Effects of cytochalasin D on fusion of cells by HVJ (Sendai virus)

Experimental Cell Research 116 (1978) 167-178

EFFECTS

OF CYTOCHALASIN

D ON FUSION

(SENDAI

OF CELLS

BY HVJ

VIRUS)

Cell-cell fusion is separable from cell-virus fusion YASUKO MIYAKE,

JEMAN KIM and YOSHIO OKADA

Research Institute for Microbial Diseases, Osaka University, Yumudu-kumi, Suita. Osuka 565, Japun

SUMMARY Fusion of cells mediated by HVJ was inhibited completely with 5 pg/ml or more of cytochalasin D (CD). With cytochalasin, HVJ-cell interaction at 0°C proceeded as well as without cytochalasin; HVJ was adsorbed to cell surfaces and the cells agglutinated together. Then the virus particles were enfolded with cell membranes, which resulted in the disappearance of hemadsorption activity on the cell surfaces. When the cell-virus complex was incubated at 37”C, the early reactions proceeded as well as without cytochalasin; the hemadsorption activity reappeared on the cell surfaces, the viral envelopes fused with cell membranes at the same degree as without cytochalasin, and a stage sensitive to sodium azide appeared as in a control without cytochalasin. But cell-to-cell fusion did not occur in the presence of cytochalasin; cells were dissociated freely from the cell aggregates during incubation. This indicates that cell-to-cell fusion was inhibited but HVJ envelope to cell membrane interactions proceeded well on incubation at 37°C. These findings suggest that viral envelope-ceil membrane fusion and cell
Cytochalasins, fungal metabolites, are known to inhibit cell movement, such as cytokinesis, membrane ruffling, cell locomotion [ 11,phagocytosis [2], and capping of lymphocytes mediated by antiserum or lectins [3]. Many of these drug effects seem to be caused by reversible disruption of microfilaments associated with cell membranes. Asano & Okada [4] have recently reported that cytochalasins B and D (CB and CD) could also inhibit the HVJ-induced cell fusion phenomenon. They also mentioned that the CD effect on cell fusion inhibition was greater than that of CB. Since sugar transport is known to be inhibited by CB [5], but not by CD [6], it was suggested that the inhibition of cell fusion was probably

due to the action of the drugs on microtilaments. CD was also shown to have no effect on the ATP level in cells, thus the inhibition observed was not due to depletion of intracellular ATP [4]. In the present report, more detailed experiments concerning the effects of CD on the cell fusion reaction steps are presented. With CD, virus-cell interactions on incubation at 0°C occur as well as without CD. On incubation at 37”C, viral envelope-cell membrane fusion also occurs, but the viral fusion is not followed by cell-cell fusion. In our last paper, Maeda et al. reported a similar inhibition by a high concentration of saccharides, such as glucose, mannose, galactose, or sucrose [7]. Inhibition patterns Exp Cdl Rcs 116 (1978)

Miyake, Kim and Okada

168

with CD and saccharides are also compared in the present paper. And the participation of a cytoskeleton system in cell-cell fusion is discussed. MATERIALS

sodium azide. The cell-virus mixture was incubated at 0°C for S min and then at 37°C for 30 min with shaking. After these incubations, the cells were observed under a nhase contrast microscope to examine their viability.‘During incubation at 37”*Cin the presence of HVJ, the cells degenerated due to the lytic activity of HVJ, but no cells degenerated in the absence of HVJ [l2].

AND METHODS

Cell The strain used was a mutant of Ehrlich ascites tumor cells 181. Cells were harvested from the abdomens of mice -and washed 34 times to remove ascites fluid and blood cells. Thev were suspended in 9 vol of a balanced salt solution (BSS): 140 mM NaCl, 54 mM KCI, 0.34 mM Na,HPO,, 0.44 mM KH,PO,, buffered with 10 mM Tris-HCI at pH 7.6, containing 2 mM CaCl,.

