SANDRA R. BATES AND GEORGE H. ROTHBLAT. The Wistar Institute ofAnatomy and .... VSV was obtained from Fred Clark, The Wistar Institute. The virus was.
Vol. 9, No. 6 Printed in U.S.A.
JOURNAL OF VIROLOGY, June 1972, p. 883-890 Copyright ( 1972 American Society for Microbiology
Incorporation of L Cell Sterols into Vesicular Stomatitis Virus SANDRA R. BATES AND GEORGE H. ROTHBLAT The Wistar Institute of Anatomy and Biology, Phliladelphia, Pennsylvania 19104 Received for publication 23 February 1972
The incorporation of host cell sterol into vesicular stomatitis virus can be effectively studied in an L cell system. The end product of de novo sterol synthesis in the L cell is desmosterol, and as the concentration of cholesterol in the medium is increased the cells incorporate the exogenous cholesterol and the synthesis of desmosterol decreases. L cells which contained desmosterol as their sole sterol produced virus whose sterol content was similarly composed of only desmosterol. Virus grown in L cells which had a constantly changing sterol ratio also contained a mixture of cholesterol and desmosterol, but the virus was found to be more enriched in cholesterol than in the L cells in which it was grown. Viral stability, growth, and plaquing efficiency were tested and found not to be affected by the alteration of its sterol composition, i.e., by partially or completely replacing cholesterol with desmosterol.
Previous studies have characterized the lipids in the virus envelope and compared viral lipids to those found in host cell membranes (7, 8). Most of these studies have dealt with phospholipids, whereas very little attention has been devoted to the sterols present in the viral membranes. Studies of sterol function as reflected in their specificity in viral membranes would be greatly facilitated if the host cell system contained two different sterols which could be used for viral envelope formation. Most tissue culture cells contain cholesterol (cholest-5-en-3,B-ol) [G. H. Rothblat. Cellular sterol metabolism. In G. H. Rothblat and V. J. Cristofalo (ed.), Growth, Nutrition and Metabolism of Cells in Culture, Academic Press Inc., New York, in press], which is derived from serum added to the growth medium or is the product of de novo synthesis. Recent studies with the L cell (11), however, have shown that whereas these cells cannot synthesize cholesterol, they do synthesize desmosterol (cholest-5,24-diene-33-ol), the immediate precursor of cholesterol. Moreover, the addition of cholesterol to these cells wilt inhibit de novo synthesis of sterol (10). Thus, a tissue culture system is available in which the type of sterol present in the cell can be varied by the presence or absence of exogenous cholesterol. When cholesterol is not present in the medium, the L cells contain desmosterol which is the product of de novo synthesis. When cholesterol is added to the medium, de novo synthesis of demosterol is inhibited and the cholesterol is utilized by the cell
for membrane synthesis (G. H. Rothblat, In Growth, Nutrition and Metabolism of Cells in Culture, in press). The current investigation was conducted to determine whether L cells could provide an experimental system for studies of the interrelationships between host-cell and viral sterols. The envelope virus chosen for this study was vesicular stomatitis virus (VSV). This virus matures by budding through membranes and, in the L cell, buds principally from the surface membrane (15). The chemical composition of VSV grown in L cells has been extensively studied by other investigators (8) who have determined that the virus contains 20% total lipids and 5.4% sterols. MATERIALS AND METHODS Cells. L cell mouse fibroblasts were used throughout this study. Cells were grown at 37 C in Eagle minimum essential medium (MEM) (Flow Auto-Pow) supplemented with