J Virol, January 1998, p. 452-459, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Sindbis Virus Induces Apoptosis through a
Caspase-Dependent, CrmA-Sensitive Pathway
Victor E.
Nava,1
Antony
Rosen,2,3
Michael
A.
Veliuona,1
Rollie J.
Clem,1
Beth
Levine,4 and
J. Marie
Hardwick1,5,6,*
Department of Molecular Microbiology and
Immunology,1 Johns Hopkins University School of
Public Health, and
Departments of
Medicine,2
Cell Biology and
Anatomy,3
Pharmacology and Molecular
Sciences,5 and
Neurology,6 Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205, and
Department
of Medicine, Columbia University College of Physicians and Surgeons,
New York, New York 100324
Received 13 June 1997/Accepted 16 October 1997
 |
ABSTRACT |
Sindbis virus infection of cultured cells and of neurons in mouse
brains leads to programmed cell death exhibiting the classical characteristics of apoptosis. Although the mechanism by which Sindbis
virus activates the cell suicide program is not known, we demonstrate
here that Sindbis virus activates caspases, a family of death-inducing
proteases, resulting in cleavage of several cellular substrates. To
study the role of caspases in virus-induced apoptosis, we determined
the effects of specific caspase inhibitors on Sindbis virus-induced
cell death. CrmA (a serpin from cowpox virus) and zVAD-FMK
(N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone)
inhibited Sindbis virus-induced cell death, suggesting that cellular
caspases facilitate apoptosis induced by Sindbis virus. Furthermore,
CrmA significantly increased the rate of survival of infected mice.
These inhibitors appear to protect cells by inhibiting the cellular
death pathway rather than impairing virus replication or by inhibiting
the nsP2 and capsid viral proteases. The specificity of CrmA indicates
that the Sindbis virus-induced death pathway is similar to that induced
by Fas or tumor necrosis factor alpha rather than being like the death
pathway induced by DNA damage. Taken together, these data suggest a
central role for caspases in Sindbis virus-induced apoptosis.
| |
INTRODUCTION |
Sindbis virus is an alphavirus of
the Togaviridae family which causes encephalitis in mice and
thus serves as a model for closely related human encephalitic viruses.
Infection of a variety of cultured cell types with Sindbis virus
triggers programmed cell death (33). The induction of
apoptosis in neurons of mouse brains and spinal cords correlates with
the neurovirulence of the virus strain and with mortality in mice,
suggesting that induction of apoptosis may be a primary cause of death
of young mice (34). In support of this hypothesis,
overexpressed inhibitors of apoptosis, such as Bcl-2 and IAP, can
protect cultured cells from Sindbis virus-induced apoptosis, and Bcl-2
efficiently reduces mortality in mice (17, 31, 32). These
findings also raise the possibility that endogenous inhibitors of
apoptosis inhibit Sindbis virus-induced cell death, leading to a
persistent virus infection (33, 61). Encephalitis and/or a
fatal stress response may be a consequence of neuronal apoptosis
(21, 59). Alternatively, there may be multiple pathways that
work independently to cause fatal disease.
A crucial role for the caspase family of cysteine proteases in the
execution phase of programmed cell death is supported by genetic
(24, 52, 66), biochemical (29, 57), and
physiological (25) evidence. A current model proposes a
cascade of events by which caspases proteolytically activate other
caspases (35, 39, 46). More recent evidence suggests that
different death stimuli trigger the activation of a subset of upstream
caspases that possess long prodomains at their N termini (3, 41,
62). These prodomains serve to target proteases to specific
protein complexes, where the prodomains are removed by proteolysis to produce active proteases. These caspases proteolytically activate other
downstream caspases (with shorter prodomains) that cleave key
substrates to ultimately produce the characteristic apoptotic phenotype
of cell shrinkage, membrane blebbing, chromatin condensation, oligonucleosomal DNA fragmentation, and cell death (42, 53). A growing list of proteolytic substrates of the caspases have been
identified, including protein kinase C delta (18), the retinoblastoma tumor suppressor (56), fodrin (12,
38), lamins (30, 47), the nuclear immunophilin FKBP46
(1), Bcl-2 (7), and several autoantigens
(5), and they all are cleaved after an aspartate residue (P1
position). The precise role of these cleavage events is not known, but
they may either inactivate key cellular functions or produce cleavage
products with pro-death activity. The cleavage product of Bcl-2 is
potently proapoptotic (7), and cleavage of a novel protein
designated DFF was recently shown to trigger DNA fragmentation during
apoptosis (36). These proteolytic events also serve as
biochemical markers of apoptosis. Furthermore, cell death can be
inhibited with pseudosubstrate inhibitors of the caspases, such as the
cowpox virus serpin CrmA (19, 48), and synthetic peptides
such as zVAD-FMK (67). The key feature of these inhibitors
is an aspartate at the P1 position, consistent with their specificity
for caspases.