Anti-HVJ envelope antibody Reconstituted envelopes were prepared by Hosaka’s method using Nonidet P40 [ 131and used as the antigen. About IO4 HAU of reconstituted envelopes were injected subcutaneously into rabbits. Three weeks later a booster injection in complete. Freund adjuvant was given. Booster injections were given at intervals until a suitable titer was obtained. Blood was taken 7-10 days after the last injection. The resulting antiserum showed a hemagglutination inhibitory activity of 1024 : I, when 2 HAU of HVJ was used as antigen.

Virus HVJ, Z strain was propagated in embryonated eggs. The infected chorioallantoic fluid was partially purified by two cycles of differential centrifugation [9]. Then the virus was suspended in BSS without Ca ions. The virus titer was measured as hemagglutinating units (HAU) using Salk’s pattern method [IO].

Drug CD (Shionogi Co., Osaka, Japan) was kindly provided by Dr A. Asano (Institute for Protein Research, Osaka University). CD and sucrose were dissolved in BSS and used for the experiments of cell fusion inhibition.

Fusion of cells 0.5 ml of a 10% suspension of Ehrlich ascites tumor cells in BSS-Ca 2 mM and 0.5 ml of a preparation containing 2 000 HAU of HVJ in BSS without Ca were mixed in a tube. After incubation at 0°C for 5 min, the mixture was incubated at 37°C for 30 min with shaking in a water bath. Then the sample was cooled and an aliquot was observed under a phase contrast microscope and the number of cells was counted.

Hemadsorption test Cells which had been treated with HVJ in a tube were washed twice with IO ml of BSS at 0°C to remove free virus and then resuspended in one drop of BSS. Five drops of a 10% suspension of human red blood cells were added and the-mixture was incubated at 0°C for I5 min. Free red blood cells were removed by centrifugation and the tumor cell-red blood cell complex was treated with saponin. Then the amount of hemoglobin liberated from the red blood cells was estimated from the absorption at A=540 nm [I I].

Cell lysis by HVJ The reaction mixture was the same as that for “fusion of cells”, except that the medium contained 5 mM h/J Cdl It<,,!116(1978)

Detection of viral antigen on cell surfaces by fluorescent antibody technique Cells which had been treated with HVJ at 37°C for 30 min were washed with BSS at 0°C to remove free virus, and then rabbit anti-HVJ envelope serum was added and incubated at 0°C for 20 min. The cells were washed with BSS at 0°C and FITC-conjugated antirabbit IgG goat antibody (Miles-Yeda, Ltd) was added at 0°C. After incubation at 0°C for 20 min. thev were washed at 0°C and observed under a fluorescence microscope or passed through a fluorescence-activated cell sorter, FACS II (Becton Dickinson Co., Mountain View. Calif.) F141. Using this apparatus, fluorescence intensity for individual cells was measured quantitatively and automatically.

Ferritin-conjugated envelope antibody

anti-HVJ

IgCi fraction of the above anti-HVJ envelope serum was obtained by twice repeated precipitation in 40% saturated ammonium sulfate solution and then applied to DEAE-cellulose column equilibrated with 15.mM phosphate buffer pH 6.3 [IS]. The purified IgC was conjugated with ferritin (Nutritional Biochemical Corp.) using toluene-2,4-diisocyanate [ 161.

Detection of viral antigens on cell surfaces by electron microscopy After the completion of cell fusion reaction, cells were washed with SSC (150 mM NaCI, 15 mM sodium citrate) to remove free virus and treated with ferritinconiueated anti-HVJ envelope antibodies. Thev were fixed with 5 % glutaraldehyde -4 % paraformaldehyde in 0.1 M cacodylate buffer pH 7.2 at 0°C for 40 min and then post-fixed with 2% osmium tetroxide in 0.1 M collidine buffer, pH 7.4, at room temperature for 60 min [17]. Samples were dehydrated and embedded in Epon 812.

