90
Biochimico et Biophysics Acta, 424 (1976) 90-97 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56707
SPHINGOLIPIDS
OF INFLUENZA
VIRUSES
RICHARD T.C. HUANG Institute
of Virology,
Justus Liebig-University
of Giessen (G.F.R.)
(Received July 25th, 1975)
Summary Total lipid of four egg grown influenza viruses (AT -Asia, Az -England, As-Taiwan and fowl plague virus) were extracted with chloroform-methanol. After mild alkali treatment of the extracts, glycosphingolipids and sphingomyelin were separated by a silicic acid column, and finally purified by thin layer chromatography. Fatty acid, sphingosine and carbohydrate components of individual lipid classes were then analysed by gas-liquid chromatography. Nearly identical results were obtained with all viruses investigated. Approximately 20% of the total lipid was monohexosylceramide, distributed equally between glucosyl- and galactosyl- analogues. Lactosylceramide and oligohexosylceramides were found in much smaller concentrations (approx. 2%). About 15% of the total lipid was attributed to sphingomyelin. A large proportion of fatty acids (around 25% in sphingomyelin and 60% in glycolipids) belonged to the long chain (C, 9 -C2 6 ) normal- and 2-hydroxy series. C1 8 sphingosine was found to be the only base present in all lipid classes investigated.
Introduction The surface of enveloped viruses possesses a lipid bilayer, which is sandwiched between proteins. Such a membrane is found ubiquitously in biological systems, where a stable structural boundary is necessary for survival. In the past, information has accumulated with regard to the molecular architecture of biological membranes. In virus systems the fatty acids of phospholipids have been determined [ 1,2] . A comparable study on sphingolipids, in particular glycosphingolipids, has not as yet been adequately performed. With the intention of exploring specific characteristics which might associate with viral membranes, we have proceeded to analyse the sphingolipids of four influenza viruses. The results obtained indicated that lipids of influenza viruses contain typical membrane components, which are rich in sphingolipids with long chain normal and 2-hydroxy fatty acids.
91
Materials and Methods Virus. Four influenza strains, As-Asia, AZ-England, AZ-Taiwan and fowl plague virus were propagated in ~horio-~antoi~ cavities of chick embryos. Allantoic fluid with haemagglutinating t&es* of around 2-’ was harvested. Purification of viruses was achieved by sucrose density gradient centrifugation [ 31, where rate zonal and isopycnic procedures were applied sequentially in the same run. Fowl plague virus was propagated and purified in this laboratory, whereas other three viruses were kindly provided by Behringwerke AG., Marburg. Lipid standards. Glucosylceramide was extracted from the spleen of a Gaucher patient according to the method described by Klenk and Rennkamp [4]. Galactosylceramide was obtained from human brain by the same method. Lactosylceramide was obtained by neuraminidase action (receptor destroying enzyme from Behringwerke AG., Marburg) of hematoside mixtures obtained from butter milk [ 51. Sphingomyelin was obtained from human brain as described elsewhere [ 61. Extraction of lipids. Virus concentrates (200 ml from each of A2 -strains, and 100 ml from fowl plague virus) which had haemag~u~nating titres of 2-l 5 or 2-l 6 were dialysed against running water to remove the sucrose present. Dialysates were freeze dried and extracted twice with 50 ml of chloroform / methanol (2 : 1). Extracts were pooled and evaporated to dryness in a rotary evaporator. Total lipid which remained was determined ~avime~ic~ly. Fractionation of lipids. To facilitate isolation of sphingolipids, glycerolipids were hydrolysed by alkali essentially as described elsewhere [6]. Preliminary separation of individual lipid classes was achieved by silicic acid column ~hromato~aphy. Silicic acid, mesh 70-230, ASTM (Merck), was slirried in chloroform and packed in a column of 2 X 12 cm. After equilibration with chloroform, lipid samples (32-84 mg) were applied in chloroform. Elution of fractions was performed with 80 ml of each of the following solvent mixtures; ~hlorofo~ eluted all “neutral lipids” (cholesterol, triacylglycerol, free fatty acid and cholesteryl ester), 25% of methanol in chloroform eluted all monohexosylceramide and lactosylceramide. 50% Methanol in chloroform eluted oligohexosylceramide and methanol eluted sphingomyelin. The purity of the fractions were monitored by thin layer ~hromato~aphy using silicic acid plates (0.25 mm) and chloroform/methanol/water (60 : 35 : 8) as developing solvents. All spots were visualized by spraying with 5% aqueous sulfuric acid and heating at 150°C. Identification of each lipid classes was accomplished by cochromatography with standard lipids. For final purification of each lipid classes, 20-30 mg of each lipid fraction was applied as a narrow band on a silicic acid plate (0.25 mm) and developed with the same solvent mixture. The lipid bands, which had been visualized by spraying with water, were scraped off with the aid of a razor blade and extracted with chloroform/methanol (1 : 1). Quantitution of individual sphingolipids. Spingholipids were estimated titre indicates the extent of two fold delution of the virus still cause agglutination of erythrocytes.