A role for caspases in viral infections is suggested by the finding
that baculovirus infection activates an apoptotic cysteine protease in
insect cells that is inhibited by the virus-encoded caspase inhibitor
p35 (2). Similar work with mutant adenoviruses has suggested
that the adenovirus protein E1A activates caspase 3 (CPP32), generating
cleaved products of poly(ADP-ribose) polymerase (PARP) (4).
In addition, PARP cleavage is detected during infection of mouse
neuroblastoma cells with Sindbis virus (60). To further study the role of these proteases in Sindbis virus-induced programmed cell death, we confirmed that Sindbis virus activates cellular caspases
and demonstrated the participation of a subset of caspases in the
execution of the apoptotic process.
| |
MATERIALS AND METHODS |
Plasmids and viruses.
Recombinant Sindbis virus expressing
CrmA (pMV19) was generating by cloning a BstEII-flanked PCR
product from a plasmid (provided by David Pickup) into the Sindbis
virus vector dsTE12Q as previously described (8, 31). A
control virus, (pSZ20), CrmA/stop was generated by inserting a nonsense
oligonucleotide linker into pMV19 after codon 55 of the CrmA open
reading frame. Recombinant viruses encoding bcl-xL and
bcl-xL/stop were described previously (8).
Wild-type (AR339) and recombinant Sindbis viruses were quantitated by
standard plaque assay on baby hamster kidney (BHK) cells.
Cell lines and cell viability.
Low-passage-number (<15) BHK
and mouse neuroblastoma cells (N18) were maintained free of
Mycoplasma contamination in Dulbecco's minimal essential
medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U
of penicillin per ml, and 100 µg of streptomycin per ml in 5%
CO2 at 37°C. Cells were seeded at 4 × 104/well in a 24-well dish and infected ~12 h later at a
multiplicity of infection (MOI) of 10. Cell viability was determined by
trypan blue exclusion, with blind counting of at least 500 cells per sample, as described elsewhere (17).
Progeny virus production.
Subconfluent BHK cell monolayers
were infected at an MOI of 10 in culture medium containing 1% fetal
bovine serum for 1 h. Exactly 1 h before each time point, the
cells were washed with 1× phosphate-buffered saline (PBS) to remove
accumulated virus, and the virus-containing supernatants were harvested
1 h later and analyzed in duplicate plaque assays. Cell
viabilities for the same infected wells were determined as described
above.
Animal studies.
The right cerebral hemispheres of 1-day-old
CD1 mice (Charles River) were inoculated with 5 × 103
PFU of CrmA and CrmA/stop recombinant viruses in 30 µl of Hanks' balanced salt solution. For mortality experiments, four separate litters were inoculated with each virus, and mortality was determined by daily observation of the mice for 21 days after infection. For virus
titration experiments, three mice per experimental group were
sacrificed at days 1, 2, 3, 6, and 10 after inoculation. Brains were
dissected and stored at 
70°C, and freeze-thawed tissues were used
to prepare 10% homogenates in Hanks' balanced salt solution for
plaque assay quantitation on BHK cells.
Detection of viral proteins.
BHK cells were seeded at 5 × 105 per well in six-well dishes and infected with
recombinant Sindbis viruses (MOI, 10). After three washes with 1× PBS,
cells were starved in 1 ml of 10% serum-supplemented methionine- and
cysteine-free medium for 1 h and metabolically labeled with 50 µCi of 35S-Translabel (ICN) per well for 45 min. Before
being harvested, the cells were washed three times with ice-cold PBS
and lysed on ice with 300 µl of a buffer containing 1% Nonidet P-40,
1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 50 mM Tris
(pH 7.5), 15 mM NaCl, and 25 µg of aprotinin per ml. To detect Sindbis virus structural proteins, 20 µl of lysate was resolved by
SDS-15% polyacrylamide gel electrophoresis (PAGE), amplified with 1 M
salicylic acid, and analyzed by autoradiography. For immunoprecipitation of Sindbis virus nonstructural proteins, labeled cell lysates were immunoprecipitated with rabbit anti-Sindbis virus
antiserum raised against nsP2 (provided by Charlie Rice) at a 1:100
dilution in a total volume of 200 µl for each 60 µl of lysate as
previously described (15).
Immunoblotting.
Lysates from BHK and N18 cells infected at
an MOI of 10 were prepared as described above, analyzed by SDS-PAGE,
and immunoblotted (ECL; Amersham) with anti-CrmA or anti-Bcl-x
antibodies (provided by David Pickup and Craig Thompson, respectively).
