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Inhibition of sendai virus-induced hemolysis by concanavalin A
VIROLOGY
86,
138-147
Inhibition
(1978)
of Sendai
Virus-Induced
S. TOYAMA,’ Department
of Serology
and ImmunoloRy
SUM1
Hemolysis
TOYAMA,
November
A
H. UETAKE
for Virus Research,
Institute Accepted
AND
by Concanavalin
Kyoto
University,
Kyoto,
Japan
29, 1977
Sendai virus-induced hemolysis was markedly inhibited when human erythrocytes (RBC) were incubated with Sendai virus in the presence of concanavahn A (Con A). Pretreatment of RBC or Sendai virion with Con A was also found to lead to the inhibition of hemolysis, but the inhibition in these two cases did not appear to he of sufficient magnitude to account for the inhibition of hemolysis which occurred when RBC were incubated with the virion in the presence of Con A. On the other hand, the concentration of Con A inhibiting the hemolyzing capacity of the RBC-virion complex was close to that observedunder conditions where Con A and the virion were added simultaneously to RBC. The effect of Con A was enhanced by treating the RBC-Con A complex or virion-Con A complex with anti-Con A antiserum, but the effect of Con A on the RBC-virion complex was slightly reversed by the addition of the antiserum. Divalent succinyl Con A had reduced ability to inhibit the hemolysis. The process of hemoglobin release was not affected by Con A. These results indicate that the inhibition is primarily the result of the crosslinking of Con A receptors in the RBC-virion complex. INTRODUCTION
Poste and Allison (1973) have suggested that clustering of membrane proteins is related to membrane fusion. This is supported by observations in several systems. The clustering of membrane glycoproteins during the process of Sendai virus-induced cell fusion has also been demonstrated by freeze-fracturing electron microscopy (B&hi and Howe, 1972; B&hi et al., 1973; Knutton, 1976). However, the direct experimental evidence for involvement of protein clustering in the virus-induced cell fusion has not been obtained. The use of plant lectins provides useful information on the behavior of surface glycoproteins in the cell fusion, since (1) lectins are capable of combining with specific carbohydrate determinants on the surface membrane of cells, and (2) lectins, by inducing crosslinking, might restrict the movement of proteins within the membrane (see Nicolson, 1974). Since it has been generally accepted that Sendai virus-induced hemolysis is a secondary result of the envelope fusion (Apostolov ‘Author addressed.
to whom
requests
for reprints
should
be
138 0042~6822/78/0861-0138$02.00/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
and Almeida, 1972; Apostolov and Waterson, 1975; Homma et al., 1976), hemolysis induced by Sendai virus can be used as a marker for investigating envelope fusion. Furthermore, the use of human erythrocytes has the advantage for investigating the molecular events and the structural rearrangement induced in the plasma membrane during the fusion process, because the properties of their surface membrane are known in more detail than most other mammalian cell surface membranes. Based on these considerations, the effect of Con A on Sendai virus-induced hemolysis has been examined to clarify the relationship between rearrangement of surface glycoproteins and the envelope fusion. The results indicate that Con A prevents envelope fusion by restricting the mobility of glycoproteins on the surface membrane of RBC and/or envelope of the Sendai virion. MATERIALS
AND
METHODS
Sendai virus, strain Z, and inlluenza virus, strain PR8, were grown and partial.ly purified as previously described (Toyama et al., 1977). lz51-labeled viruses were prepared according to the method of Virus.
CON
A INHIBITION
Stanley and Haslam (1971). Erythrocytes. Human erythrocytes from samples of blood group 0 were washed four times in Hepes-buffered saline (HBS, 20 mM Hepes, 107 n&f NaCl, 6.8 m&f KCl, pH 7.2). Lectins. Concanavalin A (Con A) was purchased as a 3x crystallized material from Miles-Yeda (Rehovot, Israel). Ricinus communis agglutinin, Phaseolus vulgar-is agglutinin, Mommordia charantia agglutinin, Pisum sativum agglutinin, and succinyl Con A were supplied by Dr. T. Osawa of Tokyo University. Iodination of Con A. Con A was labeled with 1251by a lactoperoxidase-catalyzed iodination in the presence of o-methyl-Dmannoside. Con A (approx 10 mg) was dissolved in PBS (2 ml) containing 5 mM glucose, 100 mM a-methyl-D-mannoside, 35 pg of lactoperoxidase (Sigma)/ml, 50 pg of oxidase (P-L Biochemicals, glucose Inc.)/ml, and 506 &i of Naiz51. After incubation at room temperature for 60 min, the reaction mixture was dialyzed extensively against repeated changes (each 3 liters) of 1.0 M NaCl at 4” for 5 days. Assay for hemolysis. The reaction mixture consisted of 0.5 ml of RBC suspension (5%, v/v), 0.1 ml of virus suspension, and 0.4 ml of HBS. The mixture was incubated at 0’ for 20 min to allow virus adsorption and then transferred to a 37’ water bath. After a 30-min incubation, the reaction was stopped by the addition of 3.0 ml of cold phosphate-buffered saline without Ca2+ and Mg2+ (PBS) of Dulbecco and Vogt (1954). The hemoglobin in the supernatant, after centrifugation at 500 g for 10 min, was estimated by its optical density at 540 nm. Assay for inhibition of hemolysis. The reaction mixture was prepared in a manner similar to that used for assay of hemolysis, except that the appropriate volume of HBS was replaced by solutions composed of the various additives. Hemolysis was measured as described above. The relationship between fraction of nonhemolysed cells and virus concentration is shown in Fig. 1. A linear correlation exists between the log of the fraction of nonhemolysed cells and the concentration of virus. This is in agreement with the results of
OF
139
HEMOLYSIS
L
1
I
,
I
1
I
20 30 40 50 60 HAU of Sendai virus FIG. 1. Dependence of the extent of hemolysis the concentration of Sendai virus. 10
on
Neurath et al. (1972) and suggests that a single particle is sufficient to lyse a RBC. Based on these results, the amount of virus used in this assay was selected to give 70 to 80% hemolysis. Usually this corresponded to about 20 HAU/ml. Con A binding studies. ‘251-labeled Con A was incubated with 108 RBC in 1.0 ml of PBS. After incubating at 0” for 30 min, the cells were centrifuged at 1,500 g for 5 min and washed two times with PBS. The final pellets were resuspended in 1.0 ml of PBS for the determination of bound radioactivity using a Nuclear-Chicago Model 1185 automatic gamma counter (Nuclear-Chicago Corp.). Blanks, for correction of nonspecific binding, contained 0.1 Ma-methylD-mannoside in the incubation mixture. Surface labeling of RBC. RBC (1.6 x l@ cells) were suspended in 2.0 ml of PBS and incubated for 60 min at 37” with 70 pg of lactoperoxidase, 100 pg of glucose oxidase, 5 mM glucose, and 500 $i of Na’251. The unbound isotope was removed by seven washes in PBS. Isolation of Con A- binding proteins. 1251labeled RBC or Sendai virions were lysed with 0.5% (v/v) NP40 for 10 min at room temperature. Debris was removed by centrifugation at 1,500 g for 10 min. Con A was added to the supernatant at a final concentration of 50 pg/ml. After incubation at 4’ for 30 min, the resulting complex was made to react with optimal amounts of anti-Con
140
TOYAMA.
