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Interaction of concanavalin A with enveloped viruses and host cells
VIROLOGY
60, 507-515
Interaction
(1972)
of Concanavalin
A with Host
YOSHIO Research
Institute
AND
Diseases,
Accepted
Viruses
and
Cells
OKADA
for Microbial
Enveloped
July
JEMAN
KIM
University,
Osaka
Osaka,
Japan
31, 1992
HVJ (Sendai virus) and herpes simplex virus, which are both enveloped viruses, have receptors for concanavalin A, but poliovirus, which has no envelope, has no receptors. The HA activity of HVJ was not lost on exposure to concanavalin A; it seems that the sites on HVJ adsorbing concanavalin A differ from the active site of its hemagglutinin. After t,reatment with excess concanavalin A, HVJ or herpes virus became noninfectious. This state was reversed by washing these viruses with a-methylmannoside, which is a specific inhibitor of concanavalin A. Embryonated eggs, a host of HVJ, and FL cells, a host of herpes virus and poliovirus can adsorb concanavalin A onto their cell surfaces, and this prevents infection with these three viruses. This protection by concanavalin A was reversed by o(methylmannoside. INTRODUCTION
Concanavalin A (Con A), a phytohemagglutinin prepared from jack bean (Sumner and Howell, 1936) is known to bind with a variety of transformed cells and causes cell agglutination (Inbar and Sachs, 1969 a, b; Eckhart et al., 1971). Recently, it was shown that normal cells, like transformed cells can bind with Con A (Cline and Livingston, 1971; Ozanne and Sambrook, 1971). During analysis of the interaction of Con A wit’h cells, we found that HVJ could adsorb Con A with resultant formation of large aggregates. HVJ is an enveloped virus, and maturation of enveloped viruses is completed at the cell surface or on the nuclear membrane through a budding process. The surface of enveloped viruses, like that of cells, is usually surrounded by glycoproteins or glycolipids. These substances are probably more limited in variety on viruses than on cells. Moreover, the surface of mature virus particles is in a static state while that of cells is in a dynamic state. Thus, analysis of en1 This work reported at the 19th Meeting of the Society of Japanese Virologists in Tokyo, in October 1971. 507 Copyright All rights
@ 1972 by Academic Press, Inc. of reproduction in any form reserved.
veloped viruses using phytohemagglutinins may provide information on the functions of polysaccharide chains on membranes. The present paper is the first of a series on the interaction of phytohemagglutinins with virus and describes the effects of Con A on enveloped viruses, a nonenveloped virus and host cells. Oram et al. (1971), Becht et al. (1972), and Calafat and Hageman (1972 independently reported the interaction of Con A with enveloped viruses. MATERIALS
AND
METHODS
CmcunauuZin A (Cm A). Con A (Miles Laboratories Inc.) was diluted with Ca- and Mg-free phosphate-buffered saline (PBS) to the desired concentrations. Viruses. HVJ (Z strain), also named Sendai virus, herpes simplex virus (HF strain), kindly provided by Dr. S. Nii, and poliovirus (type 1, Sabin’s attenuated strain) kindly given by Dr. K. Kurimura were used. HVJ was grown in embryonated eggs and then the chorioallantoic fluid (CAF) was pooled and concentrated by centrifugation as described previously (Okada, 1962). The virus was suspended in phosphated-buffered
508
OKADA
AND
saline and used for experiments on aggregation with Con A. For experiments using infectivity as an indicator, infected CAF wa.s diluted with phosphate-buffered saline. Stocks of herpes virus and polivirus were prepared by infection of FL cells. After the appearance of complete cytopathic effects by the viruses, the culture fluid was harvested by low speed centrifugation, and stored at -20”. Hemagglutination (HA) titration. For HA titration, chicken red blood cells (RBC) were used. Salk’s pattern method (Salk, 1944) was used with 0.5 ml of 0.5 % RBC and 0.5 ml of virus serially diluted 2-fold with phosphatebuffered saline. Chicken RBC are so insensitive that with a concentration of 200 pg/ml of Con A no HA is detectable. Infectivity titrations. To measure the infectivity of HVJ, lo-day-old embryonated eggs were used. Virus samples were injected into the chorioallantoic cavity of eggs and incubated for 3 days at 35.5”. Then the eggs were cooled and the chorioallantoic fluid was harvested and its HA activity was measured. For assay of the infectivity of herpes virus or poliovirus, volumes of 0.1 to 0.2 ml of virus
FIG. 1. Aggregate reversion
by addition
formation in a mixture of a-methylmannoside
of highly (left).
KIM
samples were inoculated onto FL cell-monolayers in Falcon plates of 6 cm diameter and incubated for 1 hr at 35”. Then the monolayers were washed once with phosphatebuffered saline. With herpes virus, Nii’s method (1969) was used : medium containing antiherpes virus human serum was added to the plate, and 3 days later plaques were counted. With poliovirus, MEM + 10 % calf serum medium containing 0.9 % Bacto agar was added to the plates, and 3 days later plaques were counted. RESULTS
Appearance of Aggregates in Mixtures of HVJ and Con A. When concentrated HVJ was mixed with Con A, visible aggregates promptly appeared. They increased in size with time of incubation, reaching a maximum in 1 hr at room temperature. Figure 1 shows aggregates formed after mixing equal volumes of HVJ (3.2 X lo4 HAU/0.5 ml) and Con A (200 pg/ml). No aggregates formed in the presence of ar-methylmannoside (0.25 mole), and this compound also dispersed already formed aggregates.
concentrated
HVJ
and
Con
A (right),
and
its
CONCANAVALIN
A ON HVJ AND HSV
509
With the appearance of aggregates, the HA titer of the sample decreased, as shown in Table 1. On mixing HVJ and Con A in an optimum ratio, almost all the HA activity combined with the aggregates, and almost all the original HA titer was recovered when the precipitate was dispersed by addition of ol-methylmannoside (0.25 mole). These observations clearly indicate that the envelope of HVJ has multiple receptor sit,esfor Con A, and that aggregation occurs as the result of formation of an HVJ-Con A complex. Interaction of Red Blood Cells (RBC) with Con A In 1936, Sumner and Howell reported evidenee that RBC from various animals are agglutinated by Con A. Hemagglutinating viruses are titrated with RBC. Therefore, a t.ype of RBC must be chosen which is not very sensitive to agglutination with Con A but is sensitive to HVJ hemagglutinin. RBC from various animals were harvested and suspended in phosphate-buffered saline (PBS) to give OD~X, = 0.65 measured with a Carl-Zeiss photometer. Equal volumes of these RBC and various concentrations of Con A were mixed and incubated at 35” for 30 min with shaking. Then the total number of free cells and cell aggregates was counted and compared with the original number of cells. As shown in Fig. 2, RBC from guinea TABLE Assoc~~~ro~ APPEARING
OF
HA IN
1
ACTIVITY A
MIXTURE CON A
Samp!e HVJ + PBS HVJ + Con A Supernatant” Precipitate” Precipitate” + a-methylmannoside”
WITH
AGGREGATES AND
OF HVJ
HA
titer/OS ml 32,000 256 64
256 24,000
(1The precipitate and supernatant of an HVJCon A mixture were obtained by centrifugation at 1500 rpm for 10 min. b The precipitate was resuspended in the original volume of PBS or PBS containing or-methylmannoside (0.5 mole).
