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Journal of Virology, December 1999, p. 10296-10302, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Induction of Apoptosis by Sindbis Virus Occurs at
Cell Entry and Does Not Require Virus Replication
Jia-Tsrong
Jan
and
Diane E.
Griffin*
Department of Molecular Microbiology and
Immunology, Johns Hopkins University School of Hygiene and Public
Health, Baltimore, Maryland 21205
Received 17 February 1999/Accepted 28 July 1999
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ABSTRACT |
Sindbis virus (SV) is an alphavirus that causes encephalitis in
mice and can lead to the apoptotic death of infected cells. To
determine the step in virus replication during which apoptosis is
triggered, we used UV-inactivated SV, chemicals that block virus fusion
or protein synthesis, and cells that do and do not express heparan
sulfate, the initial binding molecule for SV infection of many cells.
In initial experiments, UV-inactivated neuroadapted SV (NSV) induced
apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate
in the presence of cycloheximide. When fusion of prebound
UV-inactivated NSV was rapidly induced at the plasma membrane by
exposure to acidic pH, apoptosis was induced in CHO cells with or
without heparan sulfate in the presence or absence of cycloheximide in
a virus dose-dependent manner. In N18 neuroblastoma cells, the relative
virulence of the virus strain was an important determinant of apoptosis
induced by UV-inactivated SV. Treatment of N18 cells with monensin to
prevent endosomal acidification an hour before, but not 2 h after,
exposure to live NSV blocked the induction of cell death, as did
treatment with NH4Cl or bafilomycin A1. These studies
indicate that SV can induce apoptosis at the time of fusion with the
cell membrane and that virus replication is not required.
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INTRODUCTION |
The interactions between viruses and
host cells have several possible outcomes: transformation, persistent
productive infection, latency, or cell death. Sindbis virus (SV), a
mosquito-borne alphavirus, is an enveloped, plus-strand RNA virus that
causes infection of neurons and age-dependent encephalomyelitis in mice
(20, 24). SV causes persistent infections in most mosquito
cell lines, but it induces apoptosis in most vertebrate cell lines, a
property that may be relevant to its maintenance in natural
transmission cycles (15). Viral and host cell factors that
regulate the process of apoptosis determine the outcome of infection
for the cell and ultimately for the host. Studies of SV infection of
the central nervous system in vivo and of primary dorsal root ganglion
neurons in vitro have shown that neurons become increasingly resistant to SV-induced apoptosis as they mature, which correlates with the age
dependence of fatal infection (27, 28). The mechanism of
this increased resistance is not known, but a number of studies have
shown that apoptosis induced by SV can be blocked by caspase inhibitors
and by members of the Bcl-2 family of antiapoptotic proteins (8,
26, 27, 36), so a variety of cellular inhibitors of apoptosis can
prevent or slow SV-induced apoptosis. Neurovirulent strains of SV can
overcome some cellular inhibitors of apoptosis and cause cell death
(49), suggesting that a balance of cellular factors
regulating cell death and viral factors regulating virulence determines
the outcome of infection. However, how this apoptotic pathway is
triggered during SV infection is not understood.
For some viruses, specific viral proteins (e.g., adenovirus E1A
[41], parvovirus NS [7, 25], reovirus
sigma 1 [48], retrovirus Env and Vpr [4,
43], or chicken anemia virus VP3 [38]) have
been associated with the induction of apoptosis. Some of these proteins
act at the cell membrane, presumably through interaction with a
cellular receptor for the virus, while others act intracellularly
(4, 33, 48). SV, like other alphaviruses, encodes four
nonstructural proteins (nsPs) and three major structural proteins: an
internal capsid protein and two surface glycoproteins, E1
and E2 (44). E1 and E2 exist on the surface of the virion as
a heterodimer that is trimerized to form spikes which attach the virus
to cells to initiate infection. Receptor-mediated endocytosis brings
the virion into the endosome, where exposure to acidic pH induces a
conformational change in the E1-E2 heterodimer, leading to fusion of
the viral envelope with the endosomal membrane. Delivery of the capped
and polyadenylated genomic RNA to the cytoplasm is followed by
translation of the nsPs and formation of membrane-bound replication
complexes. At approximately the time that structural protein synthesis
is initiated, the synthesis of nonstructural viral and cellular
proteins ceases. Virions are assembled at, and bud from, the plasma
membrane (44). Although a role for nsPs in the establishment
of persistent SV infection has been suggested (11),
infection of cells with SV replicons has shown that the structural
proteins dramatically accelerate cell death (14). The most
recent data gathered from transient-transfection assays suggest that
the induction of apoptosis may involve the transmembrane regions of the
surface glycoproteins (23).
To begin to determine the step in the SV life cycle that triggers the
signal for cell death, we used UV-inactivated SV, chemicals that block
virus fusion or protein synthesis, and cell lines that do and do not
express heparan sulfate (the initial virus binding molecule) for
studies of SV-induced apoptosis. We found that UV-inactivated SV is
able to induce apoptosis. This was most easily demonstrated when the
cells lacked heparan sulfate and when virus-cell membrane fusion was
rapidly and synchronously induced by exposure of cells with bound
virions to acidic pH. Apoptosis was blocked by treating cells exposed
to infectious virus with inhibitors of endosomal acidification. It is
proposed that the process of apoptosis is initiated by an interaction
between the viral glycoprotein(s) and a cell surface
receptor and that it is specifically triggered during acid-induced
virus fusion with the cell membrane.
