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HVJ (Sendai virus)-induced envelope fusion and cell fusion are blocked by monoclonal anti-HN protein antibody that does not inhibit hemagglutination activity of HVJ
Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/821100409-12%02.00/0
Experimental
Cell Research
141 (1982) 409-420
HVJ (SENDAI VIRUS)-INDUCED ENVELOPE FUSION AND CELL FUSION ARE BLOCKED BY MONOCLONAL ANTI-HN PROTEIN ANTIBODY THAT DOES NOT INHIBIT HEMAGGLUTINATION ACTIVITY OF HVJ NAOYUKI
MILJRA,’
‘Institute for Molecular and ‘Department
TSUYOSHI
UCHIDA’
2 and YOSHIO OKADAL 2
and Cellular Biology, Osaka University, Suita, Osaka of Cell Fusion, National Institute for Basic Bidlogy, Okazaki. Aichi 444. Japan
565,
SUMMARY Two kinds of monoclonal antibodies aeainst HN orotein of HVJ were isolated. In comoetitive binding assay. binding of one of these altibodies to’HN protein did not inhibit binding of the other antibody to the same molecule. One of the antibodies. named HN-1 antibodv. inhibited hemagghitination activity of HVJ and also blocked neuraminidase activity of the viruswhen fetuin and Ehrlich ascites tumor cells were used as substrates, but it did not inhibit the activity when neuramine-lactose.was used as substrate. The other antibody, HN-2, did not inhibit hemagglutination activity or netiraminidase activity, but blocked HVJ-induced viral envelope-cell fusion, cell-cell fusion and hemolysis. The mechanism by which HN-2 antibody blocked the fusion process is discussed.
The envelope of HVJ (Sendai virus) contains two glycoproteins, HN and F, which form projections from the surface of the viral membrane. These glycoproteins are involved in the interactions between the virus and the host cell. The.HN protein has hemagglutination and neuraminidase activities and is responsible for adsorption of the virus to the host cell. The other glycoprotein, F, is involved in virus-induced cellcell fusion, viral envelope-cell fusion and hemolysis. The fusing activity of the F protein is activated by proteolytic cleavage of a precursor (FO) [4, 161. HVJ induces two kinds of membrane fusions. One is fusion between the viral envelope and the cell membrane and the other is fusion between two cell membranes. Cell-cell fusion does not occur when viral
envelope-cell fusion does not occur, i.e., when HVJ is incubated at 4°C or HVJ containing F,, protein is used. Moreover, cellcell fusion occurs rapidly after viral envelope-cell fusion has occurred, but it is separable from the latter: Cytochalasin D or 0.25 M sucrose inhibits cell-cell fusion, but not viral envelope-cell fusion [9]. So it is considered that cell-cell fusion requires viral envelope-cell fusion. The function of HN protein in the fusion process is not well understood. Its only known action is to bind to sialic acids of the receptor on the host cell and aggregate cells. To obtain further information on the function of HN protein, we attempted to obtain monoclonal antibodies against HN protein of HVJ by the hybridoma technique of Kohler & Milstein [7]. Exp Cell
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In this paper we isolated two hybridomas against HN protein of HVJ and investigated the effects of their monoclonal antibodies on the biological functions of HVJ. One monoclonal antibody inhibited hemagglutination and neuraminidase activities completely, whereas the other antibody did not block these activities at all. However, the latter antibody inhibited HVJ-induced cellcell fusion, envelope-cell fusion and hemolysis. The mechanism by which HN-2 antibody blocked the fusion process is discussed.
MATERIALS
AND METHODS
Cells S&!/O-Ag14 myeloma cells [17] and MPCll-45.6 TG1.7 myeloma cells were cultured in Dulbecco’s moditied Eagle medium (DMEM) supplemented with 15% fetal calf serum (FCS) (Flow Lab.). LLC-MK, cells and L cells were maintained in Eagle’s minimum essential medium containing 10% calf serum.
Virus Hemagglutinating virus of Japan (HVJ, Sendai virus), Z strain. was grown in the allantoic fluid of IO-dayold fertihzed eggs. The virus was purified by differential centrifmzation. as described oreviouslv rl11. The virus from theallantoic fluid contams F,, 2 protein, but the virus from the culture medium of LLC-MK, cells infected with HVJ contains F, protein.
PuriJication of viral proteins The HN protein and the F protein were purified by DEAE-cellulose and CM-cellulose chromatography by the method of Nakanishi [LO]. The M protein was ouritied as described previously r151. HVJ whole antigens were obtained by lysing- puri&ed HVJ with 10 mM Tris-HCI (pH 8.3). 0.5 M KCI, 1% Nonidet P-40 and removing aggregates by centrifugation at 1OOOOOgfor60min.
Immunization Purified HVJ was sonicated with a Sonifiep Cell Disruptor (model W185, Ultrasonics Inc., New York) for 5 min. BALB/c mice, 8 weeks old, were injected intraperitoneally with 0.2 mg of sonicated virus in Freund’s complete adjuvant. One month later, 0.2 mg of the virus in saline was injected intravenously into each Exp
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mouse. Three days later the spleen cells were fused with SP2/0 cells.
Fusion and cloning Spleen cells (2x 10R)from immunized mice were fused with SP2/0 cells (2x10') as follows. The two cell populations were mixed and precipitated by centrifugation at 300 g for 5 min in a 50 ml centrifuge tube. To the pellet 0.5 ml of 50% (w/v) polyethylene glycol 4000 and I5 % (v/v) dimethylsulfoxide in DMEM was added. and the tube was rolled gentlv. The concentration of polyethylene glycol was Fhen gradually reduced bv adding 1 ml of DMEM every 30 set for 5 min with gentle roiling of the tube. _ After fusion, the cells were seeded into 96 wells of tissue culture plates and cultured in HAT (1 x IO-’ M hypoxanthine, 4~10~’ M aminopterin, 1.6~10~” M thymidine) selection medium. Of 193 initial hybridoma cultures. 140 mew well within 10 davs. Cells were grown for a toLl of 14 days in HAT medium and then eraduallv adaoted to DMEM with 15% FCS bv oassage for- 7 days in medium without aminopterin.‘but containing hypoxanthine and thymidine. Cells secreting antibodies were cloned in soft agar (0.24%. Seakem LE agarose) at least twice.
Screening for hybridomas by a solidphase antibody-binding assa) Solid-phase antibody-binding assay was carried out by the method of Kennett [6] and Staehelin [ 181 with some modifications. For coating polyvinyl chloride microtiter plates (Dvnatech Laboratories Inc., Alexandria, Va). 100 /.LI of -HN protein, F protein, M protein or HVJ whole antigen solution (about 0.3 ma/ml) was placed in each welland kept at room temperature overnight. The next day the wells were washed and tilled with 120 ~1 of 2 % horse serum in borate-buffered saline (0.15 M NaCl, 0.03 M H,BO,. DH 8.0) and kept at room temperature overnight-to block all protein-butding sites. The plates were washed four times with saline. Hybridoma culture supernatants (50 ~1) were incubated in duplicate wells at room temperature overnight. The plates were then washed four times with saline, and then 100 ~1 of ‘““I-labelled rabbit antimouse Fab antibody (100 000 cpm; 200 ng of antibody in borate-buffered saline containing 0.2% bovine serum albumin) was added to well. After 12 h the plates were washed four times with saline and cut into individual wells, and the radioactivity of each well was counted in a gamma counter (Beckman Gamma 9000). Anti-HN hybridoma was obtained by screening the culture supernatant by solid-phase binding assay to HN protein. Anti-NP hybridoma was obtained as follows. The supernatant which gave a positive reaction on binding assay to HVJ whole antigens was screened by binding assay to HN protein, F protein or M protein. The supematant that reacted with HVJ whole antigens, but not with HN protein, F protein or M protein, was considered to contain either anti-NP protein antibody or anti-P protein antibody. Anti-NP hybridoma was obtained by the radioimmunoprecipitation method from the clones producine this antibody.
Monoclonal Ascites Cloned hybrid cells were cultured on a large scale and 2x lofi cells were injected into the peritoneal cavity of BALB/c mice that had received 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chemical Co.) intraperitoneally 2 weeks previously. After 7-10 days the ascitic fluid was harvested.
Purijication
of monoclonal antibodies
Monoclonal antibodies were purified with HN proteinconjugated Sepharose 49 for anti-HN hybridoma, with HVJ whole antigens-conjugated Sepharose 49 for antiNP hybridoma and with Protein A-Sepharose CL-49 (Pharmacia) for MPC 11 myeloma.
Isoelectric focusing Isoelectric focusing of purified antibodies was done in a 5 % polyacrylamide slab gel containing 2.4 % Ampholine (pH 3.5-9.5) using an LKB multiphor apparatus at a constant power of 15 W for 3 h.
Preparation of labelled viral antigens LLC-MK2 cells were infected with HVJ for 60 min. Then the virus-infected cells were cultured at 37°C in Eagle’s minimum essential medium containing 10% calf s&urn and 10 &i of L-[3”S]methionine (1 160 Ci/ mmol; Amersham) per ml. The medium was harvested after 72 h and virus was purified as described above. Labelled virus was lvsed in 0.01 M Tris-HCI (pH 8.3). 0.5 M KCI, 1% Nonidet P-40 for 30 min at 4°C. Insoluble aggregates were removed by centrifugation at 100000 g for 60 min. The soluble labelled viral proteins were used in immunoprecipitation assay.
