Studies on the lipids of virus-infected cells

VIROLOGY

19,

(1963)

.%%.%%I

Studies I. Lipid

on the Lipids

of Virus-Infected

Cells

Analysis of a Soluble Hemagglutinin from Chorioallantoic Membranes Infected with Vaccinia Virus’

CHARLES R. GAUSH2 Department

AND

J. S. YOUNGNER

of Microbiology, University of Pittsburgh Pittsburgh 13, Pennsylvania Accepted

December

School

of Medicine,

26, 1962

A soluble hemagglutinin from chorioallantoic membranes infected with vaccinia virus was prepared by the flotation method. The lipid analysis of this hemagglutinin, as well as normal and virus-infected chorioallantoic membranes, is described. The results show that the hemagglutinin has a high neutral lipid (glyceride) content which is reflected as an increase in the triglyceride concentration in infected membranes. The results also indicate that phosphatidylcholine is the major phosphatide of the hemagglutinin, confirming earlier assumptions of its presence. Chorioallantoic membranes infected with vaccinia virus contain less phospholipid than control membranes, a finding in agreement with data obtained from other virus-host cell systems.

strated two hemagglutinins in vaccinia virus suspensions, one of which was separable The hemagglutinating activity of vaccinia virus was described by Nagler (1942) and from the virus particle, Seitz filterable, not Burnet (1946). The phenomenon was origi- sedimented by high speedcentrifugation and nally attributed to the elementary bodies, heat labile. This fraction was called the but Burnet and Stone (1946) then dis- soluble hemagglutinin (SHA) . Recent studies on the vaccinia SHA were covered that virus particles could be dissociated from the hemagglutinin and this concerned with its separation from the virus finding was confirmed by Chu (1948). Early particle (Youngner and Rubinstein, 1959; chemical studies indicated that the hemag- McCrea and O’Loughlin, 1959). In a more glutinin was alcohol soluble and that sus- extensive investigation, Youngner and Rupensions of certain lipids would produce binstein (1962) reported that the soluble hemagglutination (Stone, 1946a). Clostrid- hemagglutinin appeared to be a normal izcm welchii toxin type A and cobra venom tissue agglutinin, since under certain condestroyed the hemagglutinin of vaccinia ditions it could be detected in uninfected virus, indicating the presence of a phospho- chlorioallantoic membrane (CAM) suspenlipid as an integral component of the antigen sions. The study of vaccinia hemagglutinins is a (Stone, 1946b). Gillen et al. (1950) demonsegment of a larger area of interest, the lipid ‘Part of a thesis submitted by the senior author metabolism of virus-infected cells. Cohn to the School of Medicine of the University of (1952) reported a significant decrease in Pittsburgh in partial fulfillment of the requirements phospholipid phosphorus in deembryonated for the degree of Doctor of Philosophy. This study CAM after infection with influenza virus alwas aided by grants Al-03414 and 2E-80 from the though the P3aspecific activity of this fracUnited States Public Health Service. tion increased. Similarly, Cornatzer et al. ’ Present address : Department of Microbiology, School of Medicine, State University of South (1953) demonstrated a decrease in total Dakota, Vermillion, South Dakota. lipid and an increase in the specific activity INTRODUCTION

573

574

GAUSH AND YOUNGNER

of the phospholipid fraction of rabbit papilloma compared to normal rabbit skin. Alterations in lipid metabolism were also shown by the work of Todd et al. (1958) and Randall et al. (1960)) who studied the effects of fowlpox virus infection in chick skin. Information concerning the lipids of cells infected with vaccinia virus was lacking and a study was therefore initiated to attempt to clarify this aspect of the problem. A lipid analysis of cells infected with vaccinia virus was also of interest in view of the nature of the soluble, hemagglutinin and its ease of preparation. The present report describes the lipid composition of the soluble hemagglutinin as well as virus-infected and control CAM. METHODS

