Eur. J. Biochem. 6.5. 155- 160 (1976)
Metabolism of Preexisting Lipids in Baby Hamster Kidney Cells during Fusion from Within, Induced by Newcastle Disease Virus Heino DIRINGER and Rudolf ROTT Institut fur Virologie, Justus Liebig-Univerdtat, GieBen
(Received December 8, 1975/January 22, 1976)
The effect of fusion from within induced by Newcastle disease virus on the metabolism of phospholipids, neutral lipids and glycolipids has been studied in baby hamster kidney cells. Hematoside, the only ganglioside in these cells, is rapidly broken down shortly after the onset of virus-induced neuraminidase activity resulting in an increased level of lactosylceramide. No specific breakdown of any phospholipid or neutral lipid could be related to cell fusion.
Cell fusion is a common cytopathic or cytotoxic response to infection by a wide range of viruses (see [1,2] and can be separated into two distinct mechanisms. Fusion from within [3] occurs during the replication of certain enveloped viruses and requires the synthesis of virus specific macromolecules. Fusion from without [4] can be achieved by addition of a high amount of virus - not necessarily infective - to the cells to be fused. During the past years this last method has been used extensively to study the metabolism of heterokaryons (see [5,6]). According to Poste and Allison [7] intermembranous clustering of proteins plays an important role in cell fusion. Two observations, however, indicate that protein interaction might not be the only essential process for fusion but that lipids are also involved. Firstly, it has been demonstrated that protein-free phospholipid vesicles can fuse [8 - 101. Secondly, fusion can be induced by lyso-lecithin [I 1 - 131. Ahkong et al. [14], therefore, have proposed that perturbation of the bilayer structure of membrane lipids (perhaps by the formation of lyso-lecithin [15- 171) increases the fluidity of the lipid region thereby allowing intermembranous aggregation of proteins or glycoproteins [7]. We were interested in the question of whether perturbation of the lipid layer is a matter of a biochemical degradation process of the host cell lipids caused by a virus-induced enzyme activity. As a model baby hamster kidney cells, line 21-F, infected with Newcastle disease virus were chosen, a system in which extensive fusion from within rapidly occurs [3]. We prelabelled the host cell lipids with either [32P]phosphateor tritiated palmitate and measured the radioactive label of in-
dividual neutral lipids, phospholipids, and glycolipids during the course of cell fusion in comparison with uninfected cells.
MATERIALS A N D METHODS
Virus and Experiment The Newcastle disease virus strain “Italien” was used throughout. Baby hamster kidney cells line 21-F [18] were cultivated in 10-cm glass Petri dishes using Eagle’s minimum essential medium with Earle’s balanced salt solution and 10% fetal calf serum. Just before the cells reached confluency each dish received 10 ml of fresh medium containing either 60 pCi of carrier-free [32P]phosphate or 5 pCi (500 mCi/mmol) of [jHIpalmitate (both from Amersham/Buchler). A stock solution of [3H]palmitate was prepared by sonication of the dry radioactive compound with serum, diluting the solution with medium and passing it through a millipore filter [19]. After a 12-h labelling period the radioactive medium was removed and the cells were washed twice with phosphate-buffered saline, pH 7.2. The cells on half of the dishes were infected with virus at a multiplicity of about 10 plaqueforming units/cell. The rest of the cells were mockinfected with allantoic fluid of uninfected chicken eggs. Isohtion and Analysis o f Phospholipids and Neutral Lipids At indicated times pairs of Petri dishes were taken, the medium was removed, and the cells were washed twice with phosphate-buffered saline. Lipids were
156
Lipids in BHK Cells Infected with Newcastle Disease Virus
Fig. 1. The morphologic uppeurunce of' cells hejore ( A ) infection with Newcastle disease uirus und 4 h ( B ) ,5 h ( C ) and 7 h (D)ujter injection. Fusion became detectable 4 h after infection and was clearly established after 5 h
extracted with 4 ml of chloroform/methanol (2/1) and partitioned with 0.9 ml of phosphate-buffered saline [20].Thelipid-containing organicphase wasevaporated to dryness and redissolved in 2 ml of chloroform. Aliquots were taken for the determination of radioactivity and phosphorus content [21]. For determination of radioactivity in individual phospholipids the lipid extract was chromatographed on thin-layers of silica gel [22]. The lipid-containing areas were localized by iodine vapor or by autoradiography and scraped into counting vials. When neutral lipids were investigated the lipid extract was passed through a small column of silica gel
and the lipids were eluted with chloroform [23]. They were chromatographed on silica gel plates with light petroleum/diethyl ether/acetic acid (S0/20/1). The chromatograms were scanned in a radioactive chromatogram scanner.