* HaemagglUtinating
preparationswhich
92
directly on a thin layer plate by visual comparison of virus lipids (30 mg/ml in chloroform/methanol 1 : 1) with serially diluted solutions of standard lipids (from 0.1 mg to 1.0 mg in 0.1 ml of chloroform/methanol 1 : 1) essentially as described by Hakomori et al. 171. In several instances, lipids were qu~tita~d by gas chromatography of fatty acid methyl esters, using heptanoic acid methyl ester as the internal standard. The results agree within ?5% to those obtained above. ~~d~o~ys~s of Eipids. Approxima~ly 10 mg of the lipids were hydrolysed in sealed 5 ml ampules with 2 ml of a mixture of methanol/concn. HCl/water (70 : 4 : 4) for 6 h [8]. At the end of this period fatty acids, which had been converted quantitatively to methyl esters, were extracted twice with equal volumes of heptane. The lower layer (methanol/HCl layer), which contained the methyl sugars and the sphingosine, was evaporated to dryness. The residual HCl was removed by repeated addition of methanol to the residue and continued evaporation. Analysis of fatty acids. Fatty acids were analysed in a gas chromatograph, model Perkin-Elmer F 20 B. The column (2 m X 0.27 cm) was 1% silicone ester-30 on chromosorb G, 80-100 mesh, AW-DMCS. Nitrogen gas, which was used as the moving phase, was supplied at a pressure of 2. 5 Kg/cm2. Column temperature was programmed from 160” -260°C at 2°C per minute. Injector temperature was 300” C. Normal fatty acids from C1 4 to CZ 6 were identified by comparing with authentic standards from Merck. Hydroxy fatty acids were identified by comparing with those obtained from cerebrosides of human brain, the structure of which had been previously described [ 9,101. For better separation of fatty acids according to degrees of unsaturation a column (2 m X 0.27 cm) of 5% ethyleneglycol succinate on gas chrom A, 80-100 mesh was used. Analysis of s~hingosi~~ and sugars. The residue which remained in the lower methanol/NC1 phase after removal of methanol and HCl, was silylated according to the method of Gaver and Sweeley [8] and gas chromatographed in a SE-30 column, 200 X 0.27 cm. Column temperature was programmed from 140 to 170°C at 2°C per min to elute the sugars, and subsequently from 170”~240°C at 7.5”C per min to analyse the sphingosine. The condition provided the possibility to analyse sugar and sphingosine components simultaneously on the same chromatogram. Results The method [3] of sequential zonal rate and isopycnic centrifugation were employed to obtain highly purified viruses. The preparations contained only virus particles as judged by electron microscopy and by polyacrylamide gel electrophoresis of the proteins. The amounts of total lipid in all virus samples were comparable. The yields ranged from 32-42 mg in 100 mg of the total protein. For further analysis total lipid was treated with alkali to hydrolyse glycerolipids, which otherwise could not be readily separated from sphingolipids. After such a treatment with alkali all strains of influenza viruses investigated gave similar lipid patterns (Fig. 1). Spots of monohexosylceramide and sphingomyelin were the most prominent in all virus samples. In comparison, cells of chorioallantoic membrane where the bulk of the virus
93
Fig. 1. Sphingolipid pattern of influenza viruses and of the chorioalIantoic membrane of the fowl plague virus infected chick embryos I, AZ-A&: II, AZ-England; III, AZ-Taiwan; IV, fowl plague virus: V, enriched glycolipid fraction from the chorioallantoic membrane; VI, total lipid of the chorioallantoic membrane. Thin layer plate: Kieselgel (0.25 mm). Developing solvent: chloroform/methanol/water 60 : 35 : 8. visualized by spraying with 5% H2SO4 and heated at 15O’C.
propagated, contained only small proportions of glycolipids (Fig. 1, Table I). Spots corresponding to monohexosylceramide yielded glucose and galactose in approximately the same quantity on hydrolysis. It was therefore concluded that they were mixtures of equal amounts of glucosylceramide and galactosylceramide. Lactosylceramide was present in only very small amounts in A2 -Taiwan and fowl plague virus, but could not be clearly detected in the other two viruses. The spots were identified as lactosylceramide, since they cochromatographed with the standard lactosylceramide and yielded galactose and glucose in equimolar quantities on hydrolysis. Fractions containing oligohexosylceramide were separated into at least four components on thin layer plates. Due to limited quantities of material available, each component could
94 TABLE I COMPOSITION (% OF TOTAL LIPID) OF SPHINGOLIPIDS CHORIOALLANTOIC MEMBRANE OF FOWL PLAGUE -~ Lipids
-----.___.