TOYAMA,
AND
A rabbit serum for 30 min at 4”. Immune complexes were precipitated by a slight excess of goat anti-rabbit Ig during an 18-hr incubation period at 4”, and the precipitates were washed four times in PBS. After the last wash, the precipitates were dissolved by boiling for 3 min in 0.063 M Tris-HCl, pH 8.4, containing 2% SDS, 10% glycerol, 0.01 M 2-mercaptoethanol, and 0.001% bromophenol blue. Polyacrylamide gel electrophoresis. Electrophoresis in the presence of sodium dodecyl sulfate (SDS) was done according to Laemmli (1970) on 10% acrylamide slabs. After completion of the electrophoresis, the gels were stained with Coomassie brilliant blue and destained (Weber and Osborn, 1969). Autoradiography. Gels were dried under vacuum on Whatman 3MM chromatography paper, and the dried gels were exposed to Fuji Medical X-ray fihn. Preparation of anti-Con A serum. Rabbits were inoculated in each footpad with Con A (approximately 1 ml of total emulsion containing 3 mg of Con A) emulsified in an equal volume of complete Freund’s adjuvant (Difco, Detroit, Michigan). The second immunization was administered in the same manner 2 weeks later. The booster injection was given intravenously 2 weeks after the second injection. The animals
UETAKE
were bled 10 days after the booster injection. The sera were inactivated at 56” for 30 min and stored at -20’. RESULTS
Inhibition of Sendai virus-induced Hemolysis by Plant Lectins Various lectin preparations were tested for their inhibitory activity against Sendai virus-induced hemolysis. In this ‘assay, Sendai virus and lectins were mixed simultaneously with erythrocytes (RBC). The results shown in Fig. 2, where the effects of various lectins on hemolysis were shown to be a function of lectin concentration, indicate that although variations were seen between the relative efficacy of the lectins, hemolysis was inhibited by a variety of lectins irrespective of their binding specificities. Of the lectins tested, Con A was found to be the most effective in reducing hemolysis. The amount of Con A required to cause 50% inhibition of hemolysis was 1.2 pg/ml. Therefore, Con A was used for further analysis, although it was not clear whether the inhibition of hemolysis by various lectins resulted from a common mechanism. The inhibition of hemolysis by Con A could be reversed by the addition of (Ymethyl-D-mannoside (results not shown). There are three possible targets to which
60-
Micrograms
of
10 lectin
/ml
FIG. 2. Inhibition of Sendai virus-induced hemolysis by various lectins. Sendai virus and RBC were incubated in the presence of various concentrations of lectins at 0’ for 20 min. The mixture was then transferred to a 37” water bath. Hemolysis was measured 30 min after incubation. Concanavalin A (0); Ricks communis aggulutinin ((3); Phaseolus vulgaris aggulutinin (0); Momordia charantia aggulutinin (Q; and Pisum sativum aggulutinin (0).
CON
A INHIBITION
Con A molecules bind to inhibit hemolysk (1) the RBC, (2) the virion, and (3) the RBC-virion complex. To determine which target played a principal role for the inhibition of hemolysis, the following experiments were designed. Effect of Con A on RBC RBC were pretreated with various concentrations of Con A, and then their response to Sendai virus was examined. As shown in Fig. 3, the pretreatment of RBC with Con A reduced their ability to respond to hemolysis. However, the amount of Con A required to achieve 50% inhibition was more than 100 pg/ml. Thus, the inhibition of hemolysis by Con A under the conditions where RBC were incubated simultaneously with Con A and the virions may not be explained as a result of simple binding of Con A to RBC. Addition of anti-Con A antiserum produced additional inhibition of hemolysis in the Con A-treated cells over cells treated with Con A alone (Fig. 3). Effect of Con A on Free Virion Sendai virion was treated with various concentrations of Con A, and their ability
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HEMOLYSIS
to induce hemolysis was tested. Figure 4 shows that the treatment with Con A resulted in a decrease of hemolyzing activity of the virion. Con A at 17 pg/ml inhibited the hemolysis by 50%. At a lower dose range of Con A, the decrease in hemolyzing activity of the virion does not appear to be of sufficient magnitude to account for the inhibition of hemolysis which occurs when RBC are incubated simultaneously with Con A and the virion. The treatment of the virion-Con A complex with anti-Con A antiserum enhanced the effect of Con A, as found in the case where the RBC-Con A complex was treated with the antiserum (Fig. 4). Effect of Con A on the RBC-Virion Corn” plex To test the possibility that the primary target for the effect of Con A at a lower dose range was the RBC-virion complex, the effect of Con A on the RBC-virion complex was examined. As shown in Fig. 5, Con A could inhibit the hemolyzing capacity of the complex. The concentration of Con A to inhibit hemolysis by 50% was close to that observed under conditions where Con A and the virion were added lOOF--
I. ’
80 i
5 ‘Z 3 60P .s EaI 40-
.-5 1
P 60c .E
? 2
0. 1.0
10 Micrograms
100 of
Con
Alml
3. Effect of Con A on RBC. RBC were treated with various concentrations of Con A at 0” for 30 min. The cells were then washed twice with HBS and incubated with Sendai virus. After incubating for 20 min at O”, the reaction mixture was divided into two portions: One set was incubated at 37” for 30 min in the presence of lo-’ dilution of anti-Con A antiserum (0); the other set was incubated in the absence of the antiserum (0) FIG.
.q 1.0
.
. . . . . .(
1
10
100
Micrograms
of
Con
A /ml
FIG. 4. Effect of Con A on free virion. Various concentrations of Con A were incubated for 30 min with concentrated virus suspensions (usually 2500 HAU/ml) at 0’. After this incubation, aliquots were removed and diluted to preclude any carryover effect of Con A on the hemolysis. The ability of Con Atreated virion to induce hemolysis was tested in the presence of lo-’ dilution of anti-Con A antiserum (0) or in the absence of the antiserum (0).
142
TOYAMA,
TOYAMA,
simultaneously to RBC. The possibility that the observed inhibition resulted from the sum of the effect of Con A on RBC and on the virion seems unlikely. When RBC and the virion were pretreated separately with the same amount of Con A and interacted in the absence of Con A, the amount of Con A required to achieve 50% inhibition of hemolysis was more than 10 pg/ml. In contrast to the foregoing findings that the effect of Con A was enhanced by treating the RBC-Con A complex or virion-Con A complex with anti Con A antiserum (Figs. 3 and 4), the effect of Con A on the RBC-virion cmplex was slightly reversed by the addition of the antiserum (Fig. 5). The maximal level of inhibition by the antiserum was less than 30%. This inhibition may be explained by assuming that, at the time of the addition of antiserum, part of the Con A molecules bound to the complex have some free binding sites which are still available for the inhibition of hemolysis after transfer to 37”, and that these free binding sites of Con A molecules are blocked by the antibody. These results indicate that the inhibition of hemolysis is primarily the result of the binding of Con A to the RBC-virion complex, and that the mode of action of Con A 100 -
I
1
I-
AND UETAKE
on the RBC-virion complex is different from that on RBC or on virion. Effect of Succinyl Con A on Hemolysis Divalent succinyl Con A was used in experiments to establish the positive correlation of the valence of Con A with ability to inhibit hemolysis. The results are shown in Fig. 6. Although Sendai virus-induced hemolysis was inhibited when RBC were interacted with the virus in the presence of succinyl Con A, the amount of divalent succinyl Con A to inhibit hemolysis by 50% was sevenfold greater than that of tetravalent native Con A. In contrast to the effect of native Con A on the RBC-virion complex (Fig. 5), succinyl Con A produced only a small inhibition in the hemolysis of the RBC-virion complex (Fig. 6). Similarly, pretreatment of RBC or Sendai virion with succinyl Con A at 100 pg/ml produced only 10 and 50% inhibition of hemolysis, respectively (cf. Figs. 3 and 4). All of these experiments suggest that cross-linkage of Con A receptors plays a large role in the inhibition of hemolysis, and that the main effects do not result from preventing the interaction between surface glycoproteins of RBC and viral envelope proteins for steric reasons. Effect of Con A on the Release of Hemoglobin Klemperer (1960) has shown that NDV-
80-
I
. . ..I 1.0 Micrograms
I 10 of ConAlml
I 100
FIG. 5. Effect of Con A on the RBC-virion complex. After RBC were incubated with Sendai virus for 20 min at O”, various concentrations of Con A were added to the reaction mixtures. The mixtures were further incubated for 30 min. Hemolysis was measured 30 min after incubation at 37” in the presence of IO-” dilution of anti-Con A antiserum (@) or in the absence of the antiserum (0).
I
I
1.0 Micrograms
10 of succinyl
100 Con A I ml
FIG. 6. Effect of succinyl Con A on the hemolysis. Co-incubation of Sendai virus and RBC with succinyl Con A (0); incubation of the RBC-virion complex with succinyl Con A (0).