T’irus. The virus employed in these studies was designated vaccinia strain Canada and was the same stock used in previous studies (Youngner and Rubinstein, 1959, 1962). One-tenth milliliter of virus suspension containing lo3 plaque-forming units (PFU) was inoculated on the dropped chorioallantoic membranes of 1l-day-old embryonated eggs. Vaccinia-infected eggs and controls inoculated with phosphate-buffered saline (PBS) were incubated at 37” for 72 hours. The harvested membranes were washed several times in cold PBS, and a 25% suspension (w/v) was prepared in PBS from infected membranes by homogenizing at one-half maximum speed in a Virtis homogenizer. An aliquot of this suspension was stored at -62” as stock virus and the remainder was utilized for the separation of the soluble hemagglutinin. Infected and control membranes were homogenized directly in absolute ethyl alcohol for the extraction of total lipids. Flotation procedure. A soluble hemagglutinin was prepared from virus-infected CAM as described by Youngner and Rubinstein (1962). The CAM homogenate was centrifuged at 2600 rpm (International PR2) and the resulting supernatant fluid (2600 super) was spun at 10,000 rpm (Spinco model L), after which this supernatant fluid was adjusted to density 1.063 with sodium chloride. This solution (10 +

NaCl) was centrifuged at 30,000 rpm for 3 hours and the creamy top layer (30 cr ; SHA) , the supernatant fluid (30 super) and the sediment (30 sed) were separated. All centrifugations were carried out at O-5”. Hemagglutination titrations. Hemagglutination titrations were performed as described previously (Youngner and Rubinstein, 1962). Inpctivity assay. Virus titers were determined by plaque assays on primary monkey kidney monolayers. Virus was adsorbed to the monolayers at 37” for 4 hours when 3 ml of a nutrient agar overlay was added to each culture. The cultures were then incubated for 6 days at 37” in a humidified atmosphere containing 5% COZ . Total lipids. Total lipids were extracted from CAM homogenates or soluble hemagglutinin with ethyl alcohol and ether. Approximately 7 volumes of alcohol were added to the membranes or SHA, which were then homogenized. This homogenate was transferred to a brown bottle and allowed to stand for 1 hour. Three volumes of diethyl ether were then added to the homogenate, which was allowed to stand at room temperature (RT) for another hour. After centrifugation at 2000 rpm for 20 minutes, two additional extractions were performed on the sediment at RT using 10 volumes of Bloor’s solvent per volume of sediment. The three extracts were pooled, filtered through glass fiber paper (Reeve-Angel, 934-AH) and evaporated to dryness in a rotary evaporator at 37-40” under an atmosphere of nitrogen. The residue was re-extracted with moist diethyl ether and was washed 6-9 times with demineralized water. The washed extract was transferred to a tared weighing bottle and dried under nitrogen at 40”. The lipid residue was placed in a desiccator (previously flushed with nitrogen) over Drierite and evacuated for 18 hours at room temperature. The total lipid thus obtained was weighed on an analytical balance. Separation of neutral lipid from phospholipid. Neutral lipids, including fatty acids, were separated from phospholipids on columns of silicic acid (Borgstrom, 1952). A slurry of silicic acid (1 g/25 mg total lipid) in chloroform was prepared and poured

SOLUBLE

HEMAGGLUTININ

into a 22 x 200 mm chromatographic column. The total lipids dissolved in chloroform were poured into the column and allowed to flow through by gravity. Neutral lipids were eluted with 300-400 ml chloroform and phospholipids with 300-400 ml absolute methanol. The two fractions were evaporated to dryness and weighed. Separation of neutral lipid classes. Neutral lipids were separated into 5 classes of compounds using the method of Horning et al. (1960). The neutral lipid fraction was dissolved in a 6% solution of benzene in nhexane and was allowed to flow into a silicic acid column (1 g “Unisil” silicic acid per 20 mg neutral lipid) that had been prepared in the same solvent. Individual lipid classes were eluted with the following solvents: 6% benzene in hexane (B-H), hydrocarbons; 18-240/o B-H, sterol esters; 100% benzene, triglycerides ; 100% benzene, sterols : 100% chloroform, mono- and diglycerides. The column was monitored by drying aliquots from each tube and observing the presence of residual lipids. The identity of the compound in each fraction was determined on silicic acid coated glass fiber paper by chromatographing with known lipid standards. The solvent system was composed of 9 parts diisobutyl ketone and 1 part n-heptane. The location of the separated lipids was determined by staining the paper with an acid protoporphyrin solution (Sulya and Smith, 1960) and viewing the wet chromatogram under ultraviolet light (3660 A). The lipids in eluates representing one fraction were determined gravimetrically. Separation of phospholipid classes. Chromatographic separation of phospholipids was achieved on silicic acid columns using a method described by Hanahan et al.

