J. gen. Virol. (1985), 66, 1171-1177. Printedin Great Britain
1171
Key words: Sindbis virus/liposomes/maturation
The Ratio of Plasma Membrane Cholesterol to Phospholipid and the
Inhibition of Sindbis Virus Maturation by Low NaCI Medium
By R O B E R T F. G A R R Y . 1. D E N I S E A . B O S T I C K , 1 R I C H A R D
AND M A R I L Y N N R. F. WAITE2~"
SCHRAM 2
1Department oJ Microbiology and Immunology, Tulane School oJ Medicine, 1430 Tulane Avenue,
New Orleans, Louisiana 70112 and 2Department of Microbiology, University of Texas, Austin,
Texas 78712, U.S.A.
(Accepted 16 January 1985)
SUMMARY
Sindbis virus maturation is inhibited by low NaCI medium in chick embryo cells and
in one strain of BHK cells, but not in another strain of BHK cells which has a different
passage history. The plasma membrane of the cells in which Sindbis virus maturation is
resistant to low NaCl medium has a higher ratio of cholesterol to phospholipid than the
other cells. Cholesterol-containing liposomes, but not cholesterol-free liposomes, can
release Sindbis virus from low NaCl-inhibited cells. These results suggest that low
NaCl medium may block Sindbis virus maturation by a mechanism which is influenced
by the ratio of plasma membrane cholesterol to phospholipid.
Alphaviruses have been employed extensively as models for study of the insertion of proteins
into cellular membranes and for the acquisition of membranes by viruses (Brown, 1980: Simons
et al., 1982). Sindbis virus, prototype of the group, contains equimolar amounts of two envelope
proteins, El and E2 (Mr of each is approx. 50000), and a capsid protein, C (Mr approx. 30000)
(Schlesinger et al., 1972). In addition to E1 and E2, the virion envelope contains host-derived
cholesterol and phospholipid arranged in a bilayer structure (Pfefferkorn & Hunter, 1963).
Hydrophobic tails of one or both envelope proteins penetrate this bilayer and interact with the C
protein (Harrison et al., 1971 ; Rice et al,, 1982).
Lowering the NaC1 concentration of the cellular growth medium from 138 mM to 68 mM
inhibits the maturation of Sindbis virus from chick embryo (CE) cells at a stage after the
nucleocapsids have bound to the plasma membrane (Waite & Pfefferkorn, 1968, 1970; Waite et
al., 1972: Bell et al., 1978; Strauss et al., 1980). Returning low NaCl-inhibited cells to normal
medium rapidly reverses the inhibition; the number of virus particles produced within minutes
after medium shift is the same or greater than that released by cultures incubated in normal
medium throughout (Waite & Pfefferkorn, 1968; Pierce et al., 1974; Garry et al., 1979).
Experiments using antiserum specific for E 1 and E2 or lactoperoxidase radioiodination indicate
that low NaCI medium inhibits virus release at a step after the cleavage of PE2, a polyprotein
precursor to E2 and E3 (a non-structural protein) (Bell et al., 1978). E3, which is normally shed
into the medium of Sindbis virus-infected cells after its cleavage from PE2, fails to accumulate in
cells exposed to low NaCI medium (Bell et al., 1978; Mayne et al., 1984).
Waite & Pfefferkorn (1968) demonstrated that Sindbis virus maturation was similarly
inhibited in both CE cells and in baby hamster kidney (BHK) cells incubated in low NaCI
medium (Waite & Pfefferkorn, 1968), In contrast, Pierce et al. (1974) reported that, although
virus maturation was blocked by low NaC1 medium in CE cells, the inhibition did not occur in
BHK cells. To determine whether the differences in the effects of low NaC1 medium might be
accounted for by differences between BHK cell strains, the effects of low NaC1 medium on
Sindbis virus maturation were examined in several strains of BHK cells with different passage
histories. As reported previously (Waite & Pfefferkorn, 1968: Pierce et al., 1974), virus
t Present address: University of Texas Health Science Center at San Antonio. 7703 Floyd Curl Drive, San
Antonio, Texas 78284, U.S.A.