Isolation and Analysis oj' Glycolipids Radioactive glycolipids were isolated from the cells after the addition of 25 pg of non-radioactive glucosyl-, lactosylceramide, and hematoside to each plate. Lipids were extracted with 3 ml of chloroform/methanol(2/1) followed by 3 ml of chloroform/methanol (1/2). The
H. Diringer and R. Rott
combined extracts were taken to dryness and glycolipids were isolated according to Saito and Hakomori [24]. Individual glycolipids were separated by thinlayer chromatography on silica plates with chloroform/ methanol/ammonia (65/25/4). The thin-layer chromatograms were scanned, afterwards sprayed with 50% sulfuric acid and heated at 140 "C to visualize the lipids. Reference glycolipids were isolated according to Hakomori and Strycharz [25].
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RESULTS
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In baby hamster kidney cells infected with Newcastle disease virus (strain "Italien") cell fusion starts about 4 - 5 h post infection and is completed within the next 2-3 h (Fig. 1) [27]. To see whether these pronounced and rapid morphologic changes can be correlated to any breakdown of cellular lipids, cells were prelabelled with radioactive lipid precursors, chased and infected with virus. Control cells were mock-infected. After various time intervals cell samples were taken and analyzed for their radioactive lipid content. In the experiments reported here infection occurred immediately after the chase. The results are, however, the same when cells were infected up to 12 h after the chase. Fig. 2 shows the results of an experiment in which phospholipids were prelabelled with [32P]orthophosphate. The specific activity of the total phospholipid fraction decreases slightly during the course of the experiment in virus-infected and mock-infected cells. When individual phospholipid fractions such as lecithin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin were separated by thin-layer chromatography no difference in their relative content of radioactivity could be observed between mock-infected and virus-infected cells at any time of the experiment. In addition no increased levels of lyso-lecithin or lyso-phosphatidylethanolamine could be detected in the infected cells after autoradiography of thin-layer chromatograms. Neutral lipids and glycolipids were prelabelled with tritiated palmitate. When neutral lipids were separated from the polar lipids by silicic acid column chromatography no difference in the time-dependent loss of radioactivity in the total neutral lipid fraction could be observed between controls and infected cells (Fig. 3A). Equal amounts of neutral lipids from cell samples taken 1 h and 7 h after infection were separated into individual lipid fractions by thin-layer chromatography. The superimposed radiochromatograms
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Fig. 2. Specific aclicity o/ content
1
t o t d phospholipid j i a c t i o n ( A ) and
relative
of rarlioaciicephosphate ( B ) plotted against time. ( A )Specific
activity of the total phospholipid fraction of control (U) and virus-infected (0 --O) cells in dependence of time. (B,C) The relative content of radioactive phosphate in individual phosphoand virus-infected (M) cells. From lipids in control (-) top to bottom: lecithin, phosphatidylethanolamine,phosphatidylinositol, phosphatidylserine, and sphingomyelin. In this and the following figures the arrow indicates the time when fusion was clearly visible ( 5 h post-infection)
(Fig. 3B) only showed a somewhat decreased relative content of tritium in the free fatty acid fraction of the "late" sample. A similar result was found in corresponding control cells. Glycolipids only represent a small fraction of the cellular lipids. Baby hamster kidney cells contain only the ganglioside hematoside and the two neutral glycolipids lactosyl- and glucosylceramide [28,29]. These lipids can be separated from phospholipids and neutral lipids by florisil chromatography [25] and can be resolved further by thin-layer chromatography. The radioactive chromatogram of such a lipid isolated from virus-infected baby hamster kidney cells is given in Fig. 4. It can be seen that the non-labelled carrier lipids added before the isolation were recovered in the glycolipid fraction and that radioactivity is associated with these carriers as well as with some unidentified material running near the front. Again no difference
Lipids in BHK Cells Infected with Newcastle Disease Virus
158
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Time after infection (h)
A
Triglycerides
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Alkyl -diglycerides
