Previous Article | Next Article 
Journal of Virology, July 2000, p. 6425-6432, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Sindbis Virus Entry into Cells Triggers Apoptosis
by Activating Sphingomyelinase, Leading to the Release of
Ceramide
Jia-Tsrong
Jan,1,
Subroto
Chatterjee,2 and
Diane E.
Griffin1,*
W. Harry Feinstone Department of Molecular
Microbiology and Immunology, Johns Hopkins University School of Hygiene
and Public Health, Baltimore, Maryland 21205,1
and Department of Pediatrics, Johns Hopkins University School
of Medicine, Baltimore, Maryland 212872
Received 24 November 1999/Accepted 12 April 2000
 |
ABSTRACT |
Sindbis virus (SV) causes acute encephalomyelitis by infecting and
inducing the death of neurons. Induction of apoptosis occurs during
virus entry and involves acid-induced conformational changes in the
viral surface glycoproteins and sphingomyelin (SM)-dependent fusion of
the virus envelope with the endosomal membrane. We have studied
neuroblastoma cells to determine how this entry process triggers cell
death. Acidic sphingomyelinase was activated during entry followed by
activation of neutral sphingomyelinase, SM degradation, and a sustained
increase in ceramide. Ceramide-induced apoptosis and SV-induced
apoptosis could be inhibited by treatment with Z-VAD-fmk, a caspase
inhibitor, and by overexpression of Bcl-2, an antiapoptotic cellular
protein. Acid ceramidase, expressed in a recombinant SV, decreased
intracellular ceramide and protected cells from apoptosis. The data
suggest that acid-induced SM-dependent virus fusion initiates the
apoptotic cascade by inducing SM degradation and ceramide release.
| |
INTRODUCTION |
Alphaviruses, enveloped plus-strand
RNA viruses, are important causes of mosquito-borne viral arthritis and
encephalitis worldwide (25). Neurons are the primary target
cell in the central nervous system of hosts that develop encephalitis.
Sindbis virus (SV), the prototypic alphavirus, causes neuronal
infection in mice (22), and studies of this infection have
provided important insights into the molecular mechanisms underlying
virus-induced encephalomyelitis.
Like other alphaviruses, SV has three major structural proteins, two
surface glycoproteins, E1 and E2, and a capsid protein that surrounds
the genome. E1 and E2 heterodimerize and then trimerize to form spikes
on the virion surface that mediate virus binding and entry
(55). E2 is the primary determinant of binding to cellular
receptors, and E1 is responsible for cholesterol-dependent binding to
liposomal membranes and contains the hydrophobic domain essential for
virus-cell fusion (8, 43). Alphavirus fusion occurs in the
endosome and requires an acid-induced conformational change in the
E1-E2 heterodimer and a target membrane that contains sphingomyelin
(SM) (8, 46, 59).
SV infects many types of cells in culture and induces apoptosis in
neurons and other vertebrate cells in vitro and in vivo (31,
32). The study of SV-induced apoptosis offers a powerful system
for defining important cellular mechanisms regulating virus-induced cell death since recombinant viruses can be used to express modifiers of the apoptotic process in virus-infected cells. Studies using these
recombinant SVs have shown that SV-induced apoptosis can be slowed or
prevented by caspase inhibitors and by Bcl-2 family member proteins
(10, 30, 31, 45). However, the mechanism of induction of
apoptosis by SV is largely unknown.
In the best-defined systems, tumor necrosis factor (TNF)- and Fas
ligand-induced cell death, apoptosis is initiated at the cell membrane
through cross-linking of a transmembrane protein belonging to the TNF
receptor family. Cross-linking initiates a cascade of intracellular
events resulting in death of susceptible cells. Many viruses that cause
acute infections and host cell destruction induce apoptosis. Some
viruses initiate this process at the time of binding or entry (7,
20, 50), while others initiate the process after infection is
established and viral proteins are produced (4). Previous
studies of SV-induced apoptosis have shown that cell death is most
efficiently induced when the structural proteins are present
(17), that transient overexpression of the transmembrane
portions of either E1 or E2 induces apoptosis (24), and that
induction of apoptosis does not require virus replication but does
require virus fusion with the cell membrane (23).
Since alphaviruses require sphingolipids containing ceramide (i.e., SM)
to be present in the cell membrane as a cofactor for fusion
(46), we investigated the role of the SM pathway in
SV-induced apoptosis and have found that SV infection rapidly induced
activation of acidic sphingomyelinase (aSMase) to hydrolyze SM and
release ceramide, a well-defined intracellular mediator of apoptosis
(47). Mg2+-dependent neutral sphingomyelinase
(nSMase) was activated at later times, leading to a prolonged increase
in intracellular ceramide. Recombinant SV expressing acid ceramidase
(AC) decreased levels of ceramide and delayed virus-induced cell death.
Our results suggest that SV-induced apoptosis can be triggered by
activation of aSMase associated with the endosomal membrane during the
SM-requiring process of virus-cell fusion, leading to the release of
ceramide and induction of apoptosis.
| |
MATERIALS AND METHODS |
Cell culture and biological reagents.
Mouse neuroblastoma
cell line N18 (3), baby hamster kidney cell line BHK-21,
type A Niemann-Pick disease (NPD) and control fibroblasts, and rat
prostate carcinoma cell line AT3 transfected with the expression vector
pZipNeo (AT3Neo) or the recombinant vector pZipBcl-2 (AT3Bcl-2)
(31) were grown in Dulbecco's modified Eagle medium
supplemented with 10% fetal bovine serum. Cell viability was assessed
by trypan blue exclusion. Fumonisin B1, C2-ceramide, diacylglycerol, phosphatidic acid, bis-benzimide (Hoechst 33258), 6-dimethylaminopurine (DMAP), and okadaic acid (OKA) were obtained from
Sigma Chemical Company (St. Louis, Mo.). Z-VAD-fmk was obtained from
Alexis (San Diego, Calif.).
Virus preparation.
Neuroadapted SV (NSV), derived by serial
passage of wild-type SV (strain AR339) in mouse brain (18),
was plaque purified, grown, and assayed in BHK-21 cells. For
purification, virus was precipitated in 10% (wt/vol) polyethylene
glycol 8000 in 0.5 M NaCl, pelleted, suspended in NET buffer (10 mM
Tris, 3 mM EDTA, 150 mM NaCl, pH 7.4), and banded in a continuous
15-to-40% potassium tartrate gradient. Banded virus was dialyzed
against 0.05M Tris-Cl (pH 7.4) and stored in aliquots at 
70°C.