Radioimmunoprecipitation and sodium dodecyl sulfate (SDS)-polyawlamide gel electrophoresis The monoclonal antibody was added to labelled viral antigen solution (2OOOOt!lcpml0.5 ml). The reaction mixture was incubated at 4°C for 12 h. Then 30 ~1 of Protein A-Sepharose CL-49 was added and incubation was continued at 4°C for 4 h. Sepharose was washed three times with saline containing 1% Nonidet P-40 and then 50 ~1 of sample buffer containing 0.063 M Tris-HCI, pH 6.8, 3% SDS, 10% glycerol and 50 mM dithiothreitol was added. The supernatant was subjected to electrophoresis in a 10% polyacrylamide SDS gel as described by Laemmli [8]. The gel was dried and exposed to Kodak X-Omat S tilm for 2 days.
Radio-iodination of antibodies and HVJ Antibodies and HVJ were radioiodinated with chloramine T [3]. Labelled antibody was separated from free iodide by Sephadex G-25 chromatography and dialysed against BSS overnight. The specific activities of HN-1, HN-2 and NP-1 antibodies were 2.7~ 10” cpm/ mg, 3.0~ 10” cpmlmg and I .4x lo9 cpm/mg, respective-
antibodies
against HVJ HN protein
411
ly. Labelled HVJ was separated from free lz51 by BioGel A-150m (SO-100 mesh, Bio-Rad Lab.) chromatography and dialysed against BSS overnight. The specific activitv of HVJ was 4.2~ 103 cum/HAU and the hemagglutination and cell-fusing activities of HVJ were not changed by radio-iodination.
Competitive binding assay of monoclonal antibody to the antigenic determinant on the HN molecule of HVJ A mixture of 200 ng of ““I-1abelled antibody and 2000 HAU HVJ in 0.2 ml of BSS containing 0.2% bovine serum albumin was incubated at 37°C for 60 min and then layered on 4 ml of 10% (w/v) sucrose in BSS. The mixture was centrifuged in a Hitachi SW-50 rotor at 40000 rpm for 60 min and the radioactivities of the precipitate and the supernatant were measured with a gamma counter. For competitive binding assay, 200 HAU of HVJ in 0.1 ml of BSS was incubated with 0.1 ml of BSS or BSS containing excess (lOO-fold the saturation dose) cold antibody at 37°C for 60 min. Then ‘251-labelled antibody was added and the mixture was incubated at 3PC for 60 min further. The solution was layered on 4 ml of 10% sucrose in BSS and centrifuged as mentioned above.
Assay of hemagglutination units (HAU) and hemagglutination inhibition (HA!) titer Salk’s pattern method [ 131 was used. The virus sample was mixed with an equal volume of 0.5% (v/v) chick red blood cell suspension and after several hours the titer was determined. In assay of HAI. antibody solution at a maximum concentration of 1 mg/ml was diluted 2-fold serially. Then 2 HAU/ml of HVJ was added to the antibody solutions and the mixtures were incubated at room temperature for 5 h. Then an equal volume of red blood cell suspension was added. The hemagglutination inhibition (HAI) titer was expressed as the degree of dilution of antibody that inhibited HVJ-induced hemagglutination at 1 HAU/ml.
Assay of neuraminidase activity The virus sample was mixed with the substrate and incubated at 37°C for 30 min. Then the reaction was stopped by boiling the mixture for 1 min. The substrates used were (a) Ehrlich ascites tumor (EAT) cells, 4x 107/ml; (b) fetuin (Difco), 8 mglml; and (c) N-acetyl neuramine (NANAtlactose ( Sigma), 0.34 mg/ml. The reaction was performed at pH 7.5 and pH 6.0. Sialic acid was determined by the method of Aminoff[l].
Kinetics of adsorption and release of HVJ ‘z”I-Labelled ?IVJ (32 HAU in 0.4 ml) was mixed and incubated with 1.2 ml of 10% (v/v) EAT cell suspenExp Cell
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141 /I9821
412
Miura,
Uchida and Okada
a basic c-
acidic -
Labeled antibody added(x 10” cpm)
b 1
2
3
4
5
Fig. 2. Competitive binding assay of monoclonal antibodies to the antigenic determinants on HN protein of HVJ. 200 HAU of HVJ was preincubated with BSS (0 or A), excess cold HN-1 antibody (0) or HN-2 antibody (A) and then incubated with various doses of 1251-labelled (a) HN-I antibody, or (b) HN-2 antibody. HVJ was precipitated by centrifugation and the radioactivity of bound antibody was measured. Then the mixtures were centrifuged at 20@0 rpm for 2 min at 4°C and pelleted ceils were washed once and suspended in 2 ml of BSS containing I mM CaCI,. The suspensions were incubated at 3PC and 300 ~1 samples were taken at the indicated times and centrifuged at 2000 rpm for 2 min at 4°C. Pelleted cells were washed once with BSS and the two supematants were combined. Radioactivities of cell-associated and released virus were measured in a gamma counter.
Cell fusion
Fig. 1. (a) Isoelectric focusing pattern (pH 3.5-9.5) of purified monoclonal antibodies. A, B, C. results with HN-1, HN-2 and NP-1 antibodies. (b) Autoradiogram of SDS-polyacrylamide electrophoregram of immunoprecipitation with [35S]methionine-labelled viral proteins. I, HVJ marker proteins: 2, no antibodies added; 3, HN-I antibody: 4. HN-2 antibody; 5, NP-I antibody. sion in BSS containing I mM CaCI, at 4°C. At the indicated times, 300 ~1 samples were taken and centrifuged at 2000 rpm for 1 min at 4°C. Pelleted cells were washed once with BSS at 4°C and the radioactivity of cell-associated HVJ was counted in a gamma counter. One ml of HVJ (““I-HVJ and non-labelled HVJ; final titer 500 HAtJ/ml) was mixed with 1 ml of 10% EAT cell suspension in BSS containing 2 mM CaCI, and the mixtures were incubated at 4°C for 15 min. Exp Cell
Res 141 (1982)
Ehrlich ascites tumor cells were washed [ 1 I] and suspended in 9 vol of balanced salt solution (BSS) (140 mM NaCI, 5.4 mM KCI, 0.34 mM Na,HPO,, 0.44 mM KH,PO,, 10 mM Tris-HCI pH 7.5). A mixture of 0.4 ml of 10% (v/v) EAT cell suspension in BSS containing 2 mM CaCI, and 0.4 ml of HVJ sample (500 HAU/ml) was incubated at 4°C for 10 min and then at 37°C for 3C min with shaking in a water bath.
Hemolytic
activity
A mixture of I ml of virus sample and 2 ml of 2% (v/v) chick red blood cell suspension was kept at 4°C for 10 min and then incubated at 37°C for 60 min without agitation. After incubation, the mixture was centrifuged at 2000 rpm for IO min and the optical density of the supernatant at 540 nm was measured spectrophotometrically.
Cytotoxic assay of liposomes HVJ spikes
with
The formation of fragment A-containing liposomes with HVJ spikes and cytotoxic assay of the liposomes were done, as described previously [20].
Monoclonal
antibodies
200
.
a
against HVJ HN protein
413
I
C
1
f-
.
.
.
:,
.
t.zr .Ol
.,
1.0
10
.01
:1
1:o
lb
Antibody concentration(yg/ml) 3. Effects of monoclonal antibodies on HVJassociated neuraminidase activity. HVJ ((a) 250 HAU/ ml; (6) 64 HAU/ml; (c) 128 HAU/ml) was preincubated with BSS (control) or various concentrations of 0, HN-1 antibody; A, HN-2 antibody; or n , NP-1 antibody, and then mixed with (a) EAT cells (4x lO’/ml) at pH 7.5; (6) fetuin (8 mg/ml) at pH 6.0; or
Fig.
RESULTS Isolation of two monoclonal antibodies against HN protein of HVJ Hybridomas produced by fusion of SP2/0 cells with splenic lymphocytes from mice immunized with sonicated HVJ might produce antibodies to any one of the viral proteins. Therefore, it was necessary to identify the antigen to which the hybridoma produced antibodies. Of thirty hybridomas, two hybridomas producing anti-HN protein antibodies (named HN-1 and HN-2) and one hybridoma producing anti-NP protein antibody (named NP-1) were obtained. After at least two clonings, the cells were injected intraperitoneally into BALB/c mice. The monoclonal antibodies were purified from the ascitic fluid by affinity chromatography, as described in the Materials and Methods. Fig. la shows the pattern of isoelectric focusing of the three purified antibodies. Each gave the characteristic pattern of monoclonal protein. The specificities of these antibodies were determined by radioimmunoprecipitation and polyacrylamide
(c) NANA-lactose (0.34 mg/ml) at pH 6.0. The reaction mixture was incubated at 37°C for 30 min, and free sialic acid was determined by the method of Aminoff [ll. The OJh9,, of the controls were (a) 0.23; (b) 0.64; and (c) 0.51 respectively. Each control was taken as 100%.