Inhibition of cell fusion by CD

169

RESULTS Inhibition of cell fusion reaction by CD Cytochalasin stock, dissolved in DMSO at a concentration of 1 mglml, was diluted to appropriate concentrations with BSS medium containing 1 mM CaCl, and added to test tubes containing 0.5 ml of 10% suspension of Ehrlich ascites tumor cells and 0.5 ml of 2000 HAU of HVJ. The tubes were incubated at 0°C for 5 min and then incubated at 37°C for 30 min. After incubation the degree of cell fusion was estimated. Control experiments including the same concentrations of DMSO as the experiments but omitting CD were also performed. As shown in fig. 1, CD inhibited the HVJ-induced cell fusion and the effect was a function of the concentrations used. DMSO showed no inhibition at the concentrations used. Cell fusion was inhibited completely by 5 pglml of CD under these conditions. This inhibition was known to be reversible [4]. With CD, HVJ-cell interaction at 0°C proceeded as well as without cytochalasin. As shown in fig. 2B-1 (see also 2A-l), virus was adsorbed onto cell surfaces and the cells agglutinated together. Disappearance of hemadsorption activity of the virus-cell complex, which had been reported [ll], was also observed under the conditions with CD on incubation at 0°C. However, on incubation at 37”C, cells were released freely from the cell aggregates and no cell fusion occurred (fig. 2B-2, compare with fig. 2A-2). The non-fused cells had viral envelope antigens on their surfaces and a ring-shaped fluorescence image was observed when stained by the fluorescent antibody technique (fig. 3A). When red blood cells were added to the cells, the red blood cells were adsorbed promptly onto the cell surfaces and rosettes were produced by the

0

0

I'

I 1.25

2.5

? 10

5

Fig. I. Abscissa: (upper)% of DMSO; (lo~ev) pg/ml of CD; ordinafo: fusion index.

Fusion index=

total cell no. in control without HVJ cell no. after fusion with HVJ -1.0

Extent of cell fusion with IO00 HAU of HVJ in BSSCa medium containing various CD and DMSO concentrations (0); or DMSO only (0).

cells and red blood cells (fig. 3B). This hemadsorption was strong and its activity was neutralized by anti-HVJ envelope antibody. As reported previously [ll], when cells adsorbing virus particles are incubated at 37°C for 30 min under the standard conditions without CD, a few of the virus are liberated from the cell surfaces by destruction of the receptors, but most of the virus are irreversibly fused with the cells. The above findings suggest that the viral envelope-cell membrane fusion is possible in the presence of CD, while cell-to-cell fusion is inhibited completely. The overall features of inhibition by CD seem to be similar to those of inhibition by saccharides [71. Kinetics of reappearance of hemadsorption activity on incubation at 37°C As reported previously [ll], at an early Erp

Cell

Kr.,

II6

(1978)

170

Miyake, Kim and Okada

Exp Cd Rcs 116 11978)

Inhibition of cell fusion by CD stage of viral adsorption onto the cell surfaces, one side of the virus attaches to the cell surface receptors and the opposite side of the virus is free. When human red blood cells are added at this stage, the cell-virus complex can adsorb red blood cells, i.e., a red blood cell is caught by the free side of the virus; this is called “hemadsorption”. On incubation at 0°C the area of the virus surface attached to the cell surface grow quickly and there is no free side. At this stage, hemadsorption is not observed in the cell-virus complex. When the cell-virus complex is incubated at 37°C the hemadsorption activity promptly reappears and increases with the incubation time, reaching a maximum after incubation for 5-10 min. On further incubation, the hemadsorption activity decreases gradually. During the course of incubation at 37”C, both the “virus-cell” and the “cell-cell” fusions occur. With CD, the hemadsorption activity of the cell-virus complex disappeared at 0°C as well as in a control without cytochalasin. On incubation at 37°C hemadsorption activity reappeared. But as shown in fig. 4, the late hemadsorption decreasing phase was unclear in the sample with CD; the hemadsorption activity continued during the incubation for 30 min and the maximum activity reached a higher degree than the control without CD. This kinetic pattern closely resembled the case of cell fusion inhibition by saccharides reported previously [7]. The late phase may be correlative with mix-

