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J. sen. Virol. (1987), 68, 2359-2369. Printed in Great Britain 2359 Key words: VSV/erythrocyte membrane/haemagglutination/haernolysis Characterization of Membrane Components of the Erythrocyte Involved in Vesicular Stomatitis Virus Attachment and Fusion at Acidic pH By P A O L A M A S T R O M A R I N O , l* C I N Z I A C O N T I , 1 P A O L A G O L D O N I , z B E R N A R D H A U T T E C O E U R 2 AND N I C O L A O R S I 1 l lstituto di Microbiologia, Facoltgt di Medicina, Universith 'La Sapienza', Piazzale Aldo Moro 5, 00185 Roma, Italy" and Zlnstitut Pasteur, Rue du Dr Roux, 75724 Paris Cedex 15, France (Accepted 1 June 1987) SUMMARY Goose erythrocyte membranes were isolated and tested for their ability to compete with red cell receptors for vesicular stomatitis virus (VSV) attachment and fusion at acidic pH. Crude membranes, solubilized with Triton X-100, Tween 80 and octyl-/~-Dglucopyranoside, showed a dose-dependent inhibitory effect on virus binding and haemolysis. The chemical nature of the active molecules was investigated by enzyme digestion and by separation of purified components. Only the lipid moiety, specifically phospholipid and glycolipid, was found to inhibit VSV attachment; a more detailed analysis of these molecules showed that phosphatidylinositol, phosphatidylserine and GM3 ganglioside were responsible for the inhibitory activity and could therefore represent VSV binding sites on goose erythrocyte membranes. Removal of negatively charged groups from these molecules by enzymic treatment significantly reduced their activity, suggesting that electrostatic interactions play an important role in the binding of VSV to the cell surface. Enzymic digestion of whole erythrocytes confirmed the involvement of membrane lipid molecules in the cell surface receptor for VSV. INTRODUCTION During the initial phase of infection, the interaction of viruses with their host cells leads to several events such as attachment, endocytosis and/or membrane fusion. Attachment to cells is believed to involve an interaction between viruses and specific components of the plasma membrane. Most enveloped viruses (Dales, 1973; Lonberg-Holm & Philipson, 1974) enter cells by adsorptive endocytosis and are subsequently delivered to intracellular vacuoles and lysosomes. The final and critical step in the penetration of the viral genome into the cytoplasm is a low pH-induced membrane fusion event between the viral and the lysosomal membranes. Viral haemagglutination (HA) is equivalent to the first step of infection (attachment) while haemolysis (He) is a useful model for viral fusion with plasma or lysosomal membranes. Vesicular stomatitis virus (VSV) binds to goose erythrocyte membrane producing HA, He and fusion (Mifune et al., 1982) at a mildly acidic pH. The interaction of VSV with the erythrocyte membrane therefore mimics what happens in the lysosomes where the low pH environment triggers the fusion. The He process seems to be mediated by a hydrophobic peptide segment of the G envelope glycoprotein of VSV (Schlegel & Wade, 1984), but the cellular counterpart involved in this phenomenon has not yet been characterized. For Vero cells supporting viral replication, Schlegel et al. (1983) demonstrated that the chloroform-methanol fraction of solubilized cell membranes inhibits the binding of VSV to saturable, high-affinity sites on the cell surface, and phosphatidylserine appeared to be the most potent lipid tested. Infectivity and HA of rhabdoviruses are inhibited by phospholipids and by the lipid component of low density lipoproteins, probably because of a chemical similarity between these compounds and the receptors for the virus in the cell membrane (Halonen et al., 1974; Seganti et al., 1983 ; Superti et al., 1984). 