Virus was UV inactivated at 4°C with a germicidal lamp (254 nm) at a
distance of 5 cm for 30 min. Inactivation was confirmed by plaque assay
on monolayers of BHK-21 cells.
Lipid studies.
N18 cells were pelleted, washed twice with
ice-cold phosphate-buffered saline (PBS), and extracted with
chloroform-methanol-1 N HCl (100:100:1, vol/vol/vol). Lipids in the
organic phase were dried in a vacuum dryer and subjected to mild
alkaline hydrolysis (0.1 N methanolic KOH for 1 h at 37°C) to
remove glycerophospholipids. Samples were reextracted, and the organic
phase was dried. Detergent solution (20 µl of 7.5%
n-octyl-
-glucopyranoside with 5 mM cardiolipin in 1 mM
diethylenetriamine-pentaacetic acid) was added, and the sample was
sonicated. Ceramide was measured using the
sn-1,2-diacylglycerol kinase assay reagent system and
labeling for 30 min with 1 mCi of [
-32P]ATP in 10 µl
of 5 mM ATP (57) (Amersham, Arlington Heights, Ill.).
Ceramide-1-phosphate and sphingosine-1-phosphate were resolved by
thin-layer chromatography on Silica Gel 60 plates (Whatman, Clinton,
N.J.) using a solvent of chloroform-methanol-acetic acid (65:15:5) and
detected by autoradiography. Incorporated 32P was
quantified by scraping the spots and counting radioactivity in a liquid
scintillation counter. Diacylglycerol was quantified in a similar
manner to ceramide, except that the alkaline hydrolysis step was omitted.
Changes in SM levels were measured by labeling cells to isotopic
equilibrium with [3H]choline chloride (79.2 Ci/mmol; 1.0 µCi/ml; Dupont New England Nuclear) for at least four cell doublings.
After infection with NSV, cellular lipids were extracted, dried, and
subjected to alkaline hydrolysis as described for ceramide measurement.
SM was resolved from residual phosphatidylcholine and
lysophosphatidylcholine by thin-layer chromatography using a solvent of
chloroform-methanol-acetic acid-water (50:30:8:4), identified by iodine
vapor staining, and quantified by liquid scintillation counting.
SMase assays.
Activities of aSMase and nSMase were measured
according to previously described methods with minor modifications
(49). In brief, 5 × 106 N18 cells were
scraped from the culture plates, washed with PBS, and disrupted by
repeated passage through a 25-gauge needle. Nuclei and cell debris were
pelleted at 800 × g for 5 min. Supernatant fluid was
collected, and the protein concentration was measured using the Bio-Rad
(Hercules, Calif.) protein detection kit. To measure SMase activity, 50 µg of protein was incubated for 90 min (the reaction was linear for
up to 120 min) at 37°C in buffer (200-µl final volume) containing
250 mM sodium acetate and 1 mM EDTA, pH 5.0, for aSMase or 250 mM
Tris-Cl, pH 7.4, with or without 6 mM MgCl2 for nSMase, and
0.75 µl of [methyl-14C]SM (0.2 mCi/ml; 56.6 mCi/mmol). Radioactive phosphorylcholine produced from
[14C]SM was extracted with 800 µl of
chloroform-methanol (2:1, vol/vol) and 100 µl of H2O.
[14C]phosphorylcholine in the aqueous phase was measured
by scintillation counting.
Assessment of apoptosis.
A total of 5 × 106 NSV-infected or C2-ceramide-treated N18
cells were scraped from the culture plates and washed with PBS. Genomic
DNA was isolated with DNAZOL and incubated with RNase (1µg/ml) at 50°C for 1 h. DNA (10 µg) from each sample was
electrophoresed through a 2.0% agarose gel in TAE buffer (40 mM Tris
acetate, 2 mM EDTA) and stained with ethidium bromide.
Morphological changes in the nuclear chromatin were visualized by
staining with the DNA-binding fluorochrome bis-benzimide.
In brief,
cells were pelleted, washed with PBS, and resuspended
in 50 µl of 3%
paraformaldehyde in PBS. After 10 min cells were
washed and resuspended
in 15 µl of PBS containing 16 µg of bis-benzimide
per ml. Five
hundred cells were scored for the presence of apoptotic
chromatin
changes using a Nikon Eclipse E800 fluorescence microscope.
Cells with
two or more chromatin fragments were considered
apoptotic.
Construction of recombinant SV vectors encoding the AC gene.
A plasmid, pACFL, encoding the full-length human AC gene was provided
by Konrad Sandhoff (Institute of Organic Chemistry and Biochemistry,
Bonn, Germany). The plasmid was digested with BamHI and
SalI. The fragment containing the 1,185-bp AC open reading frame was blunt end-ligated into the BstEII site of the
double subgenomic SV vector (dsTE12) (10) in forward and
reverse orientations. dsTE12 DNA containing AC was linearized with
XhoI and transcribed in vitro using SP6 RNA polymerase.
Stocks of recombinant viruses were generated by transfecting the
full-length RNA into BHK-21 cells and collecting the supernatant fluid
when more than 70% cytopathic effect was observed. Virus titers were
determined by plaque assay on BHK-21 cells.
| |
RESULTS |
SV infection induces ceramide release.
To determine whether SV
infection leads to increased ceramide production, N18 cells were
infected with NSV at a multiplicity of infection (MOI) of 50, which
results in synchronous infection of all the cells. Levels of
intracellular ceramide were measured, and an increase was detectable by
2 h after initiation of infection (Fig.
1A) and peaked between 2 and 6 h at
2.2 times control levels (Fig. 1B). Diacylglycerol levels remained at
80 to 100% of control levels. Sphingosine levels increased slightly
(Fig. 1A), possibly due to in vitro catabolism of ceramide. Levels of
ceramide decreased between 6 and 10 h after NSV infection (Fig.
1B), a time of rapid increase in virus production (56).
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 1.
Generation of ceramide in response to NSV infection. (A)
N18 cells were infected with NSV (MOI = 50), and at the indicated
times (p.i., postinfection), lipid extracts of the cells were assayed
for ceramide and sphingosine by the diacylglycerol kinase reaction and
resolved by thin-layer chromatography followed by autoradiography. Std,
C2-ceramide, 5 nmol. (B) Quantitation of ceramide-1-P and
sphingosine-1-P in NSV-infected cells. Values are percentages of the
levels present in mock-infected cells ± standard deviations
(error bars).
|
|
Ceramide mimics SV infection in inducing apoptosis.