(1000-6439 r~ 1985 SGM
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1172
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I
I
I
(a)
l O 0 0 i ~
!
I
I
I
I
I-
1000
(c)
100
loo
10
/
I
I
0
50
100
I
I
I
I
N
I
1500
50 100 1500
50 100 150
Na + concentration (mM)
Fig. 1. Effect of medium containing low concentrations of NaCI on maturation of Sindbis virus from
(a) CE, (b) BHK-S and (c) BHK-R cells. Subconfluent monolayers of CE, BHK-S and BHK-R cells
were infected with Sindbis virus at a multiplicity of 5 p.f.u./cell. After 1 h for virus adsorption the cells
were incubated at 37 °C in medium supplemented with 5~~ dialysed calf serum and containing the
indicated concentrations of Na +. At 5.5 h post-infection samples of the altered media were harvested
for virus titration. All cells were then exposed to normal medium (138 myt-Na +) for 0.5 h to allow release
of virus whose morphogenesis was inhibited in the low NaC1 medium. Virus titrations were carried out
by plaque assays on CE cell monolayers as previously described (Pfefferkorn & Hunter, 1963). O, Virus
released during incubation in medium of the indicated NaC1 concentration; [5], total virus yield
including that released into medium of lowered NaCI concentration and into normal medium.
maturation in CE cell cultures was inhibited more than 95 ~ when the NaC1 concentration of the
medium was reduced below 68 mM (Fig. 1). This block in maturation was rapidly reversed by
incubation in medium containing normal amounts of NaCI. Total virus yields (low plus normal
NaCI harvests) from CE cells incubated in low NaCI medium then exposed to normal m e d i u m
may exceed those from CE cells grown under isotonic conditions throughout infection (Pierce et
al., 1974; Garry et al., 1979). A similar inhibitory effect of low NaCI medium on Sindbis virus
maturation was observed in a strain of B H K cells (BHK-sensitive; BHK-S) originally obtained
through E. Pfefferkorn in which Sindbis virus morphogenesis had previously been shown to be
sensitive to low NaCI medium (Waite & Pfefferkorn, 1968). The effects of low NaC1 m e d i a on
Sindbis virus maturation in CE cells and BHK-S cells contrasted sharply with the effect of these
media on Sindbis virus maturation in certain other independently-passaged strains of B H K
cells. One of these strains (BHK-resistant; B H K - R ) from the A m e r i c a n Type Culture Collection
(ATCC-CCL10) was selected for further study. Although total virus production by B H K - R
cultures incubated in low NaC1 medium was reduced, there was no selective inhibition of virus
maturation (Fig. 1).
Both BHK-S and B H K - R were obtained as BHK-21 cells and proved to be hamster cells by a
karyotype analysis (data not shown). Both showed slight variation in the number of hamster
chromosomes per nucleus as is typical for BHK-21 cells. B H K - R cultures contained
significantly more 'giant' cells, showed more karyotypic variation, and displayed more orderly
orientation in cell culture than BHK-S. The significance of the differences between the two
strains of B H K cells with respect to their sensitivity to low NaC1 medium is not known.