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MATERIALS AND METHODS |
Cell lines.
Chinese hamster ovary (CHO-22) cells
(18) and mutant CHO-18.4m cells lacking cell surface
sulfated proteoglycans (21) were grown in Dulbecco's
modified Eagle's medium (DMEM) (Gibco, Grand Island, N.Y.)
supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1.5×
nonessential amino acids (Gibco). Mouse neuroblastoma (N18) cells
(2) and baby hamster kidney (BHK-21) cells (American Type
Culture Collection, Manassas, Va.) were grown in DMEM supplemented with
10% FBS.
Virus strains, purification, and inactivation.
AR339 was
originally isolated from mosquitoes (45) and obtained from
the American Type Culture Collection. A neuroadapted strain of SV (NSV)
was derived by serial passages of AR339 in mouse brain (16).
The heat-resistant small-plaque strain of SV (HRSP) was derived from
serial passages of the HR strain (derived from AR339) in chicken embryo
fibroblasts (6). Virus stocks were grown and assayed in
BHK-21 cells and contained 108 to 109 PFU/ml.
Radiolabeling and purification of virions.
BHK-21 cells were
infected at a multiplicity of infection (MOI) of 5, and 1 h later,
cells were fed with methionine- and cysteine-free MEM to which was
added 10 µCi of [35S]methionine-cysteine
(Tran35S-label; ICN) per ml. When a >90% cytopathic
effect was evident, supernatant fluid was harvested and clarified. For
purification, virus was precipitated in 10% (wt/vol) polyethylene
glycol 8000 in 0.5 M NaCl, resuspended in NET buffer (10 mM Tris, 3 mM
EDTA, 150 mM NaCl [pH 7.4]), and banded in a 15 to 40% potassium
tartrate gradient as previously described (42). Banded virus
was collected, dialyzed against 0.05 M Tris-HCl (pH 7.4), and stored in
aliquots at 
70°C.
Virus binding assay.
Cells were grown in 12-well plates and
washed twice in cold binding medium (RPMI 1640 without
NaHCO3, 0.2% bovine serum albumin, 20 mM HEPES [pH
7.4]). Radiolabeled virus (4,000 cpm per well in 0.4 ml of cold
binding medium) was added, and the plates were rocked gently at 4°C.
At various times after addition of virus, supernatant fluids were
collected from triplicate wells and cells were washed twice. The wash
fluids were added to the supernatant fluids and counted to determine
the amount of unbound virus. Cells were lysed in 1% sodium dodecyl
sulfate (SDS) and counted to determine the amount of bound virus
(46).
UV inactivation of SV.
SV was inactivated by exposing the
virus to a germicidal lamp (wavelength, 254 nm) at a distance of 5 cm
for 30 min at 4°C. Inactivation was confirmed by the inoculation of
BHK-21 cells before use and, in individual experiments, by monitoring
the exposed cells for synthesis of nsPs and the supernatant fluids from
these cells for the presence of infectious virus at 24 h.
Assessment of DNA degradation.
A total of 2 × 106 cells infected with untreated SV or UV-treated SV were
harvested after a 48- to 72-h exposure to the virus. Genomic DNA was
isolated with DNAzol (Gibco BRL), incubated with RNase (1 µg/ml) at
50°C for 1 h, and then loaded onto a 1.2% agarose gel
containing ethidium bromide. After electrophoresis, the gel was
photographed and examined for the presence of DNA laddering.
Drug treatments and assessment of viral protein synthesis.
Confluent cells were pretreated with NH4Cl (20 mM),
monensin (10 µM), bafilomycin A1 (10 µM), or cycloheximide
(CHX) (10 µg/ml; Sigma Chemical Co., St. Louis, Mo.) beginning 1 h before or 2 h after exposure to SV. At various times after
infection, 2 ml of labeling medium containing
[35S]methionine-cysteine (50 µCi/ml) was added to the
cells for 1 h. After removal of the labeling medium, cells were
washed twice with phosphate-buffered saline (PBS) and lysed with
radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-Cl [pH 7.5],
150 mM NaCl, 10 mM EDTA, 0.1% SDS, 1% Triton X-100, 1%
deoxycholate). Equal amounts of cell lysates were analyzed on an
SDS-15% polyacrylamide gel.
To monitor the synthesis of nsPs as evidence of infection, N18 cells
were infected with NSV diluted in DMEM at an MOI of 50 or were exposed
to UV-inactivated NSV at an MOI equivalent of 500 at 4°C for 2 h. Cells exposed to live virus were washed, fed with DMEM-2% FBS, and
shifted to 37°C. Cells exposed to UV-inactivated virus were treated
with DMEM (pH 5.0) at 37°C for 1 h, and then they were washed
and fed with DMEM-2% FBS. At 24 h, cells were washed with PBS,
fixed with acetone-methanol (1:1), and stained overnight at 4°C with
rabbit anti-nsP1 (diluted 1:500), followed by fluorescein
isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (1:2,000)
at 37°C for 2 h.