Eagle basal medium vitamins. The medium was supplemented with delipidized calf serum protein (DLP), obtained by extraction of calf serum (Flow Laboratories) by the cold ether extraction procedure of Scanu et al. (12), as modified by Albutt (1). This technique yields a mixture of soluble serum proteins that contain virtually no sterol. L cells can be routinely grown in MEM supplemented with DLP; under these growth conditions they synthesize all of the sterol necessary for continued growth. The major (>90%) sterol present in these cells has been shown by gas-liquid chromatography (GLC) to be desmosterol, whereas a second minor sterol has been detected 883
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BATES AND ROTHBLAT
and tentatively identified as A I 72 24-cholestatriene-30ol (11). Less than 1% of the total sterol had a relative retention time (RRT) similar to that of cholesterol (11). Lipid-free serum proteins were relipidized by procedures which have been described previously (10). A single addition of the lipids dissolved in ethanol was made to the protein solution to yield a final constant level of protein (5 mg/ml), lecithin (20 pg/mi), and ethanol (0.5%) with varying levels of unesterified cholesterol. This concentration of ethanol had no effect on cell growth. Monolayers of L cells were harvested by the following procedure. They were washed two times with buffered salt solution (3), dispersed by treatment with 0.25% trypsin (Grand Island Biological Company), pelleted by low-speed centrifugation, and washed twice by buffered salt solution. They were stored at -4C. In this report, cells grown on DLP in the absence of exogenous cholesterol, and therefore containing desmosterol, are designated "desmosterol L cells." Cells grown in the presence of cholesterol, and therefore containing cholesterol, are designated "cholesterol L cells." Virus. The Indiana serotype of VSV was obtained from Fred Clark, The Wistar Institute. The virus was cloned by plaque-purification procedures three times in desmosterol L cells (desmosterol VSV). Two types of stock virus were prepared: (i) virus obtained from L cells grown in 40 pAg of cholesterol per ml (cholesteroldesmosterol VSV), and (ii) virus produced from desmosterol Lcells (desmosterol VSV). L cells were grown in milk dilution bottles in medium containing 40 ug of cholesterol per ml and 20 jpg of lecithin per ml (cholesterol L cells) or in a medium containing 20 pAg of lecithin per ml alone (desmosterol L cells). When the cells reached confluency, the medium was removed, and plaque-purified desmosterol VSV was added at an input multiplicity of 1.0 in 1.0 ml of MEM supplemented with 0.25% albumin. The virus was allowed to adsorb at 37 C for 45 min. The monolayers were then washed three times with 5 ml of MEM and covered with 15 ml of MEM supplemented with 0.25% albumin. The virus from each cell type was harvested after the infected monolayers had been incubated for 7 hr at 37 C. All stock virus was stored at -40 C. Plaque assay. L cell monolayers were grown in MEM supplemented with 5% calf serum on Falcon plastic petri dishes (60 by 15 mm). At confluency, the medium was removed, and 0.2 ml of virus, diluted in 0.25% albumin MEM, was placed on the monolayers. Virus was allowed to adsorb for 45 min, after which the unadsorbed virus was removed by washing the cell monolayer twice with 5 ml of MEM. The monolayers were then overlaid with 5 ml of 0.95% agarose in MEM (without phenol red). After a 48-hr incubation period, the monolayer was stained with a 1:450 dilution of neutral red, and plaques were counted. All titers were determined as the average of duplicate plates at each dilution. Purification of virus. Virus to be used for sterol analysis was obtained from monolayers of L cells grown in 2-liter Blake bottles. Confluent cell monolayers,