CON
A INHIBITION
induced hemolysis was inhibited by bovine serum albumin that prevented cell swelling but did not affect the initial action of the virus. Based on these findings, experiments were designed to test the possibility that Con A affected the process of hemoglobin release. As shown in Table 1, Sendai virusinduced hemolysis was inhibited when RBC-virion complexes were incubated at 37” in the presence of 30% bovine serum albumin, but the inhibition of hemolysis could be reversed if the complexes were subsequently resuspended in HBS buffer without albumin and incubated at 0”. This indicates that bovine serum albumin inhibits the release of hemoglobin without affecting envelope fusion. The release of TABLE
1
EFFECT OF CON A ON THE RELEASE HEMOGLOBINS Treatment First incubation at 37” Albumin + + + + + + +
Con A (pg/d) 0 0 0 100 0 0 0 0
OF Am nm
Second incubation at 0” Albumin
+ -
Con A k/ml) 0 0 0 100 50 10 5
0.720h 0.051’ 0.663” 0.010’ 0.710 0.708 0.680 0.738
a RBC (2.5%, v/v) were interacted with Sendai virus for 20 min at 0” in HBS-buffer containing 30% bovine serum albumin. The mixtures were then transferred to a 37” water bath and incubated for 30 min (first incubation). The cells were chilled to O’, received various concentrations of Con A, and incubated for 30 min. The Con A-treated cells were centrifuged, resuspended in cold HBS buffer, and incubated at 0” for 10 min (second incubation). The released hemoglobin was estimated as described under Materials and Methods. ‘RBC were interacted with Sendai virus in the absence of bovine serum albumin and hemolysis was assayed after the first incubation. * ‘After the first incubation, the cells were centrifuged and resuspended in HBS buffer containing 30% tiovine serum albumin. “After the first incubation, the cells were centrifuged and resuspended in HBS buffer without Con A treatment. ‘Before the fast incubation, the cells were interacted with Sendai virus in the presence of Con A.
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was unaffected when hemoglobin RBC-virion complexes which had been incubated at 37” in the presence of albumin were treated with Con A before transfer to HBS buffer and incubation at 0” (Table 1). These results indicate that Con A does not affect the process of hemoglobin release. Effect of Con A on Virus Adsorption
Since the treatment of the RBC-virion complex with Con A was found to inhibit hemolysis, the possibility that Con A might inhibit hemolysis by interfering with virus adsorption seemed unlikely. However, when RBC were interacted with Sendai virus in the presence of high concentrations of Con A, this possibility could not be excluded. To test this point, the effect of Con A on virus adsorption was examined. When RBC were incubated simultaneously with Con A and Sendai virion, the adsorption of the virus to RBC decreased with an increase in the amount of Con A (Fig. 7). In contrast, the adsorption of influenza virus to RBC was unaffected under these conditions (Fig. 7). Of the five lectin preparations which were used in the experiment shown in Fig. 2, only Con A was I
1.0
I
10 Micrograms
I
1
100 of Con A Iml
FIG. 7. Effect of Con A on virus adsorption. RBC (2.5%, v/v) were incubated with ““I-labeled Sendai virus (13 HALJ/ml) or ‘““I-labeled influenza virus (13 HALT/ml) in the presence of various concentrations of Con A. After incubating for 20 min at 0”, unadsorbed virus was removed by two washings at 4” with PBS. The final pellets were resuspended in 1 ml of PBS for the determination of bound radioactivity, using a Nuclear-Chicago model 1185 automatic gamma counter. Sendai virus (0); influenza virus (m).
144
TOYAMA,
TOYAMA,
found to be effective in reducing the virus adsorption (results not shown). These results indicate that Con A can inhibit Sendai virus adsorption at a higher dose range, and that this inhibition is specific both for Con A and for Sendai virus. To determine whether the observed inhibition of adsorption was due mainly to the binding of the Con A to RBC or to virion, RBC and Sendai virion were pretreated with Con A; then the ability of Con A-treated RBC to adsorb the virion and the ability of the virion to adsorb to RBC were tested in the absence of Con A. The results are shown in Table 2. The pretreatment of RBC with Con A failed to inhibit the adsorption of the virion. Prior treatment of the virions with Con A reduced their ability to adsorb to RBC, but the extent of inhibition was less than that observed when RBC were incubated with the virions in the presence of Con A. Thus, the binding of Con A to the virion, although important and even necessary for the inhibition, may not be the only factor determining the inhibition. Binding of Con A to RBC As described in the preceding section, the treatment of RBC with 100 pg/ml of Con A did not show any significant inhibition of Sendai virus adsorption. To test the possibility that most of the Con A receptor sites on RBC had not been occupied by Con A at that concentration, and thus no inhibition occurred, binding of Con A to RBC was studied quantitatively. The ability of ‘251-labeled Con A to bind to RBC is shown in Fig. 8. The same data TABLE
2
EFFECT OF CON A ON VIRUS Sample-
RBC”
Sendai
virus6
ADSORPTION
Concentration of Con A Wml)
Percentage inhibition
100 50 10
1.4 1.1 1.0
100 10
35.4 6.9
a Con A-treated RBC were prepared for Fig. 5. b Con A-treated Sendai virions were described for Fig. 6.
as described prepared
AND
UETAKE
plotted in a double-reciprocal fashion according to the method of Steck and Wallach (1965) indicate that RBC have approximately 1.1 X 105 binding sites. The apparent associated constant was calculated to be 3.5 x lo6 M-l. These values are in agreement with the ones reported previously (Schnebli and Bachi, 1975) but are in conflict with those reported by Phillips et al. (1974). Assuming 1.1 X lo5 Con A-binding sites per RBC, the percentage of the Con A receptors that would be occupied at a given concentration of Con A was calculated. At 100 pg/ml Con A, about 70% of the Con A receptors were occupied. Thus, the results of the Con A-binding experiments indicate that even if most of Con A receptors have been occupied, RBC retain their ability to adsorb Sendai virus. The results also show that at the concentration sufficient for complete inhibition of the hemolysis under the conditions where RBC are incubated simultaneously with the virion and Con A (5 pg/mh Con A occupies only about 15% of its receptors. Receptors for Con A on the Membrane of RBC and Sendai Virion To clarify the nature of receptor molecules for Con A on the membrane of RBC and Sendai virion, 1251-labeled proteins obtained from RBC and Sendai virion which were specifically precipitated with Con A and anti-Con A antibody were analyzed by SDS-polyacrylamide gel electrophoresis. As expected from previous reports (Findley, 1974; Tanner and Anstee, 19761, the results shown in Fig. 9 demonstrate that the principal high-affinity receptor for Con A on the surface membrane of RBC was the Band 3 glycoproteins (also called minor glycoproteins and component a). Two minor bands were also observed, but the nature of these bands was not clear. As also shown in Fig. 9, the two viral proteins precipitated by Con A were HN and F glycoproteins. Similar results were obtained when metabolically labeled virus was used instead of surface-labeled virus. DISCUSSION
as
When RBC are incubated with Sendai virus in the presence of Con A, the Sendai
CON
I
,
1
-0.04
-0.02
0.02
0.04
A INHIBITION
0.06
OF
HEMOLYSIS
145
0.08
1lCConAl
FIG. 8. Binding of Con A to RBC. The binding reactions were performed as described under Mater&&z and Methods. The data were plotted by the method of Steck and Wallack (1965). The inset shows a standard plot of the data.
virus-induced hemolysis is inhibited at low concentrations of Con A. Three possibilities may be considered to explain this inhibition, since Sendai virus-induced hemolysis is a secondary result of the envelope fusion (Apostolov and Almeida, 1972; Apostolov and Waterson, 1975; Homma et aZ., 1976): (1) Con A inhibits hemolysis by interfering with virus adsorption; (2) Con A inhibits the process of the envelope fusion; and (3) Con A inhibits the process of hemoglobin release. The present study strongly supports the second possibility. At high concentrations of Con A, the first possibility may play some role in the inhibition of hemolysis. The present data clearly show that the RBC-virion complex is the principal target for the inhibition of hemolysis by Con A, and that the mode of action of Con A on the RBC-virion complex appears to be different from that on RBC or on Sendai virion. The results also indicate that crosslinkage of Con A receptors plays a major role in the inhibition of the hemolysis, and that the main effects do not result from preventing the interaction between glycoproteins on the surface of RBC and viral envelope proteins for steric reasons. Although the exact mechanism by which Con A molecules bind to inhibit the hemolyzing capacity of the virion-RBC complex is not
FIG. 9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Con A-binding proteins from RBC and Sendai virion. The samples were subjected to electropboresis on slab gels and processed for autoradiography as described under Materials and Methods. The origin is at the top. (A) ‘““I-labeled RBC, (B) Con A-binding proteins from RBC, (C) ““I-labeled Sendai virion, (D) Con A-binding proteins from Sendai virion.
