Bioscience Reports, Vol. 11, No. 5, 1991
Semliki Forest Virus Envelope Proteins
Function as Proton Channels
Andreas Schlegel, 1'2 Adames Omar, ~ Pia Jentsch, 1'2 Andreas
MorelP and Christoph Kempf L3
Received July 8, 1991; revised September 25, I991
It has been shown that isolated nucleocapsids of Semliki Forest virus (SFV) contract upon low pH
exposure (Soederlund et al., 1972). This contraction of the nucleocapsids has been used as an
indicator to demonstrate that the spike proteins of SFV can translocate protons into the interior of the
virus particle upon low pH (5.8) exposure. Spikeless virus particles obtained after bromelain
digestion, which were used as a control, did not translocate protons. This implies that the ectodomain
of the spike plays a crucial role for the proton translocation.
KEY WORDS: Semliki Forest virus (SFV); proton channel; virus envelope.
INTRODUCTION
Cell-cell fusion induced by enveloped viruses at low pH is a complex, well
regulated, naturally occurring phenomenon (Hoekstra and Kok, 1989; Hoekstra,
1990, Spear, 1987; Stegmann et al., 1989; White and Blobel, 1989). Several
enveloped viruses, for example, Semliki Forest virus (SFV), influenza virus, and
vesicular stomatitis virus (VSV), enter susceptible cells via endocytosis. Within
the endosomes the viruses encounter an acidic environment which triggers fusion
of the viral envelopes with the endosomal membrane. This leads to the release of
the nucleocapsid, containing the viral genome, into the cytoplasm and to infection
of the cell. Transcription of the virus genome is followed by translation which
results in the production of viral envelope proteins. These proteins are then
transported to the cell surface. If at this stage of infection, the cells are exposed
to a mildly acidic, extraceUular pH, analogous to the pH in the endosome,
cell-cell fusion can be induced (Garoff et al., 1982; Mann et al., 1983; White et
al., 1983). This virus-induced fusion is also referred to as fusion from within
(FFWI). Several dynamic changes occur at the cell membrane in SFV-induced
~Central Laboratory Blood Transfusion Service, Swiss Red Cross, Wankdorfstrasse i0, 3000 BERN
22, Switzerland.
2 lnstitue of Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.
3To whom correspondence should be addressed.
2.43
0144-8463/91/1000-0243506.50/0(~ 1991 Plenum Publishing Corporation
244
Schlegel, Omar, Jentsch, Morell and Kempf
FFWI, namely: i) a conformational change of a plasma membrane protein, most
probably the viral protein E1 (Koblet et al., 1985; Omar and Koblet, 1989); ii) an
influx of protons into the cells (Kempf et al., 1987 and 1988a); iii) a
hyperpolarization of the plasma membrane potential (Kempf et al., 1988b) and
iv) Na+/K+-fluxes across the plasma membrane (Kempf et al., 1988b). All of
these events and probably more correlate with the fusion process. Several lines of
evidence suggest that these phenomena are crucial for membrane fusion.
However, it is not clear whether these events are mediated only by viral envelope
proteins or whether host membrane proteins also participate in the process.
Furthermore, it was found that a transient acidification of the cytoplasm of
SFV-infected cells at an extracellular, neutral pH is capable of triggering cell-cell
FFWI (Kempf et al., 1990). Recently, it was shown that SFV-particles which
contained only the E1 envelope protein and the transmembrane anchor of E2 are
capable of inducing membrane fusion (Omar and Koblet, 1988) indicating that
the viral envelope protein E1 plays an important role and is directly responsible
for the initial events in the fusion process. In addition it was shown that upon
exposure of virions to low pH, the E1 protein undergoes a conformational change
to become completely trypsin resistant (Kielian and Helenius, 1985). Taking
these findings together it appears that the conformational change could be linked
to the proton influx observed in FFWI. Based on this assumption, we have
investigated the possibility of proton transfer by the envelope proteins utilising
virions. We have made use of the observation that isolated nucleocapsids shrink
upon low pH exposure (Soederlund et al., 1972). This shrinkage phenomenon
was therefore used as an indicator for the determination of a proton influx into
the interior of SFV particles. In this report we present evidence that the
ectodomain of the viral spike proteins mediates translocation of protons. This
observation lends further support to the mechanism we have proposed for the
initiation of membrane fusion, namely, that a viral spike protein, most probably
El, folds back into the lipid bilayer in which it is embedded (Kempf et al., 1990).