in the content of radioactivity in the total glycolipid fraction could be detected at any time after virus infection in comparison to the controls. In contrast to glucosylceramide the relative content of radioactivity in hematoside and lactosylceramide changes during the course of the experiment (Fig. 5). In infected cells the loss of radioactivity from hematoside beginning 4 h after infection was accompanied by a concomitant increase of tritium in lactosylceramide. This change correlates with increased viral neuraminidase activity which becomes detectable 3 - 4 h after infection. These results are in agreement with studies of Klenk et al. [30] and Klenk and Huang [31] who also found a decreased amount of lipid-bound neuraminic acid in monkey kidney cells and chicken cells after infection with paramyxoviruses.
DISCUSSION
Fig. 3. R ~ i d i ( ~ ~ ~ t in i c iihr t y total neutrul lipid ,frociion and superimposed rcidiooctiae chromatogram of neutral 1ipid.s. ( A ) Radioactivity and virusin the total neutral lipid fraction of control (+a) cells in dependence of time. (B) Superimposed infected (-) radioactive chromatogram of the neutral lipids of baby hamster and 7 h (. .. . . .) after infection. Equal kidney cells taken 1 h ( -) amounts of radioactivity were chromatographed
1
Hematoside
Our results demonstrate that infection of baby hamster kidney cells with Newcastle disease virus does not affect the breakdown of cellular phospholipids and neutral lipids and therefore specific destruction of such molecules does not play an essential role in virus-induced cell fusion. These results are in agreement with the observations of Hosaka [32] studying Sendai virus induced cell fusion, with results of Falke et al.
I
Fig. 4. Chromatography qfglycoiipids. The bottom panel represents the pure carrier glycolipids. The middle panel shows the chromatogram of the glycolipid fraction of virus-infected cells shortly after infection. The top shows the radioactive scan of the chromatogram of infected cells
H. Diringer and R. Rott
159 Lactosyiceramide
Hematoside
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4
6
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Fig. 5. The relative content of' radioactivity in hemutoside and lactosylceramide of control ( M) und virus-inJected (M) cells in dependence of'time. The data plotted were obtained by measuring the relative area represented by the two lipids in radioactive scans as shown in Fig. 4 taken at various time intervals after infection
[33] using herpes virus in rabbit kidney cells as a model system, and with theexperimentsof Elsbachrtul. [34] who infected baby hamster kidney cells with Simian virus 5. In all these studies it was shown that cellular lecithin did not undergo any appreciable hydrolysis under conditions in which extensive cell fusion occurred. More recently Parkes and Fox [35]demonstrated that no correlation exists between the content of phosphatidylserine in various strains of Newcastle disease virus and its ability to induce fusion from without. Now it can be said that neither degradation of any phospholipid at all, nor enzymatic destruction of neutral lipids, nor breakdown of glycolipids seems to be a necessary event in cell fusion. One has to keep in mind, however, that fusion takes place as a rapid local event in a small proportion of the cellular membranes. Determination of the bulk lipids of whole cells, therefore, does not necessarily rule out completely that at the specific site of fusion breakdown of lipid occurs. On the other hand we could measure a drastic decrease of hematoside levels concomitant with an increase in the content of lactosylceramide, most probably occurring at the site where viral components interact with the cell membrane. This change in the glycolipid composition can be explained by the induction of neuraminidase by the virus. It corresponds with the observation of Klenk and Choppin [36] that neuraminidase-containing parainfluenza virus SV5 grown in various cells does not contain gangliosides but rather neutral glycolipids in its envelope. It is rather unlikely that such changes in the glycolipid composition are a general prerequisite for cell fusion, since myxoviruses which contain neuraminidase, do not fuse cells whereas for example herpes virus [37], mumps virus [38], measles virus [39], and
Rous sarcoma virus [40] not containing the enzyme bring about cell fusion. Breakdown of cellular lipids is, therefore, unlikely to be involved in the perturbation of the lipid bilayer structure, leading to cell fusion [13]. This process seems to be a biophysical rather than a biochemical event as far as lipids are concerned. We thank Mrs Ingrid Becker and Miss Michaela Orlich for excellent technical assistance. The work was supported by the Sondrrfi,rsc/zung.~hereic.h47 (Virologie).