0000-7756 © 1987 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 07:30:43 2360 p. MASTROMARINO AND OTHERS W e h a v e s o l u b i l i z e d goose e r y t h r o c y t e m e m b r a n e s a n d s t u d i e d t h e i r i n h i b i t o r y a c t i v i t y o n e i t h e r V S V a t t a c h m e n t or its f u s i o n w i t h r e d b l o o d cells. I n a d d i t i o n , we i s o l a t e d p r o t e i n , lipid, phospholipid and ganglioside components of membranes and identified those showing receptor a c t i v i t y for V S V . T o a s c e r t a i n t h e role o f v a r i o u s c h e m i c a l g r o u p s , we a n a l y s e d t h e effect o f enzymic modification of whole erythrocytes, solubilized membranes and their components on t h e b i n d i n g a n d f u s i o n a c t i v i t i e s o f VSV. METHODS Virus. VSV, Indiana serotype, was grown in CER (chicken embryo-related) cells, pelleted at 80000g for 2 h and resuspended in bovine albumin borate saline buffer (BABS) pH 9.0. Haemagglutination. HA studies were carried out in 96-well (V-shaped bottom) microtitre plates. An equal volume of 1 ~o goose erythrocyte suspension in 0.2 M-phosphate saline buffer pH 5-0 was added to twofold serial dilutions of VSV in BABS. In HA inhibition tests twofold dilutions of inhibitor in BABS were mixed with an equal volume of virus containing 4 HA units (HAU). After 2 h incubation at 4 °C red blood cells were added, and the plates were kept at 4 °C over crushed ice for 3 h, before titres were recorded. Haemolysis. 0.5 ml of 1~ goose erythrocytes in phosphate buffer pH 5-0 was added to 0.5 ml of a virus dilution in BABS. The tubes were incubated for 30 rain at 4 °C, followed by 40 min at 23 °C. The samples were then centrifuged at 170 g for 10 rain at 4 °C, and the absorbance of the supernatant was measured at 540 nm. The results were expressed as ~ He compared with the maximal 100K obtained by treatment with 0-1 ~ NP40. One haemolytic unit (HeU) was the viral dilution giving 5 0 ~ He. In the He inhibition test 250 gl of twofold dilutions of inhibitor were mixed with 250 gl of virus containing 8 HeU/ml. Goose erythrocytes were added after 2 h incubation at 4 °C over crushed ice. The results were expressed as percent of the maximum He obtained without inhibitor. Enzyme treatment o f erythrocytes. Enzymic treatments were carried out using a 10~ suspension of goose erythrocytes washed three times in PBS. The cells were incubated at 37 °C for 1 h, in the presence or absence of various enzymes, solubilized in PBS. They were collected by centrifugation and washed three times with PBS before being used for virus HA and He studies. Only cells treated with phospholipase Az were incubated for 5 min at 23 °C with 100 mg/ml bovine serum albumin (BSA) to extract the reaction products (Haest et at., 1981). Isolation and solubilization o f goose erythroeyte membranes. Goose erythrocyte membranes were prepared by the method of Ginsberg et al. (1976). Red cells were lysed by hypotonic shock in a buffer containing 8-5 mM-Tris-HC1 pH 7.2, 3.0 mM-NaC1, 1.0 raM-glucose, 0"1 mg/ml BSA, 2 mM-MgC12. The haemoglobin-free pellet obtained after several washings was homogenized in a Dounce homogenizer (tight pestle). The membrane fraction obtained after sedimentation at 5000 g for 10 min was purified three times by sedimentation at 15000 g. The purified membrane fraction, containing only membrane vesicles when viewed by phase-contrast microscopy, was resuspended in PBS at a protein concentration of 4 mg/ml. Membrane solubilization was performed according to the method of VandenBerg et al. (1983) by the addition of 4.0 ~ octyl-/~-o-glucopyranoside, 3 ~ Triton X-100, 1~ Tween 80 in PBS, followed by sonication. The suspension was centrifuged at 100000 g for 1 h, and the supernatant was dialysed overnight at 4 °C against PBS. Residual detergent was then removed by treatment with Bio-Beads SM-2. The dialysed, adsorbed material was considered as the solubilized erythrocyte membrane (SEM). Solubitization o f membrane proteins. Proteins were extracted from isolated plasma membranes using n-butanol according to the method of Maddy (1966). Membranes (10 mg/ml protein) in hypotonic buffer containing 0.2 mMMgC12 were mixed with 0.75 vol. butanol and shaken thoroughly for 20 s. After incubation for 20 rain on ice, the samples were centrifuged at 2500 g for 10 rain, the lower aqueous phase was collected and extensively dialysed against PBS