IN INFLUENZA VIRUSES AND IN THE VIRUS INFECTED CIiICK EMBRYOS
Viruses
___-
Monohexosyl. ceramide Lactosylceramide Oligohexosylceramide Sphingomyelin Others
AZ-Asia
AZ-England
A2JI’aiwan
Fowl plague
ChorioaIlantoic membrane
17.5 0 1.5 17.5 63.5
20 0 2 17.5 60.5
20 0.5 2 15 63.0
22.5 1.5 2 15 59.0
1.5 1.5 0 10 67.0
not be rigorously identified. However, hydrolysis of the whole fraction revealed the presence of galactose, glucose, glucosamine, galactosamine and fucose in a proportion of about 2 : 1.5 : 1 : 0.3 : 0.3. Fatty acids of all individual sphingolipids were estimated by gas-liquid chromatography. Using a nonpolar column (SE-30) and applying the technique of temperature programming, it was possible to separate all fatty acid methyl esters. The 2-hydroxy fatty acids, when unacetylated, emerged as separate peaks from the, normal fatty acids of corresponding chain lengths. Identical fatty acid patterns were obtained for the corresponding sphingolipids of the four viruses analysed. Since the percent composition of each fatty acids in the comparable sphingolipids did not vary beyond a maximum of i 5% among different viruses, the values for the fowl plague viruses is shown in Table II to represent the composition for all four viruses. For comparison, fatty acid values of the chorio-allantoic membrane, where the bulk of the virus multiply, is also included in the table. It can be ascertained from the table that the presence of a high concentration of long chain fatty acids is a common feature in all sphingolipids. Long chain (C, 9 -Cz 6 ) normal and 2-hydroxy fatty acids together accounted for approximately 60% and 25% of the total fatty chains in the glycolipids and sphingomyehn respectively (Table II). Sphingomyelin contained very little Zhydroxy fatty acids, whereas about 40% of the fatty chains in the glycolipids could be attributed to these hydroxy analogues. It can also be pointed out, that although fatty acid patterns were similar among glycolipids, lactosylceramide and oligohexosylceramide were more alike, both being rich in the 2-hydroxyCz 6 -acid, (Table II). Oleic acid and linoleic acid were the main unsaturated fatty acids found in all sphingolipids. They overlapped in one peak when chromato~aphed in a SE-30 column, but could be resolved in an ethyleneglycol succinate column into separate peaks with a ratio of about 3 to 1 in all lipid classes. Sphingosine and carbohydrate components could be simultaneously estimated in an SE-30 column. It was found that C1 s-sphingosine was the only base present in all sphingolipid classes.
c22:o ‘23:O ‘24:O ‘24:l C26:0 ‘26:O ‘l&OH CZO.OH C22.OH ‘23-GH ‘24OH c25-OH ‘26-OH
‘16:O ‘18:O
-
7*6
-
Oag :‘B”
*
7.3 ;;
’
’
7.6
-
-
14.8 8.0 9.0 1.1 -
6.9
0
48.9
7.5 0.5 4.9 0.4 0.8 1.3 1.9 2.2 16.5 1.3 15.4 1.7 1.2
23.0 10.6 7.7 3.6
40.0
19.0
41.6 7.8 1.7
30.0 17.3
7.1 0.2 4.6 6.8 0.4 1.1
0.4 0.2 7.6 2.1 36.8
-
-
3.2
20.0 7.2 2.5 -
I
47.0
1
23.2
Chorioellantoic membrane
Fowl pIague
virus
Choxioallentoic membrane
Fowl plague
ViIUS
Monohexosylceramide
OF THE FOWL
Sphingomyelin
acids
ACID COMPOSITIONS OF THE SPHINGOLIPIDS INFECTED WITH THE SAME VIRUS
OH refers to z-hydrory
W FATTY EMBRYOS
VIRUS
8.5 0.3 5.0 0.5 3.2 1.1 1.3 1.0 7.3 1.0 8.0 0.6 20.4
19.0 9.2 9.6 3.1 -
39.6
22.8
Fowl plague virus
3.5 15.3 1.7 9.5 1.4 5.9
12.9 0.8 0.4 6.2 1.4 0.6 -
18.8 12.1 0.8 0.3 6.8
37.2
29.2
MEMBRANE
7.6 0.4 4.2 0.4 2.9 1.2 0.9 1.1 7.5 1.7 6.7 0.4 26.4
18.4 8.1 8.1 1.0 2.9 -
43.9
19.6
Fowl plague virus
OF CHICK
4.0 14.0 1.8 9.8 0.2 6.3
13.8 1.0 1.1 5.9 0.7 0.7 -
19.8 13.2 0.9 0.2 4.7
37.9
27.9
Chorioalbmtoic membrane
Oligohexoeyleeramide
OF THE CHORIOALLANTOIC
Chorioallantiic membrane
AND
Lactosylceramide
PLAGUE
96
Discussion Spingolipids, p~ti~ul~ly glycosphingolipids, are typical membrane components and may participate in biological events associated with the membranes, Hence, a detailed knowledge of their structure is of great importance for understanding properties of membranes and virus particles which contain membranes. We have now analysed sphingolipids of four influenza strains, which have become available to us in large quantities. All virus strains were found to contain unusually high concen~ations of sphingolipids, in particular glycosphingolipids. The content of monohexosylceramide (17.5-22.5s of total lipid) was comparable to that found in the myelin sheath of the central nervous system [6], but was much higher than values reported for other membranes, including membranes of other viruses [ll,l2] and plasma membranes [11,13,14]. In light of the fact, that glycolipids are antigens [ 151 their enrichment in the virus particle is no~worthy. Also the existence of galactosylceramide has not been previously reported in virus particles. Other investigators found only glucosylceramide in viral membranes [11,13,14]. The failure of other workers to detect galactosylceramide might be due to insufficient quantity of material used for analysis. Lactocylcer~ide was found to be absent in A2 -Asia and AZ -England, and present only in very small concentrations in the other two strains of the virus investigated. In contrast, chorioallantoic membrane