Con
A
concentrotlon
added
(r/ml
)
FIG. 2. Sensitivity of RBC from various animals to Con A. Equal volumes of RBC in PBS of (ODtw = 0.65) and various concentrations Con A (abscissa) were mixed and incubated at 35” for 30 min with shaking. Then the total number of free cells and cell aggregates was counted under a microscope and compared with the original number of cells. Percent of the original is shown on the ordinate. GP, guinea pig; GM, green monkey; R, rabbit; H, hamster; M, mouse; HM, human; C, chicken; S, sheep.
pig, green monkey, hamster, and mousewere sensitive to Con A and those from human were less sensitive. Chicken RBC were less sensitive than human RBC and sheep RBC showed no visible agglutination under the present conditions. Next, the affect of Con A on the HA pattern of chicken and sheep RBC was tested. Samples of 0.5 ml of 0.5 % RBC were mixed with 0.5 ml of serial 2-fold dilutions of Con A in HA plates without virus and incubated at room temperature for l-3 hr. With chicken RBC, samplesmixed with up to 200 pg/ml of Con A showed a negative pattern like that of RBC only, but those mixed with more than 400 pg/ml of Con A showed a positive pattern. With sheepRBC, the negative pattern observed with RBC alone changed to a positive pattern on addition of 3 pg/ml of Con A or more, in spite of which no agglu-
510
OKADA AND KIM
tination of sheep RBC was detected in samples mixed with 200, 25, 3 cl.g/ml or no Con A, as determined by a Coulter counter. From these results, chicken RBC were chosen for HA titration of the HVJ-Con A mixture : from the above results it seems that the presence of less than 200 pg/ml of Con A should not affect HA titration of the mixture.
increase in the concentration of Con A, reaching a plateau at 500 pg/ml of Con A, 256 HAU/0.5 ml. Even in the mixture containing the highest concentration of Con A
Possible Difference of Con A Binding Sites from HA Active Sites on the HVJ Virion Next, experiments were made to see whether the decrease in the HA titer shown in Table 1 was due to loss of HA activity of the virus or whether it was an apparent decrease due only to aggregation of HVJ. Serial 2-fold dilutions of HVJ in PBS were mixed with equal volumes of a fixed concentration of Con A (200 pg/ml). After incubation for 1 hr at room temperature, the mixtures were diluted serially 2-fold with PBS for HA titration. The HA titers of this series were compared with those of a control series without Con A. As shown in Fig. 3, the rate of reduction in the HA titer was higher in samples with a high concentration of HVJ and decreased with decrease in the HVJ concentration, even though the relative concentration of Con A per virus particle increased on decreasing the HVJ concentration. With concentrations of less than 2 HAU/0.5 ml, no reduction was observed. These HA activties, even in the mixture of 4 HAU/0.5 ml of HVJ and 200 pg of Con A per milliter, were completely neutralized by anti-HVJ y-globulin. The reduction was clearly a function of the virus concentration, and it seems that aggregation of the virus was the main reason for the reduction of HA activity. With decrease in the virus concentration, the chance of collision of virus particles probably decreases and the chance of collision of virus particles with Con A molecule increases. Thus, each receptor site on the virions in samples with lower concentrations of HVJ may be blocked by adsorption of a Con A molecule, but even under these conditions the HA activity of HVJ is retained. Next, equal volumes of a constant concentration of HVJ (3.2 X lo4 HAU/O.5 ml) and various concentrations of Con A were mixed and the resulting HA tit’ers were determined. As shown in Fig. 4, the titer decreased with
0
‘5-J Dilution
of HVJ
before
mixing
wit;‘Con
A:
2”
FIG. 3. Dependency of aggregate formation with Con A on concentration of HVJ. Serially 2-fold diluted HVJ wan mixed with an equal volumeof 200 g of ConA/ml (0-O). As a control, HVJ wasmixed with PBS (O-O).
0 125 250 Con
500 A
1000 concentration
2000 added;
FIG. 4. Retention of HA activity treatment with Con A.
pg/ml
of HVJ after
CONCANAVALIN
A ON
(2 mg/ml), the HA titer did not decrease further. This result also showed that the HA activity of HVJ was retained after maximum adsorption of Con A onto the receptor sites of the virions. In studies on the identification of Con A binding sites on the virus, HVJ was solubilized with a detergent, NP40, by Hosaka’s method (1972). When the solubilized HVJ was mixed with Con A, visible aggregates appeared, and on low speed centrifugation all the HA activity sedimented with the aggregates. Recent data on the structural components of HVJ suggest that its hemagglutinin is composed of a glycoprotein unit(s). At least, one of the sites on the virion which binds with Con A may be the carbohydrate chain of the hemagglutinin, and the present results show that the active site of the hemagglutinin retains functional activity after adsorption of Con A. The active site of hemaglutinin of HVJ may differ from the Con A binding sites. Inhibition of HVJ Infection Eggs by Con A
of Embryonated
When 0.2 ml of a mixture of equal volumes of HVJ (2 HAU/0.5 ml, 106.3EIDSO/O.l ml), and Con A (80 pg/ml) was injected into eggs no virus could be recovered from the eggs after 3 day-incubation at 35”. After injection of 8 pg of Con A per 0.1 ml with virus the yield of HVJ was the same as that after injection of virus alone. The inhibition of virus infection by 80 pg of Con A was reversed by adding a-methylmannoside (0.25 mole) to the mixture before injecting it into eggs. A similar inhibition of virus growth was observed in eggs pretreated with Con A. First, 80 or 8 pg of Con A were injected per egg, and then after incubation for 1 hr, 0.1 ml of HVJ (106.3 EIDGO/O.l ml) was injected per egg. The eggs pretreated with 80 rug of Con A showed no virus growth, but those treated with 8 pg of Con A gave the same virus yield as control eggs injected with HVJ alone. This inhibition was also reversed by injection of 0.1 ml of or-methylmannoside (0.5 mole) into eggs treated with 80 pg of Con A before virus injection. In these two kinds of experiment conditions were different but the results were the same. The volume of chorioallantoic fluid of
HVJ
AND
HSV
511
lo-day-old embryonated eggs is over 1 ml. Thus, 0.1 ml of injected Con A or HVJ is diluted over IO-fold. This suggests that the efficiency of collision of HVJ with Con A in the former experiment is higher than in the latter. But the relative concentration of Con A to the host cells was probably nearly the same in the two experiments, if the number of Con A molecules was very much higher than the number of receptors on the HVJ used and the binding of Con A by the virus was very small compared with the total amount of Con A used in the former experiment. To test this possibility, a mixture of HVJ (106.3 EIDso/O.l ml) and Con A (80 pg/O.l ml) was incubated for 1 hr and then centrifuged at 20,000 rpm for 30 min. The supernatant (0.2 ml/egg) was injected into eggs and after incubation for 1 hr 0.1 ml of HVJ (106.3 EID& was also injected into these eggs. No HVJ was recovered from the eggs. This clearly showed that the supernatant had the same inhibitory activity as 80 pg Con A/O.1 ml. These observations indicate that adsorption of Con A onto host cells in CAM converts the cells to a state in which their susceptibility to HVJ infection is lost. The mechanism of this is discussed later. Next, evidence was obtained that HVJ itself changed to a noninfectious state on adsorption of Con A onto its receptors. Equal volumes of HVJ (2 HAU/O.5 ml, 106,3EIDso/ 0.1 ml) and 80 pg of Con A/0.1 ml were mixed and incubated at room temperature for 1 hr. Then the mixture was serially diluted lo-fold with phosphate-buffered saline and 0.2 ml of each dilution was injected into eggs for infectivity assay. As a control, two series of lo-fold serial dilutions of virus and Con A sample were prepared separately; 0.1 ml per egg of each dilution of Con A was injected into eggs 1 hr before injection of 0.1 ml of each dilution of HVJ. After incubation for 3 days at 35”, infectivity was examined. As shown in Table 2, no virus was obtained from eggs injected with any of the dilutions of the HVJ-Con A mixture in the former series. But in the latter series Con A caused no inhibition except in eggs injected with undiluted Con A and HVJ. Confirming the result of the preceding experiment, this rcsult also showed that inoculation of eggs with SO pg of Con A inhibited virus growth but
OKADA
512
Loss OF INFECTIVITY
OF
AND KIM
TABLE 2 HVJ ON ADSORPTION
OF CON
Series l5 Dilution of HVJ-Con A Mixture loo 10-l 10-Z 10-E 10-d
lo-6 10-e
A
Series 2a No. infected eggs
Dilution of
No. infected eggs
Total No. eggs used
Con A
HVJ
Total No. eggs used
O/5 O/5 O/5 O/5 O/5 O/5 O/5
10” 10-l 10-z 10-3
10” 10-l 10-Z 10-a
10-4
10-4
lo-6 10-6
10-S 10-S
O/5 5/5 515 5/5 515 5/5 4/5
0 In series 1, equal volumes of HVJ (106** EID6a/O.l ml) and Con A (80 pg/O.l ml) were mixed. After incubation for 1 hr at room temperature, the mixture was serially diluted lo-fold with PBS and 0.2 ml of each dilution was injected into eggs. In series 2, Con A (original cont. 80 fig/O.1 ml) was serially diluted lo-fold and 0.1 ml was injected into eggs. After incubation for 1 hr, 0.1 ml of HVJ (original cont. 104.3 EIDho/O.l ml) serially diluted lo-fold wa8 alsoinjected into the eggs.