MATERIAL AND METHODS
Cells and media: Aedes albopictus cells, clone C6/36 (Igarashi, 1978), were
grown at 28~ in Mitsuhashi-Maramorosch (MM-medium) medium containing
20% fetal calf serum and supplemented with 100/ag streptomycin and 100 U of
penicillin per ml. The cells were passaged weekly by 1 : 10 dilutions.
Virus Propagation
SFV was propagated in Aedes cells. The cells were infected with 1 plaque
forming unit (PFU) per cell. Twenty to 24 hours post infection (hpi) the medium
was harvested and cellular debris was removed by centrifugation. The supernatant containing the virus was aliquoted and stored at -80~ Virus titers were
determined on Vero cells by plaque assays according to established methods.
SFV Envelope Proteins
245
Preparation of Radiolabelled Virus
Aedes albopictus cells were infected with SFV at a multiplicity of infection
(MOI) of approximately 10 plaque forming units (PFU) per cell. Six hours post
infection (hpi) the supernatant was removed and replaced with MM-medium
which was diluted 1:10 with phosphate buffered saline (PBS), containing 0.18%
(w/v) bovine serum albumin (BSA) and 10/~Ci 3sS-Met per ml. At 24 hpi the
medium was harvested and cellular debris was removed by centrifugation at
3000 g for 15 minutes. Virus was concentrated by centrifugation at 80,000 g for 3
hours through a cushion of 12% (w/w) sucrose in PBS. The pellet was
resuspended and analysed by SDS polyacrylamide gel electrophoresis (SDSPAGE; Laemmli, 1970). The gels were stained with Coomassie blue or dried and
exposed to an Amersham Hyperfilm /3-max. Protein concentrations were determined according to the method of Lowry et al. (1951).
Preparation of Spikeless Particles
The proteolytic enzyme bromelain (Calbiochem) was used to produce
spikeless particles. The bromelain was activated with 5 mM dithiothreitol (DTT)
and 2 mM EDTA in digestion buffer (50 mM NaCI, 100 mM Tris, pH 7.2) for 60
rain at 37~ SFV (2 mg/ml), suspended in digestion buffer, was mixed with an
equal volume of activated bromelain (3 mg/ml) and incubated for 24 hours at
37~ The spikeless particles were isolated by centrifugation at 195,000 g for 2.5
hours through 10% (w/w) sucrose in 250 mM NaCI, 50 mM Tris, pI-I 7.4 and
analysed by SDS-PAGE.
Isolation of Nucleocapsids
In order to isolate the nuc~eocapsids, uncoating of viral particles (removal of
the viral envelope) was performed by mixing the viral particle suspension (intact
SFV or spikeless particles) with an equal volume of 4% (w/v) CHAPS
(3-[(3-Cholamidopropyl)-dimethylammonio]-propanesulfonate). Complete uncoating was obtained after stirring at 4~ for 30 min.
Low pH Exposure
Freshly prepared virus was used in all experiments. Freezing and thawing
was avoided in order to minimise damage to the viral envelope thereby causing
leakiness of the lipid bilayer. Low pH exposure of SFV and spikeless particles
was performed by adjusting the pH of the samples to 5.8 with 0.1 M HC1 in
100 mM NaC1. After 30 min incubation at 4~ the samples were neutralised with
an equivalent amount of 0.1 M NaOH in 100 mM NaC1. The particles were then
uncoated and applied to the sucrose gradients for sedimentation and electron
microscopy analysis of the nucleocapsids. In the control experiments, NaCI was
added to the virions at neutral pH corresponding to the amount of HC1 and
NaOH used above for acidification and neutralisation.
246
Schlegel, Omar, Jentsch, Morell and Kempf
Purified nucleocapsids isolated from spikeless particles after uncoating were
exposed to low pH or left at neutral pH and analysed in the same manner as
above.