contained about as much monohexosylceramide and dihexosylceramide (Table I), Since lactosylceramide is a component of intracellular membranes rather than of plasma membranes [ 163 , the absence of it in the virus particle seems to be a further indication that lipids of influenza viruses originate primarily from the plasma membranes 1131. Oligohexosylceramides were present a in slightly higher concentration than the lactosylceramide. It was thought that they represented a mixture of blood group glycolipids and the Forssman antigen [15] since they contained glucosamine, galactosamine and fucose which also occur in these glycolipids. In the past, the presence of such antigens in viral membranes was detected by irnrn~olo~~~ means [ 17-191. To determine the composition of fatty acids, including those containing long chain normal and 2-hydroxy analogues, it is important to use a nonpolar column and to employ temperature programming. Polar stationary phases, such as those used by other authors [ll-131 would allow better separation of fatty acids according to degree of unsaturation, but did not permit detection of long alkyl chains, due to delayed and extended retention times. It was found that all sphingolipids contained high proportions of long chain (C, 9-C, 6 ) normal and 2-hydroxy fatty acids. Long chain normal groups of viruses [ 11, but of up to c&4 were previously found in the phospholipid 2-hydroxy fatty acids were never encountered in virus particles. Long chain fatty acids are characteristic membrane components, which are also present in many other biological systems such as the central nervous system [9,10], milk fat globules [20,21] and chylomicrons [22]. Our data show, that despite wide differences in biological origin and function, membranes of influenza viruses possess a feature common in most biological membranes, i.e. enrichment of sphingolipids with very long fatty acids. Since the Van der Waals forces opera-
97
tive between closely apposed fatty chains increase in direct proportion to the chain length [ 231, the rigidity and stability of the virus membranes [ 231 would seem to depend to a large extent on the presence of such long chain lipids. The elevated amounts of 2-hydroxy fatty acids in the viral membranes is particularly noteworthy. Such hydroxylated acids have been usually encountered in the past primarily in the central nervous susystem [lO,ll] and in smaller concentrations in the kidney [ 171. Other membranes contained none or only small proportions of these components. The finding implies a special need of the virus to possess a rigid structure. The detection of C1 8 -sphingosine as the sole base component in all sphingolipids of the influenza viruses may also merit attention. In other membranes sphingosines of different chain lengths, substitution and branching are often present as complex mixtures [24]. The present result compares well with that of Laine et al. [2], who also found C 1 8 -sphingosine as the only base in Semliki forest virus. Acknowledgements This work was supported by the Sonderforschungsbereich 47, Virologie of the Deutsche Forschungsgemeinschaft. The interest and support of Professor R. Rott is greatly appreciated. References 1 Blough, H.A. (1971) J. Gen. Viral. 12.317-320 2 Laine. R., Kettunen, M.L.. Gahmberg. C.G.. Kaiiriainen. L. and Renkonen. 0. (1972) J. Virol. 10. 433-438 3 Chucholowius, H.-W., Rott, R. (1972) Proc. Sot. Exp. Biol and Med. (N.Y.) 140.245-247 4 Klenk, E. and Rennkamp. F. (1942) Hoppe-Seyler’s 2. Physiol. Chem. 273,253-268 5 Huang, R.T.C. (1973) Biochim. Biophys. Acta 306.82-84 6 Klenk. E. and Hung, R.T.C. (1969) Hoppe-Seyler’s Z. Physiol. Chem, 350,373-378 7 Hakomari. S.I.. Saito, T. and Vogt, P.K. (1971) Virology 44,609-621 8 Gaver, R.C. and Sweeley, C.C. (1965) J. Am. Oil Chem. Sot. 42, 294-299 9 Klenk. E. and Schorsch. U. (1968) Hoppe-Seyler’s Z. Physiol. Chemie 349,653-658 10 Klenk, E. and Rivera, M.E. (1969) Hoppe-Seyler’s Z. Physiol. Chemie 349.1589-1592 11 Renkonen, 0.. Kaiiriainen. L., Simons, K. and Gahmberg. C.G. (1971) Virology 46.318-326 12 Blougb, H.A. and Merlic. J.P. (1970) Virology 40. 685-692 13 Klenk, H.-D. and Choppin, P.W. (1970) Proc. Natl. Acad. Sci. 66, 57-64 14 Klenk, H.-D. and Choppin, P.W. (1971) J. Virol. 7. 416-417 15 Martensson, E. (1969) Progr. Chem. Fats Lipids, 10, 367-407 16 Weinstein, D.B. and Marsh. J.B. (1969) J. Biol. Chem. 244. 4103-4111 17 Springer, G.F. and Tritel. H. (1962) Science 138.687-688 18 Isacson, P. and Koch. A.E. (1965) Virology 27,129-138 19 Rott. R.. Drzeniek, R.. Saber, S. and Reichert, E. (1966) Arch. Ges. Virusforsch. 19. 273-288 20 Morrison, W.R. (1969) Biochim. Biophys. Acta 176.537-546 21 Morrison, W.R. and Hay. T.D. (1970) Biochim. Biophys. Acta 202.460-467 22 Huang. T.C. and Kuksis. A. (1967) Lipids 2.443-452 23 O’Brien, J.S. (1967) J. Theoret. Biol. 15. 307-324 24 Karlsson, K.-A. (1970) Chem. Phys. Lipids 5, 6-43
Biochimico et Biophysics Acta, 424 (1976) 90-97 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56707
SPHINGOLIPIDS
OF INFLUENZA
VIRUSES
RICHARD T.C. HUANG Institute
of Virology,
Justus Liebig-University
of Giessen (G.F.R.)