gel electrophoresis analysis. As shown in fig. 1 b , HN protein was specifically precipitated by HN-1 and HN-2 antibodies (fig. lb, 3; 4) and NP protein was precipitated by NP-1 antibody (fig. lb, 5). However, under these conditions, M protein was precipitated non-specifically (fig. 1b , 2). When L251-labelled glycoproteins of HVJ purified from the chorioallantoic fluid of hen eggs were used as antigens, the autoradiogram showed that HN-1 and HN-2 antibodies bound to HN protein, but not to F,, 2 protein (data not shown). Ouchterlony analysis and binding to protein A showed that HN-1, HN-2 and NP-1 antibodies were IgG (data not shown). To examine the functional uniformity of the purified antibodies, the antibodies were radioiodinated with [‘251]Na. ‘251-Labelled monoclonal antibodies were then treated with excess HVJ at 37°C for 60 min and bound antibodies were separated from unbound antibody by centrifugation. The ratio of bound antibody to total antibody was calculated. The ratios for HN- 1, HN-2 and NP-1 antibodies at pH 7.5 were 90, 95, and Exp
Cell
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Miura,
Uchida and Okada
4%. respectively. Those for HN-1 and HN2 antibodies at pH 6.0 were 85 and 90%, respectively. Competitive binding assay was used to determine whether the monoclonal antibodies were directed toward the same or different antigenic determinants on the HN molecule of HVJ. HN-2 antibody bound to the HN molecule of HVJ did not inhibit binding of HN- 1 antibody to the HN protein (fig. 2a) and HN-1 antibody bound to HN protein only partially inhibited the binding of HN-2 antibody (fig. 2b). These tindings suggest that HN-1 and HN-2 antibodies were directed toward different antigenic determinants. Effects of monoclonal antibodies on hemagglutination activity of HVJ One of the known functions of HN protein is hemagglutination activity. The hemagglutination inhibition (HAI) titer by antiHN antibody was determined by the conventional Salk’s pattern method, as described in the Materials and Methods. The titer of HN- 1 antibody was 16 000, whereas those of HN-2 and NP-1 antibodies were less than 1. These results indicated that HN- 1 antibody inhibited hemagglutination activity of HVJ, whereas HN-2 and NP-1 antibodies did not. Effects of antibodies on newaminidase activity of HVJ Another function of HN protein is neuraminidase activity. Enzymatic activity was determined at pH 7.5 and pH 6.0 with three different substrates. After preincubation of HVJ with buffer or the antibody at 4°C for 5 h, the substrate was added and the reaction mixture was incubated at 37°C for 30 min. Fig. 3 shows the percent activity of neuraminidase at various concentrations of Exp
Cell
Res 141 (1982)
.-0 -0 a
a 0
Time(min) Kg. 4. Time course of HVJ adsorption to EAT cells. ‘*“I-Labelled HVJ (80 HAU/ml) was preincubated with BSS (0, control), HN-1 antibody (0, 30 r.&ml), HN-2 antibody (A, 30 &ml) or NP-I antibody (W. 30 pg/ ml) at 4°C for 5 h. The HVJ-antibody solution (0.4 ml) was added to I.2 ml of 10% (v/v) EAT cell susoension in BSS containing 1 mM CaCI, and the mixture was incubated at 4°C. At the indicated times, 300 ~1 samples were taken and centrifuged at 2000 rpm at 4°C. Cell-associated radioactivity was counted in a gamma counter.
antibodies. The neuraminidase activity at pH 6.0 was about twice that at pH 7.5, but the percentage activities of the control with the three antibodies were the same at pH 7.5 and pH 6.0. When EAT cells or fetuin was used as a substrate (fig. 3a, b), HN-1 antibody at a concentration of more than 3 pg/ml inhibited the neuraminidase activity completely, whereas HN-2 antibody and NP-1 antibody did not affect the activity. But when neuramine-lactose was used as a substrate, HN-1 and HN-2 antibodies increased the activity (fig. 3~). As a control, HVJ was preincubated with antibody at 4°C and then incubated at 37°C for 30 min without substrates. We confirmed that under these conditions anti-HN antibody itself was not used as substrate (data not shown).
Monoclonal a
antibodies
against HVJ HN protein
415
b
i
30
so
9’0
Time(min) Fig. 5. Time course of cell-associated and released HVJ. ‘9-Labelled HVJ (500 HAU/ml) was preincubated with BSS (0, control), HN-1 antibody (0, 3Oc(g/ ml), HN-2 antibody (A, 30 pg/ml) or NP-1 antibody (W, 30 pg/ml) at 4°C for 5 h. One ml of the HVJantibody solution was added to 1 ml of 10% (v/v) EAT cell suspension and the mixture was incubated at 4°C for 15 min. The mixture was centrifuged at 2000
rpm for 2 min and pelleted cells were washed once and suspended in BSS containing 1 mM CaCl, and then incubated at 3PC. At the indicated times, 300 ~1 samples were taken and centrifuged at 2 000 rpm for 1 min and pelleted cells were washed once with BSS. TCAprecipitable counts in (a) the pellet (cell-associated HVJ); (b) the supematant (released HVJ) were measured in a gamma counter.
Effects of antibodies and release of HVJ
From the described experiments, we ascertained that HVJ treated with HN-1 antibodies did not bind to cells and that virus treated with HN-2 antibodies adsorbed to cells well and was not released rapidly.
on adsorption
First, to examine the effect of monoclonal antibodies on HVJ adsorption to a cell, ‘251-labelled HVJ was preincubated with antibodies and then mixed with EAT cells. The mixtures were incubated at 4°C. Fig. 4 shows time course of HVJ adsorption to cells. HN- 1 antibody inhibited adsorption of HVJ to cells completely. But HN-2 and NP-1 antibodies did not inhibit the adsorption. Next, to examine the effect of antibodies on HVJ release from a cell, 1251-labelled HVJ was preincubated with antibodies and then mixed with EAT cells. After the mixtures were incubated at 4°C for 15 min, cells were washed and suspended and then incubated at 37°C. Fig. 5 shows time course of cell-associated and released HVJ. When HVJ was bound with HN-2 antibody, cellassociated HVJ decreased and virus was released at almost the same rate as in control.
Table 1. Eflects of antibodies on cytotoxicity offragment A-containing liposomes with HVJ spikes
Sample
No. of surviving colonies per dish
Buffer Liposomes Liposomes+ HN-1 antibody Liposomes+HN-2 antibody Liposomes+MPCIl antibody
260 33 214 162 54
Fragment A-containing
liposomes with HVJ spikes were formed as described previously 1201. After preincubation with BSS or antibody (30 @g/ml), the liposome solutions were incubated with L cells, as described in the text. MPC 11 monoclonal protein was used as control antibody because liposomes with HVJ spikes were contaminated with NP protein. The liposome suspension had 400 nglml fragment A and 400 HAU/ml. Colony counts are averages of those in duplicate plates. Exp Cell
RPS 141 f/982)
Miura,
4 16
Uchida
and Okada
Fig. 6. Photographs of HVJ-induced cell fusion in the absence and presence of antibodies. HVJ (500 HAU/ ml) was preincubated with (n. b) BSS: (c. d) HN-I antibody: (e.Jt HN-2 antibody or (p. /I) NP-I antibody at a concentration of IO pglml, and then an equal Exp
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volume of 10% EAT cells (2x107/ml) H /as a.dded . When the suspensions were incubated at 4°C ‘for 1!O min (u, c, e. g). EAT cells were aggregated (a. (?. g). Then the mixtures were incubated at 37°C for 30 min (b, d. ,fi h) and fused cells appeared (b. /I). x 144.
Monoclonal
antibodies
against HVJ HN protein
417
.
O/ 0.1 Ani%dy
2
1I
OI .ol
'I,
.l
1.0
10
h-fthdy concantratior$g/mD Fig. 7. Effects of monoclonal antibodies on the fusion index. HVJ (500 HAU/ml) was preincubated with various concentrations of l , HN-1 antibody, A, HN-2 antibody; or n , NP-1 antibody. Then EAT cells were added and the mixtures were incubated as described for fig. 6. Total cell numbers after incubation were counted and the fusion index was calculated from the following formula [ 121 Fusion index=
Number of cells in control without virus -1.0. Number of cells after fusion
Effects of antibodies on HVJ-induced cell-cell fusion The cell-cell fusion induced by HVJ is attributed to two glycoproteins, HN and F proteins. To examine whether the antibodies affect the HVJ-induced cell-cell fusion, we incubated HVJ with the antibody at 4°C for 5 h and then added EAT cells to the reaction mixture. The mixture was kept at 4°C for 10 min, and then incubated at 37°C for 30 min with shaking. Fig. 6 shows the appearance of cells after incubation at 4°C for 10 min and after incubation at 37°C for 30 min. HVJ induced cell aggregation on incubation at 4°C (fig. 6a) and fused cells appeared on incubation at 37°C (fig. 66). NP-1 antibody (10 pg/ml) did not affect cell aggregation or cell fusion
10 1.0 .z.mmbation(rglmO
Fig. 8. Effects of monoclonal antibodies on HVJinduced hemolysis. HVJ (500 HALJ/mJ) was preincubated with BSS (control) or various concentrations of 0, HN-1 antibody; A, HN-2 antibody: or n , NP-1 antibodv. Then 2 vol of 2 % chick red blood cells were added. *The mixture was incubated at 4°C for 10 min and then at 37°C for 60 min. and then centrifuged. The optical density of the supernatant at 540 nmwas measured spectrophotometrically. The OD,,,, of the control ofU.28 was taken as 100%.