REFERENCES I. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11.
12.
13. 14. 15. 16.
Poste, G. (1 970) Adv. Virus Res. 16, 303 - 356. Poste, G. (1972) Int. Rev. Cytol. 33, 157-252. Kohn, A. (1965) Virology, 26, 228-245. Bratt, M. A. & Gallaher, W. R. (1969) Proc. Nut1 Acad. Sci. U.S.A. 64, 536 - 543. Harris, H. (1970) Cell Fusion, Clarendon Press. Oxford. Watkins,J. F. (1971) in Pc.rspectives in Virohxy (Polard,M., ed.) vol. 8, pp. 159 - 178. Academic Press, New York and London. Poste, G. & Allison, A . C. (1973) Biochim. Biophys. Actu, 300, 421 -465. Haywood, A. M. (1974) J . Mol. Biol. 87, 625-628. Papahadjopoulos, D., Poste, G., Schaeffer, B. E. & Vail, W. J . (1974) Biochim. Biophys. Acta, 352, 10-28. Prestegard, J. H. &Fellmeth, B. (1974) Biochemistr.y, 13, 11221126. Ahkong, Q. F., Cramp, F. C., Fisher, D., Howell. J . I., Tampion, W.. Verrinder, M. & Lucy, J. A. (1973) Nut. New Biol. 242, 215-217. Croce, C. M., Sawicki, W., Kritchevsky, D. & Koprowski, H. (1971) Exp. Cell Res. 67, 421-435. Poole, A. R.. Howell, J. 1. & Lucy, J. A. (1970) Nature (Lond.) 227, 810-813. Ahkong, Q. F., Fisher, D., Tampion, W. & Lucy, J. A. (1975) Nature (Lond.) 253, 194- 195. Guttler, F. & Clausen, J . (1969) Biochem. J . 115, 959-968. Howell, J. I . & Lucy, J. A. (1969) FEBS Lett. 4, 147-150.
160
H. Diringer and R. Rott: Lipids in BHK Cells Infected with Newcastle Disease Virus
17. Rubin, H. (1967) in Specificity of Cell Surjaces (Davies, B. D. & Warren, L., eds) pp. 181- 194, Prentice Hall, Englewood Cliffs, New Jersey. 18. Holmes, K. V. & Choppin, P. W. (1966) J . Exp. Med. 124,501 520. 19. Robbins, P. W. & Macpherson, I. A. (1971) Proc. R. Soc. Lond. I77,49 - 58. 20. Folch, J., Lees, M. & Sloane Stanley, G. H. (1957) J . Biol. Chem. 266, 497 - 509. 21. Bartlett, G. R. (1959) J. Biol. Chmn. 234, 466-468. 22. Siakotos, A . N. & Rouser, G. (1965) J . Am. Oil. Chem. SOC. 42, 913-919. 23. Klenk, H.-D. & Choppin, P. W. (1969) Virology. 38, 255-268. 24. Saito, T. & Hakomori, S. (1971) J. Lipid Res. 12, 257-259. 25. Hakomori, S. &Strycharz, G. D. (1968) Biochemistry, 7,12791286. 26. Drzeniek, R., Seto, J. T. & Rott, R. (1966) Biochim. Biophys. Aria, 128, 547 - 558. 27. Nagai, Y., Ogura, H. & Klenk, H.-D. (1976) Virology (in press). 28. Diringer, H., Kulas, H.-P., Schneider, L. G. & Schlumberger. H. D. (1973) Z . Naturfbrsch. 28c, 90-93.