(Received July 25th, 1975)
Summary Total lipid of four egg grown influenza viruses (AT -Asia, Az -England, As-Taiwan and fowl plague virus) were extracted with chloroform-methanol. After mild alkali treatment of the extracts, glycosphingolipids and sphingomyelin were separated by a silicic acid column, and finally purified by thin layer chromatography. Fatty acid, sphingosine and carbohydrate components of individual lipid classes were then analysed by gas-liquid chromatography. Nearly identical results were obtained with all viruses investigated. Approximately 20% of the total lipid was monohexosylceramide, distributed equally between glucosyl- and galactosyl- analogues. Lactosylceramide and oligohexosylceramides were found in much smaller concentrations (approx. 2%). About 15% of the total lipid was attributed to sphingomyelin. A large proportion of fatty acids (around 25% in sphingomyelin and 60% in glycolipids) belonged to the long chain (C, 9 -C2 6 ) normal- and 2-hydroxy series. C1 8 sphingosine was found to be the only base present in all lipid classes investigated.
Introduction The surface of enveloped viruses possesses a lipid bilayer, which is sandwiched between proteins. Such a membrane is found ubiquitously in biological systems, where a stable structural boundary is necessary for survival. In the past, information has accumulated with regard to the molecular architecture of biological membranes. In virus systems the fatty acids of phospholipids have been determined [ 1,2] . A comparable study on sphingolipids, in particular glycosphingolipids, has not as yet been adequately performed. With the intention of exploring specific characteristics which might associate with viral membranes, we have proceeded to analyse the sphingolipids of four influenza viruses. The results obtained indicated that lipids of influenza viruses contain typical membrane components, which are rich in sphingolipids with long chain normal and 2-hydroxy fatty acids.
91
Materials and Methods Virus. Four influenza strains, As-Asia, AZ-England, AZ-Taiwan and fowl plague virus were propagated in ~horio-~antoi~ cavities of chick embryos. Allantoic fluid with haemagglutinating t&es* of around 2-’ was harvested. Purification of viruses was achieved by sucrose density gradient centrifugation [ 31, where rate zonal and isopycnic procedures were applied sequentially in the same run. Fowl plague virus was propagated and purified in this laboratory, whereas other three viruses were kindly provided by Behringwerke AG., Marburg. Lipid standards. Glucosylceramide was extracted from the spleen of a Gaucher patient according to the method described by Klenk and Rennkamp [4]. Galactosylceramide was obtained from human brain by the same method. Lactosylceramide was obtained by neuraminidase action (receptor destroying enzyme from Behringwerke AG., Marburg) of hematoside mixtures obtained from butter milk [ 51. Sphingomyelin was obtained from human brain as described elsewhere [ 61. Extraction of lipids. Virus concentrates (200 ml from each of A2 -strains, and 100 ml from fowl plague virus) which had haemag~u~nating titres of 2-l 5 or 2-l 6 were dialysed against running water to remove the sucrose present. Dialysates were freeze dried and extracted twice with 50 ml of chloroform / methanol (2 : 1). Extracts were pooled and evaporated to dryness in a rotary evaporator. Total lipid which remained was determined ~avime~ic~ly. Fractionation of lipids. To facilitate isolation of sphingolipids, glycerolipids were hydrolysed by alkali essentially as described elsewhere [6]. Preliminary separation of individual lipid classes was achieved by silicic acid column ~hromato~aphy. Silicic acid, mesh 70-230, ASTM (Merck), was slirried in chloroform and packed in a column of 2 X 12 cm. After equilibration with chloroform, lipid samples (32-84 mg) were applied in chloroform. Elution of fractions was performed with 80 ml of each of the following solvent mixtures; ~hlorofo~ eluted all “neutral lipids” (cholesterol, triacylglycerol, free fatty acid and cholesteryl ester), 25% of methanol in chloroform eluted all monohexosylceramide and lactosylceramide. 50% Methanol in chloroform eluted oligohexosylceramide and methanol eluted sphingomyelin. The purity of the fractions were monitored by thin layer ~hromato~aphy using silicic acid plates (0.25 mm) and chloroform/methanol/water (60 : 35 : 8) as developing solvents. All spots were visualized by spraying with 5% aqueous sulfuric acid and heating at 150°C. Identification of each lipid classes was accomplished by cochromatography with standard lipids. For final purification of each lipid classes, 20-30 mg of each lipid fraction was applied as a narrow band on a silicic acid plate (0.25 mm) and developed with the same solvent mixture. The lipid bands, which had been visualized by spraying with water, were scraped off with the aid of a razor blade and extracted with chloroform/methanol (1 : 1). Quantitution of individual sphingolipids. Spingholipids were estimated titre indicates the extent of two fold delution of the virus still cause agglutination of erythrocytes.