inoculation of 8 rg of Con A or less had no effect on the susceptibility of eggs to HVJ infection. The lack of viral infection in eggs injected with more than lo-fold diluted HVJCon A mixture may be due to modification of the character of HVJ by Con A adsorption. The change in the character of HVJ resulting in great decreasein infectivity may not correspond directly to the aggregation of virus by Con A described in the preceding section. The effective number of virus particles decreasewhen the virus particles aggregate, but the result shown in Fig. 3 indicates that the degree of viral aggregation is a function of the virus concentration, and at a viral concentration of 2 HAU/0.5 ml aggregation is minimal. Even under the most effective conditions for viral aggregation using highly concentrated HVJ, the decreasein the effective number of HVJ particles due to viral aggregation could not be expected to be more than lOO-fold, from the results shown in Table 1 and Figs. 3 and 4. Thus, the extreme decrease of about one millionfold in infectivity of HVJ (10 6.3EID60) in the mixture with Con A (80 pg) suggests that HVJ virions change to a state that is unable to infect host cells owing to adsorption of Con A on their envelopes. When free Con A was removed from the HVJ-Con A mixture, the virus titer became 1Oo.5EIDso . This means that the results
shown in Table 2 can be explained as follows : (1) the lack of virus growth in eggs inoculated with undiluted HVJ-Con A mixture was mainly due to loss of susceptibility of host cells to HVJ due to Con A which was not adsorbed on the viral envelopes, and (2) the lack of viral growth on inject,ion of diluted Con A-HVJ mixture was due to a change in HVJ caused by Con A. Interaction betweenHerpes Simplex Virus and CMkA Herpes virus is an enveloped virus and easily induces plaque formation in monolayers of FL cells. Therefore, reduction in the plaque number was used as an indicator of the interaction of virus with Con A. First, the effect of Con A on FL cells was tested. Con A (1.6 or 160 pg in 0.2 ml) was added to FL cell monolayers in plates of 6 cm diameter. As a control, 0.2 ml of PBS was added to similar plates. The plates were incubated at 35” for 1 hr, washed t’wice with PBS and infected with 0.2 ml of herpes virus stock. After incubation for 1 hr at 35” to allow adsorption, the plates were washed with phosphate-buffered saline and then cultured for 3 days in medium containing antiherpes human serum. As shown in Table 3, no plaques appeared in plates treated with 160 pg of Con A. The mean number of plaques in plates treat,ed with 1.6 pg of Con ,4 was 369
CONCANAVALIN
A ON TABLE
Loss
Amount
0 1.6 160 0 160 160 (incubated 160 (washed
OF SENSITIVITY
OF FL
CELLS
of Con A added to FL monolayers infection &g/plate)
with
HVJ
513
HSV
3
TO HERPES VIRUS CON Aa before
AND
INFECTION
ON TREATMENT
Mean
virus
plaque
WITH
No./plate
517 (l@J%‘o) 369 (71%) 0 (0%) 715 (loo%‘o) 0 (0%) 160 (22%) 646 (90%)
in standard mediumb) cY-methylmannosidec)
5 After exposure to Con A for 1 hr at 35”, FL monolayers were washed twice and inoculated with herpes virus. * After exposure to Con A, FL cells were washed twice with PBS and incubated for 5 hr at 35” in standard culture medium. Then herpes virus was added. c After exposure to Con A, FL cells were treated with a-methylmannoside (2 mg/ml) for 15 min, and then herpes virus was added.
per plate. This was slightly less than the value of 517 plaques per plate in control plates. Inhibition by Con A was completely reversed by treating the cells with cr-methylmannoside (0.5 mole) for 15 min. These observations show that Con A was adsorbed onto the surface of FL cells and protected the cells from infection with herpes virus. The FL cells treated with 160 pg of Con A were still intact after incubation for 3 days and no degenerated cells appeared. Moreover, reversion of the protective effect of Con A against virus infection was observed during incubation of the cells in standard culture medium without a-methylmannoside. Thus the protection was not due to degeneration of cells resulting from adsorption of Con A. Next, the effect of Con A on herpes virus itself was examined. Equal volumes of herpes virus stock preparation and solution containing 8 rg or 80 pg of Con A/O.1 ml were mixed and incubted for 1 hr at room temperature. Then the mixtures were diluted 50-fold with PBS. Volumes of 0.2 ml of the diluted mixtures were inoculated onto FL monolayers: 0.16 and 1.6 pg of Con A per plate. As shown in Table 4, the number of plaques was markedly less in the sample mixed with 8 pg of Con A (23 plaques per plate) than in the control (492 plaques per plate), and no plaques appeared in the sample mixed with 80 pg of Con A, in which FL
TABLE Loss
OF INFECTIVITY ON TREATMENT
4 OF HERPES VIRUS WITH CON A
Amount of Con A Total amount of (I.rg/O.l ml) added inoculum of to equal volumes Con A per plate’ of herpes virus (pg in 0.2 ml) 0 8 80 0 80 80 +
cr-methylmannosideb
0 0.16 1.6 0 1.6 1.6
No. plaques/ plate
492 0@3%) 23 (5%) 0 (0%) 715 (100%) 0 (0%) 636 (8970)
a Equal volumes of stock herpes virus and Con A were mixed; after incubation for 1 hr at room temperature the mixture was diluted 50.fold with PBS. Then 0.2 ml of the diluted mixture was added to FL monolayers. b or-Methylmannoside (0.5 mole) was added to 0.1 ml of mixture which had been incubated for 1 hr. Then after incubation for another hour the mixture was diluted 50-fold with PBS.