J. VIROL.
containing approximately 6 X 107 cells, were obtained by growing cells in medium with 2.5 mg of DLP per ml and the appropriate lipid supplements. Upon confluency, the medium was removed, the monolayers were washed once with MEM, and 15 ml of stock virus inoculum (desmosterol VSV) was added. Throughout this study, all virus inocula were prepared in MEM supplemented with 0.25% albumin. After a 45-min adsorption period, the viral solution was poured off and 150 ml of 0.25% albumin in MEM was added to each 2-liter bottle. The supernatant virus was harvested after incubation, clarified of ceUular debris by centrifugation at 4,000 X g, and stored at -40 C. VSV was purified by the method of Sokol et al. (13). This procedure involves precipitation of the viral supernatant fraction with zinc acetate, sedimentation of the precipitate by centrifugation, suspension of the pellet in ethylenediaminetetraacetic acid, followed by filtration of this material through a Sephadex G-75 column, and collection of the viral band after centrifugation through a 10 to 60% (w/w) linear sucrose density gradient. Gradient centrifugation at 61,000 X g for 90 min at 4 C produced two distinct bands: an upper band, which was shown by electron microscopy and virus titration to be the complete virus; and a lower, more diffuse band, which contained fragmented material. No T band, as described by Huang et al. (6), was detected in these preparations. The upper viral band was collected and stored at -40 C for further sterol analysis. Sterol assay. Cellular lipids were obtained by extracting washed cell pellets three times with 5 ml of boiling chloroform-methanol (2:1, v/v). Viral lipids were obtained by extracting the banded virus with 40 volumes of chloroform-methanol (2:1) (4). Viral and cellular lipids were saponified with 10% ethanolic KOH at 60 C for 30 min. After saponification, four volumes of saline were added and the aqueous layer was extracted with one volume of petroleum ether and one volume of ethyl ether (2X) to obtain nonsaponifiable lipids. The sterols in the nonsaponifiable lipids were qualitatively and quantitatively analyzed by GLC using a no. 402 Hewlett Packard gas chromatograph equipped with a hydrogen flame ionization detector. The two columns used were: 6 ft (ca. 1.8 m), 100- to 120-mesh Supelcoport coated with 3% XE-61; and 4 ft (ca. 1.2 m), 80- to 100-mesh Supelcoport coated with 3% SE-30. The following temperatures were used: column oven, 230 C; flash heater, 290 C; and flame detector, 250 C. The gas flow rates were: air, 250 ml/ min; carrier gas (helium), 100 ml/min; and hydrogen, 30 ml/min. Retention times (relative to cholestane) were determined with known standards. RRT on on XE-61 column were: cholesterol, 2.59, and desmosterol, 3.16; and on SE-30 column: cholesterol, 1.88, and desmosterol, 2.09. Specific radioactivities of isolated sterols were determined with an effluent stream splitter. Samples were collected at the outlet port, and the condensed sterols were counted by liquid scintillation techniques. Recoveries were monitored with standards of known specific activity. Sterol was quantitated by measurement of peak areas with coprostanol as an internal standard. All solvents used for extraction were reagent grade
VOL. 9,1972
and redistilled prior to use. All solvents were dried under a stream of nitrogen. Isotopes. Radioactive compounds used were cholesterol4-'4C (58 mCi/mmole, New England Nuclear), and sodium acetate-2-14C (55 mCi/mmole, Amersham/Searle). Labeled sterols were purified before use by thin-layer chromatography by using Silica Gel G plates developed with petroleum ether-ethyl etheracetic acid (75:24: 1, v/v). When necessary, labeled compounds were adjusted to desired specific activity by the addition of unlabeled carrier. Radioactivity was measured in a Packard or an Intertechnique spectrophotometer, using either 0.6% 2,5-diphenoloxazole and 0.02% dimethyl-1,4bis-2-(5-phenyloxazolyl) benzene in toluene, or Aquasol (New England Nuclear). When necessary, counts were corrected for quenching by use of internal standards. Viral growth curves. Growth curves of VSV were obtained with monolayers of both desmosterol and cholesterol L cells. Ten Falcon plastic plates (60 by 15 mm) were seeded with 5 X I05 cells per plate and grown for 1 day on DLP at 5 mg/ml. After removal of the DLP, one-half of the plates were refed with medium containing DLP plus 20 /tg of lecithin per ml (desmosterol cells), and the other half with medium