171

Fig. 3. (A) A fluorescent micrograph of the viral anti-

gens associated with cells; (B) a phase contrast micrograph showing hemadsorption activity of the cells. (A, i3) Cells were incubated with 1000 HAU of HVJ at 37°C for 30 min in the medium containing 5 pg/ml CD. After washing (A) cells were stained by indirect fluorescent antibody technique using anti-HVJ envelope serum; (II) human red blood cells were added.

ing of viral envelope components with the cell membrane components. It should be noted that the dullness in the late phase and the lack ‘of cell-cell fusion were both apparent in the presence of CD and saccharides.

Electron microscopical observation Cells which had been treated with HVJ at Fig. 2. Phase contrast micrographs showing virus-cell interactions in medium with 5 pg/ml CD and 0.5 % of 37°C for 30 min were observed by electron DMSO (B series), or with 0.5% of DMSO only (A series). Cells agglutinated together on addition of 1000 microscopy using ferritin-conjugated antiHAU of HVJ at 0°C (A-l and B-l). When the cell ag- HVJ envelope antibodies. It was confirmed gregates were incubated at 37°C for 30 min, the cells fused in medium without CD (A-2), whereas the cell that the fluorescence image seen in fig. 3A aggregates separated into single cells in medium with was due to the fusion of viral envelopes CD (E-2). When the cell aggregates were incubated at 37°C with 5 mM NaN,, cells degenerated both in the with cell membranes. The viral envelopes presence and absence of CD (B-3 and A-3). had been integrated into the cell membranes

172

Miyuke, Kim and Okada

and saccharides, but they are not completely the same. One of the differences concerns the appearance of an NaN,-sensitive stage. As reported previously [12, 181, when 2,4-dinitrophenol, NaN,, or EDTA is present, cells are lysed by the action of HVJ during incubation at 37°C. Namely, a stage sensitive to those reagents appears during the course of cell fusion reaction. This stage does not appear in cell fusion inhibition by saccharides. With CD, cell-cell fusion was inhibited completely and the non-fused cells were intact. When 5 mM NaN, was added to the 0 5 10 20 30 cell-virus mixture containing CD and incuFig. 4. Abscissa: incubation time (min);ordinate: hem- bated at 37°C for 30 min, all cells were adsorption activity of cells (OD,,,,). Iysed, as shown in fig. 2B-3. The degree Kinetic curves of reannearance of hemadsorntion of degeneration of individual cells was mild activity of cells. Cells were incubated with 2 000 HAU of HVJ at 37°C for various lengths of time in a medium compared with that of the control without containing 5 pg/ml CD and 0.5 % DMSO (0); or 0.5 % DMSO only (0). After washing, hemadsorption ac- cytochalasin (fig. 2A-3), but it showed a tivity was measured. Each plot is an average of dupli- clear contrast to the appearance of viable cate samples. cells in samples with saccharides [7]. To demonstrate more clearly the difference beand the viral antigens were distributed tween the conditions with CD and with sacpatchily over the cell surfaces (fig. 5A, B) charides, the degree of NaN, sensitivity and and non-fused viral particles were not ob- the degree of cell fusion inhibition under served on the cell surfaces as in a control various concentrations of these two inhibiwithout CD [7]. tors were compared in fig. 6. In the present In the case of cell fusion inhibition by experiment, sucrose was used as a sacchaglucose, cell-viral envelope fusion was also ride, and cell fusion inhibition and cell lysis observed but the pattern was different; the inhibition were closely correlated. The patspecific envelope morphology remained in- tern closely resembled that with glucose retact in the cell membrane and the nucleo- ported previously [7]. In the case of CD capsid was present inside the envelope [7]. in all concentrations used, cell lysis was However, in the case of CD, the viral nucleocapsid had been released out from the Fig. 5. Electron micrographs of Ehrlich ascites tumor cells treated with HVJ at 37°C for 30 min in the presfusion site. This suggested that the viralof CD. After treatment, cells were chilled and cell interactions proceeded further in the ence exposed to ferritin-conjugated anti-HVJ envelope antipresence of CD than in that of glucose. bodies. (B) High-power view of the area enclosed in Appearance

of a NaN+ensitive

stage

As described above, the cell fusion inhibitions show similar patterns with both CD E.Y/, Cd/