To
determine whether ceramide alone could induce apoptosis, N18 cells were
treated with a ceramide analog, C2-ceramide, which has two
carbons rather than the 18 carbons of ceramide and is cell permeable,
and assessed for apoptosis. C2-ceramide mimicked SV
infection by inducing oligonucleosomal DNA fragmentation (Fig. 2A) and the appearance of typical
apoptotic nuclear changes (Fig. 2B). The induction and extent of
apoptotic morphology depended on the dose of ceramide, with 40 µM
C2-ceramide being the minimum required for inducing
apoptosis of all cells (data not shown). Treating cells with 50 µM
dihydroceramide, the immediate precursor of ceramide, which lacks the
double bond at C-4-C-5 of the sphingosine backbone, did not result in
apoptosis (Fig. 2C). Furthermore, other cell-permeable analogs of lipid
second messengers, including sphingosine, 1,2-diacylglycerol, and
phosphatidic acid, did not induce apoptosis (Fig. 2C).
View larger version (53K):
[in this window]
[in a new window]
|
FIG. 2.
Induction of apoptosis in N18 cells by NSV and
C2-ceramide. (A) Gel electrophoresis of DNA from cells
infected with NSV (MOI = 5) or treated with 50 µM
C2-ceramide or diluent (0.1% ethanol) (control). After
36 h (NSV) and 48 h (C2-ceramide), genomic DNA
was analyzed by agarose gel electrophoresis and staining with ethidium
bromide. (B) Morphological alterations of the chromatin of N18 cells
treated with 0.1% ethanol (control) or 50 µM
C2-ceramide. After 36 h cells were fixed and stained
with bis-benzimide. (C) Quantitation of nuclear morphological changes
of N18 cells 48 h after treatment with 0.1% ethanol (control) or
50 µM C2-ceramide, dihydro-C-2-ceramide, or
phosphatidic acid. A total of 500 cells per slide were scored for
apoptotic chromatin changes. Values represent means ± standard
deviations (error bars).
|
|
Mechanism of ceramide generation.
Ceramide can be generated by
hydrolysis of membrane SM by SMase or by biosynthesis from sphingosine
by ceramide synthase (40), and both pathways can lead to
apoptosis. TNF alpha (TNF-
), Fas ligand, and ionizing radiation
activate the apoptotic pathway by increasing intracellular ceramide
through hydrolysis of SM (11, 28), while the
chemotherapeutic agent daunorubicin activates ceramide synthase
(5). To determine the mechanism of SV-induced ceramide
generation, we measured SM levels during NSV infection. As the ceramide
level increased, there was a concomitant decrease in content of
cellular SM (Fig. 1B and 3A). Cellular SM
levels decreased soon after infection and reached a minimum of 4 to
6 h, suggesting that ceramide generated in the infected cells was derived from hydrolysis of membrane SM.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 3.
Changes in SM levels and effect of fumonisin B1 on
ceramide generation in response to NSV infection. (A) SM degradation in
N18 cells labeled with [3H]choline and then infected with
NSV (MOI = 50). Lipids were extracted, and SM was resolved by
thin-layer chromatography and quantified by liquid scintillation
counting. The values represent means ± standard deviations (SD)
(error bars) of the SM levels in NSV-infected cells as a percentage of
mock-infected cells × 100. (B) N18 cells treated with 100 µM
fumonisin B1 (FB1) and then mock infected or infected with NSV
(MOI = 50). At 6 and 12 h after infection, cellular lipids
were extracted and assayed for ceramide as described in the legend to
Fig. 1. Values represent means ± SD (error bars).
|
|
To confirm this observation cells were treated with fumonisin B1, a
potent inhibitor of ceramide synthase (
41). Preincubation
of
N18 cells with fumonisin B1 delayed, but did not prevent, NSV-induced
ceramide elevation (Fig.
3B). Intracellular ceramide levels increased
by 12 h after NSV infection in the presence of fumonisin B1,
whereas
fumonisin B1 alone did not induce the elevation of ceramide
levels.
Fumonisin B1 may delay NSV-induced ceramide generation by
inhibiting
virus entry or by having a toxic effect on N18 cells. This
result,
combined with the slightly increased sphingosine level (Fig.
1B),
indicated that ceramide in infected cells was not generated by
synthesis from
sphingosine.
To determine the involvement of SMase in the generation of
ceramide, a micellar assay system was used. aSMase and nSMase were
distinguished by adjusting the buffer pH to the activation requirements
of the SMase. Membrane-associated, Mg
2+-dependent and
cytosolic, Mg
2+-independent nSMases (
9,
48) were
distinguished by using
buffers with and without Mg
2+.
aSMase activity increased quickly and peaked at a maximum of
1.7-fold
control 90 min after infection. Mg
2+-dependent nSMase
activity also increased, but with a peak (1.5-fold
control) at 4 h
(Fig.
4A). An additional peak at 8 h
may represent
activity of a different nSMase or secondary activation by
downstream
mediators. Mg
2+-independent nSMase activity
increased only slightly after NSV
infection.
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 4.
Activation of aSMase and nSMase during NSV infection.
(A) NSV (MOI = 50) was bound to N18 cells at 4°C for 2 h.
The temperature was shifted to 37°C to initiate entry and at the
indicated times lysates were assayed for SMase activity at pH 5.0 (aSMase) and pH 7.4 (nSMase) with and without Mg2+. Data
are expressed as a percentage of control values (NSV infected/mock
infected × 100) (means ± standard deviations [SD] [error
bars]). (B) UV-inactivated NSV induces generation of ceramide. N18
cells with or without UV-inactivated NSV (MOI equivalent = 500)
bound at 4°C were shifted to 37°C and incubated with medium of pH
5.0, pH 6.0, or pH 7.4. Five hours later, lipids were extracted and
ceramide levels were determined. (C) NH4Cl blocks ceramide
elevation in response to NSV infection. Lipids were extracted from N18
cells treated with 20 mM NH4Cl for 1 h prior to
infection with NSV (MOI = 50), and ceramide levels were
determined. (D) NH4Cl protects against NSV-induced cell
death. N18 cells pretreated with 20 mM NH4Cl for 1 h
were infected with NSV (MOI = 5). Cell viabilities were determined
by trypan blue exclusion. (B to D) Values represent means ± SD
(error bars).
|
|
UV-inactivated NSV can trigger apoptosis when cells with virus
bound to the surface are transiently exposed to a low-pH
environment
to induce virus-cell fusion (
23). To determine
whether ceramide
was generated during such fusion, intracellular
ceramide levels
were assessed (Fig.