cells were exposed to a total of 1.6 pg of Con A. When the mixture of virus and Con A was treated with a-methylmannoside, the original number of plaques was again seen. It was not clear whether the reduction in plaque number was due only to decrease in the effective number of virus particles by viral aggregation or to modification of the
514
OKADA
character of the virus itself by adsorption of Con A as in the case of HVJ in the preceding section. However, it is at least clear that herpes virus like HVJ has receptors for Con A on its envelope. Ej’ect of Con A on Infection Poliovirus
of FL Cells with
Poliovirus type 1 was used as a nonenveloped virus, and FL cells were used as the host. First, the effect of Con A on FL cells in a poliovirus-FL cell system was tested. FL monolayers in plates of 6 cm diameter were inoculated with 0.2 ml of solutions containing 8 or 80 pg of Con A/0.1 ml: 16 or 160 pg of Con A per plate. After incubation for 1 hr at 35”, the plates were washed with PBS and infected with 0.2 ml of poliovirus stock. After incubation for 1 hr at 35 ’ to allow viral adsorption, the plates were washed with PBS and overlayed with nutrient agar. As shown in Table 5, there was no reduction in the number of plaques in plates treated with 16 pg of Con A, but the number was much reduced in plates treated with 160 pg of Con A. This reduction was reversed by treatment with cu-methylmannoside. This shows that adsorption of Con A onto FL cells protected the cells from infection with poliovirus. Next, equal volumes of poliovirus and solution containing 8 or SO pg of Con A/0.1 ml were mixed, incubated for 1 hr at room temperature and then diluted 50-fold. Volumes of 0.2 ml of the diluted mixtures were inoculated onto FL monolayers: 0.16 and 1.6 pg of Con A per plate. As shown in Table 6, plaque formation was not affected by these TABLE Loss
Amount
0 16 160 160 +
5
OF SENSITIVITY OF FL CELLS TO POLIOVIRUS INFECTION ON CON A TREATMENT of Con A added Wplate)
to FL
cells
Mean plaque No./plate 138
(100%) 63%) 15 (11%) 93 (67%)
121 a-methylmannosidea
a After incubation for 1 hr with Con A, FL monolayers were washed twice with PBS and treated with a-methylmannoside (2 mg) for 1 hr before virus inoculation.
AND
KIM TABLE INFECTIVITY
6
0~ POLIOVIRUS A TREATMENT
Amount of Con A Total amount of Con A per plate5 b4dO.1 ml) mixed with equal (fig in 0.2 ml) volumes of poliovirus 0 8 80
0 0.16 1.6
AFTER
CON
Plaque No./ plate
181
1% 162
(100%) Cl@)%) (90%)
a Equal volumes of poliovirus and the indicated concentrations of Con A were mixed and incubated for 1 hr at room temperature. Then the mixtures were diluted 50-fold with PBS, and 0.2 ml of the diluted mixtures were added to FL monolayers.
concentrations of Con A. This indicates that there is not direct’ reaction between poliovirus and Con A under these conditions. DISCUSSION
In the present experiments, HVJ and herpes virus were used as enveloped viruses and poliovirus as a nonenveloped virus, and embryonated eggs and FL cells were used as hosts. All these except poliovirus had receptors for Con A on their surfaces, and by covering the receptors with Con A their biological characters seemed to be modified, The present results indicated the presence of polysaccharide chains able to combine with Con A molecules on the surfaces of enveloped viruses, including hemagglutinating and nonhemagglutinating viruses. From these results together with reports of the interaction of Con A with arbovirus by Oram et al. (1971), with myxoviruses by Becht et al. (1972) and with murine RNA tumor viruses by Calafat and Hageman (1972)) this seems to be a characteristic of enveloped viruses in general. On covering its receptors with Con A, HVJ changed to noninfectious state. The reason for this loss of infectivity is not known, but there are two possible explanations: (1) This loss of infectiousness is probably not due to block of the step of adsorption of virus onto host cells, because the virus specific HA activity was retained after Con A treatment If the main reason is due to a modification of HVJ particle itself, this loss of infectivit’y
CONCANAVALIN
A ON
suggests that polysaccharide chains of the virus envelope have an essential role in the infection process and the modification of them by Con A adsorption causes the loss of infectiousness of HVJ. (2) Under the present conditions the sites of adsorption of the virus particles combined with Con A molecules were the Con A-receptors on the host cells not the natural viral receptors. On this basis, the present result seems to suggest that when HVJ is adsorbed onto other receptors of the host than the natural viral receptors (which include sialic acid as the structural component), it could not cause infection. Both embryonated eggs and FL cells were protected from viral infection by adsorption of Con A. However, on infection with an enveloped virus it is possible that the inoculated virus is adsorbed onto Con A molecules combined with the host cell surface not onto natural viral receptors of the host and so is noninfectious. This possibility does not explain why Con A protected FL cells from infection with poliovirus, since poliovirus does not interact directly with Con A and so could not be adsorbed onto Con A molecules combined with the cell surface. The changes in surface structure by Con A was not followed by degeneration of the cells but was reversed on incubat,ion of the cells in standard medium onlv at 35”. ACKNOWLEDGMENTS This Ministry
work was supported by a grant of Education of Japan.
from
the
REFERENCES BECHT, H., ROTT, R., and KLENK, H. D. (1972). Effect of concanavalin A on cells infected with enveloped RNA viruses. J. Gen. Fivirol. 14, l-8. CALAFAT, J., and HAGEMAN, P. C. (1972). Binding of concanavalin A to the envelope of two murine RNA viruses. J. Gen. Fi’iroZ. 14,103-106.
HVJ
AND
HSV
515
CLINE, M. J., and LIVINGSTON, D. C. (1971). Binding of aH-concanavalin A by normal and transformed cells. Nature (London) New Biol. 232, 155-156. ECKHART, W., DULBECCO, R., and BURGER, M. M. (1971). Temperature-dependent surface changes in cells infected or transformed by a thermosensitive mutant of polioma virus. Proc. Nat. Acad. Sci. U.S.A. 68,283-286. HOSAKA, Y., and SIMIZU, Y. K. (1972). Artificial assembly of envelope particles of HVJ (Sendai virus). I. Assembly of hemolytic and fusion factors from envelopes solubilieed by Nonidet P 40. Virology. 49, 627-639. INBAR, M., and SACHS, L. (1969a). Interaction of the carbohydrate-binding protein concanavalin A with normal and transformed cells. Proc. Nut. Acad. Sci. U.S.A. 63, 1418-1425. INBAR, M., and SACHS, L. (1969b). Structural difference in sites on the surface membrane of normal and transformed cells. Nature (London) 223, 71&712. NII, S. (1969). Plaque formation by three variants of the Miyama strain of herpes simplex virus. Biken J. 12, 251-255. OKADA, Y. (1962). Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich’s ascites tumor cells. Exp. Cell Res. 26, 9% 107. OR.~M, J. D., ELLWOOD, D. C., APPLEYARD, G., and STANLEY, J. L. (1971). Agglutination of an arbovirus by concanavalin A. Nature (London) New Biol. 233, 50-51. OZANNE, B., and S.*MBROOK, J. (1971). Binding of radioactively labelled concanavalin A and wheat germ agglutinin to normal and virus-transformed cells. Nature (London) New Biol. 232, 156-160. S.~LK, J. E. (1944). A simplified procedure for titrating hemagglutinating capacity of influenza virus and the corresponding antibody. J. Immunol. 49, 87-98. SUMNER, J. B., and HOWELL, S. F. (1936). The identification of the hemagglutinin of the jack bean with concanavalin A. J. Bacterial. 32, 217237.
