a specific activity of 2-3 X 1 O5 cpm/mg lipid. VW-Liposome Binding Assay Purified VSV (100 pg protein) and 100 pg “C-liposomes were suspended in 300 pl PBS and incubated at room temperature for different time periods. The mixture was then layered above 2.0 ml glycerol:PBS (1 :l) in a 10 ml Nalge polycarbonate tube and centrifuged in a type 40 fixed-angle centrifuge head in a Beckman ultracentrifuge. The tubes were centrifuged at 38,000 rpm for 45 min at 4°C. After the supernatant was completely aspirated, the viral pellet was solubilized in 1% Triton X-i 00 and the amount of bound “‘C-liposomes was determined in a liquid scintillation counter. When control preparations of ‘%-liposomes (no VSV) were centrifuged as described above, only l%-3% of the input radioactivity was found in the pellet fraction. Binding experiments were performed in duplicate and had a variability 5 10%. Percoll Gradient Separation of VSV-Phosphotidylserine Complexes 14C-liposomes were incubated with or without VSV at room temperature for 30 min according to the protocol given above. Approximately 70,000 cpm of either liposomes or liposomes and VSV were applied to the top of an 8 ml 40% Percoll (Pharmacia) solution which was made isotonic with sucrose. The Percoll gradient was then generated by centrifugation in a Beckman type 40 fixed-angle centrifuge head at 30,000 rpm for 20 min at 5°C. The gradient was collected in 0.3 ml fractions from the bottom of the tube and assayed for radioactivity

by liquid scintillation. In addition, applied to parallel Percoll gradients vsv.

samples to locate

of ?S-VSV the position

were also of unbound

Methanolysis PS and PE were subjected to methanolysis as previously described (Lester and Steiner, 1968). Methanol:toluene (1 :l) solutions of lipids (1 mg/ml) were mixed 1 :I with 0.2 N KOH in methanol and kept at O’C for 2 hr. The solutions were then neutralized with 1 N acetic acid and evaporated to dryness under NI at 4°C. The samples were then redissolved in 4 ml chloroform:methanol(2:1) and 1 ml distilled H20. After cooling to 4’C. the samples were centrifuged at 3000 x g for 10 min. Small aliquots of the aqueous phase were used in the VSV binding assay. Enzymes and Lipids Phospholipase C (B. cereus and C. perfringens) and neuraminidase were obtained from Calbiochem. Pronase E and trypsin were obtained from Sigma. PS, PE, PC and the GM, and GDI gangliosides were obtained from Supelco Laboratories. Phosphatidylinositol, phosphatidylinositol monophosphate and diphosphate, phosphatidylglycerol, cerebrosides, phosphatidic acid and sphingomyelin were obtained from Sigma. Enzyme Treatment of Vero Monolayers and OG Detergent Extract Vero cells were treated with proteases in the following manner. Confluent Vero monolayers were rinsed twice with 4°C DMEM. Then 1 .O ml DMEM was added to each 35 mm culture dish and the cultures were kept at 4°C for 10 min. Various concentrations of papain or pronase E (Sigma protease type XIV) were added, and the cultures were incubated for 20 min at 4°C. The monolayers were gently rinsed with cold DMEM containing 10% calf serum and 1% aprotinin and used in the VSV binding assay. Phospholipase C (8. cereus) was incubated with confluent monolayers for 30 min at 37°C in serum-free medium. The cultures were then washed four times with DMEM containing 10% calf serum, cooled to 4°C for 15 min, and used in the binding assay. Undiluted, dialyzed OG extract was incubated with various enzymes at 37°C for 30 min. The enzymes were then inactivated by heating to 100°C for 10 min, and the extract was diluted 1 :lOO for use in the binding assay. The inhibitory activity present in the OG extract was stable to 1 OO’C heating (Table 1). Enzyme-treated samples were compared with control, heated samples to estimate residual binding inhibitory activity. Preparative Thin-Layer Chromatography of Phospholipids Vero cells (5 g wet weight) were extracted with 10 ml 50 mM OG for 15 min at 0°C. The extract was then centrifuged at 100,000 X g for 60 min at 4°C and the supernatant was dialzyed against 100 ml PBS. The dialyzed extract (8 ml) was extracted with 90 ml chloroform:methanol(2:1) for 18 hr at 4“C. The chloroform-methanol solution was evaporated at room temperature under nitrogen gas. Half of the dried extract was solubilized with 500 ~1 chloroform:methanol (2:l) and applied to a preparative thin-layer chromatography plate impregnated with phosphor (Supelco). The lipids were chromatographed in chloroform:methanol:ammonium hydroxide (65:35:4) to separate phosphatidylserine and phosphatidylinositol from other phospholipids. Separated lipids were visualized with ultraviolet light and compared to lipid standards, and the phosphatidylserine and phosphatidylinositol regions were scraped from the plate. All other phospholipids were combined into a single fraction. The lipids were eluted from the silica gel with chloroform:methanol: water (1:2:0.8), dried at room temperature and resolubilized in 50 mM OG, and samples were tested for inhibitory activity. Electron Microscopy Purified VSV (100 pg viral protein) was added to 100 pg PC or PC/ PS liposomes in 1.0 ml PBS for 60 min at 23°C. Samples of the mixture (10 pl) were applied to glow-discharged, Formvar-coated grids for 1 min at 23°C and the excess fluid was removed with filter