Since low NaC1 medium blocks Sindbis virus maturation at a step following the binding o f
nucleocapsids to the plasma m e m b r a n e (Waite et al., 1972), we compared the composition of the
plasma membranes of infected and uninfected CE, B H K - S and B H K - R cells and the
compositions of the membranes of virus released from these cell types (Table 1). The ratio of
cholesterol to phospholipid was significantly higher in the plasma membranes of infected B H K R cells compared to either infected CE cells or B H K - S cells. B H K - R cells had more protein,
while BHK-S cells had somewhat lower levels of protein, than the CE cells. Measurements of the
cholesterol and phospholipid contents of CE cell plasma m e m b r a n e s obtained by previous
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38.2 _+ 2.7
12.3 ± 0.3
50.1 ± 1-8
BHK-R
30.8 +_ 2"6t 42.6 ± 3'[
12.7 ± 1-0 17.2 ± 1'5t
56-4 ± 3.9 40.0 ± 1.6]
BHK-S
36.3 ± 2-8
11.8 +_ 0.3
51.0 + 3.6
CE
BHK-R
31-8 ± 0-8]. 42.8 ± l.l
11.9 ± 0.4
16.3 + 1-7~
56.3 ± 0.5 41.4 ± 2-4"}"
BHK-S
A
~k
A
BHK-S
BHK-R
51.2 _+ 3.1"]" 52-3 + 3"8t 53.1 ± 4-4~
17.1 + 0"9t 16.5 ± 0.9~ 17.5 ± 1-1~
31.8 ± 2.4]" 31.2 ± 2.7] 29.4 ± 0.3]"
CE
Sindbis virus m e m b r a n e
* Plasma m e m b r a n e s were prepared from infected cells by two cycles of gradient centrifugation by the method of B o s m a n n el al. (1968) as previously described
(Bose & Brundige, 1972). Between 3 and 5 °.o of total cellular glucose 6-phosphatase activity, a standard marker enzyme of the endoplasmic reticulum (Bosmann et al.,
1968: Bose & Brundige, 1972), was present in the plasma m e m b r a n e preparations indicating that m a x i m u m contamination with endoplasmic reticulum accounted
for no more than 10°o by weight. Sindbis virus was purified by three cycles of velocity gradient centrifugation (Waite & Pfefferkorn, 1968). Virus preparations
contained only Sindbis virus proteins and no host proteins detectable by SDS polyacrylamide gel electrophoresis. Protein was determined by the method of Lowry et
al. ( 1951 ). This value for Sindbis virus m e m b r a n e s was obtained by assuming that E 1, E2 and C were present in the virion in a 1 : 1 : 1 ratio and had a total Mr of
138 000 while the capsid protein has an M~ of 30000. Lipids were extracted by the method of Folch et al. (1956). Neutral lipids were chromatographed on silica gel
T L C plates in a solvent system of hexane :ether : formic acid ( 180 : 150 : 3). Spots corresponding to cholesterol were located by co-chromatography with an authentic
standard (Sigma), scraped from the plates, extracted with chlorolbrm, evaporated to dryness, resuspended in chloroform and quantified as described by Zlatkis et al.
(1953). Total phosphorus and the amount of phospholipid were determined by the method of Bartlett (1959). All values are expressed as the m e a n _ the standard
error of the m e a n (mg/100 mg total weight). Total weights were the s u m of the weights of the protein, phospholipid and cholesterol. Statistical significance was
determined by comparison with the corresponding value obtained in uninfected CE cell plasma m e m b r a n e s by Student's t-test (n = 8 or 9 for plasma m e m b r a n e
preparations: n = 6 for virus preparanons) (Schefler, 1969).
t P < 0.01 when compared to value obtained in uninfected CE cell plasma membranes. Values which did not differ significantly from the corresponding value
obtained in uninfected CE cells have no symbol.
Protein
Cholesterol
Phospholipid
CE
Plasma m e m b r a n e
(infected)
Plasma m e m b r a n e
(uninfected)
Table 1. Composition of plasma membranes.lrom CE, BHK-R and BHK-S and of Sindbis virus membranes prepared fi'om each cell type*
ta~
%
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workers (Renkonen et al., 1971 ; Quigley et al., 1972; Pessin & Glaser, 1980) were very similar to
the values reported here. Differences in the composition of plasma membranes from CE cells
and certain strains of BHK cells have been observed in previous studies (David, 1971) and
different strains of BHK cells have been shown to differ in the relative content of plasma
membrane cholesterol and phospholipid (Renkonen et al., 1971). The plasma membranes of
infected CE, BHK-S and BHK-R cells contained somewhat lower ratios of cholesterol to
phospholipid than the plasma membranes of uninfected cells. Lower ratios of cholesterol to
phospholipid were also observed after infection of BHK cells by Semliki Forest virus, another
ulphavirus (Renkonen et al., 1971). The levels of protein and the ratio of cholesterol to
phospholipid were higher in the Sindbis virus envelopes than in the plasma membranes of any of
the infected cell types examined. These values for the protein and lipid compositions of
alphavirus membranes were comparable to previously reported values (Renkonen et al., 1971;
David, 1971). Despite differences in the cholesterol to phospholipid ratios in the plasma
membranes of the different cell types, the ratios in virions grown in the different cells were
similar. Because Sindbis virus infection does not significantly increase the relative overall levels
of plasma membrane cholesterol, the process of virus budding must selectively occur in or
generate regions of plasma membrane with higher ratios of cholesterol to phospholipid.