Western blot analysis.
Cells were washed twice with PBS and
lysed in RIPA buffer. The protein concentrations were determined by the
Bradford protein assay (Bio-Rad, Hercules, Calif.). Proteins (50 to 100 µg) were electrophoresed in an SDS-15% polyacrylamide gel, blotted
onto a Hybond-C extra membrane (Amersham, Arlington Heights, Ill.), and
reacted with antibody. Rabbit polyclonal antiserum to SV
(19) was used to detect the E1, E2, and capsid proteins.
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RESULTS |
Induction of apoptosis in CHO-18.4m cells by UV-inactivated
NSV.
To begin to determine the step in SV replication that induces
apoptosis, we investigated whether virus replication was required. CHO-18.4m cells, which lack heparan sulfate (21) and
therefore allow virus to interact directly with a postulated receptor,
and parental CHO-22 cells were used. Cells were treated with
UV-inactivated NSV at a BHK cell MOI equivalent of 500 in the presence
and absence of CHX to inhibit the synthesis of viral and cellular
proteins (Fig. 1). UV-inactivated NSV
induced the death of CHO-18.4m cells, but not that of CHO-22 cells, in
the presence of CHX. CHX alone did not induce significant cell death in
these cell lines within 3 days. The death of CHO-18.4m cells induced by
UV-inactivated NSV was due to apoptosis, as indicated by the presence
of DNA laddering (Fig. 2). These data
suggest that the induction of cell death did not require virus
replication or virus protein synthesis, was optimized by interaction of
the virus with a cell surface molecule other than heparan sulfate, and,
surprisingly, appeared to require the inhibition of cellular protein
synthesis by CHX.
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FIG. 1.
Effect of UV-inactivated NSV (UV-NSV) on CHO-22 and
CHO-18.4m cells in the presence or absence of CHX. Cells were or were
not pretreated with CHX (10 µg/ml) for 1 h, after which
UV-inactivated NSV (MOI equivalent = 500) was added with
additional CHX. Cell viabilities were assessed at 1, 2, and 3 days by
trypan blue exclusion. The bar indicates standard deviation.
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FIG. 2.
Integrity of DNA of CHO-18.4m cells after exposure to
NSV. Lane 1, cells infected with viable NSV (MOI equivalent = 5, harvested at 48 h); lane 2, uninfected cells; lanes 3, 4, 6, and
7, cells exposed to UV-inactivated NSV (MOI equivalent = 500) in
the presence of CHX (10 µg/ml) (lane 3, 48 h; lane 6, 72 h)
or without CHX (lane 4, 48 h; lane 7, 72 h); lanes 5 and 8, cells exposed to CHX alone (lanes 5, 48 h; lane 8, 72 h). DNA
was analyzed on a 1.2% agarose gel stained with ethidium bromide.
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To determine whether apoptosis could be enhanced by inducing
coordinated fusion of UV-inactivated virus at the plasma membrane,
CHO-18.4m and CHO-22 cells, with UV-inactivated NSV bound to the
surface at 4°C, were exposed to pH 6.0 and shifted to 37°C, and
their viabilities were assessed at 48 h (Fig.
3). With this experimental
paradigm, both
CHO-18.4m and CHO-22 cells pretreated with CHX
to inhibit viral and
cellular protein synthesis died, although
CHO-18.4m cells were killed
more efficiently. Neither CHX nor
acidic medium, alone or together,
induced cell death.
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FIG. 3.
Effect of exposure to pH 6.0 on viabilities of CHO-22
and CHO-18.4m cells after treatment with UV-inactivated NSV (UV-NSV) in
the presence or absence of CHX. Cells were pretreated with CHX (10 µg/ml) for 1 h, UV-inactivated NSV (MOI equivalent = 500)
was added, and then the plates were kept at 4°C to allow virus
binding for 2 h. Culture medium was then replaced with fresh
medium adjusted to pH 6.0, and cells were shifted to 37°C.
Viabilities were assessed 48 h later by trypan blue exclusion.
Bars indicate standard deviations.
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To determine the effects of altering the amount of bound UV-inactivated
NSV and of inducing fusion at the plasma membrane
more rapidly by
exposure to a lower pH, gradient-purified UV-inactivated
NSV at MOI
equivalents ranging from 0 to 1,000 was bound to CHO-22
and CHO-18.4m
cells at 4°C for 2 h. The pH was then shifted to
5.0 for an
hour, and the temperature was shifted to 37°C (Fig.
4A). Cell death induced by the
acid-induced fusion of UV-inactivated
NSV with the plasma membrane was
dose dependent, was equally efficient
in the two types of CHO cells,
and was not influenced by pretreatment
with CHX. An MOI equivalent of
at least 500 was required for the
death of all cells, but some cell
death also occurred at an MOI
equivalent of 250. Cell death was
characterized as apoptosis by
the presence of DNA laddering (Fig.
4B).
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FIG. 4.
UV-inactivated NSV induces apoptosis in the absence of
CHX in CHO-22 and CHO-18.4m cells exposed to pH 5.0. (A)
UV-inactivated, gradient-purified NSV at various MOI equivalents was
allowed to bind to cells for 2 h at 4°C. Culture medium was then
replaced with medium of pH 5.0 and incubated at 37°C for 1 h.