(fig. 6g, h). HN-1 antibody inhibited both cell aggregation and cell fusion (fig. 6c, d), and HN-2 antibody did not block cell aggregation, but inhibited cell fusion (fig. 6e, f). Fig. 7 shows the fusion indices at various concentrations of antibodies. Clearly, HN-1 and HN-2 antibodies at concentrations of more than 3 pg/ml blocked cell fusion, but NP-1 antibody did not inhibit cell fusion. HN-1 antibody blocked cell fusion by inhibiting cell aggregation, but HN-2 antibody inhibited cell fusion by another mechanism. Effects of antibodies on HVJ-induced hemolysis It is well known that HVJ has hemolytic activity, so the effects of monoclonal antibodies on HVJ-induced hemolysis were examined. HVJ was preincubated with various concentrations of antibodies and then red blood cells were added and the reaction mixtures were incubated at 37°C for 60 min. HN-1 and HN-2 antibodies clearly inhibited the hemolytic activity as well as the cellfusing activity (fig. 8). Exp Cell
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Effects of the antibodies on erl\?elopecell fitsion As shown in the described experiments, HVJ treated with HN-2 antibodies was associated with EAT cells to the same extent as control (fig. 5a). The next question was whether when HVJ bound with HN-2 antibodies, envelope-cell fusion could occur. Liposomes with HVJ spikes can fuse with the cell membrane efficiently by envelopecell fusion [20]. Fragment A of diphtheria toxin introduced into the cytoplasm can kill cells, so fragment A-containing liposomes with HVJ spikes were formed and preincubated with the monoclonal antibodies at 4°C for 5 h. Assay of the cytotoxicity of the liposomes to L cells was performed, as described in Materials and Methods. Table 1 shows the results. MPC 11 monoclonal antibody slightly inhibited the cytotoxicity of the liposomes, but HN-1 and HN-2 antibodies inhibited the toxicity 80 % and 57 %, respectively. This finding indicates that HN-2 antibody inhibited envelope-cell fusion.
DISCUSSION In this work we examined the effects of two monoclonal antibodies on HVJ-associated biological functions. The two monoclonal antibodies were directed toward different antigenic determinants on the HN molecule (fig. 2). We found that HN-1 antibody inhibited hemagglutination activity of HVJ completely, but that HN-2 antibody did not block the activity at all. We then tested the inhibition of HVJ neuraminidase by antiHN antibody for dependence on substrate size using monoclonal antibodies (fig. 3). HN-1 antibody inhibited neuraminidase activity with EAT cells or fetuin, but not with neuramine-lactose as substrate. This findExp Cell
Res 141 (1982)
ing suggests that steric factors accounted for the inhibition by the antibody. Moreover, HN-1 antibody increased the activity with neuramine-lactose as substrate. And HN-2 antibody increased the activity with the small substrate to a great extent. The enhancement of activity by binding of the antibody to HN protein might be explained by a conformational change in HN protein. However, HN-2 antibody did not increase the activity with macromolecular substrates, so in the case of HN-2 antibody, the enhancement of activity was counteracted by steric hindrance of substrate. From these results we assume that the antigenie site recognized by HN- 1 antibody was near the center for hemagglutination activity and neuraminidase activity, whereas that recognized by HN-2 antibody was remote from the center. It is known that HN protein is responsible for virus adsorption to a host cell [2, 14, 15, 191 and for virus release from the cell. We examined whether HVJ bound with HN-2 antibodies adsorbed cells slowly or whether it was released rapidly from the cells. We ascertained that when bound with HN-2 antibodies, HVJ showed neither delayed adsorption nor accelerated release (figs 4, 5) and therefore it had the same chance to fuse with a cell as did intact HVJ. Then we examined the effects of antibodies on HVJ-induced cell-cell fusion, envelope-cell fusion and hemolysis and showed that HN-2 antibody inhibited cellcell fusion, envelope-cell fusion and hemolysis. In the cell fusion experiment (fig. 6), HVJ treated with HN-2 antibody could aggregate cells at 4°C but could not induce cell fusion after warming to 37°C. Moreover, after 30 min of incubation at 37°C the cells were completely disaggregated (fig. 6f). HVJ treated with HN-2 antibody still had neuraminidase activity (fig. 30).
Monoclonal
When fusion does not occur, neuraminidase can destroy the virus receptor and allow the cells to disaggregate. The rate of release of HN-2 antibody-treated HVJ was the same as that of control virus (fig. 5b). After 30 min about half of the virus initially bound remained associated with the cells (fig. 5a). This virus may be bound to cells via a site other than the receptor-binding site, despite being unable to fuse with cell membranes, due to the presence of HN-2 antibody. In the assay of cell-fusing and hemolytic activities, HN-2 antibody inhibited tile activity almost as much as did HN-1 antibody. Moreover, in the cytotoxic assay of fragment A-containing liposomes with HVJ spikes (table l), HN-2 antibody blocked the cytotoxicity of the liposomes, but to a lesser extent than HN-1 antibody. These findings could be explained as follows. The liposomes bound with HN-2 antibodies may associate with L cells without fusing with the cell membrane. Some of the bound liposomes may enter the cells by endocytosis at 37°C and, in a few cases, fragment A may be released from the endocytic vesicles into the cytoplasm and kill the cells. The present work showed that HN-2 antibody, which did not inhibit cell aggregation or neuraminidase activity, inhibited cellcell fusion, envelope-cell fusion and hemolysis. The mechanism by which HN-2 antibody blocked the fusion process is not known, but there are four possible mechanisms. First, a second site on the HN molecule, distinct from the binding site, may be involved in the fusion process. HN2 antibody may block the second site. Second, an unknown function other than hemagglutination activity may be impaired due to a conformational change of HN protein and the fusion process may not occur. In these two cases HN protein would play some important roles in the fusion process
antibodies
against HVJ HN protein
4 19
besides being an adsorbing agent and neuraminidase. Third, binding of HN-2 antibody may block the cooperative function of HN protein and F protein. However, Huang [5] recently found that liposomes containing HVJ F protein and influenza HA+NA proteins fused with cell membranes. Because it is expected that proteins from different viruses have no specific interactions between them, this finding suggests that the third possibility is less probable. Fourth, although HN-2 antibody binds to HN protein, it may interfere sterically with the expression of F protein. In our preliminary experiment, fragment A-containing liposomes with HN protein can kill selectively SSPE (Subacute Sclerosing Panencephalitis) cells. These cells contain F protein of measle virus in the cell membrane. HN-2 antibody blocked the cytotoxicity of the liposomes containing HN protein. In this case steric hindrance of F protein by HN-2 antibody bound to HN protein seems unlikely to account for the inhibition of fusion. For understanding the relation between the structure and the function of glycoproteins of HVJ, it is necessary to obtain more monoclonal anti-HN protein antibodies and many monoclonal anti-F protein antibodies. Use of these monoclonal antibodies, in combination with CNBr-fragmented peptides or proteolytic fragments of the proteins, should clarify the structure and the function of the proteins. This work was supported by research grants from the Ministry of Education of Japan and IBM Japan.
REFERENCES 1. Aminoff, D, Biochem j 81 (1961) 384. 2. Choppin, P W & Scheid, A, Rev infect dis 2 (1980) 40. Exp
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Res 141 (1982)
3. Greenwood, F C, Hunter. W H & Glover. .I S. Biochem j 89 (1963) 114. 4. Homma. M & Ohuchi, M, J virol 12 (1973) 1457. 5. Huang, R T, Rott, R, Wahn, K, Klenk, H D & Kohama, T, Virology 107 (1980) 313. 6. Kennett, R H, Denis, K A, Tung, A S & Klinman, N R, Curr top microbial immunol81 (1978) 77. 7. Kohler, G & Milstein, C, Nature 256 (1975) 495. 8. Laemmli, U K, Nature 227 (1970) 680. 9. Miyake, Y, Kim, J & Okada, Y. Exp cell res 116 (1978) 167. 10. Nakanishi, M, Uchida, T, Kim, J & Okada, Y, Exp cell res 142 (1982) 95. 11. Okada, Y, Koseki, I, Kim, J, Maeda, Y, Hashimoto, T, Kanno, Y & Matsui, Y, Exp cell res 93 (1975) 368. 12. Okada, Y & Tadokoro, J, Exp cell res 26 (1962) 108.