Rc,.\ 116 11978)

the rectangle. Viral envelopes were integrated into the cell membrane and viral antigens were distributed patchily over the cell surface (+). No specific structures of viral envelopes and nucleocapsids were seen. Zeiotic protuberances [23] caused by CD were also seen at low magnification(Z). (A) x9000; (B) ~60000.

174

Miyake, Kim and Okada

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of CD; ordinufe: of cell fusion (%). (A) Extent of cell lysis with NaN,. Cells were incubated with 1000 HAU of HVJ with 5 mM NaN, at 37°C for 30 min in the medium containing various CD concentrations (0); or sucrose (A). Viability was measured under a phase contrast microscope; (B) extent of cell fusion without NaN3. Cells were incubated with 1000 HAU of HVJ at 37°C for 30 min in the medium containing various CD (0) or sucrose (A) concentrations. Fusion index of control sample without CD or sucrose was taken as 100%.

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observed in samples containing NaN,. This showed that the virus-cell interactions in the presence of CD proceeded at least to the NaN,-sensitive stage, but not with saccharides. This coincides with the electron microscopical observation that the virus-cell interactions seem to proceed further with CD than with saccharides. The efficiency of viral envelope-cell membrane fusion

In this section, the amount of viral antigens fused with cell membranes after incubation at 37°C for 30 min with CD was estimated quantitatively. Although we can estimate the amount of virions associated with cells by isotope-labelling (as in [7]), in this method the amount of virions integrated into the cytoplasm, probably by cell fusion, was also included. Using a newly developed apparatus, FACS II, we can estimate only the amount of viral envelopes present on cell surfaces. This apparatus can estimate the

I 0

I 50

I 100

I

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Fig 7. Abscissu: rel. fluorescence intensity/cell: or” .. dinare: ret. no. or cells.

Fluorescence distributions of cells treated with 0, 100, 300, or 1000 HAU of HVJ, respectively. Cells were incubated with various concentrations of HVJ at 37°C for 30 min in the medium containing 5 pg/ml CD. After washing, cells were stained by indirect fluorescent antibody technique using anti-HVJ envelope serum and analyzed on FACS II.

size of individual cells after exposure to a laser beam by their scattering value, and the fluorescence intensity of individual cells after exposure to a laser beam of UV range. The cells were washed and stained by fluorescent antibody technique using antiHVJ envelope antibodies after treatment with 0, 100, 300, and 1000 HAU of HVJ, respectively, at 37°C for 30 min under the conditions with 5 pg/ml of CD. Then the fluorescence intensity of the individual cells was estimated by passing them through

Inhibition of cell fusion by CD 1 r

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HAU

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175

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/ a

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1 10

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. 1000

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Fig. 8. Abscissa:

HVJ titer (HAU); ordinute: (left) mean fluorescence intensity/cell; (right) fusion index. HVJ dose dependency of the fluorescence intensity of the cells treated with various HVJ concentrations. The mean values were calculated from the data of fig. 7 and plotted against the HVJ titer (0). The fusion index at each virus titer was also indicated (m).

1 0

I 50

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I 150

I 200

U 250

Fig. 9. Abscissu: rel. fluorescence intensity/cell; ordinate: rel. no. of cells.

Fluorescence distributions of cells incubated with HVJ in the presence or absence of CD. Cells were incubated with 100 HAU of HVJ in the medium with and without 5 pg/ml of CD. These cells and nontreated control cells were incubated and stained as described in fig. 7 and analysed on FACSII. The fluorescence profile of the cells distributed only in the size range of mononuclear cells was taken.