Sedimentation Analysis of Nucleocapsids
Sedimentation analysis of nucleocapsids was performed by centrifugation at
195,000g for 2 hours on 15 to 30% (w/v) linear sucrose gradients in TBS
(150 mM NaC1, 10 mM Tris, pH 7.4). Fractions of 200 ~1 were collected from the
bottom of the tubes. The radioactivity in the fractions was measured by liquid
scintillation counting and the sucrose concentration determined by refractometry.
The S-values of the nucleocapsids were then calculated from these measurements.
Electron Microscopy
The nucleocapsids obtained after detergent uncoating were concentrated by
centrifugation through 12% (w/w) sucrose in capsid buffer (100 mM NaC1, 50 mM
Tris, pH 7.4) in an airfuge at 135,000g for 20 minutes. The pellets were
resuspended in capsid buffer, transferred to carbon coated copper grids and
stained with 2% potassium phosphotungstate pH 7.2 and analysed by electron
microscopy (Soederlund et aI., 1972).
RESULTS
The Effect of Low pH Exposure of SFV on the Nucleocapsids
We have previously shown that SFV induced FFWI upon low pH exposure is
accompanied by a proton influx into the syncytium forming cells (Kempf et al.,
1987; Kempf et al., 1988a). By lowering the intracellular pH while keeping the
extracellular pH neutral and constant, we found that the low intracellular pH per
se can induce cell-cell fusion. This indicated that proton influx into the cells is a
crucial step in triggering the membrane fusion mechanisms (Kempf et al., 1990).
However, in this FFWI model it was not clear whether the viral or the cellular
proteins were responsible for the proton influx. One method of determining
whether the viral proteins alone can translocate protons across the lipid bilayer is
to utilise untreated intact virions. It has been shown that isolated nucleocapsids
undergo a contraction or shrinkage upon low pH exposure resulting in a decrease
of the diameter and a higher sedimentation rate (Soederlund et al., 1972). Thus,
if the envelope proteins of SFV are capable of promoting proton translocation
across the envelope lipid bilayer into the virus interior, then it should be possible
to detect both a change in the nucleocapsid diameter and in their sedimentation
behavior. We have therefore exposed SFV to low pH and compared the
sedimentation constant and the size of nucleocapsids with those from untreated
SFV. As shown in Fig. 1A, the nucleocapsids from low pH treated virions have
an S-value of 162 as compared to 153 S of the nucleocapsids of untreated virions.
SFV Envelope Proteins
247
These values are significantly different as determined by the Students t-test
(t = 3.19; t0.95 = 1.94).
Electron microscopic examination revealed that the nucleocapsids were
ellipsoid in shape (Fig. 1C). A similar result was reported earlier by Soederlund et
al. (1972). Therefore, we measured two diameters perpendicular to one another,
one at the major and the other at the minor axes, for every particle. The mean
values and standard deviations were determined for both diameters and are
summarised in Fig. lB. The centres of the circles represent the mean values, the
radii correspond to the standard deviations. Using the geometric mean of the
major and the minor axes as the nucleocapsid diameter, we found 51.74 + / 4.80 nm for the nucleocapsids of untreated SFV and 40.03 + / - 5 . 2 1 nm for the
nucleocapsids of SFV which were exposed to low pH. This difference is highly
significant, as determined by the Student t-test: t = 17.3, t0.95 = 1.65).
These results clearly show that exposure of SFV virions to low pH causes
contraction of the nucleocapsids similar to that found for isolated nucleocapsids.
This can only be explained by a proton influx into the virion. The proton influx
could be either mediated by the viral spike proteins or be due to a leakiness of
the lipid envelope. Leakiness or structural changes in the lipid envelope have
been observed after freezing and thawing of virions but not with freshly prepared
virions. However, to entirely eliminate this possibility we have used spikeless
particles obtained by proteolytic digestion. These particles were tested for their
ability to translocate protons.