Exp Cell
Res 141 (1982)
13. Salk. J E, J immunol49 (1944) 87. 14. Scheid, A, Caliguiri, L A, Compans, R W & Choppin, P W, Virology 50 (1972) 640. 15. Scheid, A & Choppin, P W, J virol 11 (1973) 263. 16. - Virology 57 (1974) 475. 17. Shulman. M, Wilde, C D & Kohler, G, Nature 276 (1978) 269. 18. Staehelin, T, Durrer, B, Schmidt, J, Takacs, B, Stocker. J, Miggiano, V, Stahli, C, Rubinstein, M, Levy, W P, Hershberg, R & Peska, S, Proc natl acad sci US 78 (1981) 1848. 19. Tozawa, H, Watanabe, M & lshida, N, Virology 55 (1973) 242. 20. Uchida, T, Kim, J, Yamaizumi, M, Miyake, Y & Okada, Y, J cell biol80 (1979) 10. Received January 8, 1982 Accepted April 7, 1982
Printed
in Sweden
Experimental
Cell Research
141 (1982) 409-420
HVJ (SENDAI VIRUS)-INDUCED ENVELOPE FUSION AND CELL FUSION ARE BLOCKED BY MONOCLONAL ANTI-HN PROTEIN ANTIBODY THAT DOES NOT INHIBIT HEMAGGLUTINATION ACTIVITY OF HVJ NAOYUKI
MILJRA,’
‘Institute for Molecular and ‘Department
TSUYOSHI
UCHIDA’
2 and YOSHIO OKADAL 2
and Cellular Biology, Osaka University, Suita, Osaka of Cell Fusion, National Institute for Basic Bidlogy, Okazaki. Aichi 444. Japan
565,
SUMMARY Two kinds of monoclonal antibodies aeainst HN orotein of HVJ were isolated. In comoetitive binding assay. binding of one of these altibodies to’HN protein did not inhibit binding of the other antibody to the same molecule. One of the antibodies. named HN-1 antibodv. inhibited hemagghitination activity of HVJ and also blocked neuraminidase activity of the viruswhen fetuin and Ehrlich ascites tumor cells were used as substrates, but it did not inhibit the activity when neuramine-lactose.was used as substrate. The other antibody, HN-2, did not inhibit hemagglutination activity or netiraminidase activity, but blocked HVJ-induced viral envelope-cell fusion, cell-cell fusion and hemolysis. The mechanism by which HN-2 antibody blocked the fusion process is discussed.
The envelope of HVJ (Sendai virus) contains two glycoproteins, HN and F, which form projections from the surface of the viral membrane. These glycoproteins are involved in the interactions between the virus and the host cell. The.HN protein has hemagglutination and neuraminidase activities and is responsible for adsorption of the virus to the host cell. The other glycoprotein, F, is involved in virus-induced cellcell fusion, viral envelope-cell fusion and hemolysis. The fusing activity of the F protein is activated by proteolytic cleavage of a precursor (FO) [4, 161. HVJ induces two kinds of membrane fusions. One is fusion between the viral envelope and the cell membrane and the other is fusion between two cell membranes. Cell-cell fusion does not occur when viral
envelope-cell fusion does not occur, i.e., when HVJ is incubated at 4°C or HVJ containing F,, protein is used. Moreover, cellcell fusion occurs rapidly after viral envelope-cell fusion has occurred, but it is separable from the latter: Cytochalasin D or 0.25 M sucrose inhibits cell-cell fusion, but not viral envelope-cell fusion [9]. So it is considered that cell-cell fusion requires viral envelope-cell fusion. The function of HN protein in the fusion process is not well understood. Its only known action is to bind to sialic acids of the receptor on the host cell and aggregate cells. To obtain further information on the function of HN protein, we attempted to obtain monoclonal antibodies against HN protein of HVJ by the hybridoma technique of Kohler & Milstein [7]. Exp Cell
Res
141 (1982)
410
Miura,
Uchidu and Oliadu
In this paper we isolated two hybridomas against HN protein of HVJ and investigated the effects of their monoclonal antibodies on the biological functions of HVJ. One monoclonal antibody inhibited hemagglutination and neuraminidase activities completely, whereas the other antibody did not block these activities at all. However, the latter antibody inhibited HVJ-induced cellcell fusion, envelope-cell fusion and hemolysis. The mechanism by which HN-2 antibody blocked the fusion process is discussed.
MATERIALS
AND METHODS
Cells S&!/O-Ag14 myeloma cells [17] and MPCll-45.6 TG1.7 myeloma cells were cultured in Dulbecco’s moditied Eagle medium (DMEM) supplemented with 15% fetal calf serum (FCS) (Flow Lab.). LLC-MK, cells and L cells were maintained in Eagle’s minimum essential medium containing 10% calf serum.
Virus Hemagglutinating virus of Japan (HVJ, Sendai virus), Z strain. was grown in the allantoic fluid of IO-dayold fertihzed eggs. The virus was purified by differential centrifmzation. as described oreviouslv rl11. The virus from theallantoic fluid contams F,, 2 protein, but the virus from the culture medium of LLC-MK, cells infected with HVJ contains F, protein.
PuriJication of viral proteins The HN protein and the F protein were purified by DEAE-cellulose and CM-cellulose chromatography by the method of Nakanishi [LO]. The M protein was ouritied as described previously r151. HVJ whole antigens were obtained by lysing- puri&ed HVJ with 10 mM Tris-HCI (pH 8.3). 0.5 M KCI, 1% Nonidet P-40 and removing aggregates by centrifugation at 1OOOOOgfor60min.
Immunization Purified HVJ was sonicated with a Sonifiep Cell Disruptor (model W185, Ultrasonics Inc., New York) for 5 min. BALB/c mice, 8 weeks old, were injected intraperitoneally with 0.2 mg of sonicated virus in Freund’s complete adjuvant. One month later, 0.2 mg of the virus in saline was injected intravenously into each Exp
Cell
Res 141 (1982)
mouse. Three days later the spleen cells were fused with SP2/0 cells.
Fusion and cloning Spleen cells (2x 10R)from immunized mice were fused with SP2/0 cells (2x10') as follows. The two cell populations were mixed and precipitated by centrifugation at 300 g for 5 min in a 50 ml centrifuge tube. To the pellet 0.5 ml of 50% (w/v) polyethylene glycol 4000 and I5 % (v/v) dimethylsulfoxide in DMEM was added. and the tube was rolled gentlv. The concentration of polyethylene glycol was Fhen gradually reduced bv adding 1 ml of DMEM every 30 set for 5 min with gentle roiling of the tube. _ After fusion, the cells were seeded into 96 wells of tissue culture plates and cultured in HAT (1 x IO-’ M hypoxanthine, 4~10~’ M aminopterin, 1.6~10~” M thymidine) selection medium. Of 193 initial hybridoma cultures. 140 mew well within 10 davs. Cells were grown for a toLl of 14 days in HAT medium and then eraduallv adaoted to DMEM with 15% FCS bv oassage for- 7 days in medium without aminopterin.‘but containing hypoxanthine and thymidine. Cells secreting antibodies were cloned in soft agar (0.24%. Seakem LE agarose) at least twice.
Screening for hybridomas by a solidphase antibody-binding assa) Solid-phase antibody-binding assay was carried out by the method of Kennett [6] and Staehelin [ 181 with some modifications. For coating polyvinyl chloride microtiter plates (Dvnatech Laboratories Inc., Alexandria, Va). 100 /.LI of -HN protein, F protein, M protein or HVJ whole antigen solution (about 0.3 ma/ml) was placed in each welland kept at room temperature overnight. The next day the wells were washed and tilled with 120 ~1 of 2 % horse serum in borate-buffered saline (0.15 M NaCl, 0.03 M H,BO,. DH 8.0) and kept at room temperature overnight-to block all protein-butding sites. The plates were washed four times with saline. Hybridoma culture supernatants (50 ~1) were incubated in duplicate wells at room temperature overnight. The plates were then washed four times with saline, and then 100 ~1 of ‘““I-labelled rabbit antimouse Fab antibody (100 000 cpm; 200 ng of antibody in borate-buffered saline containing 0.2% bovine serum albumin) was added to well. After 12 h the plates were washed four times with saline and cut into individual wells, and the radioactivity of each well was counted in a gamma counter (Beckman Gamma 9000). Anti-HN hybridoma was obtained by screening the culture supernatant by solid-phase binding assay to HN protein. Anti-NP hybridoma was obtained as follows. The supernatant which gave a positive reaction on binding assay to HVJ whole antigens was screened by binding assay to HN protein, F protein or M protein. The supematant that reacted with HVJ whole antigens, but not with HN protein, F protein or M protein, was considered to contain either anti-NP protein antibody or anti-P protein antibody. Anti-NP hybridoma was obtained by the radioimmunoprecipitation method from the clones producine this antibody.
Monoclonal Ascites Cloned hybrid cells were cultured on a large scale and 2x lofi cells were injected into the peritoneal cavity of BALB/c mice that had received 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chemical Co.) intraperitoneally 2 weeks previously. After 7-10 days the ascitic fluid was harvested.
Purijication
of monoclonal antibodies
Monoclonal antibodies were purified with HN proteinconjugated Sepharose 49 for anti-HN hybridoma, with HVJ whole antigens-conjugated Sepharose 49 for antiNP hybridoma and with Protein A-Sepharose CL-49 (Pharmacia) for MPC 11 myeloma.
Isoelectric focusing Isoelectric focusing of purified antibodies was done in a 5 % polyacrylamide slab gel containing 2.4 % Ampholine (pH 3.5-9.5) using an LKB multiphor apparatus at a constant power of 15 W for 3 h.
Preparation of labelled viral antigens LLC-MK2 cells were infected with HVJ for 60 min. Then the virus-infected cells were cultured at 37°C in Eagle’s minimum essential medium containing 10% calf s&urn and 10 &i of L-[3”S]methionine (1 160 Ci/ mmol; Amersham) per ml. The medium was harvested after 72 h and virus was purified as described above. Labelled virus was lvsed in 0.01 M Tris-HCI (pH 8.3). 0.5 M KCI, 1% Nonidet P-40 for 30 min at 4°C. Insoluble aggregates were removed by centrifugation at 100000 g for 60 min. The soluble labelled viral proteins were used in immunoprecipitation assay.
