Glycosphingolipids of Plasma Membranes of Cultured Cells

Transcription

1 Proceedings of the National Academy of Sciences Vol. 66, No. 1, pp , May 1970 Glycosphingolipids of Plasma Membranes of Cultured Cells and an Enveloped Virus (SV5) Grown in These Cells Hans-Dieter Klenk and Purnell W. Choppin THE ROCKEFELLER UNIVERSITY, NEW YORK, NEW YORK Communicated by Maclyn McCarty, February 16, 1970 Abstract. Glycosphingolipids of rhesus monkey kidney (MK), bovine kidney (MDBK), and two lines of hamster kidney (BHK21-F and Hak) cells have been compared with those of parainfluenza (SV5) virions grow in these cells. There are qualitative and quantitative differences in the neutral glycolipids and gangliosides found in the various cells. Cells with a high neutral glycolipid content (MK and MDBK) contain little or no gangliosides, and those with a relatively high ganglioside content (BHK21-F and HaK) contain little neutral glycolipid. Glycosphingolipids are found predominantly in the plasma membranes. Neutral glycolipids of the host cell membrane are incorporated into the envelope of SV5 virions, but neither gangliosides nor protein-bound neuraminic acid are found in virions. The absence of neuraminic acid from the virion may be due to the action of viral neuraminidase. Introduction. SV5, like all myxoviruses and paramyxoviruses, is assembled and released at the cell surface.' In this budding process, the viral envelope is derived from a segment of the plasma membrane which has been altered by the incorporation of viral proteins. Analysis of such membrane-enclosed viruses and the events in virus assembly is not only important to an understanding of virus structure and replication, but may also contribute to the knowledge of membrane structure and biogenesis. All the proteins of the SV5 envelope appear to be virus-specific.2 However, in studies with four cell types, their plasma membranes, and virus grown in each cell, we found that, with a few exceptions, lipids of the plasma membranes are incorporated quantitatively into the viron.3 4 This report describes the glycosphingolipids of cultured cells and their presence in SV5 virons. Glycosphingolipids are composed of sphingosine, fatty acids, and carbohydrates. They may be neutral glycolipids or neuraminic acid-containing gangliosides, and are present in wide variety in the membranes of animal cells. Their carbohydrate moieties may serve as antigenic determinants.5 Thus, if incorporated in the viral envelope, a cellular glycolipid could act as a host antigen which is a constituent of the virion. In spite of the biological importance of glycosphingolipids, little biochemical evidence is available concerning their content in cultured cells or the envelopes of membrane-enclosed viruses. Differences in ganglioside content between normal and virus-transformed cells have been described,6 7 and glycolipids have been detected in myxoviruses,8, 9 but not analyzed in detail. 57

2 58 MICROBIOLOGY: KLENK AND CHOPPIN PROC. N. A. S. We have analyzed the glycolipids of four cell types, of isolated plasma membranes, and of SV5 virions grown in these cells. In an attempt to correlate biological behavior with chemical composition in these and previous studies,3' rhesus monkey, bovine, and hamster kidney cells were selected. The plasma membranes of these cells show striking biological differences, including differing sensitivities to virus-induced cell fusion and immune cytolysis, and large differences in the amount of infective virus produced." 10, 1 1 Materials and Methods. Glycosphingolipids used as reference substances (Table 1) were kindly provided by Drs. E. Klenk, W. Gielen, and G. Tschope of the University of Cologne. Cells: Primary rhesus monkey kidney (MK) cells and three cell lines, bovine kidney (MDBK), baby hamster kidney fibroblasts (BHK21-F), and adult hamster kidney epithelioid cells (HaK), were grown as described previously.3' 10 For preparation of plasma membranes, BHK21-F and HaK cells were grown in suspension culture. For determinations on whole cells, monolayers were washed with phosphate-buffered saline, scraped off, and pelleted. Cells in suspension culture were pelleted and washed twice with the saline solution. Packed cells were suspended in 10 volumes of water and disrupted by sonication for about 3 min with