Low pH Effect on Isolated Nucleocapsid of Spikeless Particles
Proteolytic digestion of Sindbis virus, a close relative of SFV, produces
spikeless enveloped particles containing short membrane-associated peptides of
each of the two virion transmembrane glycoproteins E1 and E2 (Rice et al.,
1982). Removal of the spike proteins of enveloped viruses leads to the loss of
certain functions such as hemagglutination (Kennedy, 1974; Ravid and Goldblum, 1973/74) or ion transfer across artificial bilayer membranes (Young et al.,
1983). SFV was therefore digested with bromelain and the resulting particles were
isolated and analysed by SDS-PAGE. As shown in Fig. 2A proteolytic digestion
completely removes the ectodomains of the envelope proteins E1 and E2. The
capsid protein remains intact showing that the viral membrane is impermeable to
the protease.
Before utilising these spikeless particles as a control for proton translocation,
it was first necessary to demonstrate that these nucleocapsids retained their
contractability upon low pH treatment.
For this purpose, the spikeless SFV was uncoated with CHAPS as described
in the methods. This material was split into two aliquots. One aliquot was left at
neutral pH and the other was exposed to low pH. The sedimentation analysis is
depicted in Fig. 2B. Low pH treated nucleocapsids had an S value of
146.1 + / - 1.0 as compared to 142.4 + / - 1.2 S for the untreated controls clearly
showing that the nucleocapsids still contract upon low pH exposure (students
t-test: t = 3 . 3 4 ; t095=2.92). Furthermore the S-values of the nucleocapsids
248
Schlegel, Omar, Jentsch, Morell and Kempf
162 S ~
153 s
50
C
X\
-M
.r--!
r-
GJ
-i-i
X
4J
r
..Q
0
r-
CO
E
'\
E
o
40
30
I
I
I
I
40
80
50
ma j or ax is
top
]ottom
neutral
(rim)
fractions
--
--
~
neutral
~
acid
acid
(a)
(b)
II
Z
(c)
Fig. 1. Low pH exposure of SFV and its effect on the nucleocapsids: The sedimentation analysis on
15-30% sucrose gradients of the nucleocapsids of SFV is shown in A. The SFV particles were either
exposed to low pH or kept at neutral pH (controls). The nucleocapsids of low pH exposed SFV
clearly show a higher sedimentation rate (162S) as compared to the controls (153 S). The peak
fractions were collected, concentrated and examined by electron microscopy (C). The nucleocapsides
of the acid treated particles were smaller (CI) in size as compared to the controls (CII) (bar = 48 nm).
The diameters of the particles were measured (B). The centre of the circles represent the mean values
of the diameters and the radii correspond to the standard deviation of the diameters.
SFV Envelope Proteins
249
146 S ~
~J
142 S
\
\\
L
-H
E
el_
0
\
bottom
top
fractions
-- -- neutral
--
acid
Fig. 2. Low pH effect on isolated nucleocapsid of
spikeless particles: Spikeless SFV particles were
prepared by bromelain digestion. The absence of the
viral envelope protein bands E1 and E2 in SDSPAGE (A) show that the ectodomains of these
proteins are completely removed by the proteolytic
enzyme (right lane) as compared to the control (left
lane). The nucleocapsids of these spikeless particles
were isolated after uncoating, then exposed to low
pH or kept at neutral pH (control) and analysed on
sucrose gradients (B). The nucleocapsids of spikeless
particles retain the ability to contract upon low pH
treament.
250
Schlegel, Omar, Jentsch, Morell and Kempf
4J
C
C.
4-3
50
JO
(OO
03
X
fO
E
00
//
\k
o~
E
30
top
bottom
4~I
fractLons
neutral--
-- -
|
acid
(a)
I
40
I
I
50
I
60
major axis (nm)
neutral
Q
acid
(N
I
II
(c)
Fig. 3. Low pH exposure of spikeless particles and its effect on the nucleocapsids: The spikeless
particles were either exposed to low pH or kept at neutral pH and the nucleocapsids analysed as in
Fig. 1. The sedimentation and size of the nucleocapsids were identical showing no effect of the low
pH. A: sedimentation analysis; B: analysis of capsid diameters; C: electron microscopy, I = acid
treated, II = control.