LIPIDS

(1957). Five grams of “BioRad” silicic acid per 25 mg of phospholipid was used to prepare columns in chloroform-methanol (CM) mixtures. Generally, C-M 9: 1 eluted amino acid phosphatides; C-M 4:1, phosphatidylinositol; C-M 3: 2, phosphatidylcholine, and C-M 1:4, sphingomyelin. In some experiments, the phosphatide mixture was resolved into 5 fractions as follows: C-M 20 : 1, phosphatidylserine ; C-M 9 : 1, phosphatidylethanolamine ; C-M 4 : 1, phosphatidylinositol; C-M 1: 1 and 1: 20, phosphatidylcholine and sphingomyelin. After the lipids had been eluted from the columns, a l-ml aliquot from each tube was used for quantitative determination of organic phosphate by the method of King (1932). The phospholipids were identified by chromatographing the acid hydrolyzates on Whatman No. 1 paper in a solvent system composed of n-propanol : ethyl acetate : water, 7: 1:2. On these chromatograms, serine and ethanolamine were detected with ninhydrin; glycerol and inositol with alkaline silver nitrate (Trevalyan et al., 1950) ; and choline with phosphomolybdic acidstannous chloride (Levine and Chargaff, 1951). Infrared spectroscopy with a Beckman IR-7 instrument was also employed to identify the phosphatides.

TABLE

RESULTS

Separation and Lipid Analysis of a Vaccinia Virus Soluble Hemagglutinin A soluble hemagglutinin was prepared from vaccinia virus-infected CAM as described above. The distribution of virus and hemagglutinin in different fractions of a typical preparation is presented in Table 1. Although the 30 cr fraction had less he1

DISTRIBUTION OF PLAQUE-FORMING (PFU) AND HEMAGGLUTINATING UNITS (HAU) IN VARIOUS FRACTIONS SEPARATED BY THE FLOTATION METHOD FROM CHORIOALLANTOIC MEMBRANE SUSPENSIONS INFECTED WITH VACCINIA VIRUS Parameter

PFUa HAUa PFU/HAU a The values

given

2600 Super

10 - NaCl

3.2 x 10” 1.5 x 105 2.1 x 106

3.8 x 109 3.2 X lo4 1.2 x 105

are total

PFU and HAU

30 Cr 4.8 x 106 6.8 x lo2 7.0 x 103

per fraction.

30 Super 3.0 x 106 0

30 Sed 4.2 X 108 3.0 x 104 1.4 x 104

576

GAUSH AND YOUNGNER TABLE

2

THE EFFECT OF HEAT AND DIALYSIS ON THE 30 CR AND 30 SED FRACTIONS OF VIRUS-INFECTED CAM SUSPENSIONS OBTAINED BY THE FLOTATION METHOD

Expt. no.

Hemagglutinating units per ml

Fraction

-

(56’, Unheated Heated 30 min)

30 Cr 30 Sed

128 1,280

8 320

30 Cr 30 Sed

10,240 5,120

80 1,280

Not dialyzed

Dialyzeda

30 Cr 30 Sed

640 5,120

>20,000 5,120

30 Cr 30 Sed

2,560 5,120

>20,000 2,560

magglutinating activity than reported by Youngner and Rubinstein (1962)) the effect of heat and dialysis indicated that it was the same hemagglutinin (Table 2). The lowered activity is not explained but may be a reflection of the degree of physical aggregation of hemagglut,inin particles. Total lipids were extracted from 12.5 ml of the 30 cr fraction which contained 1.6 x lo4 units of activity and were separated on columns of silicic acid. The total lipid fraction was predominantly neutral lipid (Table 3) and was composed of 73.6% neutral lipid and 26% phospholipid. The major components of the neutral lipid fraction were glycerides, which accounted for 58% of the total lipid, while sterol and sterol esters each accounted for 6% of the total. It was t,hought that some phospholipid may have eluted with the mono- and diglycerides, making the value of this fraction erroneously high. However, an organic phosphate determination revealed that this fraction cont’ained less than 1% phospholipid.

a Dialyzed

overnight against water at 4”.