60, 507-515
Interaction
(1972)
of Concanavalin
A with Host
YOSHIO Research
Institute
AND
Diseases,
Accepted
Viruses
and
Cells
OKADA
for Microbial
Enveloped
July
JEMAN
KIM
University,
Osaka
Osaka,
Japan
31, 1992
HVJ (Sendai virus) and herpes simplex virus, which are both enveloped viruses, have receptors for concanavalin A, but poliovirus, which has no envelope, has no receptors. The HA activity of HVJ was not lost on exposure to concanavalin A; it seems that the sites on HVJ adsorbing concanavalin A differ from the active site of its hemagglutinin. After t,reatment with excess concanavalin A, HVJ or herpes virus became noninfectious. This state was reversed by washing these viruses with a-methylmannoside, which is a specific inhibitor of concanavalin A. Embryonated eggs, a host of HVJ, and FL cells, a host of herpes virus and poliovirus can adsorb concanavalin A onto their cell surfaces, and this prevents infection with these three viruses. This protection by concanavalin A was reversed by o(methylmannoside. INTRODUCTION
Concanavalin A (Con A), a phytohemagglutinin prepared from jack bean (Sumner and Howell, 1936) is known to bind with a variety of transformed cells and causes cell agglutination (Inbar and Sachs, 1969 a, b; Eckhart et al., 1971). Recently, it was shown that normal cells, like transformed cells can bind with Con A (Cline and Livingston, 1971; Ozanne and Sambrook, 1971). During analysis of the interaction of Con A wit’h cells, we found that HVJ could adsorb Con A with resultant formation of large aggregates. HVJ is an enveloped virus, and maturation of enveloped viruses is completed at the cell surface or on the nuclear membrane through a budding process. The surface of enveloped viruses, like that of cells, is usually surrounded by glycoproteins or glycolipids. These substances are probably more limited in variety on viruses than on cells. Moreover, the surface of mature virus particles is in a static state while that of cells is in a dynamic state. Thus, analysis of en1 This work reported at the 19th Meeting of the Society of Japanese Virologists in Tokyo, in October 1971. 507 Copyright All rights
@ 1972 by Academic Press, Inc. of reproduction in any form reserved.
veloped viruses using phytohemagglutinins may provide information on the functions of polysaccharide chains on membranes. The present paper is the first of a series on the interaction of phytohemagglutinins with virus and describes the effects of Con A on enveloped viruses, a nonenveloped virus and host cells. Oram et al. (1971), Becht et al. (1972), and Calafat and Hageman (1972 independently reported the interaction of Con A with enveloped viruses. MATERIALS
AND
METHODS
CmcunauuZin A (Cm A). Con A (Miles Laboratories Inc.) was diluted with Ca- and Mg-free phosphate-buffered saline (PBS) to the desired concentrations. Viruses. HVJ (Z strain), also named Sendai virus, herpes simplex virus (HF strain), kindly provided by Dr. S. Nii, and poliovirus (type 1, Sabin’s attenuated strain) kindly given by Dr. K. Kurimura were used. HVJ was grown in embryonated eggs and then the chorioallantoic fluid (CAF) was pooled and concentrated by centrifugation as described previously (Okada, 1962). The virus was suspended in phosphated-buffered
508
OKADA
AND
saline and used for experiments on aggregation with Con A. For experiments using infectivity as an indicator, infected CAF wa.s diluted with phosphate-buffered saline. Stocks of herpes virus and polivirus were prepared by infection of FL cells. After the appearance of complete cytopathic effects by the viruses, the culture fluid was harvested by low speed centrifugation, and stored at -20”. Hemagglutination (HA) titration. For HA titration, chicken red blood cells (RBC) were used. Salk’s pattern method (Salk, 1944) was used with 0.5 ml of 0.5 % RBC and 0.5 ml of virus serially diluted 2-fold with phosphatebuffered saline. Chicken RBC are so insensitive that with a concentration of 200 pg/ml of Con A no HA is detectable. Infectivity titrations. To measure the infectivity of HVJ, lo-day-old embryonated eggs were used. Virus samples were injected into the chorioallantoic cavity of eggs and incubated for 3 days at 35.5”. Then the eggs were cooled and the chorioallantoic fluid was harvested and its HA activity was measured. For assay of the infectivity of herpes virus or poliovirus, volumes of 0.1 to 0.2 ml of virus
FIG. 1. Aggregate reversion
by addition
formation in a mixture of a-methylmannoside
of highly (left).
KIM
samples were inoculated onto FL cell-monolayers in Falcon plates of 6 cm diameter and incubated for 1 hr at 35”. Then the monolayers were washed once with phosphatebuffered saline. With herpes virus, Nii’s method (1969) was used : medium containing antiherpes virus human serum was added to the plate, and 3 days later plaques were counted. With poliovirus, MEM + 10 % calf serum medium containing 0.9 % Bacto agar was added to the plates, and 3 days later plaques were counted. RESULTS
Appearance of Aggregates in Mixtures of HVJ and Con A. When concentrated HVJ was mixed with Con A, visible aggregates promptly appeared. They increased in size with time of incubation, reaching a maximum in 1 hr at room temperature. Figure 1 shows aggregates formed after mixing equal volumes of HVJ (3.2 X lo4 HAU/0.5 ml) and Con A (200 pg/ml). No aggregates formed in the presence of ar-methylmannoside (0.25 mole), and this compound also dispersed already formed aggregates.
concentrated
HVJ
and
Con
A (right),
and
its
CONCANAVALIN
A ON HVJ AND HSV
509
With the appearance of aggregates, the HA titer of the sample decreased, as shown in Table 1. On mixing HVJ and Con A in an optimum ratio, almost all the HA activity combined with the aggregates, and almost all the original HA titer was recovered when the precipitate was dispersed by addition of ol-methylmannoside (0.25 mole). These observations clearly indicate that the envelope of HVJ has multiple receptor sit,esfor Con A, and that aggregation occurs as the result of formation of an HVJ-Con A complex. Interaction of Red Blood Cells (RBC) with Con A In 1936, Sumner and Howell reported evidenee that RBC from various animals are agglutinated by Con A. Hemagglutinating viruses are titrated with RBC. Therefore, a t.ype of RBC must be chosen which is not very sensitive to agglutination with Con A but is sensitive to HVJ hemagglutinin. RBC from various animals were harvested and suspended in phosphate-buffered saline (PBS) to give OD~X, = 0.65 measured with a Carl-Zeiss photometer. Equal volumes of these RBC and various concentrations of Con A were mixed and incubated at 35” for 30 min with shaking. Then the total number of free cells and cell aggregates was counted and compared with the original number of cells. As shown in Fig. 2, RBC from guinea TABLE Assoc~~~ro~ APPEARING
OF
HA IN
1
ACTIVITY A
MIXTURE CON A
Samp!e HVJ + PBS HVJ + Con A Supernatant” Precipitate” Precipitate” + a-methylmannoside”
WITH
AGGREGATES AND
OF HVJ
HA
titer/OS ml 32,000 256 64
256 24,000
(1The precipitate and supernatant of an HVJCon A mixture were obtained by centrifugation at 1500 rpm for 10 min. b The precipitate was resuspended in the original volume of PBS or PBS containing or-methylmannoside (0.5 mole).