FACSII. As shown in fig. 7, the fluorescence profiles differed from each other in correspondence to the HVJ titer used; they shifted to the right (showing higher intensity) with the increase in HVJ titer. In fig. 8, the mean fluorescence intensity per cell is plotted against the HVJ titer. The mean fluorescence profile of the cells distributed values were calculated from the data of the in the size range of mononuclear cells was pulse height analyzer. The cell fusion level obtained. In fig. 9, the fluorescence profile is also indicated. The amount of viral an- of the cells without CD is compared with tigens on the cell surfaces was logarithmi- that of cells with CD. Both profiles overcally proportional to the dose of HVJ lapped closely and the mean fluorescence added, although cell-to-cell fusion was com- value/cell with CD calculated from the propletely inhibited. files was 97.6% of that without CD. Next, the amount of viral antigens irThese two observations clearly indicate reversibly associated with cell membranes that viral envelopes can fuse efficiently after treatment with and without CD was with cell membranes with or without the compared. For this purpose, 100 HAU of presence of CD. HVJ was used to minimize cell-to-cell fusion under standard conditions without CD, Cell membrane movement and CD because large fused cells may cause clog- As reported previously [19], on culture of ging of the nozzle of the cell sorter. The Ehrlich ascites tumor cells fused with HVJ light scattering profile of the cells treated envelopes in MEM medium at 37”C, (1) the without CD was first determined to know hemadsorption activity disappears; and (2) the size-distribution of the cells because the viral antigens disappear from almost all the sample was a mixture of non-fused mono- cell surfaces. During culture, accumulation nuclear and small fused cells. Then, the of viral antigens takes place on a specific

176

Miyake, Kim and Okada

b+-----A

100

60

60

40 20

\ 0

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4

Fig. 10. Abscissa: culture time (hours); ordinate: hemadsorption activity (%). Changes in hemadsorption activity during culture of fused cells in medium containing CD, DMSO, or sucrose. Cells were treated with 1000 HAU of HVJ at 37°C for 30 min and washed and cultured in MEM medium with 5 pg/ml CD and 0.5% DMSO (0); or with 0.5% DMSO only (0); or with 0.25 M sucrose (a), respectively. After culture for 4 h, cells were washed and hemadsorption activity was measured. Hemadsorption activity before culture was taken as 100%. Each plot is an average of duplicate samples.

area of the fused cell. In a previous report it was suggested that, on culture of the fused cells, internalization occurs in the areas of the cell membrane containing viral antigens, and then the viral antigens are enclosed in vesicles in the cytoplasm. The vesicles gather together in a specific area and the viral antigens are reexposed on the cell surface by exocytosis of the vesicles

WI. One of the differences in the effect of CD and saccharides concerned the appearance of a NaN,-sensitive stage. The other difference concerned the cell membrane movement. For fig. 10, 0.5 ml of 10% suspension of Ehrlich ascites tumor cells was treated with 0.5 ml of 2000 HAU of HVJ at 0°C for 5 min, then at 37°C for 30 min. The cells were washed and cultured in MEM medium with 5 pg/ml of CD, with 0.5% of DMSO, and with 0.25 M sucrose,

respectively. After culture for an appropriate length of time, cells were washed and then the hemadsorption activity was estimated. As fig. 10 shows, the activity with CD decreased at the same rate as with DMSO and this rate was comparable with that of a standard without CD or DMSO. With sucrose, no decrease in the activity was observed. These findings show that CD does not affect this kind of cell membrane movement. DISCUSSION Fusion of Ehrlich ascites tumor cells by HVJ has been studied for about 20 years in our laboratory. During this time, it became clear that the cell fusion reaction was pH dependent [21], energy dependent [ 181,and required Ca ions [9]. By shifting these factors from their optimums, cell fusion was inhibited and cell lysis became dominant. These findings were valuable for understanding the cell fusion mechanism, but there was a problem in that the appearance of cell lysis prevented the observation of cell membrane characters after treatment with the virus. The use of saccharides or CD for the inhibition of cell fusion helped to solve the problem; no cell lysis appeared under such conditions. This advance produced evidence that “virus-cell” fusion and “cell-cell” fusion were not a single phenomenon but were separable. In the presence of either saccharides or CD, “cell-cell” fusion was inhibited whereas “virus-cell” fusion proceeded. But the mechanisms of inhibition of cell-cell fusion by these inhibitors were not the same. Saccharides seemed to inhibit cell membrane movement thoroughly and this may have been due to the inhibition of cell-cell fusion. The viral envelope-cell membrane fusion proceeded well but the reaction stopped at an early stage. After incubation