at 4 °C. Isolation o f membrane lipids. Total lipids were extracted from membranes with chloroform-methanol (C:M; 1 : 1.5 v/v) for 1 h at 4 °C, followed by extraction with C :M (2 : 1 v/v) for 5 min and C : M : H 2 0 (32:64:5 v/v/v) for 5 rain. The two last steps were repeated twice, the first time at 55 °C, and the second time at 37 °C. The extracts were combined and dried under nitrogen. Lipids were washed again with C : M (2 : 1 v/v), the supernatant was dried under nitrogen and resuspended in PBS by sonication. To obtain phospholipids the dried residues were dissolved in C : M (2 : 1 v/v) and partitioned by adding 0.2 vol. distilled water according to Folch et al. (1957). Under these conditions, phospholipids separated into the lower phase and were solubilized by sonication in PBS before use. . Isolation o f membrane glyeolipids. Glycolipids were prepared from goose erythrocyte ghosts obtained according to Hakomori & Watanabe (1976). The packed ghosts were extracted three times with 500 vol. C :M :H20 (4 : 8:3 v/v/v) (Svennerholm & Fredman, 1980). The extracts were combined and evaporated to dryness, dissolved in a small volume of C : M (1 : 1 v/v) and centrifuged. The gangliosides in the supernatant were isolated according to Ladish & Gillard (1985) and separated from neutral glycolipids by ion-exchange chromatography on D E A E Sephadex. Gangliosides were further purified by gel filtration on a column of Sephadex LH-20 equilibrated with C : M : H 2 0 (5:5:1 v/v/v) (Byrne et al., 1985). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 07:30:43 Erythrocyte receptor for V S V at acidic p H 2361 The ganglioside fraction was separated into two different classes by column chromatography on Silica gel 60 (Merck) using C : M : H20 (62 : 25 : 4 v/v/v) as the solvent system. High-performance thin-layer chromatography of purified goose red blood cell gangliosides was performed on precoated Silica gel 60 activated at 120 °C for 1 h, and developed in C : M : 5 M-NH4OH : 27 mM-CaC12.2H20 (60:40 :4 : 5 v/v/v/v). Gangliosideswere visualized with the resorcinol-HC1 reagent as purple bands. Enzymic treatments of solubilized membranesand isolated components. Trypsin and pronase were dissolved in 50 mM-Tris-HCl pH 8-2 and 7-4 respectively. Neuraminidase (from Clostridiumperfringens), phospholipases A2 and C were dissolved in PBS. Solubilized erythrocyte membranes, total lipids, phospholipids and gangliosides were incubated in the presence or absence of various enzymes at 37 °C for 1 h. The enzymes were then inactivated by heating the samples to 100 °C for 15 min. To remove from SEM the products of digestion, samples were washed in an Amicon Centricon-I 0 M icroconcentrator using PBS as exchange buffer. Membranes were washed by three cycles of 10 : 1 concentration and volume restoration using PBS. The original activity of untreated membrane was recovered at 100~ after this process. Chemicaldeterminations. Protein concentration was determined by the method of Lowry et al. (1951) using BSA as a standard. Lipid concentration was determined by the phosphovanillin procedure described by Frings et al. (1972) using olive oil as a standard. Sialic acid was determined by the thiobarbituric acid method (Aminoff, 1959); lipid-bound sialic acid was measured by the resorcinol method described by Svennerholm & Fredman (1980). Chemicals and enzymes. Octyl-fl-D-glucopyranosidewas purchased from Calbiochem. Triton X-100 and Tween 80 were obtained from Sigma. Neuraminidase (from Vibrio cholerae)was obtained from Behring. Neuraminidase (from C. perfringens), phospholipases A2 and C, pronase and trypsin were obtained from Sigma. Cholesterol, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, cerebrosides, sphingomyelin, diphosphatidylglycerol, phosphoserine and sialic acid were obtained from Sigma. Sphingomyelin was also purchased from Fluka and Koch-Light Laboratories. RESULTS Inhibiting activity o f S E M Crude membranes of goose erythrocytes were solubilized by using Triton X-100, Tween 80 and octyl-fl-D-glucopyranoside. The detergent mixture was chosen because it can be easily removed from