Since the ratios of cholesterol to phospholipid were lower in the plasma membranes of low
NaCl-sensitive cells than in the resistant cells or virus derived from all three cell types, we
reasoned that the relative cholesterol content of the membrane might influence the effect of low
NaC1 medium on Sindbis virus budding. If so, changing the relative cholesterol content of the
plasma membrane may effect virus release from the low NaCl-sensitive cells. One way to alter
the composition of the plasma membrane rapidly and selectively is to incubate cells with
liposomes of various compositions (Inbar & Shintzky, 1974). In preliminary experiments we
found that the fusion of liposomes occurred eight to ten times more effectively with Sindbis
virus-infected cells than with uninfected cells. The enhanced fusion ofliposomes to infected cells
may be a consequence of the fusogenic properties of the virus glycoproteins inserted in the
plasma membrane. Previous studies have shown that incorporation of myxovirus or herpesvirus
glycoproteins in liposomal membranes dramatically increases fusion with plasma membranes
(Huang et al., 1980; Johnson et al., 1984). Using liposomes containing labelled cholesterol or
oleic acid, we found that treatment of infected, sensitive cells (CE or BHK-S) with liposomes
containing a high content of cholesterol resulted in sufficient incorporation of cholesterol to
increase the ratio of cholesterol to phospholipid in the plasma membrane transiently to values
comparable to those in BHK-R cells. To determine whether this modification could alter the
response of Sindbis virus-infected cells to low NaC1 medium, Sindbis virus-infected sensitive
cells (both BHK-S and CE cells) were exposed in low NaC1 solution to either cholesterol-free
liposomes or to liposomes containing a high content of cholesterol. As shown in Table 2,
cholesterol-enriched liposomes, but not cholesterol-free liposomes, caused a rapid release of
Sindbis virus from the low NaCl-inhibited cells. Liposomes containing lower ratios of
cholesterol to phospholipid did not release Sindbis virus from low NaC1 medium-inhibited cells.
It has been reported that cholesterol is required for the binding and fusion of alphaviruses with
liposomes (Mooney et al., 1975; White & Helenius, 1980). Fusion of host plasma membranes
containing Sindbis virus envelope proteins and nucleocapsids with the liposomes could directly
release virus from the intact cells. However, this is difficult to visualize topologically because of
the orientation of the envelope proteins. Increasing the ratio of cholesterol to phospholipid in the
region of the plasma membrane of the cell containing virion proteins could alter the response to
low NaCI medium. Thus, Sindbis virus release from cells in which virus release had been
inhibited due to the low NaCI medium could be a secondary consequence of the change in the
ratio of cholesterol to phospholipid in the plasma membrane.