containing DLP plus 20 ,ug of lecithin per ml and 40 ug of cholesterol per ml (cholesterol cells). After a 2-day incubation period, these media were removed and the monolayers were washed twice with MEM. Virus (0.2 ml) was added to each plate at the appropriate multiplicity and adsorbed for 45 min. Unadsorbed virus was removed by rinsing two times with MEM, after which 5 ml of MEM supplemented with 0.25% albumin was added. At timed intervals over the next 24-hr period, 0.2-ml samples of the supernatant fluid were removed from each of the five plates of cholesterol or desmosterol cells and pooled. These pooled supernatant fractions were centrifuged at 1,000 X g to remove cells and quick-frozen at -40 C for subsequent plaque titration. Since the samples were pooled, the final titer at each time point represented the average titer contained in the five plates. Heat inactivation. Capped glass vials (45 by 15 mm) containing 2 ml of desmosterol VSV or cholesteroldesmosterol VSV in MEM containing 0.25% albumin were placed in a 37 C incubator. Samples were titrated at the beginning of the experiment and at timed intervals thereafter. Plaquing efficiency. The following virus-cell combinations were tested: Virus
(0.95%7) in MEM (without phenol red). Plaquing efficiency was quantitated by determination of the number of plaques which developed on each plate. Freezing and thawing. Two milliliters of stock desmosterol VSV and VSV which contained a mixture of cholesterol and desmosterol was added to small glass vials (45 by 15 mm) in MEM containing 0.25% albumin. One set of vials was titrated immediately, and the remaining samples were quick frozen in a dry iceacetone bath. The frozen samples were then thawed at room temperature. This procedure was repeated three times, with one sample of each type of virus being removed and titrated after each cycle. RESULTS L cell sterols. Experiments were conducted to ascertain the effect of varying levels of cholesterol upon synthesis of desmosterol in L cells. Cells were grown for 1 week in medium containing different levels of cholesterol, and the relative composition of cellular sterols was analyzed. Table 1 shows the typical sterol composition of the L cells as analyzed by GLC. The data indicate that the major sterol recovered in these cells was either cholesterol or desmosterol. A third sterol was observed in some of the cell cultures and has tentatively been identified as Al, 7' 24-cholestatriene-3f-ol, the immediate precursor to desmosterol. The amount of this sterol present varied from 19% in cells grown in the absence of cholesterol to less than 1 % in cells grown in high concentrations of exogenous cholesterol. Because of the variability in appearance of this material and the difficulties encountered in quantitating the low levels recovered from the cells, the amount of this material present in the cells was not routinely used in the calculations of total cellular sterol. Figure 1 shows the per cent composition of Lcell sterols when the cells are grown in increasing amounts of exogenous cholesterol. In the absence of exogenous cholesterol, L cells contain 93.5% desmosterol, 5.7% trienol, and 0.8c%o of a sterol having the chromatographic properties of cholesterol. Although this material has a retention time similar to cholesterol in this GLC TABLE 1. Sterol compositiont of L cells
L cell
Cholesterol-desmosterol
Cholesterol
% Demostrol Exogenousholesterol oeser c/ % chlDemosero holesterol g (avg )a (avg)a (JAg/Ml)
oc
30d Desmosterol
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INCORPORATION OF L CELL STEROLS
0°8b (0-4) 83.2 (72-91)
93.5 (77-100) 15.6 (9-28)
cholesta-
triene-3c olb range) ~~~~~~~~(avg 5.7 (0-19)
1.2 (0-6)
-Desmosterol
Cell monolayers were infected with the appropriate dilution of virus (0.2 ml). At intervals of 10, 25, and 45 min after the addition of virus, sets of monolayers were washed with MEM (2X) and overlayed with agarose
Numbers in parentheses refer to range. Identity unconfirmed. Average of 11 determinations. d Average of eight determinations from cells grown on a
b
c
cholesterol for I week.
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BATES AND ROTHBLAT
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100CHOLESTEROL
\
90-0 80J
0
0
S
70-
6050-
40-
*
0
30-
I-V.
D~~~~~~~~= ESMOSTEROL --2.5
5
75
10
20 pu
/ml
30
-J
CHOLESTEROL
60-
40
CHOLESTEROL
4 508
FIG. 1. The sterol compositioni of L cells grown for I week in medium containinig differenzt concentrations of cholesterol.