the solubilized fraction by dialysis and adsorption to polystyrene beads, thus permitting further purification and isolation of putative binding sites. The presence of receptor activity for VSV in solubilized m e m b r a n e s was assessed by verifying their capacity to inhibit viral attachment and fusion at acidic pH, measured as H A and He inhibiting activity (Fig. 1). Solubilized m e m b r a n e s a p p e a r e d to be more active in inhibiting H A than He, but it must be noted that 256 H A U were necessary to produce 1009/oo He (data not shown); 4 H A U and 2 H e U of VSV correspond to 2.2 × 108 and 1.4 × 10 xa p.f.u, respectively. Plotting of ~ He against S E M concentration gave a slightly sigmoid curve with an almost linear section between 2 0 ~ and 8 0 ~ He. Complete inhibition of He activity was obtained with 66 ~tg/ml (as lipid) of SEM. In these experiments controls were included which consisted of goose erythrocytes incubated with m e m b r a n e s for 30 min at 4 °C, then for 40 min at 23 °C before the addition of virus. Controls gave results similar to untreated cells. Effect o f enzymic treatments on S E M inhibiting activity To study the chemical nature of the inhibitor, solubilized m e m b r a n e s were treated with several enzymes. Results are reported in Table 1. The inhibiting activity was resistant to denaturing conditions such as heating or freezing and thawing and to trypsin and pronase digestion. After phospholipase Az treatment S E M activity was enhanced; in contrast phospholipase C and neuraminidase digestion produced a reduction in the capacity of membranes to inhibit viral attachment and fusion. Inhibiting activity o f membrane components To characterize the role of different m e m b r a n e components protein, lipid, phospholipid and glycolipid were extracted from erythrocyte membranes and tested for their ability to compete with whole cells for virus binding (Table 2). M e m b r a n e proteins were able to inhibit virus attachment and fusion only at very high concentration. Total lipids, phospholipids and Rlvcolioids, on the other Downloaded hand, retained definite inhibitory activity.byRemoval of protein from from awww.microbiologyresearch.org IP: 88.99.165.207 On: Thu, 11 May 2017 07:30:43 P. MASTROMARINO AND OTHERS 2362 11 100 ~--,~ I I |-\ I I I I I I tl I I ? \ 50 \ @ HA I I i i\ \ 50 o 0, I 100 ! I I I I\e--t 0-5 1 2 4 8 17 33 66 133 SEM (~tg/ml lipid) + + . . . . . . . I l I I 0.5 2 8 31 ~\. \£~-;. 125 500 2000 Lipid components (/ag/ml) Fig. 1 Fig. 2 Fig. 1. Inhibition of VSV attachment and fusion by SEM. SEM were incubated with virus for 2 h at 4 °C to permit attachment to VSV envelope, but not fusion of SEM and viral envelope lipids. After addition of erythrocytes the temperature was maintained at 4 °C to allow adsorption of the virus to the cell membrane. HA was read 2 h later; He was determined after 40 rain at 23 °C as described in Methods. Fig. 2. Dose-dependent inhibition of VSV haemolysis by lipid components of goose erythrocyte membranes. VSV was incubated for 2 h at 4 °C with twofold dilutions of the total lipid (I), glycolipid (O) or phospholipid (A) fractions extracted from goose erythrocyte membranes. Haemolysis assay was performed as in Methods. T a b l e 1. Effect of enzyme treatments on inhibitory activity of solubilized erythrocyte membrane towards haemagglutination and haemolysis by VSV SEM SEM + 100 °C, 15 min + freeze-thaw + trypsin -t- pronase Enzyme concentration• 0 0 0 0.01 t 1.56 0.01 1.56 1 + phospholipase A2 + phospholipase C + neuraminidase (C. perfringens) + neuraminidase (V. cholerae) 50~ H A l t 1.56 1-56 1.56 1.56 0.1 1 5 0.1 1 5 1 10 0.01 0-1 0.25 0.5 1,56 1.56 0.57 0.39 1.56 2.82 3.12 0.78 2.64 1.56 1.56 2.34 3.12 ~ HeI 50 54 47 61 47 65 56 55 57 59 45 31 19 58 38 55 45 41 36 * Protease concentrations are in mg/ml. Phospholipase and neuraminidase concentrations are in units/ml. t Values reported represent lipid concentration ~g/ml) of SEM giving 50~ HA inhibition. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 07:30:43 Erythrocyte receptor for VSV at acidic pH 2363 Table 2. Inhibiting activity of solubilized erythrocyte membrane and isolated membrane components towards haemagglutinating and haemolytic activities of VSV* SEM lipid SEM protein Protein Total lipid Phospholipid Glycolipid 50~ HAI 1.6 1.1 400 7-8 31-2 15-6 50~ HeI 7.5 5.3 1600 15-6 280 110 * Values represent concentrations ~g/ml) giving 50~ haemagglutination (HA1) and haernolysis (HeI) inhibition. SEM caused a steady decrease in the activity of total lipids, by comparison with whole solubilized membranes. This reduction could have been a consequence of protein removal or of a structural rearrangement of lipids after the extraction procedure. Reconstitution experiments were made to ascertain whether it was possible to restore the effectiveness of whole solubilized membranes. For this purpose, extracted membrane proteins were mixed with total lipids, or with isolated phospholipids and glycolipids. Lipid and protein were mixed in the same ratio present in the whole solubilized membranes. In the first case a 50~o inhibition of binding was achieved with a mixture containing 5.4 p.g/ml of proteins and 7.8 I.tg/ml of lipids. The inhibition observed seemed due therefore only to the lipid moiety, because 7.8 p.g/ml of lipids alone give 50~o HA inhibition. When phospholipids and glycolipids were mixed at their respective highest non-inhibitory concentrations a 50 ~ inhibition was observed; the inhibitory activity of fractions was therefore additive. The addition of proteins did not modify the inhibitory potency of the mixture (results not shown). The dose-dependent inhibition of VSV He produced by different lipid components is recorded in Fig. 2 which shows that total lipids were more active than isolated phospholipid and glycolipid components. To evaluate further the role of different lipid molecules on VSV receptors on goose erythrocytes, a number of phospholipids and glycolipids were tested for their ability to inhibit HA and He by VSV. Gangliosides extracted from erythrocyte membrane were analysed by thinlayer chromatography (Fig. 3). This showed that GM3 (about 96~o of total lipid-bound Nacetylneuraminic acid, NeuNAc) was the major ganglioside in the membrane of goose erythrocytes, while only traces of components with Rv between G M 1 and G D 1a were detected. The latter were presumed to belong to the neo-lacto series of gangliosides, but we did not attempt to characterize them further. Data reported in Table 3 demonstrated that cholesterol, neutral phospholipids and neutral glycolipids were devoid of any inhibitory effect. Anionic phospholipids (phosphatidylinositol, phosphatidylserine, diphosphatidylglycerol) and gangliosides, on the other hand, inhibited viral adsorption on erythrocyte membranes and subsequent He. The inhibitory activity was due to direct action on the virus, because preincubation of erythrocytes with lipids did not affect their susceptibility to VSV-induced HA and He (data not shown). The half-maximal inhibitory concentration for HA and He is reported in Table 4. Phosphatidylinositol was the membrane component most active in the inhibition of VSV attachment and fusion; sphingomyelin showed inhibitory effects only at a relatively high concentration. These results were obtained with three different standard preparations of sphingomyelin. These were found to be free of contaminating phospholipids and fatty acids as verified by thin-layer chromatography. Palmitic and oleic acids inhibited the haemolytic activity of VSV, without any effect on the attachment capacity of the virus. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 07:30:43 2364 P. MASTROMARINO AND OTHERS 1 2 3 4 5 Fig. 3. Thin-layerchromatogram ofgangliosides. Goose erythrocyte gangliosides fractions (lanes 1 and 2) were isolated by Silica gel 60 column chromatography. Lane 1, NeuNAc GM3; lane 2, minor gangliosides (RF between GM1 and GDla); lane 3, NeuNAc GM3 from human liver; lane 4, NeuNGc GM3 from horse erythrocytes; lane 5, standard gangliosides (from top to bottom): NeuNAc GM3, NeuNGc GM3, GMI, GDla, GDIb and GTlb. T a b l e 3. Effect of individual membrane components on haemagglutinating and haemolytic activities of v s v Control virus Phosphatidylinositol Phosphatidylserine Sphingomyelin Phosphatidylethanolamine Phosphatidylcholine Diphosphatidylglycerol Cholesterol GM3 ganglioside Minor gangliosides Neutral glycolipids Galactocerebrosides type I Galactocerebrosides type II Cerebroside sulphate Phosphoserine N-Acetylneuraminic acid Palmitic acid Oleic acid HA* + + + - ~ He 100 3 10 100 97 97 2 + 99 + + + + + + + + 4 8 100 100 100 100 100 100 75 76 * +, No change in HA titre; - , HA inhibition. Enzymic modification of inhibiting molecules Substances s h o w i n g i n h i b i t o r y activity towards a t t a c h m e n t of V S V to erythrocyte m e m b r a n e s b e a r a n e g a t i v e electric charge. T o verify the role of electrostatic i n t e r a c t i o n in b i n d i n g o f VSV to the lipid c o m p o n e n t o f the erythrocyte m e m b r a n e lipids, p h o s p h o l i p i d s and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 07:30:43 Erythroeyte receptor for VSV at acidic pH 2365 Table 4. Inhibition of VSV-induced haemagglutination and haemotysis by purified lipids Concentration ~M) for 50% inhibition A t HA 1.1 20.1 513-7 5.3 6.3 17.4 > 3000 > 3000 Phosphatidylinositol Phosphatidylserine Sphingomyelin Diphosphatidylglycerol N-Acetylneuraminic GM3 Minor gangliosides Palmitic acid Oleic acid He 9 193.3 1340 21.4 25.4 139.6 1468 1773 Table 5. Effect of enzymes on the inhibitory activity of membrane components towards haemagglutination and haemolysis of VSV Lipids Lipids + I00 °C, 15 rain + phospholipase C + phospholipase A2 Enzyme concentration* 0 0 0-001 0-01 0.1 1 5 0-1 50~ HAIr 7.8 7-8 7-8 15.6 31.2 62.5 125 7.8 1 GM3 GM3+ 100°C, 15 min + neuraminidase Phosphatidylinositol Phosphatidylinositol + 100 °C, 15 rain + phospholipase C Phosphatidylserine Phosphatidylserine + 100 °C, 15 min + phospholipase C Proteins Proteins + 100 °C, 15 rain + trypsin 5 0 0 0.5 0 0 l 0 0 1 0 0 1 1-95 2.45 7.8 7.8 256 0.98 0.98 1-96 15.6 15.6 31-2 400 400 400 ~ HeI 50 50 50 37 21 9 5 55 70 74 50 51 7 41 45 21 43 47 30 50 52 53 * Trypsin concentration is in mg/ml. Phospholipase and neuraminidase concentrations are in units/ml. t Values represent concentration ~g/ml) giving 50~ HA inhibition. gangliosides were enzymically modified to remove anionic groups. After treatment of lipid, phosphatidylserine and phosphatidylinositol with phospholipase C and GM3 ganglioside with neuraminidase (Table 5) a significant reduction in their inhibitory activity towards binding and fusion of VSV to erythrocytes was observed. The effect was dose-dependent. However, the isolated anionic groups of these molecules, i.e. phosphoserine and sialic acid at 1 mM were ineffective in inhibiting HA and He (Table 3). Interestingly, phospholipase A2 digestion of whole m e m b r a n e lipids caused an e n h a n c e m e n t of inhibitory activity; this effect could have been due to the strong surface activity of lysoderivatives and fatty acids produced by the enzyme or to a rearrangement of the lipid molecules in a form more suitable for interaction with the virus. Heating or trypsin digestion of extracted m e m b r a n e proteins did not modify their inhibitory activity. Effect of enzyme treatment of goose erythrocytes on attachment and fusion of VSV The results reported in the preceding sections suggested a role for lipids, specifically gangliosides and phospholipids as part of the m e m b r a n e receptors for VSV. To confirm the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 07:30:43 2366 P. M A S T R O M A R I N O AND OTHERS Table 6. Effect of enzyme treatments of goose erythrocyte on the attachment and fusion of VSV Enzyme Phospholipase A2 Phospholipase C U/ml 0 2 10 50 125 250 0 0.4 1 Neuraminidase* Untreated~ + neuraminidase + phospholipase C + neuraminidase phospholipase C 2 0 0-25 0.4 0.8 0 0.25 1 0.25 1 HA titre 32 32 32 24 24 24 32 6 4 0 32 256 384 1024 32 256 6 ND~ ~ He 81 81 72 67 66 59 60 41 38 28 67 87 96 100 63 84 46 32 * Sialic acid released from membranes by action of V. cholerae neuraminidase was respectively 0.030, 0.035, 0.038 pmol. t Erythrocytes were digested with enzyme alone or with