The acidic medium was then replaced with medium of neutral pH. Cell
viabilities were assessed 48 h later by trypan blue exclusion. (B)
DNA extracted from cells exposed only to pH 5.0 (lane 1, CHO-18.4m
cells; lane 2, CHO-22 cells) or to UV-inactivated NSV (MOI
equivalent = 500) and pH 5.0 (lane 3, CHO-18.4m cells; lane 4, CHO-22 cells) and analyzed on a 1.2% agarose gel stained with ethidium
bromide.
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UV-inactivated NSV also induces apoptosis of N18 cells.
Strains of SV vary in their neurovirulence for mice, and these
differences are often reflected in the efficiency with which these
strains bind to and replicate in N18 cells (10, 46, 47). To
determine whether UV-inactivated SV could induce apoptosis in N18 cells
and whether this varied with virus virulence, UV-inactivated NSV,
AR339, and HRSP (high, medium, and low virulence, respectively) were
prebound to N18 cells at 4°C and then exposed to various pHs at
37°C (Fig. 5). Both UV-inactivated NSV
and UV-inactivated AR339 induced cell death at pH 5.0; however, at pH
5.5, only UV-inactivated NSV induced cell death. UV-inactivated HRSP
did not induce cell death at any pH. The lack of virus replication was
confirmed in cells treated with UV-inactivated NSV by monitoring the
supernatant fluids for infectious virus and monitoring cells for
synthesis of nsPs (Fig. 6).
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FIG. 5.
Viabilities of N18 cells bound with different strains of
UV-inactivated SV and exposed to acidic pH. N18 cells were left
untreated (Mock) or were treated with UV-inactivated NSV (UV-NSV),
AR339 (UV-AR339), or HRSP (UV-HRSP) for 2 h at 4°C at an MOI
equivalent of 500. Virus inocula were then replaced with media of pH
5.0, 5.5, 6.0, and 7.4 and incubated at 37°C for 1 h. Subsequent
incubation was in medium of neutral pH. Cell viabilities were assessed
at 48 h by trypan blue exclusion. Bars indicate standard
deviations.
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FIG. 6.
Lack of virus replication in N18 cells exposed to
UV-inactivated NSV and pH 5. N18 cells exposed to live NSV (MOI = 50) (A) or UV-inactivated NSV (MOI equivalent = 500) and pH 5 (B)
were stained 24 h later to detect the synthesis of nsP1. N18 cells
infected with live virus, but not those infected with UV-inactivated
virus, synthesized nsP1.
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Inhibition of endosomal acidification blocks NSV-induced
apoptosis.
Monensin is an ionophore that blocks endosomal
acidification and the transport of glycoproteins through
the Golgi complex. Pretreatment of N18 cells with monensin protected
them against NSV-induced cell death (Fig.
7A). Monensin did not inhibit NSV binding
(Fig. 7B), but it did inhibit virus entry, as evidenced by the absence
of viral protein synthesis (Fig. 7C). Treatment of N18 cells with
monensin 2 h after infection did not protect them against cell
death (Fig. 7D), but it did inhibit virus production (Fig. 7E) due to
the inhibition of PE2-E1 transport through the Golgi complex, as
evidenced by the failure of PE2 to be processed into E2 (Fig. 7F).
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FIG. 7.
Treatment with monensin before, but not after, infection
protects N18 cells from NSV-induced cell death. (A) Effect of monensin
pretreatment on NSV-induced cell death. N18 cells were pretreated with
monensin for 1 h and then infected with NSV (MOI = 10). (B)
Effect of monensin on virus binding. N18 cells were exposed to
35S-labeled NSV at 4°C in the presence or absence of
monensin (10 µM). (C) Effect of monensin pretreatment on viral
protein synthesis. Lysates from 35S-labeled cells were
assessed by SDS-polyacrylamide gel electrophoresis and autoradiography.
(D) Effect of monensin posttreatment on NSV-induced cell death. N18
cells were infected with NSV (MOI = 10), and monensin (10 µM)
was added 2 h later. Cell viabilities at different time points
were assessed by trypan blue exclusion. (E) Plaque assays of
supernatant fluids described for panel D. (F) Lysate from NSV-infected
cells with or without monensin posttreatment analyzed by Western blot
analysis with rabbit anti-SV antiserum and showing inhibition of PE2
processing into E2. p.i., postinfection.
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The base NH
4Cl and the vacuolar H
+-ATPase
inhibitor bafilomycin A1 (
3) also prevent endosomal
acidification. Therefore,
these drugs also inhibit the acid-induced
conformational change
of E1-E2 and the subsequent fusion of the virus
envelope and cell
membrane. Pretreatment of N18 cells with either
NH
4Cl or bafilomycin
A1 protected them against infection
and NSV-induced death (Fig.
8).
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FIG. 8.
Pretreatment with NH4Cl or bafilomycin A1
protects N18 cells against NSV-induced cell death. N18 cells were
pretreated with NH4Cl (20 mM) (A) or bafilomycin A1 (10 µM) (B) for 1 h and then infected with NSV (MOI = 10). Cell
viabilities were assessed at the indicated times by trypan blue
exclusion. (C) Viral protein synthesis in NH4Cl- or
bafilomycin A1 (Baf A1)-treated N18 cells. At the indicated times,
cells were labeled with methionine- and cysteine-free medium to which
was added [35S]methionine-cysteine (10 µCi/ml) for
1 h. Cell lysates were run on an SDS-15% polyacrylamide gel and
analyzed by autoradiography. p.i., postinfection.