THE LIPID

COMPOSITION OF THE VACCINIA VIRUS SOLUBLE HEMAGGLUTININ FLOTATION METHOD

TABLE

Parameter Total lipid Total phospholipid Total neutral lipid

3 OBTAINED BY THE

Weight (mg)

y0 of Total lipid

180.0 46.8 132.6

100.0 26.0 73.6 y. of NL

Neutral lipids (NL) 1 Hydrocarbon 2 Sterol ester 3 Triglyceride 4 Sterol 5 Mono- and diglycerides

3.18 11.13 62.54 12.19 41.87

Lipid recovered

130.91

2.40 8.39 47.16 9.19 31.57 --98.71

1.77 6.18 34.74 6.77 23.26

% of PL Phospholipids (PL) 1 Aminophosphatides 2 Phosphatidylinositol 3 Phosphatidylcholine 4 Sphingomyelin Lipid recovered

5.24 3.49 38.71 1.50 -48.94

11.19 7.45 82.71 3.20 -~ 104.55

2.91 1.94 21.51 0.83 99.91

SOLUBLE

HEMAGGLUTININ

In this experiment, the phospholipids were eluted with C-M 9:1, 4:1, 3:2, and 1:4. The acid hydrolysates of fractions 1 and 2 (Table 3) were chromatographed in duplicate on cellulose papers which were stained with alkaline silver nitrate and ninhydrin. The hydrolyzate of fraction 1 contained two ninhydrin-staining components with RI values identical to those of the serine and ethanolamine reference standards. The chromatograph of fraction 2 contained only one spot which stained heavily with silver nitrate but not with ninhydrin. Its Rf value was less than that observed for serine but was identical to that of the inositol reference standard. On the other hand, fractions 3 and 4, which were presumed to be phosphatidylcholine (PC) and sphingomyelin, respectively, could not be identified by this technique since both compounds contain choline. These two fractions could be identified by infrared spectroscopy since the sphingomyelin molecule contains an amide and a hydroxyl group which are not present in phosphatidylcholine. The amide group is characterized spectrally by strong absorption of energy at frequencies of 1550 and 1650 cm-l whereas the hydroxyl group absorbs near 3400 cm-l. The spectrum of unhydrolyzed lipid from fraction 3 contained weak absorption bands at these frequencies but was otherwise identical to the spectrum of a pure synthetic phosphatidlycholine. This proved the fraction to be composed of phosphatidylcholine and that a trace amount of sphingomyelin was also present. In contrast,, t,he lipid from fraction 4 produced very strong absorption bands at 1550, 1650, and 3400 cm-l which identified this compound as sphingomyelin. A weak band was also observed at 1745 cm-l, characteristic of a carbonyl group, indicating the presence of a trace amount of phosphatidylcholine. The phospholipid in each fraction was determined calorimetrically and the results (Table 3) indicated that phosphatidylcholine was the major phosphatide present, making UP 21.5% of the total lipids and more than 80% of the phospholipid fraction.

Lipid

577

LIPIDS

Analysis of Normal fected CAM

and Virus-ln-

The total lipids of normal and infected membranes were separated and identified as described in Methods. In these experiments, phospholipids were eluted from the columns with C-M 20:1, 9:1, 4:1, l:l, and 1:20 (v/v). The composition of the total lipid mixture is presented in Table 4. Compared to normal membranes, the total phospholipid decreased significantly from 66.2% to 47.01% in virus-infected tissue and a concomitant increase in the concentration of the neutral lipid fraction from 33.70 to nearly 53% was observed. The loss of phospholipid in the infected membranes occurred in the phosphatidylethanolamine (PE) and phosphatidylcholine (PC), including sphingomyelin, fractions where the values were reduced from 22.30 to 14.71% (PE) and from 36.94 to 26.58% (PC). There was, however, a significant increase in the concentration of phosphatidylinositol (PI). The concentration of the amino acid phosphatides was greatly reduced in the hemagglutinin lipids (2.91%) compared with CAM where this lipid class constituted 28% of the total lipid in normal and nearly 19% of the total lipid in infected tissue. The most striking change in the neutral lipid fraction occurred in the concentration of triglyceride which increased to 22.4% in virus-infected tissue and represented a 4.7-fold increase over the value found for normal tissue. The differences in the concentrations of hydrocarbon and sterol ester were not considered significant. It appeared that the lipid profile of the soluble hemagglutinin resembled that of infected CAM more closely than that of normal CAM. DISCUSSION

Although the vaccinia virus soluble hemagglutinin has been studied for a number of years, its lipid composition has not been determined owing to the difficulty of obtaining this lipoprotein in a reasonably pure form. In this investigation, silicic acid chromatography revealed that the hemagglutinin separat,ed by the method of Youngner and Rubinstein (1962) was composed of glycerides (58%) and phosphatidylcho-