710-
40-
ESOSTEROL
Zi
system, its identity has not been established and
preliminary investigations have indicated that it not be cholesterol (G. H. Rothblat, unpublished observations). At the highest levels of exogenous cholesterol used in this experiment (40 pig/ml), cell sterols were shown to contain 92% cholesterol and 8% desmosterol. This indicates that cellular desmosterol synthesis is almost totally inhibited by high levels of exogenous cholesterol. In addition, after 1 week of growth, it is possible to obtain cells with different ratios of cholesterol to desmosterol by varying the levels of exogenous cholesterol in the medium (Fig. 1). In this study, all virus growth was carried out in medium without exogenous cholesterol but supplemented with albumin. Since the albumin was added to the medium without additional lipid, it was necessary to determine whether sterol biosynthesis was reinitiated in the absence of exogenous cholesterol during the period of virus growth. Experiments were conducted to determine the extent of reinitiation of synthesis and to assay the effect of virus infection on such synthesis. L cells were grown in medium with cholesterol (30 ,ug/ml) for 1 week (two cell passages). At the end of this time, one-half of the confluent monolayers were infected with VSV at an input multiplicity of 0.1, and the cholesterol medium was removed and replaced with MEM containing 0.25% albumin. The other half of the cells were used as uninfected controls, and the original cholesterol growth medium was replaced by MEM containing 0.25% albumin. At time intervals throughout the next 24 hr, control and infected cells were harvested. The sterol composition of these cells was determined by GLC. Data presented in Fig. 2 show the per cent composition of L-cell sterols upon removal of cholesterol from the medium in relation to time. In the absence of cholesterol, there is a continuous change in cellular sterol composition. No differmay
30
20-
10-
4
8
12
17
2'4
HOURS
FIG. 2. The sterol composition of L cells grown for I week in medium containing 30 .Ag of cholesterol per ml and changed to medium containing only albumin. ences were observed between infected and control L cell sterols throughout the first 17 hr of incubation in medium containing albumin. Desmosterol content increased after the first 4 hr of incubation and continued to increase until, at 17 hr, there was 52% desmosterol and 48 % cholesterol in the cell. Between 17 and 24 hr, a difference was observed between the infected and noninfected cell cultures. In the control cultures, desmosterol continued to increase, whereas in the infected cultures there was little additional change in sterol content. This can be attributed to massive cell death which occurs at this time and which could prevent further synthesis of desmosterol. Figure 3c presents the virus growth curve obtained under these experimental conditions. Viral sterol composition. Utilizing this L cell system, experiments were conducted to determine the sterol composition of virus liberated from cells previously grown in cholesterol-containing medium and changed to one containing albumin. L cells were grown to confluency in medium containing a mixture of cholesterol (30 ,ug/ml) and lecithin (20 ,ug/ml). This medium was removed, virus was added at a multiplicity of infection of 0.1, and the medium was replaced with fresh MEM supplemented with 0.25% albumin. After 9 or 17 hr, the infectious tissue culture fluid was harvested, the virus was purified, and its sterols were analyzed as previously described.
INCORPORATION OF L CELL STEROLS
VOL. 9, 1972
LU
2 z (a
z
ar0 LL-
L*J
a
a-
2
4
6
8
10 12 HOURS
24
FIG. 3. Growth curves of VSV grown in L cells. ) L cells grown in medium containzing 40 ,ug of cholesterol per ml and 20 Mug of lecithin per ml and changed to medium containing only 0.25% albumin at zero time. L cells grown in medium containiing 20 ,ug of lecithin per ml and changed to medium conttaining only 0.25% albumin at zero time. VSV input multiplicity of 5.0 (a), 1.0 (b), and 0.1 (c). (----
In conjunction with the growth of the virus, control samples of L cells were harvested. The control cells were either L cells harvested immediately before viral infection, or uninfected L cells whose medium had also been changed from one containing cholesterol to one with albumin, and which were harvested at the same time as the virus (9 or 17 hr). The sterols from these controls were analyzed and compared with those found in the virus. The results are shown in Table 2. The differences in the cholesterol-desmosterol ratio in cells grown in cholesterol at 30 ,ug/ml as shown in Tables 1 and 2 can be attributed to differences in the state of growth of the cells and in the preparation of the DLP from experiment to experiment. Differences between individual replicate cultures within a single experiment were reproducible to within +2%.