neuraminidase and successively with phospholipase C. The concentrations of enzymes used were the maximum that did not result in any haemolysis of erythrocytes upon combined treatment. :~ It was not possible to determine agglutination of erythrocytes by VSV, because removal of N-acetylneuraminic acid and polar head of phospholipids produced spontaneous agglutination of red cells. importance of these molecules in whole erythrocytes, cells were digested with neuraminidase and phospholipases and the sensitivity of modified erythrocytes to VSV attachment and fusion was studied. After phospholipase Az/albumin treatment of erythrocytes (Table 6) agglutination by VSV was unaffected; He was, on the contrary, significantly decreased. Phospholipase C digestion produced a marked reduction in the binding and fusion of VSV with the erythrocyte membrane, thus showing that polar heads of surface phospholipids played a fundamental role in the binding of the virus. The involvement of the membrane ganglioside GM3 as a VSV receptor and the crucial role of its sialic acid moiety for activity are recorded in Table 5. We therefore tested the effect of neuraminidase pretreatment of erythrocytes on subsequent VSV attachment and fusion. As shown in Table 6, binding and He were greatly increased after removal of sialic acid from the cell surface. This effect was dose-dependent. DISCUSSION Competition binding experiments showed that the lipid components of the goose erythrocyte membrane, notably phospholipids and glycolipids, were specifically recognized by VSV during the early phases of virus-cell interaction. Total lipids extracted from erythrocyte membranes were found to be more effective than isolated components in the inhibition of VSV binding to erythrocyte surface. However reconstitution experiments, carried out by mixing the fractions together, demonstrated that it was not possible to restore the effectiveness of whole solubilized membranes. It is probable that the steric arrangement of various components in solubilized membrane is more suitable for the interaction with the virus than that obtained in reconstituted membrane liposomes. Proteins did not appear to have a role as VSV receptors. While it is known that certain glycolipids associate tenaciously with membrane proteins or glycoproteins and partition into the aqueous phase on solvent extraction (Hakomori et al., 1972), we consider it unlikely that the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 07:30:43 Erythroeyte receptor for V S V at acidic p H 2367 weak inhibitory activity of membrane proteins was due to the protein itself, but rather to a putative contamination of the extract by traces of lipid. This is supported by the observation that the activity of the extracted proteins was unaffected after heating or trypsin digestion (Table 5). This result is in agreement with the previously reported effect of trypsin treatment of goose (Seganti et al., 1982) or human erythrocytes (Bailey et al., 1984), which produced an enhancement of VSV binding and He. The importance of lipid molecules in erythrocyte receptors f~r VSV is confirmed by the reduction of agglutination of red blood cells after phospholipase C digestion. Moreover, treatment of goose erythrocyte solubilized membranes with phospholipase C and neuraminidase rendered them less effective in the inhibition of VSV binding and fusion, suggesting a role of phospholipids and sialic acid-containing glycolipids in the inhibitory activity. Schlegel et al. (1983) reported a similar effect towards the inhibitory activity of saturable VSV binding after treatment of solubilized Vero cell membranes with phospholipase C. They did not notice any effect after neuraminidase digestion. Phosphatidylinositol, phosphatidylserine, diphosphatidylglycerol and gangliosides were able very efficiently to reduce VSV attachment and fusion, probably by competition with its natural binding sites on the cell surface. A common characteristic of these substances is their negative electric charge; it is therefore possible that inhibition is mediated by electrostatic interactions. The role of polar heads