578

GAUSH AND YOUNGNER

TABLE 4 THE LII’ID COMPOSITION OF VACCIN~A VIRUS-INFECTED AND Normal CAM

Parameter

Weight of dry CAM Total lipid recovered Total lipid assayed Total phospholipid Total neutral lipid

NORMAL CHORIOALLANTOICMEMBRANES

Weight

Infected CAM

‘% of Total lipids

2.8346 g 202.00 mg

180.40 119.60 60.80

100.00 66.29 33.70

3.0617 g 467.00 mg 90.60 42.60 48.00

% of Total lipids -

100.00 47.01 52.98

Hydrocarbon Sterol ester Triglyceride Sterol Mono- and diglyceride

6.20 8.60 26.80 6.40

3.44 3.44 4.77 14.86 3.55

2.50 5.80 20.30 11.40 3.41

2.76 6.40 22.41 12.58 3.76

Neutral lipid recovered

54.204

89.14"

43.41a

90.43* 4.19 14.71 4.44 26.59 106.20"

Phosphatidylserine Phosphatidyletl~anolamine Phosphatidylinositol Phosphatidylcholine Phospholipid

Recovered

6.20

-

Weight

10.64

5.90

40.21

22.29

2.22 66.55

1.23 36.89

3.80 13.33 4.02 24.09

119.62

100.01~

45.24

a Of the neutral lipid, 10 per cent was used in the column monitoring in this value. 5 Per cent of the neutral lipid. c Per cent of fhe phospholipid.

line (21.5%) with sterols and sterol esters each accounting for 6% of the total lipids. Smaller ai~ounts of otrher lipids were also present. The high content of glyceride is consistent with the fact that triglycerides increased from 4.7% in uninfected to 22.4% in infected CAM as a result of synthesis or perhaps by accumulation in cells damaged by virus. Since an increase in the concentration of diglyceride was not observed in infected CAM, the reason for the high concentration of this compound in the hemagglutinin is not clear. It is possible that differential centrifugation may have produced a change in the distribution of diglyceride or that some lipolytic degradat.ion of the triglycerides occurred during the preparation of the 30 cr fraction. The presence of phospllatidylcholine as the only major phosphatide is in agreement with earlier observations that, the hemagglutinin was inactivated by lecithinase A (Stone, 1946b). The lipid composition of the soluble hen~aggIutinin is unlike that of serum Iipopro-

procedure and is not included

teins of similar density (Oncley, 1958) or of subcellular particulates (Spiro and McKibbin, 1956). These lipids do resemble those of rat liver cell sap after the removal of the particulates (Spiro and McKibbin, 1956) but the intracellular origin of the virus-induced agglutinin cannot be determined by this fact or the data presented in the present report. The lipid analyses of control and infected CAM suggests that a virus-induced demand for phos~~hatidylinositol occurred in the infected cells and was accompanied by a concomitant synthesis of triglyceride at the expense of choline and ethanolamine phosphatides, a view which is consistent with the known metabolic pathways for the synthesis of lipids (Rossiter and Strickland, 1960). On the other hand, Bailey et aE. (1959) have demonstrat,ed that cultured cells utilize appreciable quantities of lipid from serum used in the medium and that the composition of the lipids t,aken up was different from the lipid co~n~osition of either the serum or the

SOLUBLE

HEMAGGLUTININ

cells. Bensch et al. (1961) observed the accumulation of extracellular lipids and lipoproteins in cultured cells damaged by an amino acid analog. They suggest that the lipids are used by the cells in an attempt to compensate for cellular nutritional deficiencies. This information indicates the operation of a complex sequence of events involving the utilization, interconversion, and excretion of extracellular lipids by cells. The manner in which these events are related to the soluble hemagglutinin is not known. However, the lipid composition of the hemagglutinin seems to resemble the composition of the infected CAM more closely than it does normal CAM. The question of the origin of this agglutinin and its occurrence in other normal and virus-infected cell5 is under investigation at the present time. REFERENCES BAILEY, J. M., GEY, G. O., and GEY, M. K. (1959). Utilization of serum lipids by cultured mammalian cells. Proc. Sot. Exptl. Biol. Med. 100,