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DISCUSSION |
Virus-induced apoptotic cell death is an important determinant of
the outcome of infection. However, the viral and cellular proteins that
participate in the induction and execution of the apoptotic program are
just beginning to be identified, and for many virus-cell interactions,
multiple factors may contribute to determining the outcome. For many
types of viruses, replication in the host cell is necessary for
induction of apoptosis, while for others, replication is not necessary.
In these studies of the alphavirus SV, we showed that characteristics
of the virus strain and type of cell studied were important
determinants of how easily apoptosis was induced in the absence of
virus replication. The induction of programmed cell death was most
efficient when a virulent strain of virus was able to interact
optimally with a cellular receptor other than heparan sulfate. The data
support the hypothesis that apoptosis can be initiated during
acid-induced fusion of the viral glycoproteins with the
cell membrane.
Induction of cell death in the absence of viral replication has been
reported for primary cells or cell lines exposed to type 3 reovirus
(48), avian leukosis virus (4), bovine
herpesvirus 1 (17), and vaccinia virus (40). The
mechanism(s) by which any of these viruses induces apoptosis has not
been definitively determined. For reovirus and vaccinia virus, it is
known to be related to the sigma-1 attachment protein (48)
and to a postattachment entry step (40), respectively. The
Env protein of avian leukosis virus interacts with a member of the
tumor necrosis factor (TNF) receptor family (4). For SV to
enter a cell, a continuous and uninterrupted interaction with cell
membrane molecules, facilitated by a series of conformational changes
in the viral envelope proteins and cell membrane molecules, is
essential. SV binds cells through its surface
glycoproteins. The E2 and E1 proteins of SV are primarily responsible for binding and fusion, respectively. Conformational changes of viral envelope proteins occur with binding to the cell surface and may be required for subsequent internalization
(12). With CHO-18.4m cells, cell death due to apoptosis was
easily induced by UV-inactivated NSV, indicating that virus replication
was not essential. We hypothesize that CHO-18.4m cells were more
susceptible because they lack the heparan sulfate used for initial
attachment, allowing more direct access to a cell surface molecule
specifically involved in virus entry and induction of apoptosis.
Interactions between incoming virus and cell membrane molecules also
occur in the endosome. After virus is internalized into the endosome,
the pH in the endosome decreases through the action of vacuolar
H+-ATPase. The acidic environment of the endosome induces
further conformational changes (50). Cholesterol in the
endosomal membrane is involved in low-pH-dependent binding of
alphaviruses to the lipid membrane, and sphingolipids are essential for
low-pH-induced fusion (5, 37). Therefore, interactions
between SV and the cell membrane are changed as the entry process
progresses from binding to fusion. To determine whether SV could
trigger cell death under the acidic conditions of the endosome, we
treated cells bound with UV-inactivated virus with media of low pH. The results imply that the acid-induced interaction between virus and cell
membrane which triggers fusion and delivery of the nucleocapsid into
the cytoplasm is capable of inducing cell death, and they are
consistent with the observations that the transmembrane portion of the
E1 glycoprotein is likely to be essential for fusion
(9) and that the transmembrane portion of either the E1 or
E2 glycoprotein can induce apoptosis (23).
Monensin blocks both virus entry and the transport of the PE2-E1
proteins of Semliki Forest virus (34, 39), an alphavirus related to SV, through the Golgi complex to the trans-Golgi network. Therefore, it is a useful chemical to determine whether SV triggers cell death at the time between fusion and viral protein transport or at
the time of virus assembly and budding. Pretreatment with monensin to
block virus fusion is protective; however, treatment with monensin
later to block viral protein transport is not. Therefore, the death
signal is triggered sometime between initiation of fusion and PE2-E1
protein transport. Protection by pretreatment with NH4Cl or
bafilomycin A1, which also block decreases in endosomal pH, further
supports the conclusion that fusion or activities accompanying or
following fusion are important for the induction of apoptosis.
The role of CHX in SV-induced apoptosis is unclear and potentially
confusing. CHX was originally included to determine whether viral
protein synthesis was required for initiating apoptosis, but
surprisingly, CHX itself appeared to be required, as has been shown in
a number of experimental paradigms for inducing apoptosis (35). CHX is often required for apoptosis induced by TNF
(32). In TNF-sensitive cells, neither TNF nor CHX alone
induces cell death, but when they are combined, apoptosis occurs. CHX
treatment may decrease the amount of some short-lived protein that
otherwise protects the cell from apoptosis. In other paradigms of
apoptosis, CHX protects against apoptosis by stimulating cytoprotective
pathways (35). SV infection inhibits host protein synthesis
within a few hours after infection, so CHX would mimic this aspect of
infection. In cells with bound UV-inactivated NSV, CHX-treated cells
had consistently higher, rather than lower, viabilities than those of
untreated cells when they were exposed to pH 5.0, suggesting that the
cells were protected. It is likely that these apparently contradictory
observations are linked to the strength of the apoptotic signal being
delivered in different experimental situations. If virus fusion occurs
over a relatively extended period of time (through the endosomal
pathway) or if fusion is inefficient (higher pH), then decreasing the
availability of short-lived antiapoptotic cellular factors will enhance
the apoptotic process. However, if the apoptotic signal is delivered in
a coordinated fashion, then such antiapoptotic cellular factors will be
insufficient to prevent initiation of the apoptotic process. This
balance between cellular and viral factors also likely explains why
relatively large amounts of inactivated virus, compared to those of
live virus, are required to induce apoptosis. UV irradiation has a deleterious effect on viral proteins, as well as viral RNA, resulting in less efficient binding and probably less efficient fusion than that
which occurs with virus that has not been UV inactivated.