For detection of autoantigens, cells were washed three times with PBS
and lysed in a buffer containing 1% Nonidet P-40, 20 mM Tris (pH 7.4),
150 mM NaCl, 1 mM EDTA, and the protease inhibitors pepstatin A,
leupeptin, antipain, chymostatin, and phenylmethylsulfonyl fluoride.
Proteins were analyzed by SDS-10% PAGE prior to being subjected to
immunoblotting with affinity-purified human antibodies as described
previously (5, 6, 20).
Protease inhibitors.
BHK or N18 cells were preincubated for
2 h with 5 to 50 µM zVAD-FMK
(N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; Enzyme
Systems Products) and infected with Sindbis virus strain AR339 (MOI,
10) in the presence of zVAD-FMK. The inhibitor was replenished at ~24
h postinfection. Cells were harvested at ~48 h postinfection to
determine their viability. zFA-FMK, a fluoromethyl ketone which is
inactive against caspases, was used as a negative control.
In vitro protease assays.
A glutathione
S-transferase (GST)-CrmA fusion protein expressed from a
modified pGEX2T plasmid (provided by Emily Cheng) was purified from
bacteria, and protein concentrations were determined by the
bicinchoninic acid protein assay (Pierce). Pro-interleukin-1
(pIL-1; provided by Jennifer Lewis and Henry George) was in vitro translated by use of the TNT System (Promega). Inhibition of caspase 1 (IL-1-converting enzyme) protease activity by GST-CrmA was performed
as described previously (48); 50 pg of caspase 1 (provided by Susan Molineaux) was preincubated for 30 min at 37°C with 1 or 5 µl of purified GST-CrmA (0.5 to 2.5 µg) or GST (2.3 to 11.5 µg)
in 100 mM HEPES (pH 7.5)-10% sucrose-0.1%
3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS)-10 mM dithiothreitol and then incubated for 90 min with 2 µl
of [35S]methionine-labeled, in vitro-translated pIL-1
in a total reaction volume of 20 µl with a final concentration of
GST-CrmA or GST of >1 µM. The whole reaction product was analyzed by
SDS-PAGE and autoradiography.
To evaluate the effect of GST-CrmA on the Sindbis virus cysteine
proteinase nsP2, unlabeled, in vitro-translated Sindbis virus polyprotein nsP123 with Gly-to-Val mutations at the cleavage sites flanking nsP2 (12V; provided by Jim and Ellen Strauss) was used as the
source of active protease. A Sindbis virus construct expressing the
entire nonstructural region nsP1234 with a mutation in the active site
of nsP2 (Cys-to-Gly mutation at position 481; provided by Jim and Ellen
Strauss) was in vitro translated in the presence of
[35S]methionine and used as a substrate for active nsP2
protease. In vitro translations (TNT System; Promega) were terminated
by incubating reaction mixtures with boiled RNase A and DNase I (final concentrations, 10 µg/ml and 20 U/ml, respectively) at 37°C for 10 min. To assess the effect of CrmA on nsP2 protease activity, labeled
substrate was mixed with unlabeled active protease, as described by de
Groot et al. (14), in the presence of a molar excess (>1
µM) of GST-CrmA or GST protein alone (26). The products were analyzed by SDS-PAGE and autoradiography.
| |
RESULTS |
Caspase activation during Sindbis virus-induced programmed cell
death.
Sindbis virus induces classical apoptosis (33)
by mechanisms that are still unclear. Because cellular caspases are
thought to be important downstream mediators of apoptosis in a variety of cell death paradigms, we investigated the role of caspases in
Sindbis virus infection. To verify that caspases are activated during
infection, cells were monitored for proteolytic cleavage products of
known caspase substrates. Human sera from autoimmune patients
recognized the autoantigens NuMA, PARP, and U1-70kDa in uninfected BHK
cells and also detected their signature cleavage products of 195, 89, and 40 kDa, respectively, at 24 h postinfection (Fig.
1). These results demonstrate that
Sindbis virus infection leads to caspase activation and cleavage of
intracellular target proteins in a manner that is characteristic of
other cell death stimuli (5). Digestion of U1-70kDa and PARP
by recombinant caspase 3 in vitro produces the same proteolytic
products as those detected in apoptotic cells, suggesting that caspase
3 may be the responsible protease in dying cells (5, 50).
However, caspase 3 knockout mice remain capable of cleaving PARP
(27). Therefore, other, related caspases may also be
involved in cleaving these substrates during apoptosis.
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FIG. 1.