887
In all three experiments, the L cells show an increase in desmosterol content with time, which is consistent with the results presented in Fig. 2. The predominant sterol found in the virus after 9 and 17 hr of growth was cholesterol. In all cases the percentage of cholesterol is greater in the virus than in the control cells analyzed at the equivalent time point. Thus, virus obtained from cells previously grown in medium containing cholesterol and changed to medium containing albumin was composed of a mixture of sterols, cholesterol being predominant. Since the previous experiment had shown that a VSV population could be obtained with a mixture of sterols, experiments were performed to ascertain whether the virus would reflect the sterol composition of the desmosterol L cell, in which cholesterol was not present. L cells were grown in 2.5 mg of DLP per ml in the absence of cholesterol. In these cells, desmosterol comprised more than 90% of the total sterol present (Table 1). The sterol composition of the virus obtained by growth in these cells was greater than 98% desmosterol. No A5l 1, 24-cholestatriene-3,B-ol could be detected in the virus. The possibility of host cell contamination contributing to the viral sterol was examined with the use of radioactive label. In these experiments, isotopically-labeled, uninfected, L cells were mixed with nonradioactive virus and the amount of labeled desmosterol recovered from the virus preparation was assayed. Acetate-2-'4C (20 ,Ci), an efficient precursor to desmosterol, was added for 24 hr to four milk dilution bottles of L cells grown to confluency in medium containing 2.5 mg of DLP per ml (ca. 2.5 X 10' cells). These cells were harvested, and one-fourth of the cells were analyzed as described above to determine the specific activity of the synthesized cellular desmosterol. The remaining three-fourths of the labeled cellular material was suspended in distilled water, and the cells were disrupted in a tightly-fitting Dounce homogenizer. This material was added to 900 ml of medium containing unlabeled VSV produced during a 17-hr growth period in desmosterol L cells (9 X 10 plaqueforming units per ml.) The virus was then purified by the methods previously presented. Table 3 gives the per cent contamination of the virus at each step of the purification procedure by all of the cellular material which was synthesized from acetate during the 24-hr labeling period. Table 3 also shows the cellular desmosterol specific activity prior to its addition to the virus preparation and the specific activity of the desmosterol recovered from the purified virus. Cellular contamination of the virus, as recovered by sucrose gradient centrifugation, was 1.9% of the total
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BATES AND ROTHBLAT
J. VI ROL.
TABLE 2. L cell anid vesicuilar stomatitis virus (VSV) sterol composition Sterol composition ((I
Determination
L Cells L Cells VSV L Cells VSV
Ioum atf-ser tr
cos--ig Lto c;lange to
cholesterol-free medium (hr)
0 9
17
Chol = Cholesterol, Des bND Not determined. "
=
Expt
E)xpt 2
1
Expt 3
Chol"
Des
Chol
Des
Ciol
Des
80 68 88 54 87
20 32 12 46 13
78 70 87 55 77
22 30 13 45 23
90 ND'
10 ND ND 47 36
ND 53
64
desmosterol.
=
TABLE 3. Extent of contamination of purified VSV by '4C-lalbeled cell material Description
Radioactive counts Total cellular cutmmpent
Desmosterol specific activity (counts per mmn per jAg)
Dounced cells before addition to virus Viral pellet redissolved in EDTA after prei cipitation in zinc acetate Virus suspension after Sephadex column Virus band from sucrose gradient Purified virus sterol a
947,082
833,430 603,200
18,186 5,260
100
958
88 64 1.9 0.56
90.6
Abbreviations: VSV, vesicular stomatitis virus; EDTA, ethylenediaminetetraacetic acid.