clear, the most probable explanation for the inhibition of hemolysis is that Con A crosslinks between glycoproteins on the surface membrane of RBC and envelope proteins of Sendai virion (HN and/or F glycoproteins). Subsequently, lateral mobility of glycoproteins on the surface of RBC and viral envelope proteins is restricted to such a degree that they cannot be redistributed to initiate envelope fusion. This notion can account for the effective inhibition of he-
146
TOYAMA,
TOYAMA,
molysis by Con A at a relatively low dose, since binding of Con A between glycoproteins on the surface of RBC and viral envelope proteins may easily form a threedimensional lattice, which in turn gives rise to restriction of the mobility of both these glycoproteins. This possibility is being investigated. Pretreatment of RBC with high concentrations of Con A is also found to inhibit hemolysis. The fact that the valence of Con A is closely correlated with ability to inhibit hemolysis and that the treatment of Con A-pretreated RBC with anti-Con A antiserum produces additional inhibition of hemolysis indicates that cross-linking of glycoproteins on RBC leads to the inhibition of hemolysis. In other words, mobility of glycoproteins in the plane of membrane is required for the initiation of envelope fusion. Con A receptors are exclusively associated with the membrane-intercalated particles (Pinto da Silva and Nicolson, 1974). Because the major Con A receptor at the surface membrane of RBC is contained in a Band 3 glycoprotein (Findley, 1974; Tanner and Anstee, 1976; and the present data), it follows that a Band 3 glycoprotein is a part of the intercalated particles. Bachi et al. (1973) have reported that the clustering of the intercalated particles following penetration of Sendai virus is demonstrated by freeze-fracturing electron microscopy. Although no gross changes in the distribution of intramembrane particles has occurred during the envelope fusion (Knutton, 1976), it is quite possible that the topological rearrangement of intercalating particles may be intimately related to the initiation of envelope fusion. In support of this view, our finding that, although Con A molecules bound to RBC or Sendai virion appear to have free binding sites, the bound Con A molecules do not act to inhibit hemolysis in a manner similar to the way they operate in the RBC-virion complex, suggests that some topological rearrangement of surface glycoproteins occurs when virus particles are bound to RBC. In addition, Band 3 glycoproteins, which have been isolated by affinity chromatography on a Con A column, are potent inhibitors of Sendai virus
AND UETAKE
adsorption (Toyama et al., unpublished results). The logical consequence of these considerations is that the redistribution of intercalating particles which contain Band 3 glycoproteins plays an essential role for the initiation of envelope fusion. However, the possibility that minor Con A-binding glycoproteins on the surface of RBC may play a role for the envelope fusion cannot be excluded. The ability to induce hemolysis of Sendai virus is inactivated after treatment with high concentrations of Con A. Two mechanisms seem to be involved in this inhibition. The first is that the failure of Con Atreated virion to cause hemolysis is a secondary result of their reduced ability to adsorb to RBC. However, the magnitude of the inhibition is insufficient to explain all of the reduced ability of the virion to induce hemolysis. The second is that Con A treatment results in crosslinking between envelope proteins of the virion, and thereafter, that the crosslinking leads to the inhibition of hemolysis by preventing topological redistribution of envelope proteins. This is supported by the finding that divalent succinyl Con A molecules are much less effective in inhibiting the hemolyzing activity of the virion than tetrameric Con A, and that the treatment of Con A-treated virion with anti-Con A antiserum produces additional inhibition of hemolysis. In addition, the possibility that aggregation of the virions by Con A plays a major role in reducing their hemolyzing activity seems unlikely: About 50% of hemolyzing activity is inactivated when Sendai virions are pretreated with 20 pg/ml of Con A, whereas hemagglutinating activity of the virions is fully retained under these conditions. All of these results discussed above provide strong evidence that crosslinkage of Con A receptors plays a 1arg.e role in the inhibition of the envelope fusion. The present data complement the morphological finding of Knutton (1976), who showed that changes in the structure of the viral envelope prior to fusion involve a reorganization of membrane components to produce the particle-denuded linear E-face ridges. Thus, it may be concluded that the rearrangement of Con A receptors on the surface of
CON
A INHIBITION
RBC and on the envelope of the virion is a prerequisite for the initiation of the envelope fusion. The treatment of Sendai virions with high concentrations of Con A is found to reduce their ability to adsorb to RBC. This is in conflict with the result reported by Okada and Kim (1972). The discrepancy in the conclusion may be explained by the difference in the assay system used in the two studies. When the ability of Con A-treated Sendai virion to adsorb to RBC is tested in the absence of Con A, the extent of the inhibition of adsorption is less than that assayed under the conditions where RBC and virion are interacted in the presence of Con A. This may be interpreted as follows: When free Con A molecules are present in the reaction mixture, they bind both to Band 3 glycoproteins and to viral envelope proteins. The bound Con A molecules may hinder sterically and/or electrostatically the close contact between viral envelope proteins and their receptor proteins on the surface of RBC. ACKNOWLEDGMENTS The authors thank Dr. Osawa for generous gifts of various lectin preparations. This investigation was supported in part by a grant from the Ministry of Education, Science and Culture, Japan. REFERENCES APOSTOLOV, K., and ALMEIDA, J. D. (1972). Interaction of Sendai (HVJ) virus with human erythrocytes: A morphological study of haemolysis cell fusion. J. Gen. Virol. 15,227-234. APOSTOLOV, K., and WATERSON, A. P. (1975). Studies on the mechanism of haemolysis by paramyxoviruses. In “Negative Strand Viruses,” (B. W. J. Mahy and R. D. Barry, eds.), Vol. 2, pp. 7994322. Academic Press, New Yrok. B&HI, T., AMJET,M., and HOWE, C. (1973). Fusion of erythrocytes by Sendai virus studied by immunofreeze-etching. J. Viral. 11, 1004-1012. BACHI, T., and HOWE, C. (1972). Fusion of erythrocytes by Sendai virus studied by electron microscopy. Proc. Sot. Exp. Biol. Med. 141, 141-149. DULRECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med. 99, 167-182. FINDLAY, J. B. C. (1974). The receptor proteins for concanavalin A and Lens culnaris phytohemagglutinin in the membrane of the human erythrocytes.
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147
J. Biol. Chem. 249.4398-4403. HOMMA, M., SHIMIZU, K., SHIMIZU, Y. K., and ISHIDA, N. (1976). On the study of Sendai virus hemolysis: I, Complete Sendai virus lacking in hemolytic activity. Virology 71.41-47. KLEMPERER, H. G. (1960). Hemolysis and the release of potassium from cells by Newcastle disease virus (NDV). Virology 12, 540-552. KNIJTTON, S. (1976). Changes in viral envelope structure preceding infection. Nature (London) 264. 672-673. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T+ Nature (London) 227,680-685. NEUHATH, A. R., VERNON, S. K., HARTZELL, R. W., and RUBIN, B. A. (1972). Haemolysis by Sendai virus: Lack of requirement for neuraminidase. J. Gen. Viral. 16, 245-249. NICOLSON, G. L. (1974). The interaction of lectins with animal cell surfaces. Znt. Rev. Cytol. 39,89-190. OKADA, Y., and KIM, J. (1972). Interaction of concanavalin A with enveloped viruses and host cells. Vi-
rology 50,507-515. PHILLIPS, P. G., FURMANSKI, P., and LIJBIN, M. (1974). Cell surface interactions with concanavalin A: Location of bound radiolabeled lectin. Expt. Cell
Res. 86,301-308. PINTO DA SII.VA, P., and NICOISON, G. L. (1974). Freeze-etch localization of concanavalin A receptors to the membrane intercalated particles of human erythrocyte ghost membranes. Biochim. Bioph”vs.
Acta 363,311-319. POSTE, G., and ALLISON, A. C. (1973). Membrane fusion. Biochim. Biophys. Acta 300,421-465. SCHNERLI, H. P., and BACHI, T. (1975). Reactions of lectins with human erythrocytes. I. Factors govering the agglutination reaction. Expt. Cell Res. 91, 175-183. STANLEY, P., and HASLAM, E. A. (1971). The polypeptides of influenza virus: V, Localization of polypeptides in the virion by iodination techniques. Virology 46, 764-773. STECK, T. L., and WALLACH, D. F. H. (1965). The binding of kidney-bean phytohemagglutinin by Ehrlich ascites carcinoma. B&him. Biophys. Acta 97, 510-522. TANNER, M. J. A., and ANSTEE, D. J. (1976). A method for the demonstration of the lectin-binding components of the human erythrocyte membrane. Biochem. J. 153,265-270. TOYAMA, S., TOYAMA, S~J., and UETAKE, H. (1977). Altered cell-fusion capacity in lines of KB cells resistant to Sendai virus-induced cytolysis. Virology 76, 503-515. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol.
Chem. 244,4406-4412.