a Branson sonifier. Protein was determined on aliquots by the Lowry method, and preparations containing gm protein were lyophilized and stored at -250C until used. Plasma membranes were isolated as described previously3 by a modification of the fluorescein mercuric acetate method of Warren.12 A plasma membrane preparation from BHK21-F cells containing 42 mg protein, and three preparations from MDBK cells containing 23, 18, and 11 mg protein were analyzed. Virus: Cells were inoculated with the W3 strain of SV513 at multiplicities of 5-20 plaque-forming units/cell, and released virus only was harvested and purified as described previously.'4 Three samples of MDBK-grown virus containing 27, 22, and 10 mg protein, and two samples of BHK21-F-grown virus containing 6 and 10 mg protein were analyzed. TABLE 1. Glycosphingolipids used as reference substances. Structure Source Name Gal-Cer Ox brain Cerebroside Gal-Glu-Cer Ox spleen Gal-Gal-Glu-Cer Ox spleen GalNAc-Gal-Glu-Cer Brain, Tay-Sachs disease GalNAc-Gal-Gal-Glu-Cer Human erythrocytes Globoside Gal-GalNAc-Gal-Glu-Cer Cleavage product from gangliosides -SO3-Gal-Cer Ox brain Sulfatide -Gal-Glu-Cer Dog erythrocytes N-acetylneuraminosyl-lactose ganglioside (hematoside) NeuNGly-Gal-Glu-Cer Horse erythrocytes N-glycolylneuraminosyl-lactose ganglioside (hematoside) Gal-GaiNAc-Gal-Glu-Cer Human brain N-acetvlneuraminosyl-N-tetraose ganghioside Gal-GalNAc-Gal-Glu-Cer Human brain di(n-acetylneuraminosyl)-n-tetraose ganglioside Gal-GalNAc-Gal-Glu-Cer Human brain tri(n-acetylneuraminosyl)-ntetraose ganglioside Abbreviations: Cer = ceramide; Glu = glucosyl; Gal = galactosyl; GalNAc = N-acetylgalactosaminyl; = N-acetylneuraminosyl; and NeuNGly = N-glycolylneuraminosyl.

3 VOL. 66, 1970 MICROBIOLOGY: KLENK AND CHOPPIN 59 Isolation and fractionation of glycolipids: Lyophilized samples were extracted with chloroform-methanol-water and partitioned into an aqueous phase containing the more polar glycolipids, and an organic phase containing the less polar glycolipids and other lipids.3 Lipids were extracted from the dialyzed, lyophilized aqueous phase with chloroform-methanol (1:1).6 Neuraminic acid content was determined on aliquots as a parameter of total ganglioside content. The glycolipid mixture was either submitted to analytical thin-layer chromatography, or further purified and separated by preparative thin-layer chromatography. The organic phase was separated on a silicic acid column' into a fraction containing neutral lipids and another containing phospholipids and less polar glycolipids. The latter was dried in a flash evaporator, and glycerophospholipids were removed by alkaline saponification.'5 These samples were analyzed by thin-layer chromatography. The glycolipids were further fractionated and separated from sphingomyelin by preparative thin-layer chromatography either immediately or after chromatography on a Florisil column (7.5 gm, prepared in chloroform). On elution with 30 ml of chloroform-methanol mixtures of 10:0, 8:2, 3:7, 2:8, 1:9, and 0:10, fraction 2 contained glucosylceramide, and fractions 4 and 5, N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide. Preparative thin-layer chromatography was done on 20 X 20 cm plates coated with 0.5 mm Silica Gel H (Merck, Germany). Two solvent mixtures were employed: chloroformmethanol-water, 65: 25:4 and 60: 35: 8. Glycolipids were localized with iodine, and spots were removed and extracted with methanol. Analytical thin-layer chromatography: Glycolipids were separated with the above solvents on plates coated with 0.3 mm Silica Gel H. For detection of hexose-containing lipids, plates were sprayed with H2S04 (50% by vol), and slowly charred on a hot plate. Before turning black, glycolipids showed a purple color, whereas phospholipids appeared as brown spots. For detection of gangliosides, plates were sprayed with Bial's reagent,'6 covered with a glass plate, and heated at 110C for 15 min. Phospholipids were located with Zinzadze's reagent.' For identification of sugars, purified glycolipids were hydrolyzed in 4 N HCl for 4 hr at 100'C. Acid was removed by lyophilization, and the residue was submitted to thin-layer chromatography on coated cellulose sheets (MN Polygram Cel 300, Macherey-Nagel, Germany); solvent, ethyl acetate-pyridine-acetic acid-water (5:5:1:3); stain, alkaline silver nitrate reagent;"8 fixation, 5% sodium thiosulfate in methanol-water (1:1). Quantitative analyses: Hexose was determined by the anthrone-h2so4 method1 with glucose as standard, hexosamine by a modification of Elson-Morgan's reaction20 with glucosamine-hcl as standard, neuraminic acid with thiobarbituric acid2' after hydrolysis of glycolipids or lipid-extracted virus in 0.1 N H2S04 for 1 hr at 800C, or with resorcinol,22 using N-acetylneuraminic acid as standard. Enzyme degradation of gangliosides :23 About 100 gg ganglioside and 25 units Vibrio cholerae neuraminidase (Behringwerke, Marburg, Germany) were dissolved in 0.5 ml sterile distilled water, and incubated in a dialysis bag in 100 ml water overnight at 370C. After concentration of the dialysate in a rotary evaporator, liberated neuraminic acid was examined by thin-layer chromatography.24 The residue in the bag was lyophilized, extracted with chloroform-methanol (1: 1), and assayed for glycolipids by thinlayer chromatography. Results. The major glycosphingolipids of cells, plasma membranes, and virions were characterized by: (1) comparison of migration rates with reference substances on thin-layer chromatography, (2) determination of constituent sugars after acid hydrolysis, and (3) partial degradation of gangliosides by neuraminidase. Neutral glycosphingolipids: Most of the neutral glycolipids were found in the organic phase of the chloroform-methanol-water partition. Figure 1 shows a chromatogram of glycolipids obtained from MK and MDBK cells and SV5 virions. The glycolipid identified as glucosylceramide had a slightly higher Rf

4 60 MICROBIOLOGY: KLENK AND CHOPPIN PROC. N. A. S : : FIG. 1.-Thin-layer chromatogram of neutral glycolipids of MK and MDBK cells, plasma membranes, and SYS virions. (1) galactosylceramide, (2) galactosyl-galactosyl-glucosylceramide, (3) N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide, (4) MK cells, (5) SV5 grown in MK cells, (6) MDBK cells, (7) MDBK cell plasma membranes, (8) SV5 grown in MDBK cells. The glycolipids of the organic phase were applied. They were obtained from samples containing the following amounts of protein: MK and MDBK cells, 10 mg; MK-grown SYS and MDBK plasma membranes, 2.5 mg; MDBK-grown SV5, 1 mg. Solvent: chloroform-methanol-water (65:25:4). H2S04 spray. The glycolipids may appear as double spots.33 FIG. 2.-Thin-layer chromatogram of gangliosides of BHK21-F, HaK, MDBK, and MK cells. (1) N-acetylneuraminosyl-lactose ganglioside, (2) BHK21-F cells, (3) N-glycolylneuraminosyl-lactose ganglioside, (4) HaK cells, (5) N-acetylneuraminosyl-N-tetraose ganglioside, (6) di(n-acetylneuraminosyl)-n-tetraose ganglioside (upper band) and tri(n-acetylneuraminosyl)-n-tetraose ganglioside (lower band), (7) MDBK cells, (8) MK cells. The gangliosides of the aqueous phase were applied. Each was derived from a cell sample containing 10 mg protein. Solvent: chloroform-methanol-water (60: 35: 8). Bial's reagent. FIG. 3.-Thin-layer chromatogram of the three major gangliosides of HaK cells and their products after neuraminidase treatment. (1) N-acetyl and N-glycolylneuraminosyl-lactose ganglioside before, and (2) after neuraminidase treatment, (3) N-acylneuram-inosyl-N-tetraose ganglioside before, and (4) after neuraminidase treatment, (5) di(n-acylneuraminosyl)-ntetraose ganglioside before, and (6) after neurarninidase treatment. Solvent: chloroformmethanol-water (60:35:8). Bial's reagent. The spot in (2) did not show the typical color. value than the reference galactosylceramide (Fig. 1, lane 1), and acid hydrolysis of this glycolipid released only glucose. Galactosyl-galactosyl-glucosylceramide was identified by migration identical to the reference substance from ox spleen (lane 2), and by release of galactose and glucose. (Lactose sulfatide has a similar 1?, value. It has been found in small amounts in human kidneys' and its presence in MK cells cannot be excluded.) N-acetylgalactosaminyl-galactosyl-galactosylglucosylceramide had a value identical to the reference substance from human erythrocytes (lane 3) and yielded galactosamine, galactose, and glucose on hydrolysis, with a molar ratio of hexosamine :hexose of 0.9:3. A small amount (10%) of this glycolipid was found in the aqueous phase. Neutral glycolipids are found in relatively large amounts in MK and MDBK cells (Fig. 1 and Table 2), but are absent (or present in only trace amounts) in BHK21-F and HaK cells. MDBK cells contain glucosylceramide and N- acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramjde. MK cells contain these and galactosyl-galactosyl-glucosylceramide. Plasma membranes show the same patterns as whole cells, but the ceramides are present in concentrations 4-8 times as high (Fig. 1, Table 3). The neutral glycolipid content of virus grown in MDBK or MK cells clearly reflects that of the plasma membranes