29. Klenk, H.-D. & Choppin, P. W. (1971) J . Virol. 7, 416-417. 30. Klenk, H.-D., Compans, R. W. & Choppin, P. W. (1971) Viro/OgJi, 42. 1158 - 1162. 31. Klenk, H.-D. & Huang, R. T. C. (1973) in Tumor Lipids (Wood, R., ed.) pp. 244-249, Am. Oil. Chem. SOC.Press, Champaign, Illinois. 32. Hosaka, Y. (1960) Biken J . 3, 1-8. 33. Falke, D., Schiefer, H. & Stoffel, W. (1967) Z . Naturforsch. B22, 1360- 1362. 34. Elsbach, P., Holmes, K. V. & Choppin, P. W. (1969) Proc. Soc. Exp. Biol. Med. 130, 903-908. 35. Parkes, J. G. & Fox, C. F. (1975) Biochemistry, 14, 3725 - 3729. 36. Klenk, H.-D. & Choppin, P. W. (1970) Proc. Natl Acad. Sci. U.S.A. 66, 57 -64. 37. Ludwig, H., Becht, H. & Rott, R. (1974) 1.Virof. 14, 307-314. 38. Henle, G., Deinhardt, F. & Girardi, A. (1954) Proc. SOC.Exp. Biol. Med. 87, 381 - 393. 39. Enders, J . F. & Peebles, T. C. (1954) Proc. SOC.Exp. Biol. Med. 86, 277 - 286. 40. Moses, E. & Kohn, A. (1963) Exp. Cell Res. 32, 182-186.
H. Diringer and R. Rott, Institut fur Virologie, Fachbereich Veterinirmedizin der Justus Liebig-Universitat, Frankfurter StraRe 207, D-6300 GieRen, Federal Republic of Germany
Metabolism of Preexisting Lipids in Baby Hamster Kidney Cells during Fusion from Within, Induced by Newcastle Disease Virus Heino DIRINGER and Rudolf ROTT Institut fur Virologie, Justus Liebig-Univerdtat, GieBen
(Received December 8, 1975/January 22, 1976)
The effect of fusion from within induced by Newcastle disease virus on the metabolism of phospholipids, neutral lipids and glycolipids has been studied in baby hamster kidney cells. Hematoside, the only ganglioside in these cells, is rapidly broken down shortly after the onset of virus-induced neuraminidase activity resulting in an increased level of lactosylceramide. No specific breakdown of any phospholipid or neutral lipid could be related to cell fusion.