86,
138-147
Inhibition
(1978)
of Sendai
Virus-Induced
S. TOYAMA,’ Department
of Serology
and ImmunoloRy
SUM1
Hemolysis
TOYAMA,
November
A
H. UETAKE
for Virus Research,
Institute Accepted
AND
by Concanavalin
Kyoto
University,
Kyoto,
Japan
29, 1977
Sendai virus-induced hemolysis was markedly inhibited when human erythrocytes (RBC) were incubated with Sendai virus in the presence of concanavahn A (Con A). Pretreatment of RBC or Sendai virion with Con A was also found to lead to the inhibition of hemolysis, but the inhibition in these two cases did not appear to he of sufficient magnitude to account for the inhibition of hemolysis which occurred when RBC were incubated with the virion in the presence of Con A. On the other hand, the concentration of Con A inhibiting the hemolyzing capacity of the RBC-virion complex was close to that observedunder conditions where Con A and the virion were added simultaneously to RBC. The effect of Con A was enhanced by treating the RBC-Con A complex or virion-Con A complex with anti-Con A antiserum, but the effect of Con A on the RBC-virion complex was slightly reversed by the addition of the antiserum. Divalent succinyl Con A had reduced ability to inhibit the hemolysis. The process of hemoglobin release was not affected by Con A. These results indicate that the inhibition is primarily the result of the crosslinking of Con A receptors in the RBC-virion complex. INTRODUCTION
Poste and Allison (1973) have suggested that clustering of membrane proteins is related to membrane fusion. This is supported by observations in several systems. The clustering of membrane glycoproteins during the process of Sendai virus-induced cell fusion has also been demonstrated by freeze-fracturing electron microscopy (B&hi and Howe, 1972; B&hi et al., 1973; Knutton, 1976). However, the direct experimental evidence for involvement of protein clustering in the virus-induced cell fusion has not been obtained. The use of plant lectins provides useful information on the behavior of surface glycoproteins in the cell fusion, since (1) lectins are capable of combining with specific carbohydrate determinants on the surface membrane of cells, and (2) lectins, by inducing crosslinking, might restrict the movement of proteins within the membrane (see Nicolson, 1974). Since it has been generally accepted that Sendai virus-induced hemolysis is a secondary result of the envelope fusion (Apostolov ‘Author addressed.
to whom
requests
for reprints
should
be
138 0042~6822/78/0861-0138$02.00/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
and Almeida, 1972; Apostolov and Waterson, 1975; Homma et al., 1976), hemolysis induced by Sendai virus can be used as a marker for investigating envelope fusion. Furthermore, the use of human erythrocytes has the advantage for investigating the molecular events and the structural rearrangement induced in the plasma membrane during the fusion process, because the properties of their surface membrane are known in more detail than most other mammalian cell surface membranes. Based on these considerations, the effect of Con A on Sendai virus-induced hemolysis has been examined to clarify the relationship between rearrangement of surface glycoproteins and the envelope fusion. The results indicate that Con A prevents envelope fusion by restricting the mobility of glycoproteins on the surface membrane of RBC and/or envelope of the Sendai virion. MATERIALS
AND
METHODS
Sendai virus, strain Z, and inlluenza virus, strain PR8, were grown and partial.ly purified as previously described (Toyama et al., 1977). lz51-labeled viruses were prepared according to the method of Virus.
CON
A INHIBITION
Stanley and Haslam (1971). Erythrocytes. Human erythrocytes from samples of blood group 0 were washed four times in Hepes-buffered saline (HBS, 20 mM Hepes, 107 n&f NaCl, 6.8 m&f KCl, pH 7.2). Lectins. Concanavalin A (Con A) was purchased as a 3x crystallized material from Miles-Yeda (Rehovot, Israel). Ricinus communis agglutinin, Phaseolus vulgar-is agglutinin, Mommordia charantia agglutinin, Pisum sativum agglutinin, and succinyl Con A were supplied by Dr. T. Osawa of Tokyo University. Iodination of Con A. Con A was labeled with 1251by a lactoperoxidase-catalyzed iodination in the presence of o-methyl-Dmannoside. Con A (approx 10 mg) was dissolved in PBS (2 ml) containing 5 mM glucose, 100 mM a-methyl-D-mannoside, 35 pg of lactoperoxidase (Sigma)/ml, 50 pg of oxidase (P-L Biochemicals, glucose Inc.)/ml, and 506 &i of Naiz51. After incubation at room temperature for 60 min, the reaction mixture was dialyzed extensively against repeated changes (each 3 liters) of 1.0 M NaCl at 4” for 5 days. Assay for hemolysis. The reaction mixture consisted of 0.5 ml of RBC suspension (5%, v/v), 0.1 ml of virus suspension, and 0.4 ml of HBS. The mixture was incubated at 0’ for 20 min to allow virus adsorption and then transferred to a 37’ water bath. After a 30-min incubation, the reaction was stopped by the addition of 3.0 ml of cold phosphate-buffered saline without Ca2+ and Mg2+ (PBS) of Dulbecco and Vogt (1954). The hemoglobin in the supernatant, after centrifugation at 500 g for 10 min, was estimated by its optical density at 540 nm. Assay for inhibition of hemolysis. The reaction mixture was prepared in a manner similar to that used for assay of hemolysis, except that the appropriate volume of HBS was replaced by solutions composed of the various additives. Hemolysis was measured as described above. The relationship between fraction of nonhemolysed cells and virus concentration is shown in Fig. 1. A linear correlation exists between the log of the fraction of nonhemolysed cells and the concentration of virus. This is in agreement with the results of
OF
139
HEMOLYSIS
L
1
I
,
I
1
I
20 30 40 50 60 HAU of Sendai virus FIG. 1. Dependence of the extent of hemolysis the concentration of Sendai virus. 10
on
Neurath et al. (1972) and suggests that a single particle is sufficient to lyse a RBC. Based on these results, the amount of virus used in this assay was selected to give 70 to 80% hemolysis. Usually this corresponded to about 20 HAU/ml. Con A binding studies. ‘251-labeled Con A was incubated with 108 RBC in 1.0 ml of PBS. After incubating at 0” for 30 min, the cells were centrifuged at 1,500 g for 5 min and washed two times with PBS. The final pellets were resuspended in 1.0 ml of PBS for the determination of bound radioactivity using a Nuclear-Chicago Model 1185 automatic gamma counter (Nuclear-Chicago Corp.). Blanks, for correction of nonspecific binding, contained 0.1 Ma-methylD-mannoside in the incubation mixture. Surface labeling of RBC. RBC (1.6 x l@ cells) were suspended in 2.0 ml of PBS and incubated for 60 min at 37” with 70 pg of lactoperoxidase, 100 pg of glucose oxidase, 5 mM glucose, and 500 $i of Na’251. The unbound isotope was removed by seven washes in PBS. Isolation of Con A- binding proteins. 1251labeled RBC or Sendai virions were lysed with 0.5% (v/v) NP40 for 10 min at room temperature. Debris was removed by centrifugation at 1,500 g for 10 min. Con A was added to the supernatant at a final concentration of 50 pg/ml. After incubation at 4’ for 30 min, the resulting complex was made to react with optimal amounts of anti-Con
140
TOYAMA.