686-692. BENSCH, K. G., KING, D. W., and SOCOLOW,E. L. (1961). The source of lipid accumulation in L cells. J. Biophys. Biochem. Cutol. 9, 13b139. BORGSTR~M,B. (1952). Investigation on lipid separation methods. Separation of phospholipid from neutral fat and fatty acids. Acta Physiol. &and. 25, 101-110. BURNET, F. M. (1946). Vaccinia hemagglutinin. Nature 158, 119-120. BURNET, F. M., and STONE, J. D. (1946). The hemagglutinin of vaccinia and ectromelia virus. Australian J. Exptl. Biol. Med. Sci. 24, l-8. CHU, C. M. (1948). Studies on vaccinia hemagglutinin. I. Some physico-chemical properties. J. Hyg. 46,4248. COHN, Z. A. (1952). Quantitative distribution of phosphorus in chorioallantoic membranes as affected by infection with influenza virus. Proc. Sot. Exptl. Biol. Med. 79, 566-568. CORNATZER,W. E., GALLO, D. G., DAWSON, J. P., and FISHER, R. G. (1953). Phospholipid and protein-bound phosphorus synthesis in the rabbit papilloma. Cancer Res. 13,79b797. GILLEN, A. L., BURR, M. M., and NAGLER, F. P. (1950). Recovery of two distinct hemagglutinins of vaccinia virus. J. Zmmunol. 65,701-706. HANAHAN, D. J., DITTMER, J. C., and WARASHINA, E. (1957). A column chromatographic separation of classes of phospholipids. J. Biol. Chem. 228,

685-700. HORNING, M. G., WILLIAMS, E. A., and HORNING,

LIPIDS

579

E. C. (1960). Separation of tissue cholesterol esters and triglycerides by silicic acid chromatography. J. L@d Res. 1,482-485. KING, E. J. (1932). The calorimetric determination of phosphorus. Biochem. J. 26,292297. LEVINE, C., and CHARGAFF, E. (1951). Procedures for the microestimation of nitrogenous phosphatide constituents. J. Biol. Chem. 192, 465-

479. MCCREA, J. F., and O’LOUGHLIN, J. (1959). Separation of vaccinia hemagglutinin from infectious virus particles by chromatography on DEAE columns. Virology 8,127-129. NAGLER, F. P. 0. (1942). Application of Hirst’s phenomenon to the titration of vaccinia virus and vaccinia immune serum. Med. J. Australia

1,281-283. ONCLEY, J. L. (1958). In “The Lipoproteins: Methods and Clinical Significance” (Homburger, F. and Bernfield, P., eds.). Karger, New York. p. 14. RANDALL, C. C., GAFFORD, L. G., WALKER, B. M., and TODD, W. M. (1960). Biochemical changes and incorporation of Ps2 in separated elements of the hyperplastic lesion of fowlpox. Proc. Sot. Exptl. Biol. Med. 105,621-624. ROSSITER, R. J., and STRICKLAND, K. P. (1960). The metabolism and function of the phosphatides. In “Lipid Metabolism” (K. Bloch, ed.). Wiley, New York. p. 69-127. SPIRO, M. J., and MCKIBBIN, J. M. (1956). The lipides of rat liver cell fractions. J. Biol. Chem.

219,643-650. STONE, J. D. (1946a). Lipid hemagglutinins. Australian J. Exptl. Biol. Med. Sci. 24, 197-205. STONE, J. D. (194613). Inactivation of vaccinia and ectromelia virus hemagglutinins by lecithinase. Australian J. Exptl. Biol. Med. Sci. 24, 191-196. SULYA, L. L., and SMITH, R. R. (1960). An improved method for the detection of lipid on paper chromatograms. Biochem. Biophys. Res. Com-

mun.2, 59-62. TODD, W. M., RANDALL, C. C., and CONIGLIO, J. G. (1958). Quantitative changes in lipid composition of tissues infected with fowlpox virus. Proc. Sot. Exptl. Biol. Med. 98, 65-67. TREVALYAN, W. E., PROCTER, D. P., and HARRISON, J. S. (1950). Detection of sugars on paper chromatograms. Nature 166,444-445. YOUNGNER, J. S., and RUBINSTEIN, G. (1959). Separation of a soluble hemagglutinin from vaccinia virus by a flotation method. Virology 8, 386

388. YOUNGNER, J. S., and RUBINSTEIN, G. (1962). Separation and characterization of hemagglutinins from chorioallantoic membranes of embryonated eggs infected with vaccinia virus. Virology 16,

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