4B). Ceramide was generated when
N18 cells
with bound replication-defective UV-inactivated NSV were
exposed
to low pH, with pH 5.0 being more efficient than pH 6.0. Generation
of ceramide was dependent on the acid-induced fusion event
since
it did not occur if endosomal acidification was blocked by
treatment
of NSV-infected N18 cells with NH
4Cl (Fig.
4C).
By 48 h after
NSV infection, more than 40% of the
NH
4Cl-treated cells were alive,
while all untreated cells
were dead (Fig.
4D). NSV infection did
not stimulate the release of
choline or increase diacylglycerol
(data not shown), indicating that
phosphatidyl choline-specific
phospholipase C was not activated. The
tight coupling of NSV infection,
aSMase activation, and ceramide
generation within 1 to 2 h; dependence
of ceramide generation on
endosomal acidification; and the fact
that pH 4.5 to 5.0 is optimal for
aSMase activity suggest that
activation of aSMase occurs in the
endosome during the viral fusion
event.
Activation of nSMase is sufficient for SV to induce apoptosis.
To determine whether activation of aSMase is necessary for SV to induce
apoptosis, fibroblasts from a patient with type A NPD, an inherited
deficiency of aSMase (29), were studied. Surprisingly, NPD
fibroblasts died more rapidly after NSV infection than control fibroblasts (Fig. 5A). This was
accompanied by an increase in the level of ceramide (Fig. 5B). There
was no aSMase activity induced in NPD fibroblasts (Fig. 5C), but nSMase
was activated early to a greater extent than in control fibroblasts
(Fig. 5D), suggesting the presence of compensatory SMase mechanisms.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 5.
NSV infection of type A NPD and control fibroblasts.
Fibroblasts were infected with NSV (MOI = 50) and monitored for
viability by trypan blue exclusion (A), levels of intracellular
ceramide (B), aSMase activity (C), and nSMase activity (D) at various
times after infection. (B to D) Values represent means ± standard
deviations (error bars).
|
|
Inhibitors of protein kinases and phosphatases protect SV-infected
cells against death.
Ceramide influences protein phosphorylation
by directly activating at least two target enzymes, ceramide-activated
protein kinase (CAPK) and ceramide-activated protein phosphatase (CAPP) (16, 35). An inhibitor of protein kinase, DMAP
(38), and an inhibitor of protein phosphatase, OKA
(51), were used to evaluate the roles of protein
phosphorylation and dephosphorylation in SV-induced apoptosis triggered
by an increase in ceramide. Both DMAP and OKA were protective against
SV-induced cell death (Fig. 6A and 6B).
By 48 h after NSV infection, most untreated cells were dead, while
62% of DMAP-treated cells and 75% of OKA-treated cells survived. To
determine whether DMAP or OKA has an effect on SV entry or
intracellular replication, we examined viral protein synthesis by
labeling infected cells with [35S]methionine and
determined virus titers in the culture medium. DMAP had no effect on
viral protein synthesis or virus production, but OKA delayed viral
protein synthesis and inhibited virus production possibly due to
effects on E2 processing (36). These results imply that
protein phosphorylation is an important determinant of SV-induced cell
death and ceramide may trigger these reactions.
View larger version (48K):
[in this window]
[in a new window]
|
FIG. 6.
Effects of various modulators on the viability of
NSV-infected or ceramide-treated cells. N18 cells were or were not
pretreated with 5 mM DMAP (A) or 5 µM OKA (B) for 1 h and then
infected with NSV (MOI = 5). (C) N18 cells were treated with 100 µM Z-VAD-fmk for 6 h and then infected with NSV or treated with
C2- ceramide (C2-Cer) (50 µM). (D) AT3-Bcl-2 and AT3-Neo
cells were infected with NSV or treated with C2-Cer. Cell viabilities
were determined 48 h later by trypan blue exclusion. Values
represent means ± standard deviations (error bars).
|
|
Caspase inhibition and Bcl-2 expression protect cells against SV-
and ceramide-induced death.
To determine whether caspase is
involved in induction of death of SV-infected and ceramide-treated N18
cells, we preincubated cells with a caspase inhibitor, Z-VAD-fmk, prior
to infection with NSV or treatment with C2-ceramide.
Z-VAD-fmk effectively blocked both NSV- and ceramide-induced cell death
(Fig. 6C). To investigate the contribution of Bcl-2 to protection from
these inducers of apoptosis, AT3 cells overexpressing Bcl-2 and control AT3-Neo cells (31) were examined for their resistance to
NSV- and ceramide-induced cell death. Bcl-2 protein protected against apoptosis induced by both NSV infection and C2-ceramide
treatment (Fig. 6D).
AC protects NSV-infected cells against death by decreasing
intracellular ceramide.
AC specifically catalyzes the hydrolysis
of ceramide into sphingosine and free fatty acid (27). The
AC gene was inserted in both forward and reverse orientations into a
double subgenomic SV vector (10) (Fig.
7A). Viabilities of N18 cells infected with recombinant SV encoding AC in the forward orientation (TE12AC-F) were higher than those infected with recombinant SV carrying the reverse AC gene (TE12AC-R) at all MOIs tested (Fig. 7B). To confirm the
expression and activity of the encoded ceramidase, ceramide levels in
these cells were measured. Early in infection ceramide in
TE12AC-F-infected cells was similar to that in TE12AC-R-infected cells,
consistent with predicted translation of the subgenomic RNA beginning 3 to 4 h after infection. However, by 4 to 6 h the level of
ceramide in TE12AC-F-infected cells was lower than that in
TE12AC-R-infected cells (Fig. 7C). These results demonstrate that
SV-infected cells can be protected from death by decreasing intracellular ceramide levels and further demonstrate that ceramide is
an important mediator in inducing SV-induced cell death.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 7.
Effects of AC on NSV-infected cells. (A) Diagram of the
double subgenomic SV vector containing the AC gene. (B) N18 cells were
infected with recombinant SV TE12AC-F (forward AC gene) or TE12AC-R
(reverse AC gene) at various MOIs. Cell viabilities were determined by
trypan blue exclusion at 24 and 48 h after infection. (C) N18
cells were infected with recombinant TE12AC-F and TE12AC-R (MOI = 50), and levels of ceramide were determined. Values represent
means ± standard deviations (error bars). Some error bars are too
small to be visible.
|
|
| |
DISCUSSION |
SV activates aSMase to generate ceramide during the process of
fusion with the cell membrane.