Although these experiments demonstrate that SV can induce apoptosis at
the time of entry without a requirement for virus replication, it is
likely that other steps in the virus replication cycle can also lead to
cell death and can possibly induce apoptosis. Studies with SV replicons
have shown that expressing only the nsPs can lead to the shutoff of
cellular protein synthesis and eventually to cell death (1,
13). Furthermore, mutations in the C-terminal portion of nsP2
that decrease RNA synthesis also decrease cytopathology and increase
the likelihood of establishing persistent infection by an
as-yet-unknown mechanism (11). How fusion and entry induce apoptosis is also unknown and may be related to previously described signalling roles for Ras, NF-
B, or superoxide in SV-induced
apoptosis (22, 29-31).
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ACKNOWLEDGMENTS |
We thank Charles Rice for the gift of rabbit antibody to nsP1.
This work was supported by research grant NS18596 from the National
Institutes of Health (D.E.G.) and a predoctoral scholarship from the
National Defense Medical Center, Taiwan (J.-T.J.).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-3459. Fax: (410) 955-0105. E-mail:
dgriffin{at}jhsph.edu.
Present address: Institute of Preventive Medicine, National Defense
Medical Center, Taipei, Taiwan, Republic of China.
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REFERENCES |
| 1.
|
Agapov, E. V.,
I. Frolov,
B. D. Lindenbach,
B. M. Pragai,
S. Schlesinger, and C. M. Rice.
1998.
Noncytopathic Sindbis virus RNA vectors for heterologous gene expression.
Proc. Natl. Acad. Sci. USA
95:12989-12994[Abstract/Free Full Text].
|
| 2.
|
Amano, T.,
E. Richelson, and M. Nirenberg.
1972.
Neurotransmitter synthesis by neuroblastoma clones.
Proc. Natl. Acad. Sci. USA
69:258-263[Abstract/Free Full Text].
|
| 3.
|
Bowman, E. M.,
A. Siebers, and K. Altendorf.
1988.
Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells.
Proc. Natl. Acad. Sci. USA
85:7972-7976[Abstract/Free Full Text].
|
| 4.
|
Brojatsch, J.,
J. Naughton,
M. M. Rolls,
K. Zingler, and J. A. T. Young.
1996.
CAR1, a TNFR-related protein, is a cellular receptor for cytopathic avian leukosis-sarcoma viruses and mediates apoptosis.
Cell
87:845-855[Medline].
|
| 5.
|
Bron, R.,
J. M. Wahlberg,
H. Garoff, and J. Wilschut.
1993.
Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein.
EMBO J.
12:693-701[Medline].
|
| 6.
|
Burge, B., and E. Pfefferkorn.
1966.
Isolation and characterization of conditional lethal mutants of Sindbis virus.
Virology
30:204-213[Medline].
|
| 7.
|
Caillet-Fauquet, P.,
M. Perros,
A. Brandenburger,
P. Spegelaere, and J. Rommelaere.
1990.
Programmed killing of human cells by means of an inducible clone of parvoviral genes encoding non-structural proteins.
EMBO J.
9:2989-2995[Medline].
|
| 8.
|
Cheng, E. H.,
B. Levine,
L. H. Boise,
C. B. Thompson, and J. M. Hardwick.
1996.
Bax-independent inhibition of apoptosis by Bcl-xL.
Nature
379:554-556[Medline].
|
| 9.
|
Cleverley, D. Z., and J. Lenard.
1998.
The transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus G protein.
Proc. Natl. Acad. Sci. USA
95:3425-3430[Abstract/Free Full Text].
|
| 10.
|
Dropulic, L. K.,
J. M. Hardwick, and D. E. Griffin.
1997.
A single amino acid change in the E2 glycoprotein of Sindbis virus confers neurovirulence by altering an early step of virus replication.
J. Virol.
71:6100-6105[Abstract].
|
| 11.
|
Dryga, S. A.,
O. A. Dryga, and S. Schlesinger.
1997.
Identification of mutations in a Sindbis virus variant able to establish persistent infection in BHK cells: the importance of a mutation in the nsP2 gene.
Virology
228:74-83[Medline].
|
| 12.
|
Flynn, D. C.,
W. J. Meyer,
J. M. Mackenzie, Jr., and R. E. Johnston.
1990.
A conformational change in Sindbis virus glycoproteins E1 and E2 is detected at the plasma membrane as a consequence of early virus-cell interaction.