Con
A
concentrotlon
added
(r/ml
)
FIG. 2. Sensitivity of RBC from various animals to Con A. Equal volumes of RBC in PBS of (ODtw = 0.65) and various concentrations Con A (abscissa) were mixed and incubated at 35” for 30 min with shaking. Then the total number of free cells and cell aggregates was counted under a microscope and compared with the original number of cells. Percent of the original is shown on the ordinate. GP, guinea pig; GM, green monkey; R, rabbit; H, hamster; M, mouse; HM, human; C, chicken; S, sheep.
pig, green monkey, hamster, and mousewere sensitive to Con A and those from human were less sensitive. Chicken RBC were less sensitive than human RBC and sheep RBC showed no visible agglutination under the present conditions. Next, the affect of Con A on the HA pattern of chicken and sheep RBC was tested. Samples of 0.5 ml of 0.5 % RBC were mixed with 0.5 ml of serial 2-fold dilutions of Con A in HA plates without virus and incubated at room temperature for l-3 hr. With chicken RBC, samplesmixed with up to 200 pg/ml of Con A showed a negative pattern like that of RBC only, but those mixed with more than 400 pg/ml of Con A showed a positive pattern. With sheepRBC, the negative pattern observed with RBC alone changed to a positive pattern on addition of 3 pg/ml of Con A or more, in spite of which no agglu-
510
OKADA AND KIM
tination of sheep RBC was detected in samples mixed with 200, 25, 3 cl.g/ml or no Con A, as determined by a Coulter counter. From these results, chicken RBC were chosen for HA titration of the HVJ-Con A mixture : from the above results it seems that the presence of less than 200 pg/ml of Con A should not affect HA titration of the mixture.
increase in the concentration of Con A, reaching a plateau at 500 pg/ml of Con A, 256 HAU/0.5 ml. Even in the mixture containing the highest concentration of Con A
Possible Difference of Con A Binding Sites from HA Active Sites on the HVJ Virion Next, experiments were made to see whether the decrease in the HA titer shown in Table 1 was due to loss of HA activity of the virus or whether it was an apparent decrease due only to aggregation of HVJ. Serial 2-fold dilutions of HVJ in PBS were mixed with equal volumes of a fixed concentration of Con A (200 pg/ml). After incubation for 1 hr at room temperature, the mixtures were diluted serially 2-fold with PBS for HA titration. The HA titers of this series were compared with those of a control series without Con A. As shown in Fig. 3, the rate of reduction in the HA titer was higher in samples with a high concentration of HVJ and decreased with decrease in the HVJ concentration, even though the relative concentration of Con A per virus particle increased on decreasing the HVJ concentration. With concentrations of less than 2 HAU/0.5 ml, no reduction was observed. These HA activties, even in the mixture of 4 HAU/0.5 ml of HVJ and 200 pg of Con A per milliter, were completely neutralized by anti-HVJ y-globulin. The reduction was clearly a function of the virus concentration, and it seems that aggregation of the virus was the main reason for the reduction of HA activity. With decrease in the virus concentration, the chance of collision of virus particles probably decreases and the chance of collision of virus particles with Con A molecule increases. Thus, each receptor site on the virions in samples with lower concentrations of HVJ may be blocked by adsorption of a Con A molecule, but even under these conditions the HA activity of HVJ is retained. Next, equal volumes of a constant concentration of HVJ (3.2 X lo4 HAU/O.5 ml) and various concentrations of Con A were mixed and the resulting HA tit’ers were determined. As shown in Fig. 4, the titer decreased with
0
‘5-J Dilution
of HVJ
before
mixing
wit;‘Con
A:
2”
FIG. 3. Dependency of aggregate formation with Con A on concentration of HVJ. Serially 2-fold diluted HVJ wan mixed with an equal volumeof 200 g of ConA/ml (0-O). As a control, HVJ wasmixed with PBS (O-O).
0 125 250 Con
500 A
1000 concentration
2000 added;
FIG. 4. Retention of HA activity treatment with Con A.
pg/ml
of HVJ after
CONCANAVALIN
A ON
(2 mg/ml), the HA titer did not decrease further. This result also showed that the HA activity of HVJ was retained after maximum adsorption of Con A onto the receptor sites of the virions. In studies on the identification of Con A binding sites on the virus, HVJ was solubilized with a detergent, NP40, by Hosaka’s method (1972). When the solubilized HVJ was mixed with Con A, visible aggregates appeared, and on low speed centrifugation all the HA activity sedimented with the aggregates. Recent data on the structural components of HVJ suggest that its hemagglutinin is composed of a glycoprotein unit(s). At least, one of the sites on the virion which binds with Con A may be the carbohydrate chain of the hemagglutinin, and the present results show that the active site of the hemagglutinin retains functional activity after adsorption of Con A. The active site of hemaglutinin of HVJ may differ from the Con A binding sites. Inhibition of HVJ Infection Eggs by Con A
of Embryonated
When 0.2 ml of a mixture of equal volumes of HVJ (2 HAU/0.5 ml, 106.3EIDSO/O.l ml), and Con A (80 pg/ml) was injected into eggs no virus could be recovered from the eggs after 3 day-incubation at 35”. After injection of 8 pg of Con A per 0.1 ml with virus the yield of HVJ was the same as that after injection of virus alone. The inhibition of virus infection by 80 pg of Con A was reversed by adding a-methylmannoside (0.25 mole) to the mixture before injecting it into eggs. A similar inhibition of virus growth was observed in eggs pretreated with Con A. First, 80 or 8 pg of Con A were injected per egg, and then after incubation for 1 hr, 0.1 ml of HVJ (106.3 EIDGO/O.l ml) was injected per egg. The eggs pretreated with 80 rug of Con A showed no virus growth, but those treated with 8 pg of Con A gave the same virus yield as control eggs injected with HVJ alone. This inhibition was also reversed by injection of 0.1 ml of or-methylmannoside (0.5 mole) into eggs treated with 80 pg of Con A before virus injection. In these two kinds of experiment conditions were different but the results were the same. The volume of chorioallantoic fluid of
HVJ
AND
HSV
511
lo-day-old embryonated eggs is over 1 ml. Thus, 0.1 ml of injected Con A or HVJ is diluted over IO-fold. This suggests that the efficiency of collision of HVJ with Con A in the former experiment is higher than in the latter. But the relative concentration of Con A to the host cells was probably nearly the same in the two experiments, if the number of Con A molecules was very much higher than the number of receptors on the HVJ used and the binding of Con A by the virus was very small compared with the total amount of Con A used in the former experiment. To test this possibility, a mixture of HVJ (106.3 EIDso/O.l ml) and Con A (80 pg/O.l ml) was incubated for 1 hr and then centrifuged at 20,000 rpm for 30 min. The supernatant (0.2 ml/egg) was injected into eggs and after incubation for 1 hr 0.1 ml of HVJ (106.3 EID& was also injected into these eggs. No HVJ was recovered from the eggs. This clearly showed that the supernatant had the same inhibitory activity as 80 pg Con A/O.1 ml. These observations indicate that adsorption of Con A onto host cells in CAM converts the cells to a state in which their susceptibility to HVJ infection is lost. The mechanism of this is discussed later. Next, evidence was obtained that HVJ itself changed to a noninfectious state on adsorption of Con A onto its receptors. Equal volumes of HVJ (2 HAU/O.5 ml, 106,3EIDso/ 0.1 ml) and 80 pg of Con A/0.1 ml were mixed and incubated at room temperature for 1 hr. Then the mixture was serially diluted lo-fold with phosphate-buffered saline and 0.2 ml of each dilution was injected into eggs for infectivity assay. As a control, two series of lo-fold serial dilutions of virus and Con A sample were prepared separately; 0.1 ml per egg of each dilution of Con A was injected into eggs 1 hr before injection of 0.1 ml of each dilution of HVJ. After incubation for 3 days at 35”, infectivity was examined. As shown in Table 2, no virus was obtained from eggs injected with any of the dilutions of the HVJ-Con A mixture in the former series. But in the latter series Con A caused no inhibition except in eggs injected with undiluted Con A and HVJ. Confirming the result of the preceding experiment, this rcsult also showed that inoculation of eggs with SO pg of Con A inhibited virus growth but
OKADA
512
Loss OF INFECTIVITY
OF
AND KIM
TABLE 2 HVJ ON ADSORPTION
OF CON
Series l5 Dilution of HVJ-Con A Mixture loo 10-l 10-Z 10-E 10-d
lo-6 10-e
A
Series 2a No. infected eggs
Dilution of
No. infected eggs
Total No. eggs used
Con A
HVJ
Total No. eggs used
O/5 O/5 O/5 O/5 O/5 O/5 O/5
10” 10-l 10-z 10-3
10” 10-l 10-Z 10-a
10-4
10-4
lo-6 10-6
10-S 10-S
O/5 5/5 515 5/5 515 5/5 4/5
0 In series 1, equal volumes of HVJ (106** EID6a/O.l ml) and Con A (80 pg/O.l ml) were mixed. After incubation for 1 hr at room temperature, the mixture was serially diluted lo-fold with PBS and 0.2 ml of each dilution was injected into eggs. In series 2, Con A (original cont. 80 fig/O.1 ml) was serially diluted lo-fold and 0.1 ml was injected into eggs. After incubation for 1 hr, 0.1 ml of HVJ (original cont. 104.3 EIDho/O.l ml) serially diluted lo-fold wa8 alsoinjected into the eggs.