5 VOL. 66, 1970 MICROBIOLOGY: KLENK AND CHOPPIN 61 TABLE 2. Neutral glycolipid content of TABLE 3. Neutral glycolipid content of MDBK and MK cells. MDBK cells, plasma membranes, MDBK MK and SV5 grown in these cells. (Mug per 100 mg Plasma SV5 Glycolipid protein) Whole mem- vir- Glu-Cer cells branes ions Gal-Gal-Glu-Cer (Mig per 100 mg GalNAc-Gal-Gal-Glu-Cer Glycolipid protein) Glu-Cer GalNAc-Gal-Gal-Glu-Cer (Fig. 1 and Table 3). However, virions from MK cells contain additional, slowly migrating neutral glycolipids (Fig. 1, lane 5). These have not been completely identified, but one component has the same Rf value as galactosyl-nacetylgalactosaminyl-galactosyl-glucosylceramide. Gangliosides: The aqueous phase contained most of the gangliosides. Figure 2 shows a chromatogram of gangliosides from the four cell types. The three major gangliosides from HaK cells (Fig. 2, lane 4) were isolated by preparative thin-layer chromatography. The fastest-migrating material was found to be predominantly N-acetylneuraminosyl-galactosyl-glucosylceramide, with a small amount of N-glycolylneuraminosyl-galactosyl-glucosylceramide. These compounds formed a double spot with Rf values identical to those for the reference substances from dog and horse erythrocytes (lanes 1 and 3). Acid hydrolysis released glucose and galactose, and the products of neuraminidase degradation were galactosyl-glucosylceramide (Fig. 3), N-acetylneuraminic acid, and N- glycolylneuraminic acid. A smaller amount of this glycolipid was recovered from the organic phase. The second ganglioside was identified as galactosyl- N-acetylgalactosaminyl-(N-acylneuraminosyl-) galactosyl-glucosylceramide because: (a) it had an Rf value identical to the reference substance from human brain (Fig. 2, lane 5); (b) hydrolysis released glucose, galactose, and galactosamine; and (c) it was unaffected by neuraminidase (Fig. 3), which showed that neuraminic acid is not linked to the terminal galactose. This second ganglioside from HaK cells and N-acetylneuraminic acid were the products of neuraminidase action on the third ganglioside (Fig. 3); this indicates the presence of an additional neuraminic acid residue in the latter. Since its Rf value was different from galactosyl-n-acetylgalactosaminyl-di(n-acetylneuraminosyl-)galactosyl-glucosylceramide (Fig. 2, lane 6), this third ganglioside appears to be N- acetylneuraminosyl-galactosyl-n-acetylgalactosaminyl - (N- acylneuraminosyl-) - galactosyl-glucosylceramide. There are distinctive qualitative (Fig. 2) and quantitative (Table 4) differences between the gangliosides of the various cells. BHK21-F cells contain the most total ganglioside, which is predominantly N-acetylneuraminosyl-lactose ganglioside, and very small amounts of unidentified gangliosides. HaK cells have a lower content, but contain the three major gangliosides described above. MK and MDBK cells have patterns similar to HaK cells, but contain only small amounts of gangliosides. As with neutral glycolipids, plasma membranes of BHK21-F and MDBK cells contain the same gangliosides as whole cells, but in much higher concentrations