Inhibition of cell fusion by CD at 37°C for 30 min, the virus still kept its specific morphology except that one side of its envelope had been fused with the cell membrane and no NaN, sensitive stage appeared in the cells during incubation. With CD, cell membrane movement was not affected as shown in fig. 10, and the NaN, sensitive stage appeared as without CD. When the cell surface was observed electron microscopically after incubation at 37°C for 30 min, viral specific structures had disappeared although the viral antigens were seen to have been integrated into the cell membranes. This shows that viral envelope fusion with the cell membranes had proceeded well. Nevertheless, cell-cell fusion was completely inhibited. This suggests the participation of a function(s) of the cell itself in cell-cell fusion phenomenon. Virus-cell fusion is, of course, important but a change(s) in the cytoskeleton system induced by this fusion may be essential for cell-cell fusion. If microtilaments of the cells have been modified by CD, the viruscell fusion is not followed by cell-cell fusion. Observations concerning gelation of extracts of Ehrlich tumor cells on incubation at 25°C have been carried out in our laboratory (in preparation). It seems that the gelation proceeds with the formation of fibrous networks in the extracts. The gelation is inhibited by CB or CD, Ca ions, or inhibitors of actin-function. Participation of microfilaments and actins in gelation is considered. Recently, Asano et al. (personal communication) showed that when extracts from fused cells were incubated, no gelation appeared. The conditions affecting cell-cell fusion seem to overlap closely those of gelation, and the latter phenomenon depends on the cytoskeleton system inside cells but not on the character of the lipid layer of cell membrane itself. The conditions re-

177

quired for cell-cell fusion, such as energy dependency, Ca ion dependency, or inhibition by CD, may be well understood when the participation of the cytoskeleton system including microfilaments and actins in cell-cell fusion is considered. With regard to the virus fused with the cell, there is a contrast in the kinetics of hemadsorption activity (HA)-reappearance on incubation at 37”C, between standard conditions and those with CD or saccharides. Under the standard conditions followed by cell-cell fusion, the kinetic pattern shows two phases; one is the early HA-increasing phase and the other is the late HA-decreasing phase. The early phase proceeds but the late phase is obscure with either CD or saccharides. As a result, the HA-level of cells after treatment with the virus under these conditions is much higher than that of a control under standard conditions. This evidence seems to be related to the appearance of no cell-cell fusion. In the case of saccharides, the loss of the late HA-decreasing phase may be explained thus: the viral fusion with the cell membrane is stopped at an early stage and mixing of viral envelope components with the cell membrane components is inhibited. If the loss of the late phase with CD is also due to the inhibition of the mixing, it may be suggested that microfilament function also participates in the mixing. The mechanism of the late HA-decreasing phase has not yet been clarified. However, it is known that the HA activity of the virions depends on the HANA proteins polymerized by S-S bonds. When the HANA proteins are monomerized by treatment with glutathion, the HA activity disappears [22]. It may be possible that the late decreasing phase correlates with reduction of the S-S bonds to SH radicals by reducing conditions of the cytoplasm. E.tp Cd RP I I16 11978)

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The authors wish to thank Dr A. Asano, Institute for Protein Research, Osaka University, for valuable discussion. This work was supported by research grants from the Ministry of Education of Japan.

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23. Godman, G C, Miranda, A F, Deitch, A D & Tanenbaum, S W, J cell bio164 (1975) 644. Received January 31, 1978 Revised manuscript received April 26, 1978 Accepted April 28, 1978