SFV Envelope Proteins
251
obtained from spikeless virions were found to be lower than the S-values of
nucleocapsids recovered from untreated SFV. The reasons for this difference are
not clear. However, the shift in the S-values may have been caused by the
digestion conditions which seem to affect the general sedimentation behaviour of
the nucleocapsids. Nevertheless, it was important to find that the ability of the
nucleocapsids to contract upon the low pH exposure was not lost after bromelain
digestion. This showed that these nucleocapsids could still be used as a sensitive
indicator to detect proton translocation into the interior of spikeless virions.
Low pH Exposure of Spikeless SFV Particles
An aliquot of spikeless SFV was left untreated and another aliquot exposed
to low pH. The spikeless particles were uncoated with detergent and the
nucleocapsids subjected to sedimentation and electron microscopic analysis. As
depicted in Fig. 3A, the S-values of both preparations were identical (t = 0.32;
t0.95 = 2.92), namely 142.4 + / - 1.2 S and 142.7 + / - 0.4 S for untreated and low
pH treated particles, respectively. The electron microscopic pictures of these
nucleocapsids are shown in Fig. 3C and the electron microscopy data of these
nucleocapsids are summarised in Fig. 3B. The calculated geometric means of the
diameters were 43.72 + / - 5 . 4 9 nm for untreated and 44.75 + / - 5 . 7 4 nm for low
pH treated samples. These values are not statistically different, as determined by
Student t-test: t = 1.3 (to.95 ----- 1.65). Thus, low pH exposure has no effect on the
nucleocapsid of spikeless particles; specifically, the spikeless particles are incapable of translocating protons into their interior to cause contraction of the
nucleocapsids. In addition, after exposing both particles to low pH the nuclocapsids obtained from spikeless particles were larger in size as compared to those of
intact SFV (t = 7.0, t0.95 = 1.65; compare Fig. 3B and 1B).
Furthermore the S-values of nucleocapids recovered from spikeless particles
treated at low pH (Fig. 3A) were also larger than nuclocapsids of spikeless
particles which were exposed to low pH only after uncoating (controls, Fig. 2B;
142.7 + / - 0.43 compared to 146.08 + / - 1.00; t = 4.38; t0.95= 2.92). These results
clearly demonstrate that the envelope of bromelain digested SFV particles, in
contrast to that of intact virions, is impermeable for protons. From this we
conclude that it is not the leakiness of the envelope but rather the ectodomain of
the spike proteins which is responsible for the proton translocation across the
membrane of intact SFV.
DISCUSSION
We have used the phenomenon of low pH induced contraction of the SFV
nucleocapsid to show that exposure of isolated SFV virions to acidic pH leads to
proton translocation across the viral membrane. Proteolytic removal of the
envelope proteins abolishes the capacity of proton transfer indicating that the
process is mediated by the spike protein ectodomains. By analogy to the proton
influx noticed in SFV, it appears that the viral spike protein ectodomains at the
252
Schlegel, Omar, Jentsch, Morell and Kempf
plasma membrane are also responsible for the proton influx observed in infected
cells during cell-cell FFWI upon low extracellular pH exposure (Kempf et al.,
1987; Kempf et al., 1988a). Whether SFV spike proteins are involved in
Na+/K+-fluxes across the plasma membrane detected in the syncytium forming
cells (Kempf et al., 1988b) and similar to that observed with spike proteins in
artificial bilayers (Young et al., 1983) remains to be elucidated. In several reports
it was shown that the envelope proteins of viruses fusing in endosomes undergo
low pH induced irreversible conformational changes (for reviews see Hoekstra,
1990; White, 1990). The E1 spike protein of acid treated SFV virions show an
altered behaviour towards trypsin digestion (Kielian and Helenius, 1985) and an
increased hydrophobicity (Omar and Koblet, 1988). Moreover a low pH induced
irreversible conformational change of a plasma membrane protein (Koblet et al.,
1985), probably of the E1 viral protein, precedes the fusion of SFV infected cells
(Omar and Koblet, 1989). It is commonly assumed that these low pH induced
alterations in the conformation of the envelope protein leads to a specific
interaction of the viral spike protein E1 with the target membrane in order to
induce cell-cell or virus-cell fusion. Recently, we have proposed a new model for
the low pH reaction, based on the observation that SFV-induced FFWI can also
be triggered by a transient acidification of the cytoplasm of infected cells at an
extracellular, neutral pH. Namely, the E1 envelope protein may also react with
the lipid bilayer membrane in which it is embedded. This results in a folding back
of a part of the protein to form a proton channel (Kempf et al., 1990). Briefly, we
postulated that acidification of the extracellular environment leads to a conformational change of the spike in such a way that a part of the protein----close to the
anchor sequence--folds back into the anchoring membrane to form a proton
channel, resulting in a proton influx. A transient drop of the intracellular pH
might activate additional factors which are mandatory for SFV induced FFWI.