Cell fusion is a common cytopathic or cytotoxic response to infection by a wide range of viruses (see [1,2] and can be separated into two distinct mechanisms. Fusion from within [3] occurs during the replication of certain enveloped viruses and requires the synthesis of virus specific macromolecules. Fusion from without [4] can be achieved by addition of a high amount of virus - not necessarily infective - to the cells to be fused. During the past years this last method has been used extensively to study the metabolism of heterokaryons (see [5,6]). According to Poste and Allison [7] intermembranous clustering of proteins plays an important role in cell fusion. Two observations, however, indicate that protein interaction might not be the only essential process for fusion but that lipids are also involved. Firstly, it has been demonstrated that protein-free phospholipid vesicles can fuse [8 - 101. Secondly, fusion can be induced by lyso-lecithin [I 1 - 131. Ahkong et al. [14], therefore, have proposed that perturbation of the bilayer structure of membrane lipids (perhaps by the formation of lyso-lecithin [15- 171) increases the fluidity of the lipid region thereby allowing intermembranous aggregation of proteins or glycoproteins [7]. We were interested in the question of whether perturbation of the lipid layer is a matter of a biochemical degradation process of the host cell lipids caused by a virus-induced enzyme activity. As a model baby hamster kidney cells, line 21-F, infected with Newcastle disease virus were chosen, a system in which extensive fusion from within rapidly occurs [3]. We prelabelled the host cell lipids with either [32P]phosphateor tritiated palmitate and measured the radioactive label of in-
dividual neutral lipids, phospholipids, and glycolipids during the course of cell fusion in comparison with uninfected cells.
MATERIALS A N D METHODS
Virus and Experiment The Newcastle disease virus strain “Italien” was used throughout. Baby hamster kidney cells line 21-F [18] were cultivated in 10-cm glass Petri dishes using Eagle’s minimum essential medium with Earle’s balanced salt solution and 10% fetal calf serum. Just before the cells reached confluency each dish received 10 ml of fresh medium containing either 60 pCi of carrier-free [32P]phosphate or 5 pCi (500 mCi/mmol) of [jHIpalmitate (both from Amersham/Buchler). A stock solution of [3H]palmitate was prepared by sonication of the dry radioactive compound with serum, diluting the solution with medium and passing it through a millipore filter [19]. After a 12-h labelling period the radioactive medium was removed and the cells were washed twice with phosphate-buffered saline, pH 7.2. The cells on half of the dishes were infected with virus at a multiplicity of about 10 plaqueforming units/cell. The rest of the cells were mockinfected with allantoic fluid of uninfected chicken eggs. Isohtion and Analysis o f Phospholipids and Neutral Lipids At indicated times pairs of Petri dishes were taken, the medium was removed, and the cells were washed twice with phosphate-buffered saline. Lipids were
156
Lipids in BHK Cells Infected with Newcastle Disease Virus
Fig. 1. The morphologic uppeurunce of' cells hejore ( A ) infection with Newcastle disease uirus und 4 h ( B ) ,5 h ( C ) and 7 h (D)ujter injection. Fusion became detectable 4 h after infection and was clearly established after 5 h
extracted with 4 ml of chloroform/methanol (2/1) and partitioned with 0.9 ml of phosphate-buffered saline [20].Thelipid-containing organicphase wasevaporated to dryness and redissolved in 2 ml of chloroform. Aliquots were taken for the determination of radioactivity and phosphorus content [21]. For determination of radioactivity in individual phospholipids the lipid extract was chromatographed on thin-layers of silica gel [22]. The lipid-containing areas were localized by iodine vapor or by autoradiography and scraped into counting vials. When neutral lipids were investigated the lipid extract was passed through a small column of silica gel
and the lipids were eluted with chloroform [23]. They were chromatographed on silica gel plates with light petroleum/diethyl ether/acetic acid (S0/20/1). The chromatograms were scanned in a radioactive chromatogram scanner.
Isolation and Analysis oj' Glycolipids Radioactive glycolipids were isolated from the cells after the addition of 25 pg of non-radioactive glucosyl-, lactosylceramide, and hematoside to each plate. Lipids were extracted with 3 ml of chloroform/methanol(2/1) followed by 3 ml of chloroform/methanol (1/2). The
H. Diringer and R. Rott
combined extracts were taken to dryness and glycolipids were isolated according to Saito and Hakomori [24]. Individual glycolipids were separated by thinlayer chromatography on silica plates with chloroform/ methanol/ammonia (65/25/4). The thin-layer chromatograms were scanned, afterwards sprayed with 50% sulfuric acid and heated at 140 "C to visualize the lipids. Reference glycolipids were isolated according to Hakomori and Strycharz [25].