Studies of alphavirus fusion have
shown that acid-induced fusion with liposomal membranes requires the
presence of sphingolipids in the target membrane. The need for
sphingolipid is confined to the actual fusion event, with cholesterol
being necessary and sufficient for low-pH-dependent binding of virus to
the target membrane (8, 46). Thus, the virus membrane, and
presumably the transmembrane viral surface glycoproteins, must interact
with SM present in the outer leaflet of the membrane during the process of fusion and entry. Only small amounts of SM are required, suggesting that SM acts as a cofactor for activation of the viral fusion protein
(46). The interaction is stereospecific, and the minimal molecular characteristic of SM essential for alphavirus fusion is
presence of the ceramide portion of the sphingosine backbone, which
must have a 3-hydroxyl group and a 4,5-transcarbon double bond
(12, 42). Importantly, this 4,5-transcarbon double bond is
also required for ceramide-induced apoptosis, since a ceramide analog,
C2-dihydroceramide, which lacks this double bond, is
inefficient in inducing apoptosis (47).
All three forms of SMase are capable of initiating signaling through
production of ceramide (
60). TNF-
-induced apoptosis
has
been particularly well studied. TNF-
binding to the 55-kDa
TNF
receptor activates aSMase and nSMase through distinct pathways
initiated by separate cytoplasmic domains of TNF-R55 (
1,
2,
53,
60). Membrane-associated nSMase activation is initiated
by
interaction of a novel WD repeat protein, FAN, with the nSMase
domain
of TNF-R55 (
1). Activation of aSMase occurs later and
is
initiated through interaction of the proapoptotic adapter proteins
TRADD and FADD with the death domain of TNF-R55 and involves the
activation of phosphatidyl choline-specific phospholipase C and
an
interleukin-1 (IL-1) cleavage enzyme (ICE)-like protease (
6,
14,
19,
54).
Like TNF-
, SV also activates both aSMase and nSMase, but aSMase
activation occurs first and nSMase activation is later. Studies
in
aSMase-deficient NPD fibroblasts show that activation of nSMase
by SV
can also lead to early increases in ceramide and apoptosis.
Cell type
differences may also play a role since in the normal
fibroblasts aSMase
was not induced until 4 h after infection when
there was also a
detectable increase in nSMase activity, which
was greatly exaggerated
in the NPD fibroblasts. SV activation
of aSMase may be more analogous
to the activation of aSMase by
IL-1, which is linked to the
internalization of IL-1 receptor
1 mediated by the IL-1 receptor
activating protein (
21). After
binding to a cellular
receptor SV is rapidly internalized and
fusion of the viral envelope
and the endosomal membrane occurs
within 30 min (
39).
Preincubating cells with NH
4Cl, which has
no effect on
virus binding, prevents virus fusion by blocking
the decrease of
endosomal pH and inhibits the generation of ceramide.
It is possible
that either the E1 or E2 protein directly interacts
with SM and induces
activation of aSMase during the fusion process.
Studies of transient
expression of various viral proteins have
suggested that the
transmembrane regions of either E1 or E2 can
induce apoptosis
(
24), possibly through their participation
in this
process.
Ceramide is an early mediator for triggering SV-induced
apoptosis.
Ceramide has been shown to be involved in cell
differentiation, cell cycle arrest, and apoptosis (19).
Ceramide is implicated as a mediator in the apoptotic signaling of a
variety of inducers of apoptosis, including TNF-, Fas ligand,
neurotrophins, ionizing radiation, cytokines, heat shock, UV light,
antitumor drugs, and oxidative stress (11, 15, 19, 52, 58).
In most of these cases, ceramide is generated by hydrolysis of
membrane-associated SM and the elevation of ceramide precedes apoptosis
as it does in SV-induced apoptosis. Treatment of N18 cells with
C2-ceramide induced apoptosis. Furthermore, AC, which
degrades intracellular ceramide, protected NSV-infected cells from death.
The details of the apoptotic pathway downstream of ceramide are
not fully known because the range of immediate ceramide targets
has not
been completely elucidated. Direct targets for ceramide
action include
membrane-associated CAPK, a member of the proline-directed
family of
serine/threonine protein kinases (
26), and CAPP, a
member of
the heterotrimeric protein phosphatase 2A family (
16).
CAPK,
in response to TNF-
activation of nSMase, phosphorylates
Raf1,
activating the mitogen-activated protein kinase cascade
(
61). DMAP, a protein kinase inhibitor, abrogates or
significantly
attenuates TNF-
-induced cytotoxicity (
38).
Functions of CAPP
are inhibited by the phosphatase 2A inhibitor OKA
(
16). Both
DMAP and OKA are protective against SV-induced
cell death, indicating
that a series of kinase and phosphatase
reactions occur in the
SV-triggered apoptotic pathway. Ceramide can
also activate protein
kinase C-
and link cytokine receptors to
NF-
B activation (
37,
44). In AT3 but not N18 cells,
antisense RNA of NF-
B blocks
SV-induced apoptosis through inhibition
of constitutive NF-
B
expression (
33,
34). However,
ceramide induced by TNF does
not lead to transcription of
NF-
B-dependent genes (
13). Further
studies of the
ceramide targets, their functions, compartmentalization,
and consequent
effects are necessary for the understanding of
the downstream pathway
of SV-triggered, ceramide-induced
apoptosis.
| |
ACKNOWLEDGMENTS |
Jia-Tsrong Jan was supported by a predoctoral scholarship from
the National Defense Medical Center, Taiwan. The research was supported
by grants DK31722 (S.C.) and NS 18596 (D.E.G) from the National
Institutes of Health.
NPD and control fibroblasts were supplied by the Kennedy/Hopkins NICHD
Mental Retardation Research Center Core (HD24061).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Immunology, Johns Hopkins School of 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.
| |
REFERENCES |
| 1.
|
Adam-Klages, S.,
D. Adam,
K. Wiegmann,
S. Struve,
W. Kolanus,
J. Schneider-Mergener, and M. Kronke.
1996.
Fan, a novel WD-repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase.
Cell
86:937-947[CrossRef][Medline].
|
| 2.
|
Adams-Klages, S.,
R. Schwandner,
D. Adam,
D. Kreder,
K. Bernardo, and M. Kronke.
1998.
Distinct adapter proteins mediate acid versus neutral sphingomyelinase activation through the p55 receptor for tumor necrosis factor.