An acid load leading to fusion would short cut this event. The postulated
interaction with the opposing membrane would occur via a different part of the
peptide close to the N-terminus ("fusion peptide", Garoff et al., 1982) of the
protein. Our current data, that SFV spike protein ectodomains are required for
proton translocation across the viral envelope, substantiate this hypothesis. The
proton translocation may not only be important for the acidification of the
cytoplasm in FFWI but also for the penetration of the virus, in the endosomal
membrane-virus envelope fusion process, and in the subsequent uncoating of the
viral genome, as proposed recently for rubella virus, another member of the
togaviridae family (Mauracher et al., 1991). In addition, several theoretical
considerations lend further support to the folding back hypothesis: i) As shown in
Fig. 4A, the E1 protein contains a hydrophobic stretch of amino acids (Val
352-Cys 376) localised close to the COOH-terminal, membrane anchoring
domain (Garoff et al., 1980). Analysis of putative integral membrane protein
helices (Rao and Argos, 1986) of the E1 protein reveals that this hydrophobic
stretch and additional sequences nearby (Asp 385-Asp 401 and Ala 293-Thr 319),
are potentially membrane spanning. Thus, the peptide Val 352-Cys 376 and one
of the other mentioned peptides could fold back into the membrane in order to
render a stable insertion into the virion membrane, ii) As illustrated in Fig. 4B,
253
SFV Envelope Proteins
C-TERMTNAL SEQUENCE OF El
...... Glu Ala Thr Ala
Ala GIy_LysF\~I Thr Leu
rAla Ser Ala Set Pro Set
ILeu Cy_s Set Ala Ar~ A}a
Set Cys Glu Pro Pro Lys
Pro Tyr Ala A]a Ser His
Phe Pro Asp Met Ser G}y
Trp Val Gln LysIZSe set
[Aia Phe Ala Ile Gly A}a
IVal Val Val Thr Cys I~e
Lys Val Lys
His Phe Set
Phe Val Val
Thr Cys#Ser
Asp His lle
Ser Ash Va}
Thr Ala Leu
Gly GIy Leu
Ile Leu Val
Gly~-~
Thr
Thr I
Serj
Ala
Val
Val
Set
GIy 1
Leuj
Arg
(a)
His
355
-
Ala
378
of
ES
6
(b)
Fig. 4. Putative proton channel sequence of the SFV E1 protein:
The E1 protein was analysed for membrane associated helices
(Rao and Argos, 1986). The sequence Va1352-Cys376 (dashed
box) was found to fulfil this criteria similar to the membrane
anchor Ile313-Leu436 (solid box) (Fig. 4A). Helical wheel analysm
(Schiffer and Edmundson, 1967) further demonstrates that it has
amphipathic properties (Fig. 4B).
the helical wheel analysis (Schiffer and Edmundson, 1967) of the sequence His
355-Ala 372 reveals that five out of six Set residues are in close vicinity to one
another. Considering these amphiphilic properties of this peptide segment and
the trimeric arrangement of the spike proteins (Vogel et at., 1986), we postulate
that three such helices may form a proton channel, iii) Experiments with
synthetic, a~-helical, membrane spanning peptides containing Ser residues have
shown that such peptides may also act as ion channels (DeGrado and Lear, 1990;
Lear et aL, 1988). As an alternative hypothesis one could assume that the
sequence Val 352-Cys 376 stably tucks into the virion membrane in form of a
254
Schlegel, Omar, Jentsch, Morell and Kempf
f i - h a i r p i n , similar as p r o p o s e d f o r t h e p o r e f o r m i n g r e g i o n of p o t a s s i u m c h a n n e l s
(Yoll a n d S c h w a r z , 1991).