Strauss et al. (1980) have reported that changing the medium supplement from bovine calf
serum to foetal bovine serum altered the response of their line of BHK cells to low NaCI medium
such that Sindbis virus maturation was inhibited by low NaC1 medium. Preliminary evidence
suggests that this medium modification may decrease the ratio of plasma membrane cholesterol
to phospholipid in certain subclones of BHK cells. Differences in the medium supplementation
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1175
Table 2. Release of Sindbis virus Jrom low NaCI-inhibited cells by cholesterol-enriched liposomes*
Treatment
Low NaCI medium
LBSS
Cholesterol-free liposomes in LBSS
Cholesterol-enriched tiposomes in LBSS
HBSS
Time postinfection
(h)
0-6
6 6-5
6 6.5
6 6.5
6-6.5
r
CE
[10
65
275
1850
2040
Virus release (p.f.u./cell)
"~
BHK-S
BHK-R
74
1930
265
300
115
280
2000
355
2450
180
* CE, BHK-S and BHK-R cells were infected with Sindbis virus (m.o.i. 5) in normal medium. After 1 h for virus
adsorption cells were incubated in low NaCI medium for 6 h. This medium was removed and the cells were
incubated for an additional 0-5 h in either Hanks" balanced salt solution (HBSS), cholesterol-free liposomes in
LBSS (HBSS containing 68 mM-NaCI),or cholesterol-enriched liposomes in LBSS. Liposomes were prepared by
rotary vacuum evaporation as previously described (Straub et al., 1974). Cholesterol-free liposomes contained
phosphatidylethanolamine :sphingomyelin:stearylamine :diacetylphosphate (30 : 5:6 :3 by wt.) and cholesterolenriched liposomes contained phosphatidylethanolamine:cholesterol:sphingomyelin:stearylamine:diacetylphosphate (10:30:5:6:3 by wt.). Liposomes were dialysed against LBSS prior to use. Approximately 10 mg of
liposome suspension in 1 ml was added to each 100 mm dish (5 x 106 cells). Virus release was assayed on CE cell
monolayers as previously described (Pfefferkorn & Hunter, 1963).
were not the basis for the difference in susceptibility of the two strains of BHK cells examined in
the present report since both were cultivated in medium containing bovine calf serum.
Based on present information regarding Sindbis virus morphogenesis it is possible to envisage
several models of how a higher ratio of plasma m e m b r a n e choleslerol to phospholipid could
overcome the inhibilion of Sindbis virus maturation imposed by low NaC1 medium.
Conformational or locational changes in E2 which follow its cleavage from PE2 are known to be
affected by low NaC1 medium (Bell et al., 1978). Several cellular plasma m e m b r a n e
modifications must occur either prior to or concomitant with this change in E2 to permit virus
budding. Host-derived proteins must be excluded from the membrane. However, if this were a
step limited by low NaCI medium, then specific host proteins would have to be involved since
B H K - R cells contain somewhat more protein in their plasma membranes than BHK-S cells.
Alternatively, low N aCl medium may inhibit the sequestration of cholesterol in the region of the
budding virion. Membranes with a higher initial concentration of cholesterol might be less
susceptible to such effects. Based on differences in the response of plaque morphology variants
to low NaCI medium, Strauss et al. (1980) suggested that low NaC1 medium may inhibit Sindbis
virus maturation because of charge effects on the budding viral envelope proteins. The results
presented here are not at all inconsistent with that hypothesis. Our results suggest that charge
effects on the m e m b r a n e lipid components may also be involved in the inhibition of Sindbis
virus maturation by low NaC1 medium. BHK-S and CE cells are relatively low in membrane
cholesterol which has a neutral charge, and high in phospholipid which has a negative charge.
Insufficient cations to neutralize the negative charges of the phospholipid may be contained in
low NaCI medium. A higher ratio of neutral cholesterol to negatively charged phospholipid in
the plasma membrane, as in B H K - R cells, could make the m e m b r a n e less subject to such an
effect of lowered NaC1 in the medium.
The present studies suggest that the incorporation of a relatively high ratio of envelope
cholesterol to phospholipid is important in the maturation of Sindbis virus in vertebrate cells. It
should be noted, however, that a relatively high ratio of cholesterol to phospholipid is not an
absolute requirement for maturation or infectivity of Sindbis virus, since virus grown in Aedes
albopictus (mosquito) cells is deficient in m e m b r a n e cholesterol compared to virus grown in
vertebrate cells (Luukkonen et al., 1977).
This research was supported by a grant from the Edward G. Schlieder Educational Foundation to R. F. G. and
by grants from the National Science Foundation (Grant PCM-77-25135)and the University Research Institute of
the University of Texas at Austin to M. R. F. W. We thank Henry R. Bose, Jr for advice and support, Dennis T.
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Short communication
Brown for cell lines, Greg Shackleford for help with the statistical analyses, and Jo Anne Daniel, Geraldine
Brooks, Sandra Sperry and Kim Simon for typing the manuscript. We also gratefully acknowledge the comments
of R. Walter Schlesinger during preparation of this manuscript.
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