J. Virol.
64:3643-3653[Abstract/Free Full Text].
|
| 13.
|
Frolov, I.,
E. Agapov,
T. A. Hoffman, Jr.,
B. M. Prágai,
M. Lippa,
S. Schlesinger, and C. M. Rice.
1999.
Selection of RNA replicons capable of persistent noncytopathic replication in mammalian cells.
J. Virol.
73:3854-3865[Abstract/Free Full Text].
|
| 14.
|
Frolov, I., and S. Schlesinger.
1994.
Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.
J. Virol.
68:1721-1727[Abstract/Free Full Text].
|
| 15.
|
Griffin, D. E., and J. M. Hardwick.
1997.
Regulators of apoptosis on the road to persistent alphavirus infection.
Annu. Rev. Microbiol.
51:565-592[Medline].
|
| 16.
|
Griffin, D. E., and R. T. Johnson.
1977.
Role of the immune response in recovery from Sindbis virus encephalitis in mice.
J. Immunol.
118:1070-1075[Abstract/Free Full Text].
|
| 17.
|
Hanon, E.,
G. Meyer,
A. Vanderplasschen,
C. Dessy-Doizé,
E. Thiry, and P.-P. Pastoret.
1998.
Attachment but not penetration of bovine herpesvirus 1 is necessary to induce apoptosis in target cells.
J. Virol.
72:7638-7641[Abstract/Free Full Text].
|
| 18.
|
Hubbard, S. C.,
L. Walls,
H. E. Ruley, and E. A. Muchmore.
1994.
Generation of Chinese hamster ovary cell glycosylation mutants by retroviral insertional mutagenesis.
J. Biol. Chem.
269:3717-3724[Abstract/Free Full Text].
|
| 19.
|
Jackson, A. C.,
T. R. Moench, and D. E. Griffin.
1987.
The pathogenesis of spinal cord involvement in the encephalomyelitis of mice caused by neuroadapted Sindbis virus infection.
Lab. Investig.
56:418-423[Medline].
|
| 20.
|
Jackson, A. C.,
T. R. Moench,
B. D. Trapp, and D. E. Griffin.
1988.
Basis of neurovirulence in Sindbis virus encephalomyelitis of mice.
Lab. Investig.
58:503-509[Medline].
|
| 21.
|
Jan, J.-T.,
A. P. Byrnes, and D. E. Griffin.
1999.
Characterization of a Chinese hamster ovary cell line developed by retroviral insertional mutagenesis that is resistant to Sindbis virus infection.
J. Virol.
73:4919-4924[Abstract/Free Full Text].
|
| 22.
|
Joe, A. K.,
G. Ferrari,
H. H. Jiang,
X. H. Liang, and B. Levine.
1996.
Dominant inhibitory Ras delays Sindbis virus-induced apoptosis in neuronal cells.
J. Virol.
70:7744-7751[Abstract].
|
| 23.
|
Joe, A. K.,
H. H. Foo,
L. Kleeman, and B. Levine.
1998.
The transmembrane domains of Sindbis virus envelope glycoproteins induce cell death.
J. Virol.
72:3935-3943[Abstract/Free Full Text].
|
| 24.
|
Johnson, R. T.,
H. F. McFarland, and S. E. Levy.
1972.
Age-dependent resistance to viral encephalitis: studies of infections due to Sindbis virus in mice.
J. Infect. Dis.
125:257-262[Medline].
|
| 25.
|
Legrand, C.,
J. Rommelaere, and P. Caillet-Fauquet.
1993.
MVM(p) NS-2 protein expression is required with NS-1 for maximal cytotoxicity in human transformed cells.
Virology
195:149-155[Medline].
|
| 26.
|
Levine, B.,
J. E. Goldman,
H. H. Jiang,
D. E. Griffin, and J. M. Hardwick.
1996.
Bcl-2 protects mice against fatal alphavirus encephalitis.
Proc. Natl. Acad. Sci. USA
93:4810-4815[Abstract/Free Full Text].
|
| 27.
|
Levine, B.,
Q. Huang,
J. T. Isaacs,
J. C. Reed,
D. E. Griffin, and J. M. Hardwick.
1993.
Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene.
Nature
361:739-742[Medline].
|
| 28.
|
Lewis, J.,
S. L. Wesselingh,
D. E. Griffin, and J. M. Hardwick.
1996.
Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence.
J. Virol.
70:1828-1835[Abstract].
|
| 29.
|
Lin, K.-I.,
J. A. DiDonato,
A. Hoffman,
J. M. Hardwick, and R. R. Ratan.
1998.
Suppression of steady-state, but not stimulus-induced NF-B activity inhibits alphavirus-induced apoptosis.
J. Cell Biol.
141:1479-1487[Abstract/Free Full Text].
|
| 30.
|
Lin, K.-I.,
P. Pasinelli,
R. H. Brown,
J. M. Hardwick, and R. R. Ratan.
1999.
Decreased intracellular superoxide levels activate Sindbis virus-induced apoptosis.
J. Biol. Chem.
274:13650-13655[Abstract/Free Full Text].
|
| 31.
|
Lin, K.-I.,
S. H. Lee,
R. Narayanan,
J. M. Baraban,
J. M. Hardwick, and R. R. Ratan.
1995.
Thiol agents and Bcl-2 identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor NF-kappa B.