It was r e c e n t l y p o s t u l a t e d t h a t in influenza virus t h e M 2 - p r o t e i n , a m i n o r
viral m e m b r a n e p r o t e i n , f u n c t i o n s as a c h a n n e l ( S u g r u e a n d H a y , 1991). I n
a n a l o g y t o this h y p o t h e s i s , we also t o o k into c o n s i d e r a t i o n t h a t a m i n o r e n v e l o p e
p r o t e i n o f S F V , n a m e l y t h e 6 K p r o t e i n , c o u l d b e r e s p o n s i b l e for t h e p h e n o m e n a
d e s c r i b e d in this p a p e r . It was s h o w n t h a t t h e 6 K p r o t e i n a c t u a l l y r e p r e s e n t s a
minor component of the SFV envelope (Gaedigk-Nitschko and Schlesinger,
1990). T h u s , t h e p o s s i b i l i t y t h a t t h e 6 K p r o t e i n c o u l d r e p r e s e n t an ion c h a n n e l
which is r e g u l a t e d b y t h e e c t o d o m a i n of t h e viral s p i k e p r o t e i n s c o u l d r e p r e s e n t
an a l t e r n a t i v e h y p o t h e s i s to t h e o n e p o s t u l a t e d a b o v e .
ACKNOWLEDGEMENTS
This w o r k was s u p p o r t e d in p a r t by t h e Swiss N a t i o n a l S c i e n c e F o u n d a t i o n
( G r a n t N o . 31-25732.88 to C . K . ) .
REFERENCES
DeGrado, W. F. and Lear, J. D. (1990) Conformationally constrained 0~-helical peptide models for
protein ion channels. Biopolymers 29: 205-213.
Garoff, H., Frischauf, A.-M., Simons, K., Lehrach, H. and Delius, H. (1980) Nucleotide sequence of
cDNA coding for Semliki Forest virus membrane glycoproteins. Nature 288: 236-241.
Gaedigk-Nitschko, K. and Schlesinger, M. J. (1990) The Sindbis virus 6K protein can be detected in
virions and is acylated with fatty acids. Virology 175:274-281.
Garoff, H., Kondor-Koch, C. and Riedel, H. (1982) Structure and assembly of alpha viruses. Curr.
Top. Microbiol. lmmunol. 99:1-50.
Hoekstra, D. and Kok, J. W. (1989) Entry mechanisms of enveloped viruses. Implications for fusion
of intracellular membranes. Biosci. Rep. 9: 273-305.
Hoekstra, D. (1990) Membrane function of enveloped viruses: especially a matter of proteins. J.
Bioenerg. Biomernbrane 22:121-155.
Igarashi, A. (1978) Isolation of a Singh's Aedes albopictus cell clone sensitive to Dengue and
Chikungunya viruses. J. gen. Virol. 40:531-544.
Kempf, C., Michel, M. R., Kohler, U. and Koblet, H. (1987) Can viral envelope proteins act as or
induce proton channels? Biosci. Rep. 7: 761-769.
Kempf, C., Michel, M. R., Kohler, U. and Koblet, H. (1988a). Exposure of Semliki Forest
virus-infected baby hamster kidney cells to low pH leads to a proton influx and a rapid depletion
of intracellular ATP which in turn prevents cell-cell fusion. Arch. Virol. 99:111-115.
Kempf, C., Michel, M. R., Kohler, U., Koblet, H. and Oetliker, H. (1988b) Dynamic changes in
plasma membrane properties of Semliki Forest virus infected cells related to cell fusion. Biosci.
Rep. 8:241-254.
Kempf, C., Michel, M. R., Omar, A., Jentsch, P. and Morell, A. (1990) Semliki Forest virus induced
cell-cell fusion at neutral extracellular pH. Biosci. Rep. 10:363-374.
Kennedy, S. I. T. (1974) The effects of enzymes on structural and biological properties of Semliki
Forest virus. J. gen. Virot. 23:129-143.