Radioimmunoprecipitation and sodium dodecyl sulfate (SDS)-polyawlamide gel electrophoresis The monoclonal antibody was added to labelled viral antigen solution (2OOOOt!lcpml0.5 ml). The reaction mixture was incubated at 4°C for 12 h. Then 30 ~1 of Protein A-Sepharose CL-49 was added and incubation was continued at 4°C for 4 h. Sepharose was washed three times with saline containing 1% Nonidet P-40 and then 50 ~1 of sample buffer containing 0.063 M Tris-HCI, pH 6.8, 3% SDS, 10% glycerol and 50 mM dithiothreitol was added. The supernatant was subjected to electrophoresis in a 10% polyacrylamide SDS gel as described by Laemmli [8]. The gel was dried and exposed to Kodak X-Omat S tilm for 2 days.
Radio-iodination of antibodies and HVJ Antibodies and HVJ were radioiodinated with chloramine T [3]. Labelled antibody was separated from free iodide by Sephadex G-25 chromatography and dialysed against BSS overnight. The specific activities of HN-1, HN-2 and NP-1 antibodies were 2.7~ 10” cpm/ mg, 3.0~ 10” cpmlmg and I .4x lo9 cpm/mg, respective-
antibodies
against HVJ HN protein
411
ly. Labelled HVJ was separated from free lz51 by BioGel A-150m (SO-100 mesh, Bio-Rad Lab.) chromatography and dialysed against BSS overnight. The specific activitv of HVJ was 4.2~ 103 cum/HAU and the hemagglutination and cell-fusing activities of HVJ were not changed by radio-iodination.
Competitive binding assay of monoclonal antibody to the antigenic determinant on the HN molecule of HVJ A mixture of 200 ng of ““I-1abelled antibody and 2000 HAU HVJ in 0.2 ml of BSS containing 0.2% bovine serum albumin was incubated at 37°C for 60 min and then layered on 4 ml of 10% (w/v) sucrose in BSS. The mixture was centrifuged in a Hitachi SW-50 rotor at 40000 rpm for 60 min and the radioactivities of the precipitate and the supernatant were measured with a gamma counter. For competitive binding assay, 200 HAU of HVJ in 0.1 ml of BSS was incubated with 0.1 ml of BSS or BSS containing excess (lOO-fold the saturation dose) cold antibody at 37°C for 60 min. Then ‘251-labelled antibody was added and the mixture was incubated at 3PC for 60 min further. The solution was layered on 4 ml of 10% sucrose in BSS and centrifuged as mentioned above.
Assay of hemagglutination units (HAU) and hemagglutination inhibition (HA!) titer Salk’s pattern method [ 131 was used. The virus sample was mixed with an equal volume of 0.5% (v/v) chick red blood cell suspension and after several hours the titer was determined. In assay of HAI. antibody solution at a maximum concentration of 1 mg/ml was diluted 2-fold serially. Then 2 HAU/ml of HVJ was added to the antibody solutions and the mixtures were incubated at room temperature for 5 h. Then an equal volume of red blood cell suspension was added. The hemagglutination inhibition (HAI) titer was expressed as the degree of dilution of antibody that inhibited HVJ-induced hemagglutination at 1 HAU/ml.
Assay of neuraminidase activity The virus sample was mixed with the substrate and incubated at 37°C for 30 min. Then the reaction was stopped by boiling the mixture for 1 min. The substrates used were (a) Ehrlich ascites tumor (EAT) cells, 4x 107/ml; (b) fetuin (Difco), 8 mglml; and (c) N-acetyl neuramine (NANAtlactose ( Sigma), 0.34 mg/ml. The reaction was performed at pH 7.5 and pH 6.0. Sialic acid was determined by the method of Aminoff[l].
Kinetics of adsorption and release of HVJ ‘z”I-Labelled ?IVJ (32 HAU in 0.4 ml) was mixed and incubated with 1.2 ml of 10% (v/v) EAT cell suspenExp Cell
Res
141 /I9821
412
Miura,
Uchida and Okada
a basic c-
acidic -
Labeled antibody added(x 10” cpm)
b 1
2
3
4
5
Fig. 2. Competitive binding assay of monoclonal antibodies to the antigenic determinants on HN protein of HVJ. 200 HAU of HVJ was preincubated with BSS (0 or A), excess cold HN-1 antibody (0) or HN-2 antibody (A) and then incubated with various doses of 1251-labelled (a) HN-I antibody, or (b) HN-2 antibody. HVJ was precipitated by centrifugation and the radioactivity of bound antibody was measured. Then the mixtures were centrifuged at 20@0 rpm for 2 min at 4°C and pelleted ceils were washed once and suspended in 2 ml of BSS containing I mM CaCI,. The suspensions were incubated at 3PC and 300 ~1 samples were taken at the indicated times and centrifuged at 2000 rpm for 2 min at 4°C. Pelleted cells were washed once with BSS and the two supematants were combined. Radioactivities of cell-associated and released virus were measured in a gamma counter.
Cell fusion
Fig. 1. (a) Isoelectric focusing pattern (pH 3.5-9.5) of purified monoclonal antibodies. A, B, C. results with HN-1, HN-2 and NP-1 antibodies. (b) Autoradiogram of SDS-polyacrylamide electrophoregram of immunoprecipitation with [35S]methionine-labelled viral proteins. I, HVJ marker proteins: 2, no antibodies added; 3, HN-I antibody: 4. HN-2 antibody; 5, NP-I antibody. sion in BSS containing I mM CaCI, at 4°C. At the indicated times, 300 ~1 samples were taken and centrifuged at 2000 rpm for 1 min at 4°C. Pelleted cells were washed once with BSS at 4°C and the radioactivity of cell-associated HVJ was counted in a gamma counter. One ml of HVJ (““I-HVJ and non-labelled HVJ; final titer 500 HAtJ/ml) was mixed with 1 ml of 10% EAT cell suspension in BSS containing 2 mM CaCI, and the mixtures were incubated at 4°C for 15 min. Exp Cell
Res 141 (1982)
Ehrlich ascites tumor cells were washed [ 1 I] and suspended in 9 vol of balanced salt solution (BSS) (140 mM NaCI, 5.4 mM KCI, 0.34 mM Na,HPO,, 0.44 mM KH,PO,, 10 mM Tris-HCI pH 7.5). A mixture of 0.4 ml of 10% (v/v) EAT cell suspension in BSS containing 2 mM CaCI, and 0.4 ml of HVJ sample (500 HAU/ml) was incubated at 4°C for 10 min and then at 37°C for 3C min with shaking in a water bath.
Hemolytic
activity
A mixture of I ml of virus sample and 2 ml of 2% (v/v) chick red blood cell suspension was kept at 4°C for 10 min and then incubated at 37°C for 60 min without agitation. After incubation, the mixture was centrifuged at 2000 rpm for IO min and the optical density of the supernatant at 540 nm was measured spectrophotometrically.
Cytotoxic assay of liposomes HVJ spikes
with
The formation of fragment A-containing liposomes with HVJ spikes and cytotoxic assay of the liposomes were done, as described previously [20].
Monoclonal
antibodies
200
.
a
against HVJ HN protein
413
I
C
1
f-
.
.
.
:,
.
t.zr .Ol
.,
1.0
10
.01
:1
1:o
lb
Antibody concentration(yg/ml) 3. Effects of monoclonal antibodies on HVJassociated neuraminidase activity. HVJ ((a) 250 HAU/ ml; (6) 64 HAU/ml; (c) 128 HAU/ml) was preincubated with BSS (control) or various concentrations of 0, HN-1 antibody; A, HN-2 antibody; or n , NP-1 antibody, and then mixed with (a) EAT cells (4x lO’/ml) at pH 7.5; (6) fetuin (8 mg/ml) at pH 6.0; or
Fig.
RESULTS Isolation of two monoclonal antibodies against HN protein of HVJ Hybridomas produced by fusion of SP2/0 cells with splenic lymphocytes from mice immunized with sonicated HVJ might produce antibodies to any one of the viral proteins. Therefore, it was necessary to identify the antigen to which the hybridoma produced antibodies. Of thirty hybridomas, two hybridomas producing anti-HN protein antibodies (named HN-1 and HN-2) and one hybridoma producing anti-NP protein antibody (named NP-1) were obtained. After at least two clonings, the cells were injected intraperitoneally into BALB/c mice. The monoclonal antibodies were purified from the ascitic fluid by affinity chromatography, as described in the Materials and Methods. Fig. la shows the pattern of isoelectric focusing of the three purified antibodies. Each gave the characteristic pattern of monoclonal protein. The specificities of these antibodies were determined by radioimmunoprecipitation and polyacrylamide
(c) NANA-lactose (0.34 mg/ml) at pH 6.0. The reaction mixture was incubated at 37°C for 30 min, and free sialic acid was determined by the method of Aminoff [ll. The OJh9,, of the controls were (a) 0.23; (b) 0.64; and (c) 0.51 respectively. Each control was taken as 100%.