Cell 646

paper. The grids were then stained with 2% phosphotungstic acid in 0.4% sucrose (pH 7.1). After 30 set, excess fluid was removed with filter paper and the grids were allowed to dry. Micrographs were taken with a Phillips 400 electron microscope at 60 KV. Acknowledgments We thank Drs. J. Hanover and R. Dickson for their suggestions and discussions during the course of this research, and Dr. M. Gottesman for critical reading of the manuscript. We also thank R. Coggin for the manuscript editing and text processing, and B. Lovelace and A. Harris for tissue culture assistance. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received

August

20, 1982;

revised

November

16, 1982

Dahlberg. J. (1974). Quantitative electron microscopic penetration of VSV into L cells. Virology 58, 250-262. Dickson, adsorbed mediated

analysisof

the

Enoch, H. and Strittmatter. P. (1979). Formation and properties of 1 OOO-A-diameter, single-bilayer phospholipid vesicles. Proc. Nat. Acad. Sci. USA 76, 145-l 49. as receptor Viral. 75, 119-

Frisch-Niggemeyer, W. (1967). Die chemische Struktur der bei der Adsorption von TBE-Virus on Erythrocyten wirksamen Receptosubstanz. Arch. Hyg. 151, 585-598. Friedman, R. and Pastan, I. (1968). in cells treated with phospholipase 1371-l 378.

Specific C. Proc.

inhibition of virus growth Nat. Acad. Sci. USA 59,

Haywood, A. (1974). Characteristics of Sendai model membrane. J. Mol. Biol. 83, 427-436.

virus receptors

in a

Helenius, A., Morein, B., Fries, E., Simon?,. K., Robinson, P., Schirrmacher, V., Terhorst, C. and Strominger, J. (1978). Human (HLA-A and HLA-B) and murine (H-2K and H-2d) histocompatibility antigens are cell surface receptors for Semliki Forest virus. Proc. Nat. Acad. Sci. USA 75, 3846-3850. Hennache, B. and Boulanger. P. (1977). Biochemical cell receptor for adenovirus. Biochem. J. 766, 237-247.

study

of KB-

Hughes, J., Dike, B., Thimmig, R.. Johnson, T., Rabinowitz, S. and Dal Canto, M. (1979). Neuroblastoma cell fusion by a temperaturesensitive mutant of vesicular stomatitis virus. J. Viral. 30, 883-890. Jackson, R., Segrest, J., Kahane, I. and Marchesi, V. (1973). Studies on the major sialoglycoprotein of the human red blood cell membrane. Isolation and characterization of tryptic glycopeptides. Biochemistry 72, 3131-3138. Kathan, R., Winzler, R. and Johnson, C. (1961). Preparation of an inhibitor of viral hemagglutination from human erythrocytes. J. Exp. Med. 173, 37-45. King, A. and Cuatrecasas, P. (1981). Peptide hormone-induced receptor mobility, aggregation, and internalization. New Eng. J. Med. 305, 77-88. Lester, R. and Steiner, M. (1968). The occurrence sitide and triphosphoinositide in Saccharomyces Chem. 243,4889-4893.