* HaemagglUtinating
preparationswhich
92
directly on a thin layer plate by visual comparison of virus lipids (30 mg/ml in chloroform/methanol 1 : 1) with serially diluted solutions of standard lipids (from 0.1 mg to 1.0 mg in 0.1 ml of chloroform/methanol 1 : 1) essentially as described by Hakomori et al. 171. In several instances, lipids were qu~tita~d by gas chromatography of fatty acid methyl esters, using heptanoic acid methyl ester as the internal standard. The results agree within ?5% to those obtained above. ~~d~o~ys~s of Eipids. Approxima~ly 10 mg of the lipids were hydrolysed in sealed 5 ml ampules with 2 ml of a mixture of methanol/concn. HCl/water (70 : 4 : 4) for 6 h [8]. At the end of this period fatty acids, which had been converted quantitatively to methyl esters, were extracted twice with equal volumes of heptane. The lower layer (methanol/HCl layer), which contained the methyl sugars and the sphingosine, was evaporated to dryness. The residual HCl was removed by repeated addition of methanol to the residue and continued evaporation. Analysis of fatty acids. Fatty acids were analysed in a gas chromatograph, model Perkin-Elmer F 20 B. The column (2 m X 0.27 cm) was 1% silicone ester-30 on chromosorb G, 80-100 mesh, AW-DMCS. Nitrogen gas, which was used as the moving phase, was supplied at a pressure of 2. 5 Kg/cm2. Column temperature was programmed from 160” -260°C at 2°C per minute. Injector temperature was 300” C. Normal fatty acids from C1 4 to CZ 6 were identified by comparing with authentic standards from Merck. Hydroxy fatty acids were identified by comparing with those obtained from cerebrosides of human brain, the structure of which had been previously described [ 9,101. For better separation of fatty acids according to degrees of unsaturation a column (2 m X 0.27 cm) of 5% ethyleneglycol succinate on gas chrom A, 80-100 mesh was used. Analysis of s~hingosi~~ and sugars. The residue which remained in the lower methanol/NC1 phase after removal of methanol and HCl, was silylated according to the method of Gaver and Sweeley [8] and gas chromatographed in a SE-30 column, 200 X 0.27 cm. Column temperature was programmed from 140 to 170°C at 2°C per min to elute the sugars, and subsequently from 170”~240°C at 7.5”C per min to analyse the sphingosine. The condition provided the possibility to analyse sugar and sphingosine components simultaneously on the same chromatogram. Results The method [3] of sequential zonal rate and isopycnic centrifugation were employed to obtain highly purified viruses. The preparations contained only virus particles as judged by electron microscopy and by polyacrylamide gel electrophoresis of the proteins. The amounts of total lipid in all virus samples were comparable. The yields ranged from 32-42 mg in 100 mg of the total protein. For further analysis total lipid was treated with alkali to hydrolyse glycerolipids, which otherwise could not be readily separated from sphingolipids. After such a treatment with alkali all strains of influenza viruses investigated gave similar lipid patterns (Fig. 1). Spots of monohexosylceramide and sphingomyelin were the most prominent in all virus samples. In comparison, cells of chorioallantoic membrane where the bulk of the virus
93
Fig. 1. Sphingolipid pattern of influenza viruses and of the chorioalIantoic membrane of the fowl plague virus infected chick embryos I, AZ-A&: II, AZ-England; III, AZ-Taiwan; IV, fowl plague virus: V, enriched glycolipid fraction from the chorioallantoic membrane; VI, total lipid of the chorioallantoic membrane. Thin layer plate: Kieselgel (0.25 mm). Developing solvent: chloroform/methanol/water 60 : 35 : 8. visualized by spraying with 5% H2SO4 and heated at 15O’C.
propagated, contained only small proportions of glycolipids (Fig. 1, Table I). Spots corresponding to monohexosylceramide yielded glucose and galactose in approximately the same quantity on hydrolysis. It was therefore concluded that they were mixtures of equal amounts of glucosylceramide and galactosylceramide. Lactosylceramide was present in only very small amounts in A2 -Taiwan and fowl plague virus, but could not be clearly detected in the other two viruses. The spots were identified as lactosylceramide, since they cochromatographed with the standard lactosylceramide and yielded galactose and glucose in equimolar quantities on hydrolysis. Fractions containing oligohexosylceramide were separated into at least four components on thin layer plates. Due to limited quantities of material available, each component could
94 TABLE I COMPOSITION (% OF TOTAL LIPID) OF SPHINGOLIPIDS CHORIOALLANTOIC MEMBRANE OF FOWL PLAGUE -~ Lipids
-----.___.