Kielian, M. and Helenius, A. (1985) pH induced alterations in the fusogenic spike protein of Semliki
Forest virus. J. Cell Biol. 101:2284-2291.
Koblet, H., Kempf, C., Kohler, U. and Omar, A. (1985) Conformational changes at pH 6 on the cell
surface of Semliki Forest virus-infected Aedes albopictus cells. Virology 143:334-336.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227:480-485.
SFV Envelope Proteins
255
Lear, J. D., Wasserman, Z. R. and DeGrado, W. F. (1988) Synthetic amphiphilic peptide models for
protein ion channels. Science 240:1177-1181.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein determination with
the folin reagent. J. Biol. Chem. 193:265-275,
Mann, E., Edwards, J, and Brown, D. T. (1983) Polykaryocyte formation mediated by Sindbis virus
glycoproteins. J. Virol. 45: 1083-1089.
Mauracher, C. A., Gillam, S., Shukin, R. and Tingle, A. J. (1991) pH-dependent solubility shift of
rubella virus capsid protein. Virology 181:773-777.
Omar, A. and Koblet, H. (1989) The use of sulfite to study the mechanism of membrane fusion
induced by E1 of Semliki Forest virus. Virology 168:177-179.
Omar, A. and Koblet, H. (1988) Semliki Forest virus particles containing only the E1 envelope
glycoprotein are infectious and can induce cell-cell fusion. Virology 166:17-23.
Ran, M. J. K. and Argos, P. (1986) A conformational preference parameter to predict helices in
integral membrane proteins. Biochim. Biophys. Acta 869:197-214.
Ravid, Z. and Goldblum, N. (1973/74) Analysis of antigenic activites of enzymatically digested
Sindbis virus envelopes. Intervirology 2:152-159.
Rice, C. M., Bell, J. R., Hunkapiller, M. W., Strauss, E. G. and Strauss, J. H. (1982) Isolation and
characterization of the hydrophobic COOH-terminal domains of the Sindbis virion glycoproteins.
J. Mol. Biol. 154:355-378.
Schiffer, M. and Edmundson, A. B. (1967) Use of helical wheels to represent the structures of
proteins and to identify segments with helical potential. Biophys. J. 7:121-135.
Soederlund, H., Kaeaeriaeinen, L., yon Bonsdorff, C. H. and Weckstroem, P. (1972) Properties of
Semliki Forest virus nucleocapsid. Virology 47:753-760.
Spear, P. G. (1987) Virus-induced cell fusion. In: "Cell Fusion" (Sowers, A. E., ed) Plenum
Publishing Corp., New York, USA, pp. 3-32.
Stegmann, T., Doms, R. W. and Helenius, A. (1989) Protein-mediated membrane fusion. Annu.
Rev. Biophys. Biophys. Chem. 18: 187-211.
Sugrue, R. J. and Hay, A. J. (1991) Structural characteristics of the M2 protein of influenza A
viruses: Evidence that it forms a tetrameric channel. Virology 180:617-624.
Vogel, H. H., Provencher, S. W., yon Bonsdorff, C. H., Adrian, M. and Dubochet, J. (1986)
Envelope structure of Semliki Forest virus reconstructed from cryoelectron micrographs. Nature
320: 533-535.
White, J., Kielian, M. and Helenius~ A. (1983) Membrane fusion proteins of enveloped animal
viruses. Quart Rev. Biophys. 16:151-195.
White, J. M. and Blobel, C. P. (1989) Cell-to-cell fusion. Curt. opin. cell biol. 1:934-939.
White, J. M. (1990) Viral and cellular membrane fusion proteins. Annu. Rev. Physiol. 52:675-697.
Yool, A. J. and Schwarz, T. L. (1991) Alteration of ionic selectivity of a K + channel by mutation of
the H5 region. Nature 349:700-704.
Young, J. D. E., Young, G. P. H., Cohn, Z. A. and Lenard, J. (1983) Interaction of enveloped
viruses with planar bilayer membranes: observations on Sendai, Influenza, Vesicular Stomatitis
and Semliki Forest viruses. Virology 128: 186-194.