L-cell material added. Based on the specific activity determinations, 9.3% of the desmosterol recovered from the virus was contributed by cellular material. Thus, 90.7% of the desmosterol isolated from the VSV was viral desmosterol. Biological properties of VSV. (i) Virus growth. Virus growth curves were determined in cholesterol and desmosterol L cells to ascertain whether sterol composition of the host affected growth of the virus. Host cell monolayers were obtained by growing cells for 2 days in medium containing 40 ig of cholesterol per ml and 20 ,g of lecithin per ml (cholesterol L cells) or medium containing only 20 mg of lecithin per ml (desmosterol L cells). The medium was removed, the cells were infected with VSV, and medium containing albumin was added to both cell monolayers. At timed intervals throughout the experiment, samples were removed and titrated as described above. Figure 3 shows the results of typical growth curves of VSV grown on cholesterol and desmosterol cells. After an initial lag period of 2 hr, there followed a rapid rise in titer which leveled off at about the 8th hr. There were no significant differences between virus growth curves in the two types of L cells, even when the input multiplicity of the virus was varied. (ii) Virus stability. A study was designed to
determine the effect of freezing and thawing upon the cholesterol-desmosterol virus and the desmosterol virus. After four cycles of freezing and thawing, no pronounced differences between the two types of virus were detected. With both types of virus, there was roughly a 20%70 loss of infectivity after each cycle of freezing and thawing. The thermostability of the desmosterol virus was compared to that of the virus containing a mixture of cholesterol and desmosterol. These results demonstrated that there were no significant differences in the heat stability of the two types of virus under the conditions employed. After 2 hr at 37 C, the infectivity of both types of viruses decreased by approximately 65 %. (iii) Plaquing efficiency. Table 4 shows the results of experiments designed to determine whether changes in the sterol composition of either host cell or virus would influence plaquing efficiency of the VSV. Experimental procedures are described in Materials and Methods. Plaquing efficiency was similar in all of the combinations tested. The data indicate that the plaquing eliciency of the virus increased with time of adsorption. DISCUSSION The report that L cell mouse fibroblasts have desmosterol as their major sterol (11) provided
INCORPORATION OF L CELL STEROLS
VOL. 9, 1972
TABLE 4. Plaquing efficiency of two typ es of VSV in either a cholesterol L cell or a desmosterol L cella Time (min)
VSV sterol
L cell sterol
10
Chol-Desb
Chol Des Chol Des Chol Des Chol Des Chol Des Chol Des
Des 25
Chol-Des Des
45
Chol-Des Des
Virus titer 6 .0 X 6.5 X 6.0 X 6.0 X 2.0 X
I
106 10
106 106 107
2.1 X 107 1 .7 X 107
.1
X 2.1 2.4 X
I
I
107 107
2.1 X 107 2.2 X 107
Abbreviations: VSV, vesicular sto'matitis viChol, cholesterol; Des, desmoster ol. I VSV containing a mixture of choli esterol and desmosterol. a
rus;
the basis for an experimental system inl which the role of sterols in an envelope virus might be studied. The present investigation wit] h VSV has demonstrated that the sterol compossition of a virus can be altered through the prope-r manipulation of this cell system. Examination of the host cells indi4 cated that, under the experimental conditions used for virus infection, the sterol composition of I cells was constantly changing during the peric)d of viral growth (Fig. 2). The reduction in the percentage of cholesterol and the increase in the percentage of desmosterol over the 24-hr peri( )d can be explained by a loss of cholesterol froim the cell an increase in the amount of cellular d esmosterol, or a combination of these two factor.s. It seems unlikely, however, that the decreasing percentage of cholesterol would be due solely to the efflux of cholesterol from the cell, since itt has been shown by Burns and Rothblat (2) tIhat only a small amount of sterol is excretedI into the medium in the presence of albumin. I]n addition, Sokoloff and Rothblat (L. Sokoloff;and G. H. Rothblat, manuscript in preparation) biave shown that the rate of de novo biosynthesis iis increased upon removal of exogenous cholester( .l Thus, it is probable that upon removal of cholesterol from the medium, desmosterol syynthesis is initiated in the L cells. Figure 2 als(o indicates that virus infection had little or no effect on cellular sterol composition up to thie point at which virus infection results in miassive cell death. Table 2 shows the sterol compositiorn of L cells and VSV grown in this system ofr changing sterols. In all cases the virus containe d a higher