157
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-
This was determined according to Drzeniek et al. using fetuin as a substrate [26].
60
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40
+
RESULTS
a,
c
c
In baby hamster kidney cells infected with Newcastle disease virus (strain "Italien") cell fusion starts about 4 - 5 h post infection and is completed within the next 2-3 h (Fig. 1) [27]. To see whether these pronounced and rapid morphologic changes can be correlated to any breakdown of cellular lipids, cells were prelabelled with radioactive lipid precursors, chased and infected with virus. Control cells were mock-infected. After various time intervals cell samples were taken and analyzed for their radioactive lipid content. In the experiments reported here infection occurred immediately after the chase. The results are, however, the same when cells were infected up to 12 h after the chase. Fig. 2 shows the results of an experiment in which phospholipids were prelabelled with [32P]orthophosphate. The specific activity of the total phospholipid fraction decreases slightly during the course of the experiment in virus-infected and mock-infected cells. When individual phospholipid fractions such as lecithin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin were separated by thin-layer chromatography no difference in their relative content of radioactivity could be observed between mock-infected and virus-infected cells at any time of the experiment. In addition no increased levels of lyso-lecithin or lyso-phosphatidylethanolamine could be detected in the infected cells after autoradiography of thin-layer chromatograms. Neutral lipids and glycolipids were prelabelled with tritiated palmitate. When neutral lipids were separated from the polar lipids by silicic acid column chromatography no difference in the time-dependent loss of radioactivity in the total neutral lipid fraction could be observed between controls and infected cells (Fig. 3A). Equal amounts of neutral lipids from cell samples taken 1 h and 7 h after infection were separated into individual lipid fractions by thin-layer chromatography. The superimposed radiochromatograms
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Fig. 2. Specific aclicity o/ content
1
t o t d phospholipid j i a c t i o n ( A ) and
relative
of rarlioaciicephosphate ( B ) plotted against time. ( A )Specific
activity of the total phospholipid fraction of control (U) and virus-infected (0 --O) cells in dependence of time. (B,C) The relative content of radioactive phosphate in individual phosphoand virus-infected (M) cells. From lipids in control (-) top to bottom: lecithin, phosphatidylethanolamine,phosphatidylinositol, phosphatidylserine, and sphingomyelin. In this and the following figures the arrow indicates the time when fusion was clearly visible ( 5 h post-infection)
(Fig. 3B) only showed a somewhat decreased relative content of tritium in the free fatty acid fraction of the "late" sample. A similar result was found in corresponding control cells. Glycolipids only represent a small fraction of the cellular lipids. Baby hamster kidney cells contain only the ganglioside hematoside and the two neutral glycolipids lactosyl- and glucosylceramide [28,29]. These lipids can be separated from phospholipids and neutral lipids by florisil chromatography [25] and can be resolved further by thin-layer chromatography. The radioactive chromatogram of such a lipid isolated from virus-infected baby hamster kidney cells is given in Fig. 4. It can be seen that the non-labelled carrier lipids added before the isolation were recovered in the glycolipid fraction and that radioactivity is associated with these carriers as well as with some unidentified material running near the front. Again no difference
Lipids in BHK Cells Infected with Newcastle Disease Virus
158
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B
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Time after infection (h)
A
Triglycerides
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Alkyl -diglycerides
in the content of radioactivity in the total glycolipid fraction could be detected at any time after virus infection in comparison to the controls. In contrast to glucosylceramide the relative content of radioactivity in hematoside and lactosylceramide changes during the course of the experiment (Fig. 5). In infected cells the loss of radioactivity from hematoside beginning 4 h after infection was accompanied by a concomitant increase of tritium in lactosylceramide. This change correlates with increased viral neuraminidase activity which becomes detectable 3 - 4 h after infection. These results are in agreement with studies of Klenk et al. [30] and Klenk and Huang [31] who also found a decreased amount of lipid-bound neuraminic acid in monkey kidney cells and chicken cells after infection with paramyxoviruses.