IN INFLUENZA VIRUSES AND IN THE VIRUS INFECTED CIiICK EMBRYOS
Viruses
___-
Monohexosyl. ceramide Lactosylceramide Oligohexosylceramide Sphingomyelin Others
AZ-Asia
AZ-England
A2JI’aiwan
Fowl plague
ChorioaIlantoic membrane
17.5 0 1.5 17.5 63.5
20 0 2 17.5 60.5
20 0.5 2 15 63.0
22.5 1.5 2 15 59.0
1.5 1.5 0 10 67.0
not be rigorously identified. However, hydrolysis of the whole fraction revealed the presence of galactose, glucose, glucosamine, galactosamine and fucose in a proportion of about 2 : 1.5 : 1 : 0.3 : 0.3. Fatty acids of all individual sphingolipids were estimated by gas-liquid chromatography. Using a nonpolar column (SE-30) and applying the technique of temperature programming, it was possible to separate all fatty acid methyl esters. The 2-hydroxy fatty acids, when unacetylated, emerged as separate peaks from the, normal fatty acids of corresponding chain lengths. Identical fatty acid patterns were obtained for the corresponding sphingolipids of the four viruses analysed. Since the percent composition of each fatty acids in the comparable sphingolipids did not vary beyond a maximum of i 5% among different viruses, the values for the fowl plague viruses is shown in Table II to represent the composition for all four viruses. For comparison, fatty acid values of the chorio-allantoic membrane, where the bulk of the virus multiply, is also included in the table. It can be ascertained from the table that the presence of a high concentration of long chain fatty acids is a common feature in all sphingolipids. Long chain (C, 9 -Cz 6 ) normal and 2-hydroxy fatty acids together accounted for approximately 60% and 25% of the total fatty chains in the glycolipids and sphingomyehn respectively (Table II). Sphingomyelin contained very little Zhydroxy fatty acids, whereas about 40% of the fatty chains in the glycolipids could be attributed to these hydroxy analogues. It can also be pointed out, that although fatty acid patterns were similar among glycolipids, lactosylceramide and oligohexosylceramide were more alike, both being rich in the 2-hydroxyCz 6 -acid, (Table II). Oleic acid and linoleic acid were the main unsaturated fatty acids found in all sphingolipids. They overlapped in one peak when chromato~aphed in a SE-30 column, but could be resolved in an ethyleneglycol succinate column into separate peaks with a ratio of about 3 to 1 in all lipid classes. Sphingosine and carbohydrate components could be simultaneously estimated in an SE-30 column. It was found that C1 s-sphingosine was the only base present in all sphingolipid classes.
c22:o ‘23:O ‘24:O ‘24:l C26:0 ‘26:O ‘l&OH CZO.OH C22.OH ‘23-GH ‘24OH c25-OH ‘26-OH
‘16:O ‘18:O
-
7*6
-
Oag :‘B”
*
7.3 ;;
’
’
7.6
-
-
14.8 8.0 9.0 1.1 -
6.9
0
48.9
7.5 0.5 4.9 0.4 0.8 1.3 1.9 2.2 16.5 1.3 15.4 1.7 1.2
23.0 10.6 7.7 3.6
40.0
19.0
41.6 7.8 1.7
30.0 17.3
7.1 0.2 4.6 6.8 0.4 1.1
0.4 0.2 7.6 2.1 36.8
-
-
3.2
20.0 7.2 2.5 -
I
47.0
1
23.2
Chorioellantoic membrane
Fowl pIague
virus
Choxioallentoic membrane
Fowl plague
ViIUS
Monohexosylceramide
OF THE FOWL
Sphingomyelin
acids
ACID COMPOSITIONS OF THE SPHINGOLIPIDS INFECTED WITH THE SAME VIRUS
OH refers to z-hydrory
W FATTY EMBRYOS
VIRUS
8.5 0.3 5.0 0.5 3.2 1.1 1.3 1.0 7.3 1.0 8.0 0.6 20.4
19.0 9.2 9.6 3.1 -
39.6
22.8
Fowl plague virus
3.5 15.3 1.7 9.5 1.4 5.9
12.9 0.8 0.4 6.2 1.4 0.6 -
18.8 12.1 0.8 0.3 6.8
37.2
29.2
MEMBRANE
7.6 0.4 4.2 0.4 2.9 1.2 0.9 1.1 7.5 1.7 6.7 0.4 26.4
18.4 8.1 8.1 1.0 2.9 -
43.9
19.6
Fowl plague virus
OF CHICK
4.0 14.0 1.8 9.8 0.2 6.3
13.8 1.0 1.1 5.9 0.7 0.7 -
19.8 13.2 0.9 0.2 4.7
37.9
27.9
Chorioalbmtoic membrane
Oligohexoeyleeramide
OF THE CHORIOALLANTOIC
Chorioallantiic membrane
AND
Lactosylceramide
PLAGUE
96
Discussion Spingolipids, p~ti~ul~ly glycosphingolipids, are typical membrane components and may participate in biological events associated with the membranes, Hence, a detailed knowledge of their structure is of great importance for understanding properties of membranes and virus particles which contain membranes. We have now analysed sphingolipids of four influenza strains, which have become available to us in large quantities. All virus strains were found to contain unusually high concen~ations of sphingolipids, in particular glycosphingolipids. The content of monohexosylceramide (17.5-22.5s of total lipid) was comparable to that found in the myelin sheath of the central nervous system [6], but was much higher than values reported for other membranes, including membranes of other viruses [ll,l2] and plasma membranes [11,13,14]. In light of the fact, that glycolipids are antigens [ 151 their enrichment in the virus particle is no~worthy. Also the existence of galactosylceramide has not been previously reported in virus particles. Other investigators found only glucosylceramide in viral membranes [11,13,14]. The failure of other workers to detect galactosylceramide might be due to insufficient quantity of material used for analysis. Lactocylcer~ide was found to be absent in A2 -Asia and AZ -England, and present only in very