of inhibiting molecules is confirmed by results obtained after enzymic cleavage of anionic groups. Removal of NeuNAc from GM3 ganglioside and of phosphoserine and phosphoinositol from phospholipids greatly decreased their activity. Thus electrostatic interactions seem to play a fundamental role for the inhibiting activity towards VSV of purified membrane components. However sialic acid and phosphoserine were unable to affect attachment of virus to cell receptors, indicating that in the active molecules both polar heads and hydrophobic portions are essential for inhibiting activity. The inhibitory activity shown by diphosphatidylglycerol, an anionic phospholipid found in the inner mitochondrial membrane and not present on the erythrocyte surface, demonstrated that molecules bearing head groups with negative charges and hydrophobic lipid portions can act as VSV receptors at acidic pH. This suggests that binding of VSV to the cell membrane involves (i) an electrostatic interaction between positive amino acid sequences of viral G envelope glycoprotein and an anionic group on the cell membrane and (ii) a tight hydrophobic linkage between a lipophilic peptide segment of G glycoprotein and a lipid portion of the receptor on the membrane. Bailey et al. (1984) have suggested that electrostatic interactions can mediate VSV binding to the cell surface. It was shown that the poly-cation DEAE-dextran increased the binding of VSV to BHK-21 cells and erythrocytes of several species; He was equally enhanced. DEAE-dextran produces an increase in positive charge on the cell surface which results in a reduction of repulsive forces between virus and cell membrane. We observed a great enhancement of attachment and fusion of VSV to neuraminidase-treated erythrocytes. Reduction of surface negative charge by the enzyme facilitates electrostatic interaction between viral envelope and the anionic components of the cell membrane. Analysis of GM3 ganglioside extracted from neuraminidase-treated erythrocytes demonstrated that the enzyme was able to digest sialic acid from GM3. This result does not preclude the possibility that GM3 can be a receptor for VSV; in fact it can be postulated that neuraminidase treatment inactivates one natural receptor site for VSV, but facilitates the binding of the viral envelope with the high affinity binding sites containing anionic phosphatidylinositol and phosphatidylserine. Results obtained after combined treatment of erythrocytes with neuraminidase and phospholipase C seem to confirm this hypothesis. Indeed erythrocytes treated with the two enzymes were less sensitive to He by VSV than after the action of phospholipase C alone (Table 6). The only neutral lipid showing inhibiting activity was sphingomyelin, although only at high concentrations. This result is in agreement with the observation made by Superti et al. (1984) who reported that sphingomyelin was able to reduce the infectivity of VSV on CER ceils and that sphingomyelinase digestion rendered cells less sensitive to infection by the virus. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 07:30:43 2368 P. M A S T R O M A R I N O AND OTHERS Phosphatidylinositol, phosphatidylserine and GM3 appeared to represent major binding sites for VSV on erythrocyte membranes at acidic pH. Schlegel et al. (1983) have proposed a similar explanation for the role of phosphatidylserine at neutral pH in Vero cells. In this case, however, no activity of phosphatidylinositol was detected. Binding and fusion activity of VSV were not strictly correlated; HA was essential but not sufficient for fusion. In fact, phospholipase A2 treatment of erythrocytes produced a decrease in their sensitivity to He by VSV, although agglutination was unaffected (Table 6). This reduction, therefore, does not seem to be due to a cleavage of virus receptor from the erythrocyte surface but to a rearrangement of membrane lipids after removal of fatty acids and lysophosphatidylcholine upon the extraction with albumin; fusion of the viral envelope with erythrocyte membrane is thereby hindered. 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