J. Leukoc. Biol.
63:678-682[Abstract].
|
| 3.
|
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].
|
| 4.
|
Anouja, F.,
R. Watiez,
S. Mousset, and P. Caillet-Fauquet.
1997.
The cytotoxicity of the parvovirus minute virus of mice nonstructural protein NS1 is related to changes in the synthesis and phosphorylation of cell proteins.
J. Virol.
71:4671-4678[Abstract].
|
| 5.
|
Bose, R.,
M. Verheij,
A. Haimovitzfriedman,
K. Scotto,
Z. Fuks, and R. Kolesnick.
1995.
Ceramide synthase mediates daunorubicin-induced apoptosis - an alternative mechanism for generating death signals.
Cell
82:405-414[CrossRef][Medline].
|
| 6.
|
Bourteele, S.,
A. Hauber,
H. Doopler,
J. Horn-Muller,
C. Ropke,
G. Schwarzmann,
K. Pfizenmaier, and G. Muller.
1998.
Tumor necrosis factor induces ceramide oscillations and negatively controls sphingolipid synthases by caspases in apoptotic Kmy-1 cells.
J. Biol. Chem.
273:31245-31251[Abstract/Free Full Text].
|
| 7.
|
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[CrossRef][Medline].
|
| 8.
|
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].
|
| 9.
|
Chatterjee, S.
1993.
Neutral sphingomyelinase.
Adv. Lipid Res.
26:25-48[Medline].
|
| 10.
|
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[CrossRef][Medline].
|
| 11.
|
Cifone, M. G.,
R. De Maria,
P. Roncaioli,
M. R. Rippo,
M. Azuma,
L. L. Lanier,
A. Santoni, and R. Testi.
1994.
Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase.
J. Exp. Med.
180:1547-1552[Abstract/Free Full Text].
|
| 12.
|
Corver, J.,
L. Moesby,
R. K. Erukulla,
K. C. Reddy,
R. Bittman, and J. Wilschut.
1995.
Sphingolipid-dependent fusion of Semliki Forest virus with cholesterol-containing liposomes requires both the 3-hydroxyl group and the double bond of the sphingolipid backbone.
J. Virol.
69:3220-3223[Abstract].
|
| 13.
|
Dbaibo, G. S.,
L. M. Obeid, and Y. A. Hannun.
1993.
Tumor necrosis factor- (TNF-) signal transduction through ceramide. Dissociation of growth inhibitory effects of TNF- from activation of nuclear factor-kB.
J. Biol. Chem.
268:17762-17766[Abstract/Free Full Text].
|
| 14.
|
Dbaibo, G. S.,
D. K. Perry,
C. J. Gamard,
R. Platt,
G. G. Poirier,
L. M. Obeid, and Y. A. Hannun.
1997.
Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-alpha: CrmA and Bcl-2 target distinct components in the apoptotic pathway.
J. Exp. Med.
185:481-490[Abstract/Free Full Text].
|
| 15.
|
Dobrowsky, R. T., and B. D. Carter.
1998.
Coupling of the p75 neutrotrophin receptor to sphingolipid signaling.
Ann. N. Y. Acad. Sci.
845:32-45[CrossRef][Medline].
|
| 16.
|
Dobrowsky, R. T., and Y. A. Hannun.
1993.
Ceramide-activated protein phosphatase: partial purification and relationship to protein phosphatase 2A.
Adv. Lipid Res.
25:91-104[Medline].
|
| 17.
|
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].
|
| 18.
|
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].
|
| 19.
|
Hannun, Y. A., and L. M. Obeid.
1995.
Ceramide: an intracellular signal for apoptosis.
Trends. Biochem. Sci.
20:73-77[CrossRef][Medline].
|
| 20.
|
Hanon, E.,
G. Meyer,
A. Vanderplasschen,
C. Dessy-Doize,
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].
|
| 21.
|
Hofmeister, R.,
K. Wiegmann,
C. Korherr,
K. Bernardo,
M. Kronke, and W. Falk.
1997.
Activation of acid sphingomyelinase by interleukin-1 (IL-1) requires the IL-1 receptor accessory protein.
J. Biol. Chem.
272:27730-27736[Abstract/Free Full Text].
|
| 22.
|
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].
|
| 23.
|
Jan, J.-T., and D. E. Griffin.
1999.
Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication.
J. Virol.
73:10296-10302[Abstract/Free Full Text].
|
| 24.
|
Joe, A. K.,
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].
|
| 25.
|
Johnston, R. E., and C. J. Peters.
1996.
Alphaviruses, p. 843-898.
In
B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus (ed.), Virology. Lippincott-Raven Press, New York, N.Y.
|
| 26.
|
Joseph, C. K.,
H. S. Byun,
R. Bittman, and R. N. Kolesnick.
1993.
Substrate recognition by ceramide-activated protein kinase. Evidence that kinase activity is proline-directed.
J. Biol. Chem.
268:20002-20006[Abstract/Free Full Text].
|
| 27.
|
Koch, J.,
S. Gartner,
C. M. Li,
L. E. Quintern,
K. Bernardo,
O. Levran,
D. Schnabel,
R. J. Desnick,
E. H. Schuchman, and K. Sandhoff.
1996.
Molecular cloning and characterization of a full-length complementary DNA encoding human acid ceramidase. Identification of the first molecular lesion causing Farber disease.
J. Biol. Chem.
271:33110-33115[Abstract/Free Full Text].
|
| 28.
|
Kolesnick, R., and D. W. Golde.
1994.
The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling.
Cell
77:325-328[CrossRef][Medline].
|
| 29.
|
Levade, T.,
R. Salvayre, and L. Douste-Blazy.
1986.
Sphingomyelinases and Niemann-Pick disease.
J. Clin. Chem. Clin. Biochem.
24:205-220[Medline].
|
| 30.
|
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].
|
| 31.
|
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[CrossRef][Medline].
|
| 32.
|
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].
|
| 33.
|
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].
|
| 34.
|
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].
|
| 35.
|
Liu, J.,
S. Mathias,
Z. Yang, and R. N. Kolesnick.
1994.
Renaturation and tumor necrosis factor-alpha stimulation of a 97-kDa ceramide-activated protein kinase.
J. Biol. Chem.
269:3047-3052[Abstract/Free Full Text].
|
| 36.
|
Liu, N., and D. T. Brown.
1993.
Phosphorylation and dephosphorylation events play critical roles in Sindbis virus maturation.