6 62 MICROBIOLOGY: KLENK AND CHOPPIN PROC. N. A. S. TABLE 4. Ganglioside- content of BHK21-F, HaK, MDBK, and MK cells, plasma membranes, and SV5 grown in these cells. Plasma Cells Whole cells membranes SV5 virions (Glycolipid-bound neuraminic acid, pxg per 100 mg protein) BHK21-F None detected HaK 20 * MDBK 9 30 None detected MK 8... None detected * Not determined. (Table 4). Gangliosides were not found in most preparations of SV5 virions, but were detected in trace amounts in one sample of BHK-grown virus. The lack of neuraminic acid-containing glycolipids in virions grown in cells that contain large amounts in their plasma membranes provides the first example of a membrane lipid that is not incorporated into the viral envelope. To determine if protein-bound neuraminic acid was present in SV5 virions, which contain about 6% carbohydrate,'4 we analyzed virus samples containing mg protein. No neuraminic acid was detected (neuraminic acid content therefore <0.03%). As discussed below, the absence of this substance from virions could be related to the presence of the viral neuraminidase. Discussion. Table 5 summarizes results for the glycosphingolipids found in the four cell types. The various cells show distinctive patterns. BHK21-F cells contain the highest amount of gangliosides, almost all in the form of N-acetylneuraminosyl lactose ganglioside, a finding similar to that of Hakomori and Murakami in another line of BHK21 cells.6 The other cells contain mono(nacylneuraminosyl)-and di(n-acylneuraminosyl)-n-tetraose gangliosides, a pattern found in cultured mouse cells.7 A striking finding is that BHK21-F and HaK cells, which contain large amounts of gangliosides, contain almost no neutral glycolipids, whereas the reverse is true with MDBK and MK cells. The relative concentrations of glycolipids in whole cells and plasma membranes indicate that these substances are located predominantly in plasma membranes, but their presence in other cellular structures cannot be excluded. It is clear that glycolipids comprise a significant portion of the total polar lipids of the plasma membranes, 30% in MDBK cells and 10% in BHK21-F cells. TABLE 5. Glycolipid content of four types of cultured cells. Cells- BHK21-F HaK MDBK MK Neutral glycolipids: Glu-Cer i i + + Gal-Glu-Cer Gal-Gal-Glu-Cer - GaJNAc-Gal-Gal-Glu-Cer Gangliosides: -Gal-Glu-Cer NeuNGly-Gal-Glu-Cer Gal-GalNAc-Gal-Glu-Cer Gal-GalNAc-Gal-Glu-Cer NeuAAc

7 VOL. 66, 1970 MICROBIOLOGY: KLENK AND CHOPPIN 63 Previous studies3 4indicated that, in general, quantitative differences in the lipid patterns of the plasma membranes were clearly reflected in the virions grown in different cells. This has now been found with neutral glycolipids, but here the qualitative differences between various cell membranes are also reflected in the virions, e.g., galactosyl-galactosyl-glucosylceramide is found in MK cells and virions grown in them, but not in MDBK cells or virions. Such differences in the lipids of virions which, though grown in different cells, contain the same virus-specific proteins again emphasize the importance of the host cell membrane in determining the lipid composition of the virion. In contrast to the incorporation of cholesterol, phospholipid, and neutral glycolipids of the plasma membrane into SV5 virions is the lack of incorporation of gangliosides into virions. A possible explanation is that neuraminidase is synthesized in infected cells and incorporated into the viral envelope. This enzyme might either cleave neuraminic acid from previously formed gangliosides, or interfere with the synthesis of gangliosides by preventing the addition of neuraminic acid residues to the terminal sugar of a growing carbohydrate chain, thus leading to the synthesis of other, neutral glycolipids. That the latter may be the case is suggested by the presence of unidentified neutral glycolipids with long carbohydrate chains in virus grown in MK cells (Fig. 1), and by the absence of neuraminidase-insensitive gangliosides, i.e., those with neuraminic acid in a nonterminal position. Whatever the precise explanation, the neuraminidase action apparently occurs only at regions of cell membrane where virus proteins are incorporated, because infected cells still contain membrane-bound neuraminic acid and because colloidal iron, which stains neuraminic acid residues, stains the surface of infected cells everywhere except where virus is budding.25 Gangliosides thus represent the only class of plasma membrane lipids that are eliminated, or drastically decreased, when the membrane is transformed into the viral envelope. It is therefore interesting that the ganglioside content of the four cell types investigated is directly proportional to the extent of fusion induced in these cells by SV5, and inversely proportional to the yield of infective virus.', 10 BHK21-F cells, with the highest content, fuse rapidly and extensively and then disintegrate, and produce little virus