small concentrations in the other two strains of the virus investigated. In contrast, chorioallantoic membrane contained about as much monohexosylceramide and dihexosylceramide (Table I), Since lactosylceramide is a component of intracellular membranes rather than of plasma membranes [ 163 , the absence of it in the virus particle seems to be a further indication that lipids of influenza viruses originate primarily from the plasma membranes 1131. Oligohexosylceramides were present a in slightly higher concentration than the lactosylceramide. It was thought that they represented a mixture of blood group glycolipids and the Forssman antigen [15] since they contained glucosamine, galactosamine and fucose which also occur in these glycolipids. In the past, the presence of such antigens in viral membranes was detected by irnrn~olo~~~ means [ 17-191. To determine the composition of fatty acids, including those containing long chain normal and 2-hydroxy analogues, it is important to use a nonpolar column and to employ temperature programming. Polar stationary phases, such as those used by other authors [ll-131 would allow better separation of fatty acids according to degree of unsaturation, but did not permit detection of long alkyl chains, due to delayed and extended retention times. It was found that all sphingolipids contained high proportions of long chain (C, 9-C, 6 ) normal and 2-hydroxy fatty acids. Long chain normal groups of viruses [ 11, but of up to c&4 were previously found in the phospholipid 2-hydroxy fatty acids were never encountered in virus particles. Long chain fatty acids are characteristic membrane components, which are also present in many other biological systems such as the central nervous system [9,10], milk fat globules [20,21] and chylomicrons [22]. Our data show, that despite wide differences in biological origin and function, membranes of influenza viruses possess a feature common in most biological membranes, i.e. enrichment of sphingolipids with very long fatty acids. Since the Van der Waals forces opera-
97
tive between closely apposed fatty chains increase in direct proportion to the chain length [ 231, the rigidity and stability of the virus membranes [ 231 would seem to depend to a large extent on the presence of such long chain lipids. The elevated amounts of 2-hydroxy fatty acids in the viral membranes is particularly noteworthy. Such hydroxylated acids have been usually encountered in the past primarily in the central nervous susystem [lO,ll] and in smaller concentrations in the kidney [ 171. Other membranes contained none or only small proportions of these components. The finding implies a special need of the virus to possess a rigid structure. The detection of C1 8 -sphingosine as the sole base component in all sphingolipids of the influenza viruses may also merit attention. In other membranes sphingosines of different chain lengths, substitution and branching are often present as complex mixtures [24]. The present result compares well with that of Laine et al. [2], who also found C 1 8 -sphingosine as the only base in Semliki forest virus. Acknowledgements This work was supported by the Sonderforschungsbereich 47, Virologie of the Deutsche Forschungsgemeinschaft. The interest and support of Professor R. Rott is greatly appreciated. References 1 Blough, H.A. (1971) J. Gen. Viral. 12.317-320 2 Laine. R., Kettunen, M.L.. Gahmberg. C.G.. Kaiiriainen. L. and Renkonen. 0. (1972) J. Virol. 10. 433-438 3 Chucholowius, H.-W., Rott, R. (1972) Proc. Sot. Exp. Biol and Med. (N.Y.) 140.245-247 4 Klenk, E. and Rennkamp. F. (1942) Hoppe-Seyler’s 2. Physiol. Chem. 273,253-268 5 Huang, R.T.C. (1973) Biochim. Biophys. Acta 306.82-84 6 Klenk. E. and Hung, R.T.C. (1969) Hoppe-Seyler’s Z. Physiol. Chem, 350,373-378 7 Hakomari. S.I.. Saito, T. and Vogt, P.K. (1971) Virology 44,609-621 8 Gaver, R.C. and Sweeley, C.C. (1965) J. Am. Oil Chem. Sot. 42, 294-299 9 Klenk. E. and Schorsch. U. (1968) Hoppe-Seyler’s Z. Physiol. Chemie 349,653-658 10 Klenk, E. and Rivera, M.E. (1969) Hoppe-Seyler’s Z. Physiol. Chemie 349.1589-1592 11 Renkonen, 0.. Kaiiriainen. L., Simons, K. and Gahmberg. C.G. (1971) Virology 46.318-326 12 Blougb, H.A. and Merlic. J.P. (1970) Virology 40. 685-692 13 Klenk, H.-D. and Choppin, P.W. (1970) Proc. Natl. Acad. Sci. 66, 57-64 14 Klenk, H.-D. and Choppin, P.W. (1971) J. Virol. 7. 416-417 15 Martensson, E. (1969) Progr. Chem. Fats Lipids, 10, 367-407 16 Weinstein, D.B. and Marsh. J.B. (1969) J. Biol. Chem. 244. 4103-4111 17 Springer, G.F. and Tritel. H. (1962) Science 138.687-688 18 Isacson, P. and Koch. A.E. (1965) Virology 27,129-138 19 Rott. R.. Drzeniek, R.. Saber, S. and Reichert, E. (1966) Arch. Ges. Virusforsch. 19. 273-288 20 Morrison, W.R. (1969) Biochim. Biophys. Acta 176.537-546 21 Morrison, W.R. and Hay. T.D. (1970) Biochim. Biophys. Acta 202.460-467 22 Huang. T.C. and Kuksis. A. (1967) Lipids 2.443-452 23 O’Brien, J.S. (1967) J. Theoret. Biol. 15. 307-324 24 Karlsson, K.-A. (1970) Chem. Phys. Lipids 5, 6-43