889
percentage of cholesterol than uninfected cells analyzed at equivalent time points. It is possible that in a dynamic system such as the one used in the present experiments a sterol gradient exists within cells. Thus internal membranes may be enriched in newly synthesized desmosterol, whereas the plasma membrane may contain a disproportionate amount of the cholesterol previously incorporated from the growth medium. It this were the case, virus budding from the plasma membrane would show an enrichment in cholesterol when compared to whole cell cholesterol-desmosterol ratios. In addition, a similar enrichment in cholesterol could occur if the two sterols were not equally distributed within the plasma membrane and if specific membrane components enriched in cholesterol were incorporated into the virus envelope. Such an enrichment of cholesterol in the virus could be accentuated by the fact that the values obtained for virus sterol composition are a summation of many different viral sterol ratios. For example, as virus is produced, it incorporates the sterol present in the cell membrane at the time of its maturation. Because of the constantly changing sterol ratio in the cell, virus budding early after infection would have more cholesterol than virus budding at a later time. Thus the virus population at any time point would represent a heterogenous mixture of virus with different ratios of cholesterol to desmosterol. A heterogenous population, however, cannot explain the enrichment in cholesterol seen in the 9-hr virus preparation as compared to zero-time cells. This study also indicates that the virus is able to incorporate desmosterol and does not have an absolute requirement for cholesterol for the formation of its envelope, since virus was produced which contained desmosterol as its major sterol and virtually no cholesterol (Table 3). The biological properties of the VSV containing desmosterol and the VSV containing a mixture of cholesterol and desmosterol were quite similar. Viral growth profiles of the two were nearly identical and resembled those of VSV in L cells reported elsewhere (14, 15). The stability of the two types of virus at 37 C was similar. The rapid decrease in titer observed in these experiments parallels the results described by Prevec and Whitmore (9). Subjection of the two types of virus to cycles of freezing and thawing resulted in a similar drop in titer. The reduction of virus titer observed in this study is consistent with that reported by Galasso
(5).
In summary, the results of these experiments demonstrate that VSV containing either a mixture of cholesterol and desmosterol or only desmosterol can be produced by varying the conditions
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under which the host L cells have been grown. The evidence also shows that partial or complete replacement of cholesterol by desmosterol has no effect on the biological properties of the virus, as measured in this study. The present investigation indicates that the ability to regulate sterol composition in L cells can provide an experimental system which might yield further information on the mechanism of viral envelope formation and on the role of sterols in biological membranes. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grants T01-GM-00142 from the National Institute of General Medical Sciences and R01-HE-09103 from the National Heart and Lung Institute, and by funds from the Department of Health, Commonwealth of Pennsylvania. This investigation was performed during the tenure of an Established Investigatorship (to G.H.R.) from the American Heart Association. The authors wish to thank H. F. Clark, F. Sokol, D. Kritchevsky, and H. G. Aaslestad for their assistance with these studies. LITERATURE CITED 1. Albutt, E. C. 1966. Study of serum lipoproteins. J. Med. Lab. Tech. 23:61-82. 2. Burns, C. H., and G. H. Rothblat. 1969. Cholesterol excretion by tissue culture cells: effect of serum lipids. Biochim. Biophys. Acta 176:616-625. 3. Cristofalo, V. J., and D. Kritchevsky. 1966. Respiration and glycolysis in the human diploid cell strain WI-38. J. Cell. Physiol. 67:125-132. 4. Folch, J., M. Lees, and G. H. Sloalne Stanley. 1957. A simple
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