TOYAMA,
AND
A rabbit serum for 30 min at 4”. Immune complexes were precipitated by a slight excess of goat anti-rabbit Ig during an 18-hr incubation period at 4”, and the precipitates were washed four times in PBS. After the last wash, the precipitates were dissolved by boiling for 3 min in 0.063 M Tris-HCl, pH 8.4, containing 2% SDS, 10% glycerol, 0.01 M 2-mercaptoethanol, and 0.001% bromophenol blue. Polyacrylamide gel electrophoresis. Electrophoresis in the presence of sodium dodecyl sulfate (SDS) was done according to Laemmli (1970) on 10% acrylamide slabs. After completion of the electrophoresis, the gels were stained with Coomassie brilliant blue and destained (Weber and Osborn, 1969). Autoradiography. Gels were dried under vacuum on Whatman 3MM chromatography paper, and the dried gels were exposed to Fuji Medical X-ray fihn. Preparation of anti-Con A serum. Rabbits were inoculated in each footpad with Con A (approximately 1 ml of total emulsion containing 3 mg of Con A) emulsified in an equal volume of complete Freund’s adjuvant (Difco, Detroit, Michigan). The second immunization was administered in the same manner 2 weeks later. The booster injection was given intravenously 2 weeks after the second injection. The animals
UETAKE
were bled 10 days after the booster injection. The sera were inactivated at 56” for 30 min and stored at -20’. RESULTS
Inhibition of Sendai virus-induced Hemolysis by Plant Lectins Various lectin preparations were tested for their inhibitory activity against Sendai virus-induced hemolysis. In this ‘assay, Sendai virus and lectins were mixed simultaneously with erythrocytes (RBC). The results shown in Fig. 2, where the effects of various lectins on hemolysis were shown to be a function of lectin concentration, indicate that although variations were seen between the relative efficacy of the lectins, hemolysis was inhibited by a variety of lectins irrespective of their binding specificities. Of the lectins tested, Con A was found to be the most effective in reducing hemolysis. The amount of Con A required to cause 50% inhibition of hemolysis was 1.2 pg/ml. Therefore, Con A was used for further analysis, although it was not clear whether the inhibition of hemolysis by various lectins resulted from a common mechanism. The inhibition of hemolysis by Con A could be reversed by the addition of (Ymethyl-D-mannoside (results not shown). There are three possible targets to which
60-
Micrograms
of
10 lectin
/ml
FIG. 2. Inhibition of Sendai virus-induced hemolysis by various lectins. Sendai virus and RBC were incubated in the presence of various concentrations of lectins at 0’ for 20 min. The mixture was then transferred to a 37” water bath. Hemolysis was measured 30 min after incubation. Concanavalin A (0); Ricks communis aggulutinin ((3); Phaseolus vulgaris aggulutinin (0); Momordia charantia aggulutinin (Q; and Pisum sativum aggulutinin (0).
CON
A INHIBITION
Con A molecules bind to inhibit hemolysk (1) the RBC, (2) the virion, and (3) the RBC-virion complex. To determine which target played a principal role for the inhibition of hemolysis, the following experiments were designed. Effect of Con A on RBC RBC were pretreated with various concentrations of Con A, and then their response to Sendai virus was examined. As shown in Fig. 3, the pretreatment of RBC with Con A reduced their ability to respond to hemolysis. However, the amount of Con A required to achieve 50% inhibition was more than 100 pg/ml. Thus, the inhibition of hemolysis by Con A under the conditions where RBC were incubated simultaneously with Con A and the virions may not be explained as a result of simple binding of Con A to RBC. Addition of anti-Con A antiserum produced additional inhibition of hemolysis in the Con A-treated cells over cells treated with Con A alone (Fig. 3). Effect of Con A on Free Virion Sendai virion was treated with various concentrations of Con A, and their ability
OF
141
HEMOLYSIS
to induce hemolysis was tested. Figure 4 shows that the treatment with Con A resulted in a decrease of hemolyzing activity of the virion. Con A at 17 pg/ml inhibited the hemolysis by 50%. At a lower dose range of Con A, the decrease in hemolyzing activity of the virion does not appear to be of sufficient magnitude to account for the inhibition of hemolysis which occurs when RBC are incubated simultaneously with Con A and the virion. The treatment of the virion-Con A complex with anti-Con A antiserum enhanced the effect of Con A, as found in the case where the RBC-Con A complex was treated with the antiserum (Fig. 4). Effect of Con A on the RBC-Virion Corn” plex To test the possibility that the primary target for the effect of Con A at a lower dose range was the RBC-virion complex, the effect of Con A on the RBC-virion complex was examined. As shown in Fig. 5, Con A could inhibit the hemolyzing capacity of the complex. The concentration of Con A to inhibit hemolysis by 50% was close to that observed under conditions where Con A and the virion were added lOOF--
I. ’
80 i
5 ‘Z 3 60P .s EaI 40-
.-5 1
P 60c .E
? 2
0. 1.0
10 Micrograms
100 of
Con
Alml
3. Effect of Con A on RBC. RBC were treated with various concentrations of Con A at 0” for 30 min. The cells were then washed twice with HBS and incubated with Sendai virus. After incubating for 20 min at O”, the reaction mixture was divided into two portions: One set was incubated at 37” for 30 min in the presence of lo-’ dilution of anti-Con A antiserum (0); the other set was incubated in the absence of the antiserum (0) FIG.
.q 1.0
.
. . . . . .(
1
10
100
Micrograms
of
Con
A /ml
FIG. 4. Effect of Con A on free virion. Various concentrations of Con A were incubated for 30 min with concentrated virus suspensions (usually 2500 HAU/ml) at 0’. After this incubation, aliquots were removed and diluted to preclude any carryover effect of Con A on the hemolysis. The ability of Con Atreated virion to induce hemolysis was tested in the presence of lo-’ dilution of anti-Con A antiserum (0) or in the absence of the antiserum (0).
142
TOYAMA,
TOYAMA,
simultaneously to RBC. The possibility that the observed inhibition resulted from the sum of the effect of Con A on RBC and on the virion seems unlikely. When RBC and the virion were pretreated separately with the same amount of Con A and interacted in the absence of Con A, the amount of Con A required to achieve 50% inhibition of hemolysis was more than 10 pg/ml. In contrast to the foregoing findings that the effect of Con A was enhanced by treating the RBC-Con A complex or virion-Con A complex with anti Con A antiserum (Figs. 3 and 4), the effect of Con A on the RBC-virion cmplex was slightly reversed by the addition of the antiserum (Fig. 5). The maximal level of inhibition by the antiserum was less than 30%. This inhibition may be explained by assuming that, at the time of the addition of antiserum, part of the Con A molecules bound to the complex have some free binding sites which are still available for the inhibition of hemolysis after transfer to 37”, and that these free binding sites of Con A molecules are blocked by the antibody. These results indicate that the inhibition of hemolysis is primarily the result of the binding of Con A to the RBC-virion complex, and that the mode of action of Con A 100 -
I
1
I-
AND UETAKE
on the RBC-virion complex is different from that on RBC or on virion. Effect of Succinyl Con A on Hemolysis Divalent succinyl Con A was used in experiments to establish the positive correlation of the valence of Con A with ability to inhibit hemolysis. The results are shown in Fig. 6. Although Sendai virus-induced hemolysis was inhibited when RBC were interacted with the virus in the presence of succinyl Con A, the amount of divalent succinyl Con A to inhibit hemolysis by 50% was sevenfold greater than that of tetravalent native Con A. In contrast to the effect of native Con A on the RBC-virion complex (Fig. 5), succinyl Con A produced only a small inhibition in the hemolysis of the RBC-virion complex (Fig. 6). Similarly, pretreatment of RBC or Sendai virion with succinyl Con A at 100 pg/ml produced only 10 and 50% inhibition of hemolysis, respectively (cf. Figs. 3 and 4). All of these experiments suggest that cross-linkage of Con A receptors plays a large role in the inhibition of hemolysis, and that the main effects do not result from preventing the interaction between surface glycoproteins of RBC and viral envelope proteins for steric reasons. Effect of Con A on the Release of Hemoglobin Klemperer (1960) has shown that NDV-
80-
I
. . ..I 1.0 Micrograms
I 10 of ConAlml
I 100
FIG. 5. Effect of Con A on the RBC-virion complex. After RBC were incubated with Sendai virus for 20 min at O”, various concentrations of Con A were added to the reaction mixtures. The mixtures were further incubated for 30 min. Hemolysis was measured 30 min after incubation at 37” in the presence of IO-” dilution of anti-Con A antiserum (@) or in the absence of the antiserum (0).
I
I
1.0 Micrograms
10 of succinyl
100 Con A I ml
FIG. 6. Effect of succinyl Con A on the hemolysis. Co-incubation of Sendai virus and RBC with succinyl Con A (0); incubation of the RBC-virion complex with succinyl Con A (0).