because of a block in virus maturation at the cell surface; whereas MK cells, which contain a small amount of gangliosides, are resistant to cell fusion and produce a high yield of infective virus with little cell damage. These and previous results,3' 4in which there was a correlation between high phosphatidylethanolamine content, and high virus yield, and little cell fusion, suggest that the lipid composition of the plasma membranes and changes in lipids that occur in virus-altered regions of these membranes may play an important role in virus yield, cell fusion, and cell death. Glycolipids may be antigenic5 and contribute to the immunological specificity of cell surfaces. When isolated from different sources, N-acetylgalactosaminylgalactosyl-galactosyl-glucosylceramide (which is a major glycolipid in MK and MDBK cells) exhibits different immunological specificities, including Forssman antigenicity, depending on the anomeric configuration of the terminal disaccharide.26' 27 Blood group and Forssman antigenic activities were found associated with myxovirus and paramyxovirus particles grown in cells which possess these antigens,^' 29 although in some cases the virus was purified only

8 64 MICROBIOLOGY: KLENK AND CHOPPIN PROC. N. A. S. by differential centrifugation. The present studies provide biochemical evidence of such glycosphingolipids in highly purified parainfluenza virus particles. These results and the finding of a glycopeptide host cell antigen in influenza virions30 31 suggest that if host cell antigens are present in enveloped viruses, carbohydrate moieties will be the antigenic determinants. The recent evidence suggesting that all the proteins of myxovirus and paramyxovirus particles are probably virus-coded2' 32supports this concept. We thank Mrs. Barbro Hammarstr6m and Miss Anna Wangel for excellent technical assistance. * Supported by research grant AI from the National Institute of Allergy and Infectious Diseases and contract AT(30-1)-3983 from the U.S. Atomic Energy Commission. 1 Compans, R. W., K. V. Holmes, S. Dales, and P. W. Choppin, Virology, 30, 411 (1966). 2 Caliguiri, L. A., H.-D. Klenk, and P. W. Choppin, Virology, 39, 460 (1969). 3 Klenk, H.-D., and P. W. Choppin, Virology, 38, 255 (1969). 4Klenk, H.-D., and P. W. Choppin, Virology, in press (1970). 6 Martensson, E., Progr. Chem. Fats Lipids, 10, 367 (1969). 6 Hakomori, S., and W. T. Murakami, these PROCEEDINGS, 59, 254 (1968). 7 Mora, P. T., R. 0. Brady, R. M. Bradley, and V. W. McFarland, these PROCEEDINGS, 63, 1290 (1969). 8 Armbruster, O., and U. Beiss, Z. Naturforsch., 13b, 75 (1958). 9 Blough, H. A., and D. E. M. Lawson, Virology, 36, 286 (1968). 10 Holmes, K. V., and P. W. Choppin, J. Exptl. Med., 124, 501 (1966). "1 Holmes, K. V., H.-D. Klenk, and P. W. Choppin, Proc. Soc. Exptl. Biol. Med., 131, 651 (1969). 12 Warren, L., M. C. Glick, and M. K. Nass, J. Cell Physiol., 68, 269 (1966). 13 Choppin, P. W., Virology, 23, 224 (1964). 14 Klenk, H.-D., and P. W. Choppin, Virology, 37, 155 (1969). 15 Eto, T., Y. Ichikawa, K. Nishimura, S. Ando, and T. Yamakawa, J. Biochem., 64, 205 (1968). 16 Klenk, E., and H. Langerbeins, Z. Physiol. Chem., 270, 185 (1941). 17 Dittmer, J. C., and R. L. Lester, J. Lipid Res., 5, 126 (1964). 18 Stahl, E., Dfinnschichtchromatographie 2nd ed. (Berlin: Springer-Verlag, 1967). 19 Scott, T. A., Jr., and E. H. Melvin, Anal. Chem., 25, 1656 (1953). 20 Gatt, R., and E. R. Berman, Anal. Biochem., 15, 167 (1966). 21 Aminoff, D., Biochem. J., 81, 384 (1961). 22 Suzuki, K., Life Sci., 3, 1227 (1964). 23 Klenk, E., and W. Gielen, Z. Physiol. Chem., 330, 218 (1963). 24 Granzer, E., Z. Physiol. Chem., 328, 277 (1962). 25 Klenk, H.-D., R. W. Compans, and P. W. Choppin, unpublished experiments. 26 Makita, A., C. Suzuki, and Z. Yosizawa, J. Biochem., 60, 502 (1966). 27 Koscielak, J., S. Hakomori, and R. W. Jeanloz, Immunochem., 5, 441 (1968). 28 Rott, R., R. Drzeniek, M. S. Saber, and E. Reichert, Arch. Ges. Virusforsch., 19, 273 (1966). 29 Isacson, P., and A. E. Koch, Virology, 27, 129 (1965). 30 Laver, W. G., and R. G. Webster, Virology, 30, 104 (1966). 31 Lee, L. T., C. Howe, K. Meyer, and H. U. Choi, J. Immunol., 102, 1144 (1969). 32 Holland, J. J., and E. D. Kiehn, Science, 167, 202 (1970). 33 Adams, E. P., and G. M. Gray, Chem. Phys. Lipids, 2, 147 (1968).