DISCUSSION
Fig. 3. R ~ i d i ( ~ ~ ~ t in i c iihr t y total neutrul lipid ,frociion and superimposed rcidiooctiae chromatogram of neutral 1ipid.s. ( A ) Radioactivity and virusin the total neutral lipid fraction of control (+a) cells in dependence of time. (B) Superimposed infected (-) radioactive chromatogram of the neutral lipids of baby hamster and 7 h (. .. . . .) after infection. Equal kidney cells taken 1 h ( -) amounts of radioactivity were chromatographed
1
Hematoside
Our results demonstrate that infection of baby hamster kidney cells with Newcastle disease virus does not affect the breakdown of cellular phospholipids and neutral lipids and therefore specific destruction of such molecules does not play an essential role in virus-induced cell fusion. These results are in agreement with the observations of Hosaka [32] studying Sendai virus induced cell fusion, with results of Falke et al.
I
Fig. 4. Chromatography qfglycoiipids. The bottom panel represents the pure carrier glycolipids. The middle panel shows the chromatogram of the glycolipid fraction of virus-infected cells shortly after infection. The top shows the radioactive scan of the chromatogram of infected cells
H. Diringer and R. Rott
159 Lactosyiceramide
Hematoside
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Time after
ifection (h)
Fig. 5. The relative content of' radioactivity in hemutoside and lactosylceramide of control ( M) und virus-inJected (M) cells in dependence of'time. The data plotted were obtained by measuring the relative area represented by the two lipids in radioactive scans as shown in Fig. 4 taken at various time intervals after infection
[33] using herpes virus in rabbit kidney cells as a model system, and with theexperimentsof Elsbachrtul. [34] who infected baby hamster kidney cells with Simian virus 5. In all these studies it was shown that cellular lecithin did not undergo any appreciable hydrolysis under conditions in which extensive cell fusion occurred. More recently Parkes and Fox [35]demonstrated that no correlation exists between the content of phosphatidylserine in various strains of Newcastle disease virus and its ability to induce fusion from without. Now it can be said that neither degradation of any phospholipid at all, nor enzymatic destruction of neutral lipids, nor breakdown of glycolipids seems to be a necessary event in cell fusion. One has to keep in mind, however, that fusion takes place as a rapid local event in a small proportion of the cellular membranes. Determination of the bulk lipids of whole cells, therefore, does not necessarily rule out completely that at the specific site of fusion breakdown of lipid occurs. On the other hand we could measure a drastic decrease of hematoside levels concomitant with an increase in the content of lactosylceramide, most probably occurring at the site where viral components interact with the cell membrane. This change in the glycolipid composition can be explained by the induction of neuraminidase by the virus. It corresponds with the observation of Klenk and Choppin [36] that neuraminidase-containing parainfluenza virus SV5 grown in various cells does not contain gangliosides but rather neutral glycolipids in its envelope. It is rather unlikely that such changes in the glycolipid composition are a general prerequisite for cell fusion, since myxoviruses which contain neuraminidase, do not fuse cells whereas for example herpes virus [37], mumps virus [38], measles virus [39], and
Rous sarcoma virus [40] not containing the enzyme bring about cell fusion. Breakdown of cellular lipids is, therefore, unlikely to be involved in the perturbation of the lipid bilayer structure, leading to cell fusion [13]. This process seems to be a biophysical rather than a biochemical event as far as lipids are concerned. We thank Mrs Ingrid Becker and Miss Michaela Orlich for excellent technical assistance. The work was supported by the Sondrrfi,rsc/zung.~hereic.h47 (Virologie).
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H. Diringer and R. Rott, Institut fur Virologie, Fachbereich Veterinirmedizin der Justus Liebig-Universitat, Frankfurter StraRe 207, D-6300 GieRen, Federal Republic of Germany