CON
A INHIBITION
induced hemolysis was inhibited by bovine serum albumin that prevented cell swelling but did not affect the initial action of the virus. Based on these findings, experiments were designed to test the possibility that Con A affected the process of hemoglobin release. As shown in Table 1, Sendai virusinduced hemolysis was inhibited when RBC-virion complexes were incubated at 37” in the presence of 30% bovine serum albumin, but the inhibition of hemolysis could be reversed if the complexes were subsequently resuspended in HBS buffer without albumin and incubated at 0”. This indicates that bovine serum albumin inhibits the release of hemoglobin without affecting envelope fusion. The release of TABLE
1
EFFECT OF CON A ON THE RELEASE HEMOGLOBINS Treatment First incubation at 37” Albumin + + + + + + +
Con A (pg/d) 0 0 0 100 0 0 0 0
OF Am nm
Second incubation at 0” Albumin
+ -
Con A k/ml) 0 0 0 100 50 10 5
0.720h 0.051’ 0.663” 0.010’ 0.710 0.708 0.680 0.738
a RBC (2.5%, v/v) were interacted with Sendai virus for 20 min at 0” in HBS-buffer containing 30% bovine serum albumin. The mixtures were then transferred to a 37” water bath and incubated for 30 min (first incubation). The cells were chilled to O’, received various concentrations of Con A, and incubated for 30 min. The Con A-treated cells were centrifuged, resuspended in cold HBS buffer, and incubated at 0” for 10 min (second incubation). The released hemoglobin was estimated as described under Materials and Methods. ‘RBC were interacted with Sendai virus in the absence of bovine serum albumin and hemolysis was assayed after the first incubation. * ‘After the first incubation, the cells were centrifuged and resuspended in HBS buffer containing 30% tiovine serum albumin. “After the first incubation, the cells were centrifuged and resuspended in HBS buffer without Con A treatment. ‘Before the fast incubation, the cells were interacted with Sendai virus in the presence of Con A.
OF
143
HEMOLYSIS
was unaffected when hemoglobin RBC-virion complexes which had been incubated at 37” in the presence of albumin were treated with Con A before transfer to HBS buffer and incubation at 0” (Table 1). These results indicate that Con A does not affect the process of hemoglobin release. Effect of Con A on Virus Adsorption
Since the treatment of the RBC-virion complex with Con A was found to inhibit hemolysis, the possibility that Con A might inhibit hemolysis by interfering with virus adsorption seemed unlikely. However, when RBC were interacted with Sendai virus in the presence of high concentrations of Con A, this possibility could not be excluded. To test this point, the effect of Con A on virus adsorption was examined. When RBC were incubated simultaneously with Con A and Sendai virion, the adsorption of the virus to RBC decreased with an increase in the amount of Con A (Fig. 7). In contrast, the adsorption of influenza virus to RBC was unaffected under these conditions (Fig. 7). Of the five lectin preparations which were used in the experiment shown in Fig. 2, only Con A was I
1.0
I
10 Micrograms
I
1
100 of Con A Iml
FIG. 7. Effect of Con A on virus adsorption. RBC (2.5%, v/v) were incubated with ““I-labeled Sendai virus (13 HALJ/ml) or ‘““I-labeled influenza virus (13 HALT/ml) in the presence of various concentrations of Con A. After incubating for 20 min at 0”, unadsorbed virus was removed by two washings at 4” with PBS. The final pellets were resuspended in 1 ml of PBS for the determination of bound radioactivity, using a Nuclear-Chicago model 1185 automatic gamma counter. Sendai virus (0); influenza virus (m).
144
TOYAMA,
TOYAMA,
found to be effective in reducing the virus adsorption (results not shown). These results indicate that Con A can inhibit Sendai virus adsorption at a higher dose range, and that this inhibition is specific both for Con A and for Sendai virus. To determine whether the observed inhibition of adsorption was due mainly to the binding of the Con A to RBC or to virion, RBC and Sendai virion were pretreated with Con A; then the ability of Con A-treated RBC to adsorb the virion and the ability of the virion to adsorb to RBC were tested in the absence of Con A. The results are shown in Table 2. The pretreatment of RBC with Con A failed to inhibit the adsorption of the virion. Prior treatment of the virions with Con A reduced their ability to adsorb to RBC, but the extent of inhibition was less than that observed when RBC were incubated with the virions in the presence of Con A. Thus, the binding of Con A to the virion, although important and even necessary for the inhibition, may not be the only factor determining the inhibition. Binding of Con A to RBC As described in the preceding section, the treatment of RBC with 100 pg/ml of Con A did not show any significant inhibition of Sendai virus adsorption. To test the possibility that most of the Con A receptor sites on RBC had not been occupied by Con A at that concentration, and thus no inhibition occurred, binding of Con A to RBC was studied quantitatively. The ability of ‘251-labeled Con A to bind to RBC is shown in Fig. 8. The same data TABLE
2
EFFECT OF CON A ON VIRUS Sample-
RBC”
Sendai
virus6
ADSORPTION
Concentration of Con A Wml)
Percentage inhibition
100 50 10
1.4 1.1 1.0
100 10
35.4 6.9
a Con A-treated RBC were prepared for Fig. 5. b Con A-treated Sendai virions were described for Fig. 6.
as described prepared
AND
UETAKE
plotted in a double-reciprocal fashion according to the method of Steck and Wallach (1965) indicate that RBC have approximately 1.1 X 105 binding sites. The apparent associated constant was calculated to be 3.5 x lo6 M-l. These values are in agreement with the ones reported previously (Schnebli and Bachi, 1975) but are in conflict with those reported by Phillips et al. (1974). Assuming 1.1 X lo5 Con A-binding sites per RBC, the percentage of the Con A receptors that would be occupied at a given concentration of Con A was calculated. At 100 pg/ml Con A, about 70% of the Con A receptors were occupied. Thus, the results of the Con A-binding experiments indicate that even if most of Con A receptors have been occupied, RBC retain their ability to adsorb Sendai virus. The results also show that at the concentration sufficient for complete inhibition of the hemolysis under the conditions where RBC are incubated simultaneously with the virion and Con A (5 pg/mh Con A occupies only about 15% of its receptors. Receptors for Con A on the Membrane of RBC and Sendai Virion To clarify the nature of receptor molecules for Con A on the membrane of RBC and Sendai virion, 1251-labeled proteins obtained from RBC and Sendai virion which were specifically precipitated with Con A and anti-Con A antibody were analyzed by SDS-polyacrylamide gel electrophoresis. As expected from previous reports (Findley, 1974; Tanner and Anstee, 19761, the results shown in Fig. 9 demonstrate that the principal high-affinity receptor for Con A on the surface membrane of RBC was the Band 3 glycoproteins (also called minor glycoproteins and component a). Two minor bands were also observed, but the nature of these bands was not clear. As also shown in Fig. 9, the two viral proteins precipitated by Con A were HN and F glycoproteins. Similar results were obtained when metabolically labeled virus was used instead of surface-labeled virus. DISCUSSION
as
When RBC are incubated with Sendai virus in the presence of Con A, the Sendai
CON
I
,
1
-0.04
-0.02
0.02
0.04
A INHIBITION
0.06
OF
HEMOLYSIS
145
0.08
1lCConAl
FIG. 8. Binding of Con A to RBC. The binding reactions were performed as described under Mater&&z and Methods. The data were plotted by the method of Steck and Wallack (1965). The inset shows a standard plot of the data.
virus-induced hemolysis is inhibited at low concentrations of Con A. Three possibilities may be considered to explain this inhibition, since Sendai virus-induced hemolysis is a secondary result of the envelope fusion (Apostolov and Almeida, 1972; Apostolov and Waterson, 1975; Homma et aZ., 1976): (1) Con A inhibits hemolysis by interfering with virus adsorption; (2) Con A inhibits the process of the envelope fusion; and (3) Con A inhibits the process of hemoglobin release. The present study strongly supports the second possibility. At high concentrations of Con A, the first possibility may play some role in the inhibition of hemolysis. The present data clearly show that the RBC-virion complex is the principal target for the inhibition of hemolysis by Con A, and that the mode of action of Con A on the RBC-virion complex appears to be different from that on RBC or on Sendai virion. The results also indicate that crosslinkage of Con A receptors plays a major role in the inhibition of the hemolysis, and that the main effects do not result from preventing the interaction between glycoproteins on the surface of RBC and viral envelope proteins for steric reasons. Although the exact mechanism by which Con A molecules bind to inhibit the hemolyzing capacity of the virion-RBC complex is not
FIG. 9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Con A-binding proteins from RBC and Sendai virion. The samples were subjected to electropboresis on slab gels and processed for autoradiography as described under Materials and Methods. The origin is at the top. (A) ‘““I-labeled RBC, (B) Con A-binding proteins from RBC, (C) ““I-labeled Sendai virion, (D) Con A-binding proteins from Sendai virion.