Matlin. K., Reggio, H., Helenius, A. and Simons, K. (1982). Pathway of vesicular stomatitis virus entry leading to infection. J. Mol. Biol. 756, 609-631. Mooney, J., Dalrymple, J., Alving. C. and Russell, P. (1975). Interaction of Sindbis virus with liposomal model membranes. J. Viral. 1.5, 225-231. Oldstone. M., Tishon, A., Dutko, F., Kennedy, S., Holland, J. and Lambert, P. (1980). Does the major histocompatibility complex serve as a specific receptor for Semliki Forest virus? J. Viral. 34, 256-265. Op den Kamp. J. (1979). Biochem. 48, 47-71.

Lipid asymmetry

in membranes.

Ann. Rev.

Pastan, I. and Friedman, R. (1968). Actinomycin D: inhibition of phospholipid synthesis in chick embryo cells. Science 7 9, 316-317. Pastan, I. and Willingham, M. (1982). Journey to the center cell: the role of the receptosome. Science 214, 504-509.

Ft.. Willingham. M. and Pastan, I. (1981). aup-Macroglobulin to colloidal gold: a new probe in the study of receptorendocytosis. J. Cell Biol. 89, 29-34.

Frisch-Niggemeyer. W. (1971). Polyphosphoinositides substances for certain groups of arboviruses. Acta 125.

Markwell, M., Svennerholm, L. and Paulson, J. (1981). Specific gangliosides function as host cell receptor for Sendai virus. Proc. Nat. Acad. Sci. USA 78, 5406-5410.

of diphosphoinocerevisiae. J. Biol.

Lonberg-Helm, K. (1981). Attachment of animal virus to cells: an introduction. In Receptors and Recognition, 8, Virus Receptors Part 2, K. Lonberg-Holm and L. Philipson, eds. (London: Chapman and Hall), pp. 3-20.

of the

Petri, W. and Wagner, R. (1980). Glycoprotein micelles isolated from vesicular stomatitis virus spontaneously partition into sonicated phosphatidylcholine vesicles. Virology 107, 543-547. Portis, A., Newton, C., Pangborn, W. and Paphadjopoulos, D. (1979). Studies on the mechanism of membrane fusion: evidence for an intermediate Ca++ -phospholipid complex, synergism with Mg++, and inhibition by spectrin. Biochemistry 18, 780-790. Rose, I. and Gallione, C. (1981). Nucleotide sequences of the mRNA’s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions. J. Viral. 39, 519-528. Rothman, J. and Lenard, 195, 743-752.

J. (1977).

Membrane

asymmetry.

Science

Schlegel, R.. Willingham. endocytosis of vesicular Commun. 102, 992-998.

M. and Pastan, I. (1981). Monensin blocks stomatitis virus. Biochem. Biophys. Res.

Schlegel, R.. Willingham, M. and Pastan, I. (1982a). Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of cr2-macroglobulin. Proc. Nat. Acad. Sci. USA 79, 2291-2295. Schlegel, R.. Willingham. M. and Pastan, I. (1982b). Saturable binding sites for vesicular stomatitis virus on the surface of Vero cells. J. Virol. 43, 871-875. Schloemer, R. and Wagner, R. (1975). Cellular adsorption function of the sialoglycoprotein of vesicular stomatitis virus and its neuraminic acid. J. Virol. 15, 882-893. Simpson, stomatitis

R.. Hauser, R. and Dales, S. (1969). Viropexis virus by L cells. Virology 37, 285-290.

of vesicular

Svensson, U., Persson, R. and Everitt, E. (1981). Virus-receptor interaction in the adenovirus system. I. Identification of virion attachment proteins of the HeLa cell plasma membrane. J. Viral. 38, 7081. Tycko, B. and Maxfield, F. R. (1982). vesicles containing a?-macroglobulin.

Rapid acidification Cell 28, 643-651.

of endocytic

Yefenof, E., Klein, G., Jondal. M. and Oldstone, M. (1976). Surface markers on human B and T-lymphocytes. IX. Two color immunofluorescence studies on the association between EBV receptors and complement receptors on the surface of lymphoid cell lines. Int. J. Cancer 17, 693-700.