J. Cell Biol.
131:1-14[Free Full Text].
|
| 32.
|
Liu, Z. G.,
H. Hsu,
D. V. Goeddel, and M. Karin.
1996.
Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-B activation prevents cell death.
Cell
87:565-576[Medline].
|
| 33.
|
Lowe, S. W., and H. E. Ruley.
1993.
Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis.
Genes Dev.
7:535-545[Abstract/Free Full Text].
|
| 34.
|
Marsh, M.,
J. Wellsteed,
H. Kern,
E. Harms, and A. Helenius.
1982.
Monensin inhibits Semliki Forest virus penetration into culture cells.
Proc. Natl. Acad. Sci. USA
79:5297-5301[Abstract/Free Full Text].
|
| 35.
|
Mattson, M. P., and K. Furukawa.
1997.
Anti-apoptotic actions of cycloheximide: blockade of programmed cell death or induction of programmed cell life?
Apoptosis
2:257-264.
[Medline] |
| 36.
|
Nava, V. E.,
A. Rosen,
M. A. Veliuona,
R. J. Clem,
B. Levine, and J. M. Hardwick.
1998.
Sindbis virus induces apoptosis through a caspase-dependent, CrmA-sensitive pathway.
J. Virol.
72:452-459[Abstract/Free Full Text].
|
| 37.
|
Nieva, J.-L.,
R. Bron,
J. Corver, and J. Wilschut.
1994.
Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane.
EMBO J.
13:2797-2804[Medline].
|
| 38.
|
Noteborn, M. H. M.,
D. Todd,
C. A. J. Verschueren,
H. W. F. M. de Gauw,
W. L. Curran,
S. Veldkamp,
A. J. Douglas,
M. S. McNulty,
A. J. van der Eb, and G. Koch.
1994.
A single chicken anemia virus protein induces apoptosis.
J. Virol.
68:346-351[Abstract/Free Full Text].
|
| 39.
|
Pesonen, M., and L. Kaariainen.
1982.
Incomplete complex oligosaccharides in Semliki Forest virus envelope proteins arrested within the cell in the presence of monensin.
J. Mol. Biol.
158:213-230[Medline].
|
| 40.
|
Ramsey-Ewing, A., and B. Moss.
1998.
Apoptosis induced by a postbinding step of vaccinia virus entry into Chinese hamster ovary cells.
Virology
242:138-149[Medline].
|
| 41.
|
Rao, L.,
M. Debbas,
P. Sabbatini,
D. Hockenberry,
S. Korsmeyer, and E. White.
1992.
The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins.
Proc. Natl. Acad. Sci. USA
89:7742-7746[Abstract/Free Full Text].
|
| 42.
|
Schmaljohn, A. L.
1983.
Protective monoclonal antibodies define maturational and pH-dependent antigenic changes in Sindbis virus E1 glycoprotein.
Virology
130:144-154[Medline].
|
| 43.
|
Stewart, S. A.,
B. Poon,
J. B. M. Jowett, and I. S. Y. Chen.
1997.
Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest.
J. Virol.
71:5579-5592[Abstract].
|
| 44.
|
Strauss, J. H., and E. G. Strauss.
1994.
The alphaviruses: gene expression, replication, and evolution.
Microbiol. Rev.
58:491-562[Abstract/Free Full Text].
|
| 45.
|
Taylor, R. M.,
H. S. Hurlbut,
T. H. Work,
J. R. Kingsbury, and T. E. Frothingham.
1955.
Sindbis virus: a newly recognized arthropod-transmitted virus.
Am. J. Trop. Med. Hyg.
4:844-846.
|
| 46.
|
Tucker, P. C., and D. E. Griffin.
1991.
Mechanism of altered Sindbis virus neurovirulence associated with a single-amino-acid change in the E2 glycoprotein.
J. Virol.
65:1551-1557[Abstract/Free Full Text].
|
| 47.
|
Tucker, P. C.,
S. H. Lee,
N. Bui,
D. Martinie, and D. E. Griffin.
1997.
Amino acid changes in the Sindbis virus E2 glycoprotein that increase neurovirulence improve entry into neuroblastoma cells.
J. Virol.
71:6106-6112[Abstract].
|
| 48.
|
Tyler, K. L.,
M. K. T. Squier,
S. E. Rodgers,
B. E. Schneider,
S. M. Oberhaus,
T. A. Grdina,
J. J. Cohen, and T. S. Dermody.
1995.
Differences in the capacity of reovirus strains to induce apoptosis are determined by the viral attachment protein  1.
J. Virol.
69:6972-6979[Abstract].
|
| 49.
|
Ubol, S.,
P. C. Tucker,
D. E. Griffin, and J. M. Hardwick.
1994.
Neurovirulent strains of alphavirus induce apoptosis in bcl-2-expressing cells; role of a single amino acid change in the E2 glycoprotein.
Proc. Natl. Acad. Sci. USA
91:5202-5206[Abstract/Free Full Text].
|
| 50.
|
Wahlberg, J. M., and H. Garoff.
1992.
Membrane fusion process of Semliki Forest virus. I. Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells.
J. Cell Biol.
116:339-348[Abstract/Free Full Text].
|
Journal of Virology, December 1999, p. 10296-10302, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