clear, the most probable explanation for the inhibition of hemolysis is that Con A crosslinks between glycoproteins on the surface membrane of RBC and envelope proteins of Sendai virion (HN and/or F glycoproteins). Subsequently, lateral mobility of glycoproteins on the surface of RBC and viral envelope proteins is restricted to such a degree that they cannot be redistributed to initiate envelope fusion. This notion can account for the effective inhibition of he-
146
TOYAMA,
TOYAMA,
molysis by Con A at a relatively low dose, since binding of Con A between glycoproteins on the surface of RBC and viral envelope proteins may easily form a threedimensional lattice, which in turn gives rise to restriction of the mobility of both these glycoproteins. This possibility is being investigated. Pretreatment of RBC with high concentrations of Con A is also found to inhibit hemolysis. The fact that the valence of Con A is closely correlated with ability to inhibit hemolysis and that the treatment of Con A-pretreated RBC with anti-Con A antiserum produces additional inhibition of hemolysis indicates that cross-linking of glycoproteins on RBC leads to the inhibition of hemolysis. In other words, mobility of glycoproteins in the plane of membrane is required for the initiation of envelope fusion. Con A receptors are exclusively associated with the membrane-intercalated particles (Pinto da Silva and Nicolson, 1974). Because the major Con A receptor at the surface membrane of RBC is contained in a Band 3 glycoprotein (Findley, 1974; Tanner and Anstee, 1976; and the present data), it follows that a Band 3 glycoprotein is a part of the intercalated particles. Bachi et al. (1973) have reported that the clustering of the intercalated particles following penetration of Sendai virus is demonstrated by freeze-fracturing electron microscopy. Although no gross changes in the distribution of intramembrane particles has occurred during the envelope fusion (Knutton, 1976), it is quite possible that the topological rearrangement of intercalating particles may be intimately related to the initiation of envelope fusion. In support of this view, our finding that, although Con A molecules bound to RBC or Sendai virion appear to have free binding sites, the bound Con A molecules do not act to inhibit hemolysis in a manner similar to the way they operate in the RBC-virion complex, suggests that some topological rearrangement of surface glycoproteins occurs when virus particles are bound to RBC. In addition, Band 3 glycoproteins, which have been isolated by affinity chromatography on a Con A column, are potent inhibitors of Sendai virus
AND UETAKE
adsorption (Toyama et al., unpublished results). The logical consequence of these considerations is that the redistribution of intercalating particles which contain Band 3 glycoproteins plays an essential role for the initiation of envelope fusion. However, the possibility that minor Con A-binding glycoproteins on the surface of RBC may play a role for the envelope fusion cannot be excluded. The ability to induce hemolysis of Sendai virus is inactivated after treatment with high concentrations of Con A. Two mechanisms seem to be involved in this inhibition. The first is that the failure of Con Atreated virion to cause hemolysis is a secondary result of their reduced ability to adsorb to RBC. However, the magnitude of the inhibition is insufficient to explain all of the reduced ability of the virion to induce hemolysis. The second is that Con A treatment results in crosslinking between envelope proteins of the virion, and thereafter, that the crosslinking leads to the inhibition of hemolysis by preventing topological redistribution of envelope proteins. This is supported by the finding that divalent succinyl Con A molecules are much less effective in inhibiting the hemolyzing activity of the virion than tetrameric Con A, and that the treatment of Con A-treated virion with anti-Con A antiserum produces additional inhibition of hemolysis. In addition, the possibility that aggregation of the virions by Con A plays a major role in reducing their hemolyzing activity seems unlikely: About 50% of hemolyzing activity is inactivated when Sendai virions are pretreated with 20 pg/ml of Con A, whereas hemagglutinating activity of the virions is fully retained under these conditions. All of these results discussed above provide strong evidence that crosslinkage of Con A receptors plays a 1arg.e role in the inhibition of the envelope fusion. The present data complement the morphological finding of Knutton (1976), who showed that changes in the structure of the viral envelope prior to fusion involve a reorganization of membrane components to produce the particle-denuded linear E-face ridges. Thus, it may be concluded that the rearrangement of Con A receptors on the surface of
CON
A INHIBITION
RBC and on the envelope of the virion is a prerequisite for the initiation of the envelope fusion. The treatment of Sendai virions with high concentrations of Con A is found to reduce their ability to adsorb to RBC. This is in conflict with the result reported by Okada and Kim (1972). The discrepancy in the conclusion may be explained by the difference in the assay system used in the two studies. When the ability of Con A-treated Sendai virion to adsorb to RBC is tested in the absence of Con A, the extent of the inhibition of adsorption is less than that assayed under the conditions where RBC and virion are interacted in the presence of Con A. This may be interpreted as follows: When free Con A molecules are present in the reaction mixture, they bind both to Band 3 glycoproteins and to viral envelope proteins. The bound Con A molecules may hinder sterically and/or electrostatically the close contact between viral envelope proteins and their receptor proteins on the surface of RBC. ACKNOWLEDGMENTS The authors thank Dr. Osawa for generous gifts of various lectin preparations. This investigation was supported in part by a grant from the Ministry of Education, Science and Culture, Japan. REFERENCES APOSTOLOV, K., and ALMEIDA, J. D. (1972). Interaction of Sendai (HVJ) virus with human erythrocytes: A morphological study of haemolysis cell fusion. J. Gen. Virol. 15,227-234. APOSTOLOV, K., and WATERSON, A. P. (1975). Studies on the mechanism of haemolysis by paramyxoviruses. In “Negative Strand Viruses,” (B. W. J. Mahy and R. D. Barry, eds.), Vol. 2, pp. 7994322. Academic Press, New Yrok. B&HI, T., AMJET,M., and HOWE, C. (1973). Fusion of erythrocytes by Sendai virus studied by immunofreeze-etching. J. Viral. 11, 1004-1012. BACHI, T., and HOWE, C. (1972). Fusion of erythrocytes by Sendai virus studied by electron microscopy. Proc. Sot. Exp. Biol. Med. 141, 141-149. DULRECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med. 99, 167-182. FINDLAY, J. B. C. (1974). The receptor proteins for concanavalin A and Lens culnaris phytohemagglutinin in the membrane of the human erythrocytes.
OF
HEMOLYSIS
147
J. Biol. Chem. 249.4398-4403. HOMMA, M., SHIMIZU, K., SHIMIZU, Y. K., and ISHIDA, N. (1976). On the study of Sendai virus hemolysis: I, Complete Sendai virus lacking in hemolytic activity. Virology 71.41-47. KLEMPERER, H. G. (1960). Hemolysis and the release of potassium from cells by Newcastle disease virus (NDV). Virology 12, 540-552. KNIJTTON, S. (1976). Changes in viral envelope structure preceding infection. Nature (London) 264. 672-673. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T+ Nature (London) 227,680-685. NEUHATH, A. R., VERNON, S. K., HARTZELL, R. W., and RUBIN, B. A. (1972). Haemolysis by Sendai virus: Lack of requirement for neuraminidase. J. Gen. Viral. 16, 245-249. NICOLSON, G. L. (1974). The interaction of lectins with animal cell surfaces. Znt. Rev. Cytol. 39,89-190. OKADA, Y., and KIM, J. (1972). Interaction of concanavalin A with enveloped viruses and host cells. Vi-
rology 50,507-515. PHILLIPS, P. G., FURMANSKI, P., and LIJBIN, M. (1974). Cell surface interactions with concanavalin A: Location of bound radiolabeled lectin. Expt. Cell
Res. 86,301-308. PINTO DA SII.VA, P., and NICOISON, G. L. (1974). Freeze-etch localization of concanavalin A receptors to the membrane intercalated particles of human erythrocyte ghost membranes. Biochim. Bioph”vs.
Acta 363,311-319. POSTE, G., and ALLISON, A. C. (1973). Membrane fusion. Biochim. Biophys. Acta 300,421-465. SCHNERLI, H. P., and BACHI, T. (1975). Reactions of lectins with human erythrocytes. I. Factors govering the agglutination reaction. Expt. Cell Res. 91, 175-183. STANLEY, P., and HASLAM, E. A. (1971). The polypeptides of influenza virus: V, Localization of polypeptides in the virion by iodination techniques. Virology 46, 764-773. STECK, T. L., and WALLACH, D. F. H. (1965). The binding of kidney-bean phytohemagglutinin by Ehrlich ascites carcinoma. B&him. Biophys. Acta 97, 510-522. TANNER, M. J. A., and ANSTEE, D. J. (1976). A method for the demonstration of the lectin-binding components of the human erythrocyte membrane. Biochem. J. 153,265-270. TOYAMA, S., TOYAMA, S~J., and UETAKE, H. (1977). Altered cell-fusion capacity in lines of KB cells resistant to Sendai virus-induced cytolysis. Virology 76, 503-515. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol.
Chem. 244,4406-4412.