Virology
196:703-711[CrossRef][Medline].
|
| 37.
|
Lozano, J.,
E. Berra,
M. M. Municio,
M. T. Diaz-Meco,
I. Dominguez,
L. Sanz, and J. Moscat.
1994.
Protein kinase C zeta isoform is critical for kappa B-dependent promoter activation by sphingomyelinase.
J. Biol. Chem.
269:19200-19202[Abstract/Free Full Text].
|
| 38.
|
Marino, M. W.,
J. D. Dunbar,
L. W. Wu,
J. R. Ngaiza,
H. M. Han,
D. Guo,
M. Matsushita,
A. C. Nairn,
Y. Zhang,
R. Kolesnick,
E. A. Jaffe, and D. B. Donner.
1996.
Inhibition of tumor necrosis factor signal transduction in endothelial cells by dimethylaminopurine.
J. Biol. Chem.
271:28624-28629[Abstract/Free Full Text].
|
| 39.
|
Marsh, M.
1984.
The entry of enveloped viruses into cells by endocytosis.
Biochem. J.
218:1-10[Medline].
|
| 40.
|
Merrill, A. H., Jr., and D. D. Jones.
1990.
An update of the enzymology and regulation of sphingomyelin metabolism.
Biochim. Biophys. Acta
1044:1-12[Medline].
|
| 41.
|
Merrill, A. H., Jr.,
D. C. Liotta, and R. T. Riley.
1996.
Fumonisins: fungal toxins that shed light on sphingolipid function.
Trends Cell Biol.
6:218-223[CrossRef][Medline].
|
| 42.
|
Moesby, L.,
J. Corver,
R. K. Erukulla,
R. Bittman, and J. Wilschut.
1995.
Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner.
Biochemistry
34:10319-10324[CrossRef][Medline].
|
| 43.
|
Mooney, J. J.,
J. M. Dalrymple,
C. R. Alving, and P. K. Russell.
1975.
Interaction of Sindbis virus with liposomal model membranes.
J. Virol.
15:225-231[Abstract/Free Full Text].
|
| 44.
|
Muller, G.,
M. Ayoub,
P. Storz,
J. Rennecke,
D. Fabbro, and K. Pfizenmaier.
1995.
PKC zeta is a molecular switch in signal transduction of TNF-alpha, bifunctionally regulated by ceramide and arachidonic acid.
EMBO J.
14:1961-1969[Medline].
|
| 45.
|
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].
|
| 46.
|
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].
|
| 47.
|
Obeid, L. M.,
C. M. Linardic,
L. A. Karolak, and Y. A. Hannun.
1993.
Programmed cell death induced by ceramide.
Science
259:1769-1772[Abstract/Free Full Text].
|
| 48.
|
Okazaki, T.,
A. Bielawska,
N. Domae,
R. M. Bell, and Y. A. Hannun.
1994.
Characteristics and partial purification of a novel cytosolic, magnesium-independent, neutral sphingomyelinase activated in the early signal transduction of 1 alpha, 25-dihydroxyvitamin D3-induced HL-60 cell differentiation.
J. Biol. Chem.
269:4070-4077[Abstract/Free Full Text].
|
| 49.
|
Quintern, L. E., and K. Sandhoff.
1991.
Human acid sphingomyelinase from human urine.
Methods Enzymol.
197:536-540[Medline].
|
| 50.
|
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[CrossRef][Medline].
|
| 51.
|
Reyes, J. G.,
I. G. Robayna,
P. S. Delgado,
I. H. Gonzalez,
J. Q. Aguiar,
F. E. Rosas,
L. F. Fanjul, and C. M. R. Galarreta.
1996.
c-Jun is a downstream target for ceramide-activated protein phosphatase in A431cells.
J. Biol. Chem.
271:21375-21380[Abstract/Free Full Text].
|
| 52.
|
Santana, P.,
L. A. Pena,
A. Haimovitz-Friedman,
S. Martin,
D. Green,
M. McLoughlin,
C. Cordon-Cardo,
E. H. Schuchman,
Z. Fuks, and R. Kolesnick.
1996.
Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.
Cell
86:189-199[CrossRef][Medline].
|
| 53.
|
Schutze, S.,
K. Potthoff,
T. Machleidt,
D. Berkovic,
K. Wiegmann, and M. Kronke.
1992.
TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown.
Cell
71:765-776[CrossRef][Medline].
|
| 54.
|
Schwandner, R.,
K. Wiegmann,
K. Bernardo,
D. Kreder, and M. Kronke.
1998.
TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase.
J. Biol. Chem.
273:5916-5922[Abstract/Free Full Text].
|
| 55.
|
Strauss, J. H., and E. G. Strauss.
1994.
The alphaviruses: gene expression, replication and evolution.
Microbiol. Rev.
58:491-562[Abstract/Free Full Text].
|
| 56.
|
Ubol, S.,
S. Park,
I. Budihardjo,
S. Desnoyers,
M. H. Montrose,
G. G. Poirier,
S. H. Kaufmann, and D. E. Griffin.
1996.
Temporal changes in chromatin, intracellular calcium, and poly (ADP-ribose) polymerase during Sindbis virus-induced apoptosis of neuroblastoma cells.
J. Virol.
70:2215-2220[Abstract].
|
| 57.
|
Van Veldhoven, P. P.,
W. R. Bishop,
D. A. Yurivich, and R. M. Bell.
1995.
Ceramide quantitation: evaluation of a mixed micellar assay using E. coli diacylglycerol kinase.
Biochem. Mol. Biol. Int.
36:21-30[Medline].
|
| 58.
|
Verheij, M.,
R. Bose,
X.-H. Lin,
B. Yao,
W. D. Jarvis,
S. Grant,
M. Birrer,
E. Szabo,
L. I. Zon,
J. M. Kyriakis,
A. Haimovitz-Friedman,
Z. Fuks, and R. N. Kolesnick.
1996.
Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis.
Nature
380:75-78[CrossRef][Medline].
|
| 59.
|
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].
|
| 60.
|
Wiegmann, K.,
S. Schütze,
T. Machleidt,
D. Witte, and M. Krönke.
1994.
Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling.
Cell
78:1005-1015[CrossRef][Medline].
|
| 61.
|
Yao, B.,
Y. Zhang,
S. Delikat,
S. Mathias,
S. Basu, and R. Kolesnick.
1995.
Phosphorylation of Raf by ceramide-activated protein kinase.
Nature
378:307-310[CrossRef][Medline].
|
Journal of Virology, July 2000, p. 6425-6432, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Bok, K., Prikhodko, V. G., Green, K. Y., Sosnovtsev, S. V.