inoculation of 8 rg of Con A or less had no effect on the susceptibility of eggs to HVJ infection. The lack of viral infection in eggs injected with more than lo-fold diluted HVJCon A mixture may be due to modification of the character of HVJ by Con A adsorption. The change in the character of HVJ resulting in great decreasein infectivity may not correspond directly to the aggregation of virus by Con A described in the preceding section. The effective number of virus particles decreasewhen the virus particles aggregate, but the result shown in Fig. 3 indicates that the degree of viral aggregation is a function of the virus concentration, and at a viral concentration of 2 HAU/0.5 ml aggregation is minimal. Even under the most effective conditions for viral aggregation using highly concentrated HVJ, the decreasein the effective number of HVJ particles due to viral aggregation could not be expected to be more than lOO-fold, from the results shown in Table 1 and Figs. 3 and 4. Thus, the extreme decrease of about one millionfold in infectivity of HVJ (10 6.3EID60) in the mixture with Con A (80 pg) suggests that HVJ virions change to a state that is unable to infect host cells owing to adsorption of Con A on their envelopes. When free Con A was removed from the HVJ-Con A mixture, the virus titer became 1Oo.5EIDso . This means that the results
shown in Table 2 can be explained as follows : (1) the lack of virus growth in eggs inoculated with undiluted HVJ-Con A mixture was mainly due to loss of susceptibility of host cells to HVJ due to Con A which was not adsorbed on the viral envelopes, and (2) the lack of viral growth on inject,ion of diluted Con A-HVJ mixture was due to a change in HVJ caused by Con A. Interaction betweenHerpes Simplex Virus and CMkA Herpes virus is an enveloped virus and easily induces plaque formation in monolayers of FL cells. Therefore, reduction in the plaque number was used as an indicator of the interaction of virus with Con A. First, the effect of Con A on FL cells was tested. Con A (1.6 or 160 pg in 0.2 ml) was added to FL cell monolayers in plates of 6 cm diameter. As a control, 0.2 ml of PBS was added to similar plates. The plates were incubated at 35” for 1 hr, washed t’wice with PBS and infected with 0.2 ml of herpes virus stock. After incubation for 1 hr at 35” to allow adsorption, the plates were washed with phosphate-buffered saline and then cultured for 3 days in medium containing antiherpes human serum. As shown in Table 3, no plaques appeared in plates treated with 160 pg of Con A. The mean number of plaques in plates treat,ed with 1.6 pg of Con ,4 was 369
CONCANAVALIN
A ON TABLE
Loss
Amount
0 1.6 160 0 160 160 (incubated 160 (washed
OF SENSITIVITY
OF FL
CELLS
of Con A added to FL monolayers infection &g/plate)
with
HVJ
513
HSV
3
TO HERPES VIRUS CON Aa before
AND
INFECTION
ON TREATMENT
Mean
virus
plaque
WITH
No./plate
517 (l@J%‘o) 369 (71%) 0 (0%) 715 (loo%‘o) 0 (0%) 160 (22%) 646 (90%)
in standard mediumb) cY-methylmannosidec)
5 After exposure to Con A for 1 hr at 35”, FL monolayers were washed twice and inoculated with herpes virus. * After exposure to Con A, FL cells were washed twice with PBS and incubated for 5 hr at 35” in standard culture medium. Then herpes virus was added. c After exposure to Con A, FL cells were treated with a-methylmannoside (2 mg/ml) for 15 min, and then herpes virus was added.
per plate. This was slightly less than the value of 517 plaques per plate in control plates. Inhibition by Con A was completely reversed by treating the cells with cr-methylmannoside (0.5 mole) for 15 min. These observations show that Con A was adsorbed onto the surface of FL cells and protected the cells from infection with herpes virus. The FL cells treated with 160 pg of Con A were still intact after incubation for 3 days and no degenerated cells appeared. Moreover, reversion of the protective effect of Con A against virus infection was observed during incubation of the cells in standard culture medium without a-methylmannoside. Thus the protection was not due to degeneration of cells resulting from adsorption of Con A. Next, the effect of Con A on herpes virus itself was examined. Equal volumes of herpes virus stock preparation and solution containing 8 rg or 80 pg of Con A/O.1 ml were mixed and incubted for 1 hr at room temperature. Then the mixtures were diluted 50-fold with PBS. Volumes of 0.2 ml of the diluted mixtures were inoculated onto FL monolayers: 0.16 and 1.6 pg of Con A per plate. As shown in Table 4, the number of plaques was markedly less in the sample mixed with 8 pg of Con A (23 plaques per plate) than in the control (492 plaques per plate), and no plaques appeared in the sample mixed with 80 pg of Con A, in which FL
TABLE Loss
OF INFECTIVITY ON TREATMENT
4 OF HERPES VIRUS WITH CON A
Amount of Con A Total amount of (I.rg/O.l ml) added inoculum of to equal volumes Con A per plate’ of herpes virus (pg in 0.2 ml) 0 8 80 0 80 80 +
cr-methylmannosideb
0 0.16 1.6 0 1.6 1.6
No. plaques/ plate
492 0@3%) 23 (5%) 0 (0%) 715 (100%) 0 (0%) 636 (8970)
a Equal volumes of stock herpes virus and Con A were mixed; after incubation for 1 hr at room temperature the mixture was diluted 50.fold with PBS. Then 0.2 ml of the diluted mixture was added to FL monolayers. b or-Methylmannoside (0.5 mole) was added to 0.1 ml of mixture which had been incubated for 1 hr. Then after incubation for another hour the mixture was diluted 50-fold with PBS.