gel electrophoresis analysis. As shown in fig. 1 b , HN protein was specifically precipitated by HN-1 and HN-2 antibodies (fig. lb, 3; 4) and NP protein was precipitated by NP-1 antibody (fig. lb, 5). However, under these conditions, M protein was precipitated non-specifically (fig. 1b , 2). When L251-labelled glycoproteins of HVJ purified from the chorioallantoic fluid of hen eggs were used as antigens, the autoradiogram showed that HN-1 and HN-2 antibodies bound to HN protein, but not to F,, 2 protein (data not shown). Ouchterlony analysis and binding to protein A showed that HN-1, HN-2 and NP-1 antibodies were IgG (data not shown). To examine the functional uniformity of the purified antibodies, the antibodies were radioiodinated with [‘251]Na. ‘251-Labelled monoclonal antibodies were then treated with excess HVJ at 37°C for 60 min and bound antibodies were separated from unbound antibody by centrifugation. The ratio of bound antibody to total antibody was calculated. The ratios for HN- 1, HN-2 and NP-1 antibodies at pH 7.5 were 90, 95, and Exp
Cell
Res
141 (1982)
414
Miura,
Uchida and Okada
4%. respectively. Those for HN-1 and HN2 antibodies at pH 6.0 were 85 and 90%, respectively. Competitive binding assay was used to determine whether the monoclonal antibodies were directed toward the same or different antigenic determinants on the HN molecule of HVJ. HN-2 antibody bound to the HN molecule of HVJ did not inhibit binding of HN- 1 antibody to the HN protein (fig. 2a) and HN-1 antibody bound to HN protein only partially inhibited the binding of HN-2 antibody (fig. 2b). These tindings suggest that HN-1 and HN-2 antibodies were directed toward different antigenic determinants. Effects of monoclonal antibodies on hemagglutination activity of HVJ One of the known functions of HN protein is hemagglutination activity. The hemagglutination inhibition (HAI) titer by antiHN antibody was determined by the conventional Salk’s pattern method, as described in the Materials and Methods. The titer of HN- 1 antibody was 16 000, whereas those of HN-2 and NP-1 antibodies were less than 1. These results indicated that HN- 1 antibody inhibited hemagglutination activity of HVJ, whereas HN-2 and NP-1 antibodies did not. Effects of antibodies on newaminidase activity of HVJ Another function of HN protein is neuraminidase activity. Enzymatic activity was determined at pH 7.5 and pH 6.0 with three different substrates. After preincubation of HVJ with buffer or the antibody at 4°C for 5 h, the substrate was added and the reaction mixture was incubated at 37°C for 30 min. Fig. 3 shows the percent activity of neuraminidase at various concentrations of Exp
Cell
Res 141 (1982)
.-0 -0 a
a 0
Time(min) Kg. 4. Time course of HVJ adsorption to EAT cells. ‘*“I-Labelled HVJ (80 HAU/ml) was preincubated with BSS (0, control), HN-1 antibody (0, 30 r.&ml), HN-2 antibody (A, 30 &ml) or NP-I antibody (W. 30 pg/ ml) at 4°C for 5 h. The HVJ-antibody solution (0.4 ml) was added to I.2 ml of 10% (v/v) EAT cell susoension in BSS containing 1 mM CaCI, and the mixture was incubated at 4°C. At the indicated times, 300 ~1 samples were taken and centrifuged at 2000 rpm at 4°C. Cell-associated radioactivity was counted in a gamma counter.
antibodies. The neuraminidase activity at pH 6.0 was about twice that at pH 7.5, but the percentage activities of the control with the three antibodies were the same at pH 7.5 and pH 6.0. When EAT cells or fetuin was used as a substrate (fig. 3a, b), HN-1 antibody at a concentration of more than 3 pg/ml inhibited the neuraminidase activity completely, whereas HN-2 antibody and NP-1 antibody did not affect the activity. But when neuramine-lactose was used as a substrate, HN-1 and HN-2 antibodies increased the activity (fig. 3~). As a control, HVJ was preincubated with antibody at 4°C and then incubated at 37°C for 30 min without substrates. We confirmed that under these conditions anti-HN antibody itself was not used as substrate (data not shown).
Monoclonal a
antibodies
against HVJ HN protein
415
b
i
30
so
9’0
Time(min) Fig. 5. Time course of cell-associated and released HVJ. ‘9-Labelled HVJ (500 HAU/ml) was preincubated with BSS (0, control), HN-1 antibody (0, 3Oc(g/ ml), HN-2 antibody (A, 30 pg/ml) or NP-1 antibody (W, 30 pg/ml) at 4°C for 5 h. One ml of the HVJantibody solution was added to 1 ml of 10% (v/v) EAT cell suspension and the mixture was incubated at 4°C for 15 min. The mixture was centrifuged at 2000
rpm for 2 min and pelleted cells were washed once and suspended in BSS containing 1 mM CaCl, and then incubated at 3PC. At the indicated times, 300 ~1 samples were taken and centrifuged at 2 000 rpm for 1 min and pelleted cells were washed once with BSS. TCAprecipitable counts in (a) the pellet (cell-associated HVJ); (b) the supematant (released HVJ) were measured in a gamma counter.
Effects of antibodies and release of HVJ
From the described experiments, we ascertained that HVJ treated with HN-1 antibodies did not bind to cells and that virus treated with HN-2 antibodies adsorbed to cells well and was not released rapidly.
on adsorption
First, to examine the effect of monoclonal antibodies on HVJ adsorption to a cell, ‘251-labelled HVJ was preincubated with antibodies and then mixed with EAT cells. The mixtures were incubated at 4°C. Fig. 4 shows time course of HVJ adsorption to cells. HN- 1 antibody inhibited adsorption of HVJ to cells completely. But HN-2 and NP-1 antibodies did not inhibit the adsorption. Next, to examine the effect of antibodies on HVJ release from a cell, 1251-labelled HVJ was preincubated with antibodies and then mixed with EAT cells. After the mixtures were incubated at 4°C for 15 min, cells were washed and suspended and then incubated at 37°C. Fig. 5 shows time course of cell-associated and released HVJ. When HVJ was bound with HN-2 antibody, cellassociated HVJ decreased and virus was released at almost the same rate as in control.
Table 1. Eflects of antibodies on cytotoxicity offragment A-containing liposomes with HVJ spikes
Sample
No. of surviving colonies per dish
Buffer Liposomes Liposomes+ HN-1 antibody Liposomes+HN-2 antibody Liposomes+MPCIl antibody
260 33 214 162 54
Fragment A-containing
liposomes with HVJ spikes were formed as described previously 1201. After preincubation with BSS or antibody (30 @g/ml), the liposome solutions were incubated with L cells, as described in the text. MPC 11 monoclonal protein was used as control antibody because liposomes with HVJ spikes were contaminated with NP protein. The liposome suspension had 400 nglml fragment A and 400 HAU/ml. Colony counts are averages of those in duplicate plates. Exp Cell
RPS 141 f/982)
Miura,
4 16
Uchida
and Okada
Fig. 6. Photographs of HVJ-induced cell fusion in the absence and presence of antibodies. HVJ (500 HAU/ ml) was preincubated with (n. b) BSS: (c. d) HN-I antibody: (e.Jt HN-2 antibody or (p. /I) NP-I antibody at a concentration of IO pglml, and then an equal Exp
Cell
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141 (1982)
volume of 10% EAT cells (2x107/ml) H /as a.dded . When the suspensions were incubated at 4°C ‘for 1!O min (u, c, e. g). EAT cells were aggregated (a. (?. g). Then the mixtures were incubated at 37°C for 30 min (b, d. ,fi h) and fused cells appeared (b. /I). x 144.
Monoclonal
antibodies
against HVJ HN protein
417
.
O/ 0.1 Ani%dy
2
1I
OI .ol
'I,
.l
1.0
10
h-fthdy concantratior$g/mD Fig. 7. Effects of monoclonal antibodies on the fusion index. HVJ (500 HAU/ml) was preincubated with various concentrations of l , HN-1 antibody, A, HN-2 antibody; or n , NP-1 antibody. Then EAT cells were added and the mixtures were incubated as described for fig. 6. Total cell numbers after incubation were counted and the fusion index was calculated from the following formula [ 121 Fusion index=
Number of cells in control without virus -1.0. Number of cells after fusion
Effects of antibodies on HVJ-induced cell-cell fusion The cell-cell fusion induced by HVJ is attributed to two glycoproteins, HN and F proteins. To examine whether the antibodies affect the HVJ-induced cell-cell fusion, we incubated HVJ with the antibody at 4°C for 5 h and then added EAT cells to the reaction mixture. The mixture was kept at 4°C for 10 min, and then incubated at 37°C for 30 min with shaking. Fig. 6 shows the appearance of cells after incubation at 4°C for 10 min and after incubation at 37°C for 30 min. HVJ induced cell aggregation on incubation at 4°C (fig. 6a) and fused cells appeared on incubation at 37°C (fig. 66). NP-1 antibody (10 pg/ml) did not affect cell aggregation or cell fusion
10 1.0 .z.mmbation(rglmO
Fig. 8. Effects of monoclonal antibodies on HVJinduced hemolysis. HVJ (500 HALJ/mJ) was preincubated with BSS (control) or various concentrations of 0, HN-1 antibody; A, HN-2 antibody: or n , NP-1 antibodv. Then 2 vol of 2 % chick red blood cells were added. *The mixture was incubated at 4°C for 10 min and then at 37°C for 60 min. and then centrifuged. The optical density of the supernatant at 540 nmwas measured spectrophotometrically. The OD,,,, of the control ofU.28 was taken as 100%.