(2009). Apoptosis in Murine Norovirus-Infected RAW264.7 Cells Is Associated with Downregulation of Survivin. J. Virol.
83: 3647-3656
[Abstract]
[Full Text]
-
Ng, C. G., Coppens, I., Govindarajan, D., Pisciotta, J., Shulaev, V., Griffin, D. E.
(2008). Effect of host cell lipid metabolism on alphavirus replication, virion morphogenesis, and infectivity. Proc. Natl. Acad. Sci. USA
105: 16326-16331
[Abstract]
[Full Text]
-
Danthi, P., Kobayashi, T., Holm, G. H., Hansberger, M. W., Abel, T. W., Dermody, T. S.
(2008). Reovirus Apoptosis and Virulence Are Regulated by Host Cell Membrane Penetration Efficiency. J. Virol.
82: 161-172
[Abstract]
[Full Text]
-
Finnegan, C. M., Rawat, S. S., Cho, E. H., Guiffre, D. L., Lockett, S., Merrill, A. H. Jr., Blumenthal, R.
(2007). Sphingomyelinase Restricts the Lateral Diffusion of CD4 and Inhibits Human Immunodeficiency Virus Fusion. J. Virol.
81: 5294-5304
[Abstract]
[Full Text]
-
Ng, C. G., Griffin, D. E.
(2006). Acid Sphingomyelinase Deficiency Increases Susceptibility to Fatal Alphavirus Encephalomyelitis. J. Virol.
80: 10989-10999
[Abstract]
[Full Text]
-
Danthi, P., Hansberger, M. W., Campbell, J. A., Forrest, J. C., Dermody, T. S.
(2006). JAM-A-Independent, Antibody-Mediated Uptake of Reovirus into Cells Leads to Apoptosis. J. Virol.
80: 1261-1270
[Abstract]
[Full Text]
-
Gulbins, E., Li, P. L.
(2006). Physiological and pathophysiological aspects of ceramide. Am. J. Physiol. Regul. Integr. Comp. Physiol.
290: R11-R26
[Abstract]
[Full Text]
-
Grassme, H., Riehle, A., Wilker, B., Gulbins, E.
(2005). Rhinoviruses Infect Human Epithelial Cells via Ceramide-enriched Membrane Platforms. J. Biol. Chem.
280: 26256-26262
[Abstract]
[Full Text]
-
Wollmann, G., Tattersall, P., van den Pol, A. N.
(2005). Targeting Human Glioblastoma Cells: Comparison of Nine Viruses with Oncolytic Potential. J. Virol.
79: 6005-6022
[Abstract]
[Full Text]
-
Summers, S. A., Nelson, D. H.
(2005). A Role for Sphingolipids in Producing the Common Features of Type 2 Diabetes, Metabolic Syndrome X, and Cushing's Syndrome. Diabetes
54: 591-602
[Abstract]
[Full Text]
-
Tseng, J.-C., Hurtado, A., Yee, H., Levin, B., Boivin, C., Benet, M., Blank, S. V., Pellicer, A., Meruelo, D.
(2004). Using Sindbis Viral Vectors for Specific Detection and Suppression of Advanced Ovarian Cancer in Animal Models. Cancer Res.
64: 6684-6692
[Abstract]
[Full Text]
-
Megha, , London, E.
(2004). Ceramide Selectively Displaces Cholesterol from Ordered Lipid Domains (Rafts): IMPLICATIONS FOR LIPID RAFT STRUCTURE AND FUNCTION. J. Biol. Chem.
279: 9997-10004
[Abstract]
[Full Text]
-
Lee, J.-T., Xu, J., Lee, J.-M., Ku, G., Han, X., Yang, D.-I, Chen, S., Hsu, C. Y.
(2004). Amyloid-{beta} peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway. JCB
164: 123-131
[Abstract]
[Full Text]
-
Shrestha, B., Gottlieb, D., Diamond, M. S.
(2003). Infection and Injury of Neurons by West Nile Encephalitis Virus. J. Virol.
77: 13203-13213
[Abstract]
[Full Text]
-
Liu, Y., Cai, Y., Zhang, X.
(2003). Induction of Caspase-Dependent Apoptosis in Cultured Rat Oligodendrocytes by Murine Coronavirus Is Mediated during Cell Entry and Does Not Require Virus Replication. J. Virol.
77: 11952-11963
[Abstract]
[Full Text]
-
Tseng, J.-C., Levin, B., Hirano, T., Yee, H., Pampeno, C., Meruelo, D.
(2002). In Vivo Antitumor Activity of Sindbis Viral Vectors. JNCI J Natl Cancer Inst
94: 1790-1802
[Abstract]
[Full Text]
-
Lee, J. M., Lee, K.-H., Weidner, M., Osborne, B. A., Hayward, S. D.
(2002). Epstein-Barr virus EBNA2 blocks Nur77- mediated apoptosis. Proc. Natl. Acad. Sci. USA
99: 11878-11883
[Abstract]
[Full Text]
-
Zrachia, A., Dobroslav, M., Blass, M., Kazimirsky, G., Kronfeld, I., Blumberg, P. M., Kobiler, D., Lustig, S., Brodie, C.
(2002). Infection of Glioma Cells with Sindbis Virus Induces Selective Activation and Tyrosine Phosphorylation of Protein Kinase C delta . IMPLICATIONS FOR SINDBIS VIRUS-INDUCED APOPTOSIS. J. Biol. Chem.
277: 23693-23701
[Abstract]
[Full Text]
-
Hay, S., Kannourakis, G.
(2002). A time to kill: viral manipulation of the cell death program. J. Gen. Virol.
83: 1547-1564
[Abstract]
[Full Text]
-
Connolly, J. L., Dermody, T. S.
(2002). Virion Disassembly Is Required for Apoptosis Induced by Reovirus. J. Virol.
76: 1632-1641
[Abstract]
[Full Text]
-
Johnston, C., Jiang, W., Chu, T., Levine, B.
(2001). Identification of Genes Involved in the Host Response to Neurovirulent Alphavirus Infection. J. Virol.
75: 10431-10445
[Abstract]
[Full Text]
-
Nargi-Aizenman, J. L., Griffin, D. E.
(2001). Sindbis Virus-Induced Neuronal Death Is both Necrotic and Apoptotic and Is Ameliorated by N-Methyl-D-Aspartate Receptor Antagonists. J. Virol.
75: 7114-7121
[Abstract]
[Full Text]
Top
Abstract
Introduction