cells were exposed to a total of 1.6 pg of Con A. When the mixture of virus and Con A was treated with a-methylmannoside, the original number of plaques was again seen. It was not clear whether the reduction in plaque number was due only to decrease in the effective number of virus particles by viral aggregation or to modification of the
514
OKADA
character of the virus itself by adsorption of Con A as in the case of HVJ in the preceding section. However, it is at least clear that herpes virus like HVJ has receptors for Con A on its envelope. Ej’ect of Con A on Infection Poliovirus
of FL Cells with
Poliovirus type 1 was used as a nonenveloped virus, and FL cells were used as the host. First, the effect of Con A on FL cells in a poliovirus-FL cell system was tested. FL monolayers in plates of 6 cm diameter were inoculated with 0.2 ml of solutions containing 8 or 80 pg of Con A/0.1 ml: 16 or 160 pg of Con A per plate. After incubation for 1 hr at 35”, the plates were washed with PBS and infected with 0.2 ml of poliovirus stock. After incubation for 1 hr at 35 ’ to allow viral adsorption, the plates were washed with PBS and overlayed with nutrient agar. As shown in Table 5, there was no reduction in the number of plaques in plates treated with 16 pg of Con A, but the number was much reduced in plates treated with 160 pg of Con A. This reduction was reversed by treatment with cu-methylmannoside. This shows that adsorption of Con A onto FL cells protected the cells from infection with poliovirus. Next, equal volumes of poliovirus and solution containing 8 or SO pg of Con A/0.1 ml were mixed, incubated for 1 hr at room temperature and then diluted 50-fold. Volumes of 0.2 ml of the diluted mixtures were inoculated onto FL monolayers: 0.16 and 1.6 pg of Con A per plate. As shown in Table 6, plaque formation was not affected by these TABLE Loss
Amount
0 16 160 160 +
5
OF SENSITIVITY OF FL CELLS TO POLIOVIRUS INFECTION ON CON A TREATMENT of Con A added Wplate)
to FL
cells
Mean plaque No./plate 138
(100%) 63%) 15 (11%) 93 (67%)
121 a-methylmannosidea
a After incubation for 1 hr with Con A, FL monolayers were washed twice with PBS and treated with a-methylmannoside (2 mg) for 1 hr before virus inoculation.
AND
KIM TABLE INFECTIVITY
6
0~ POLIOVIRUS A TREATMENT
Amount of Con A Total amount of Con A per plate5 b4dO.1 ml) mixed with equal (fig in 0.2 ml) volumes of poliovirus 0 8 80
0 0.16 1.6
AFTER
CON
Plaque No./ plate
181
1% 162
(100%) Cl@)%) (90%)
a Equal volumes of poliovirus and the indicated concentrations of Con A were mixed and incubated for 1 hr at room temperature. Then the mixtures were diluted 50-fold with PBS, and 0.2 ml of the diluted mixtures were added to FL monolayers.
concentrations of Con A. This indicates that there is not direct’ reaction between poliovirus and Con A under these conditions. DISCUSSION
In the present experiments, HVJ and herpes virus were used as enveloped viruses and poliovirus as a nonenveloped virus, and embryonated eggs and FL cells were used as hosts. All these except poliovirus had receptors for Con A on their surfaces, and by covering the receptors with Con A their biological characters seemed to be modified, The present results indicated the presence of polysaccharide chains able to combine with Con A molecules on the surfaces of enveloped viruses, including hemagglutinating and nonhemagglutinating viruses. From these results together with reports of the interaction of Con A with arbovirus by Oram et al. (1971), with myxoviruses by Becht et al. (1972) and with murine RNA tumor viruses by Calafat and Hageman (1972)) this seems to be a characteristic of enveloped viruses in general. On covering its receptors with Con A, HVJ changed to noninfectious state. The reason for this loss of infectivity is not known, but there are two possible explanations: (1) This loss of infectiousness is probably not due to block of the step of adsorption of virus onto host cells, because the virus specific HA activity was retained after Con A treatment If the main reason is due to a modification of HVJ particle itself, this loss of infectivit’y
CONCANAVALIN
A ON
suggests that polysaccharide chains of the virus envelope have an essential role in the infection process and the modification of them by Con A adsorption causes the loss of infectiousness of HVJ. (2) Under the present conditions the sites of adsorption of the virus particles combined with Con A molecules were the Con A-receptors on the host cells not the natural viral receptors. On this basis, the present result seems to suggest that when HVJ is adsorbed onto other receptors of the host than the natural viral receptors (which include sialic acid as the structural component), it could not cause infection. Both embryonated eggs and FL cells were protected from viral infection by adsorption of Con A. However, on infection with an enveloped virus it is possible that the inoculated virus is adsorbed onto Con A molecules combined with the host cell surface not onto natural viral receptors of the host and so is noninfectious. This possibility does not explain why Con A protected FL cells from infection with poliovirus, since poliovirus does not interact directly with Con A and so could not be adsorbed onto Con A molecules combined with the cell surface. The changes in surface structure by Con A was not followed by degeneration of the cells but was reversed on incubat,ion of the cells in standard medium onlv at 35”. ACKNOWLEDGMENTS This Ministry
work was supported by a grant of Education of Japan.
from
the
REFERENCES BECHT, H., ROTT, R., and KLENK, H. D. (1972). Effect of concanavalin A on cells infected with enveloped RNA viruses. J. Gen. Fivirol. 14, l-8. CALAFAT, J., and HAGEMAN, P. C. (1972). Binding of concanavalin A to the envelope of two murine RNA viruses. J. Gen. Fi’iroZ. 14,103-106.
HVJ
AND
HSV
515
CLINE, M. J., and LIVINGSTON, D. C. (1971). Binding of aH-concanavalin A by normal and transformed cells. Nature (London) New Biol. 232, 155-156. ECKHART, W., DULBECCO, R., and BURGER, M. M. (1971). Temperature-dependent surface changes in cells infected or transformed by a thermosensitive mutant of polioma virus. Proc. Nat. Acad. Sci. U.S.A. 68,283-286. HOSAKA, Y., and SIMIZU, Y. K. (1972). Artificial assembly of envelope particles of HVJ (Sendai virus). I. Assembly of hemolytic and fusion factors from envelopes solubilieed by Nonidet P 40. Virology. 49, 627-639. INBAR, M., and SACHS, L. (1969a). Interaction of the carbohydrate-binding protein concanavalin A with normal and transformed cells. Proc. Nut. Acad. Sci. U.S.A. 63, 1418-1425. INBAR, M., and SACHS, L. (1969b). Structural difference in sites on the surface membrane of normal and transformed cells. Nature (London) 223, 71&712. NII, S. (1969). Plaque formation by three variants of the Miyama strain of herpes simplex virus. Biken J. 12, 251-255. OKADA, Y. (1962). Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich’s ascites tumor cells. Exp. Cell Res. 26, 9% 107. OR.~M, J. D., ELLWOOD, D. C., APPLEYARD, G., and STANLEY, J. L. (1971). Agglutination of an arbovirus by concanavalin A. Nature (London) New Biol. 233, 50-51. OZANNE, B., and S.*MBROOK, J. (1971). Binding of radioactively labelled concanavalin A and wheat germ agglutinin to normal and virus-transformed cells. Nature (London) New Biol. 232, 156-160. S.~LK, J. E. (1944). A simplified procedure for titrating hemagglutinating capacity of influenza virus and the corresponding antibody. J. Immunol. 49, 87-98. SUMNER, J. B., and HOWELL, S. F. (1936). The identification of the hemagglutinin of the jack bean with concanavalin A. J. Bacterial. 32, 217237.