(fig. 6g, h). HN-1 antibody inhibited both cell aggregation and cell fusion (fig. 6c, d), and HN-2 antibody did not block cell aggregation, but inhibited cell fusion (fig. 6e, f). Fig. 7 shows the fusion indices at various concentrations of antibodies. Clearly, HN-1 and HN-2 antibodies at concentrations of more than 3 pg/ml blocked cell fusion, but NP-1 antibody did not inhibit cell fusion. HN-1 antibody blocked cell fusion by inhibiting cell aggregation, but HN-2 antibody inhibited cell fusion by another mechanism. Effects of antibodies on HVJ-induced hemolysis It is well known that HVJ has hemolytic activity, so the effects of monoclonal antibodies on HVJ-induced hemolysis were examined. HVJ was preincubated with various concentrations of antibodies and then red blood cells were added and the reaction mixtures were incubated at 37°C for 60 min. HN-1 and HN-2 antibodies clearly inhibited the hemolytic activity as well as the cellfusing activity (fig. 8). Exp Cell
Res
141 (1982)
4 18
Miura,
Uchida and Okadu
Effects of the antibodies on erl\?elopecell fitsion As shown in the described experiments, HVJ treated with HN-2 antibodies was associated with EAT cells to the same extent as control (fig. 5a). The next question was whether when HVJ bound with HN-2 antibodies, envelope-cell fusion could occur. Liposomes with HVJ spikes can fuse with the cell membrane efficiently by envelopecell fusion [20]. Fragment A of diphtheria toxin introduced into the cytoplasm can kill cells, so fragment A-containing liposomes with HVJ spikes were formed and preincubated with the monoclonal antibodies at 4°C for 5 h. Assay of the cytotoxicity of the liposomes to L cells was performed, as described in Materials and Methods. Table 1 shows the results. MPC 11 monoclonal antibody slightly inhibited the cytotoxicity of the liposomes, but HN-1 and HN-2 antibodies inhibited the toxicity 80 % and 57 %, respectively. This finding indicates that HN-2 antibody inhibited envelope-cell fusion.
DISCUSSION In this work we examined the effects of two monoclonal antibodies on HVJ-associated biological functions. The two monoclonal antibodies were directed toward different antigenic determinants on the HN molecule (fig. 2). We found that HN-1 antibody inhibited hemagglutination activity of HVJ completely, but that HN-2 antibody did not block the activity at all. We then tested the inhibition of HVJ neuraminidase by antiHN antibody for dependence on substrate size using monoclonal antibodies (fig. 3). HN-1 antibody inhibited neuraminidase activity with EAT cells or fetuin, but not with neuramine-lactose as substrate. This findExp Cell
Res 141 (1982)
ing suggests that steric factors accounted for the inhibition by the antibody. Moreover, HN-1 antibody increased the activity with neuramine-lactose as substrate. And HN-2 antibody increased the activity with the small substrate to a great extent. The enhancement of activity by binding of the antibody to HN protein might be explained by a conformational change in HN protein. However, HN-2 antibody did not increase the activity with macromolecular substrates, so in the case of HN-2 antibody, the enhancement of activity was counteracted by steric hindrance of substrate. From these results we assume that the antigenie site recognized by HN- 1 antibody was near the center for hemagglutination activity and neuraminidase activity, whereas that recognized by HN-2 antibody was remote from the center. It is known that HN protein is responsible for virus adsorption to a host cell [2, 14, 15, 191 and for virus release from the cell. We examined whether HVJ bound with HN-2 antibodies adsorbed cells slowly or whether it was released rapidly from the cells. We ascertained that when bound with HN-2 antibodies, HVJ showed neither delayed adsorption nor accelerated release (figs 4, 5) and therefore it had the same chance to fuse with a cell as did intact HVJ. Then we examined the effects of antibodies on HVJ-induced cell-cell fusion, envelope-cell fusion and hemolysis and showed that HN-2 antibody inhibited cellcell fusion, envelope-cell fusion and hemolysis. In the cell fusion experiment (fig. 6), HVJ treated with HN-2 antibody could aggregate cells at 4°C but could not induce cell fusion after warming to 37°C. Moreover, after 30 min of incubation at 37°C the cells were completely disaggregated (fig. 6f). HVJ treated with HN-2 antibody still had neuraminidase activity (fig. 30).
Monoclonal
When fusion does not occur, neuraminidase can destroy the virus receptor and allow the cells to disaggregate. The rate of release of HN-2 antibody-treated HVJ was the same as that of control virus (fig. 5b). After 30 min about half of the virus initially bound remained associated with the cells (fig. 5a). This virus may be bound to cells via a site other than the receptor-binding site, despite being unable to fuse with cell membranes, due to the presence of HN-2 antibody. In the assay of cell-fusing and hemolytic activities, HN-2 antibody inhibited tile activity almost as much as did HN-1 antibody. Moreover, in the cytotoxic assay of fragment A-containing liposomes with HVJ spikes (table l), HN-2 antibody blocked the cytotoxicity of the liposomes, but to a lesser extent than HN-1 antibody. These findings could be explained as follows. The liposomes bound with HN-2 antibodies may associate with L cells without fusing with the cell membrane. Some of the bound liposomes may enter the cells by endocytosis at 37°C and, in a few cases, fragment A may be released from the endocytic vesicles into the cytoplasm and kill the cells. The present work showed that HN-2 antibody, which did not inhibit cell aggregation or neuraminidase activity, inhibited cellcell fusion, envelope-cell fusion and hemolysis. The mechanism by which HN-2 antibody blocked the fusion process is not known, but there are four possible mechanisms. First, a second site on the HN molecule, distinct from the binding site, may be involved in the fusion process. HN2 antibody may block the second site. Second, an unknown function other than hemagglutination activity may be impaired due to a conformational change of HN protein and the fusion process may not occur. In these two cases HN protein would play some important roles in the fusion process
antibodies
against HVJ HN protein
4 19
besides being an adsorbing agent and neuraminidase. Third, binding of HN-2 antibody may block the cooperative function of HN protein and F protein. However, Huang [5] recently found that liposomes containing HVJ F protein and influenza HA+NA proteins fused with cell membranes. Because it is expected that proteins from different viruses have no specific interactions between them, this finding suggests that the third possibility is less probable. Fourth, although HN-2 antibody binds to HN protein, it may interfere sterically with the expression of F protein. In our preliminary experiment, fragment A-containing liposomes with HN protein can kill selectively SSPE (Subacute Sclerosing Panencephalitis) cells. These cells contain F protein of measle virus in the cell membrane. HN-2 antibody blocked the cytotoxicity of the liposomes containing HN protein. In this case steric hindrance of F protein by HN-2 antibody bound to HN protein seems unlikely to account for the inhibition of fusion. For understanding the relation between the structure and the function of glycoproteins of HVJ, it is necessary to obtain more monoclonal anti-HN protein antibodies and many monoclonal anti-F protein antibodies. Use of these monoclonal antibodies, in combination with CNBr-fragmented peptides or proteolytic fragments of the proteins, should clarify the structure and the function of the proteins. This work was supported by research grants from the Ministry of Education of Japan and IBM Japan.
REFERENCES 1. Aminoff, D, Biochem j 81 (1961) 384. 2. Choppin, P W & Scheid, A, Rev infect dis 2 (1980) 40. Exp
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3. Greenwood, F C, Hunter. W H & Glover. .I S. Biochem j 89 (1963) 114. 4. Homma. M & Ohuchi, M, J virol 12 (1973) 1457. 5. Huang, R T, Rott, R, Wahn, K, Klenk, H D & Kohama, T, Virology 107 (1980) 313. 6. Kennett, R H, Denis, K A, Tung, A S & Klinman, N R, Curr top microbial immunol81 (1978) 77. 7. Kohler, G & Milstein, C, Nature 256 (1975) 495. 8. Laemmli, U K, Nature 227 (1970) 680. 9. Miyake, Y, Kim, J & Okada, Y. Exp cell res 116 (1978) 167. 10. Nakanishi, M, Uchida, T, Kim, J & Okada, Y, Exp cell res 142 (1982) 95. 11. Okada, Y, Koseki, I, Kim, J, Maeda, Y, Hashimoto, T, Kanno, Y & Matsui, Y, Exp cell res 93 (1975) 368. 12. Okada, Y & Tadokoro, J, Exp cell res 26 (1962) 108.
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13. Salk. J E, J immunol49 (1944) 87. 14. Scheid, A, Caliguiri, L A, Compans, R W & Choppin, P W, Virology 50 (1972) 640. 15. Scheid, A & Choppin, P W, J virol 11 (1973) 263. 16. - Virology 57 (1974) 475. 17. Shulman. M, Wilde, C D & Kohler, G, Nature 276 (1978) 269. 18. Staehelin, T, Durrer, B, Schmidt, J, Takacs, B, Stocker. J, Miggiano, V, Stahli, C, Rubinstein, M, Levy, W P, Hershberg, R & Peska, S, Proc natl acad sci US 78 (1981) 1848. 19. Tozawa, H, Watanabe, M & lshida, N, Virology 55 (1973) 242. 20. Uchida, T, Kim, J, Yamaizumi, M, Miyake, Y & Okada, Y, J cell biol80 (1979) 10. Received January 8, 1982 Accepted April 7, 1982
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