Cleavage of cellular proteins by caspases during Sindbis
virus-induced apoptosis. BHK cells were mock infected or infected (MOI,
10) with wild-type Sindbis virus strain AR339. Lysates were collected
at 24 h postinfection and immunoblotted with three different
affinity-purified antibodies obtained from the sera of individuals with
autoimmune disease. The positions of intact NuMA, PARP, and U1-70kDa
proteins and their cleavage products are indicated. The results are
representative of two independent experiments. The positions of
molecular size markers (in kilodaltons) are shown on the left.
|
|
The caspases are expressed as precursors that must be cleaved to become
active proteases (40). The sequence of these activating cleavage sites, together with biochemical data, indicates that caspases
are proteolytically activated either autocatalytically or by other
caspases, suggesting that there is a cascade of proteolytic events
leading to activation of perhaps several caspases to facilitate cell
death. The death signal resulting from ligation of Fas by Fas ligand or
a Fas antibody, is propagated by recruiting FLICE/MACH/Mch5 (caspase
8), via its prodomain, to a protein complex bound to the cytoplasmic
domain of Fas (3, 41). Caspase 8 becomes activated, perhaps
autocatalytically, and subsequently cleaves and activates downstream
caspases, including caspase 3 (41, 42). In vitro, CrmA
specifically inhibits caspase 8 and caspase 1 over other caspases
tested (44, 68), implicating the involvement of these
proteases in Sindbis virus-induced apoptosis (Fig.
8). Although a role for caspase 1 in
apoptosis is still controversial, virus-induced activation of caspase 1 is consistent with induction of IL-1 during a Sindbis virus
infection (63).
Although CrmA is a cysteine protease inhibitor, its specificity for
caspases, as dictated by the presence of an Asp at the CrmA reactive
site, strongly suggests that CrmA could not interfere with either the
serine protease in the Sindbis virus capsid protein or the cysteine
protease found in nsP2. This is an important issue because these
proteases are essential for viral replication (55). Furthermore, a mutation in the protease domain of the nsP2 gene, resulting in a single amino acid change (Pro726Ser), produces a virus
that fails to kill cells and establishes a persistent infection of BHK
cells (16). However, because CrmA does not appear to have a
direct or indirect effect on nsP2 or capsid, CrmA is not likely to
impair cell killing by interfering with viral protease activity.
Several pieces of information support this conclusion. (i) The viral
protease cleavage sites do not contain an aspartate at the P1 position.
The capsid protease of Sindbis virus cleaves after the sequence TEEW,
which has no resemblance to caspase cleavage sites (e.g., DEVD or
YVAD). Based on the sequences of 10 alphaviruses, the nsP2 protease
cleaves following the consensus sequence XAG(A/G) (55), and
substitution of the P1 glycine for valine at the nsP3-nsP4 cleavage
site in Sindbis virus abolishes cleavage (14). (ii)
Experimental results presented here demonstrate that CrmA did not cause
an accumulation of viral nonstructural precursors, and the viral
structural proteins accumulated normally between 0 and 11 h
postinfection. (iii) Purified CrmA protein failed to interfere with
cleavage of the nsP3nsP4 site by nsP2 in vitro.
The role of CrmA in cowpox virus infections is to reduce the host
immune response by blocking production of IL-1, and recent evidence
suggests that CrmA may also prevent apoptosis of cowpox virus-infected
cells (48, 49). Likewise, other antiapoptotic genes encoded
by large DNA viruses appear to be required for completion of the virus
replication cycle. Deletion of p35 from baculovirus or deletion of E1B
19K from adenovirus severely reduces progeny virus production because
the cells die prematurely from apoptosis (11, 64). Several
herpesviruses encode homologs of the cellular bcl-2 gene, a
potent inhibitor of a wide variety of death stimuli (9, 23,
43). In contrast to these viruses, Sindbis virus appears to
thrive in apoptotic cells, presumably in part because the replication
cycle of Sindbis virus is as short as 4 h (Fig. 4), allowing
abundant virus production prior to cell death. In fact, overexpressed
Bcl-2 appears to suppress Sindbis virus replication both in
overexpressing cell lines and in mouse brains infected with the Sindbis
virus vector expressing Bcl-2 (31, 33, 61). Likewise, Bcl-2
can slow the replication of influenza virus, Semliki Forest virus, and
human immunodeficiency virus (45, 51, 54). Thus, some
viruses may prefer to replicate in the milieu of an apoptotic cell. In
contrast to these findings with Bcl-2, we detected only slight
reductions in Sindbis virus replication when caspase inhibitors were
present (Fig. 2 to 4 and 6). One possible explanation for this
discrepancy is that caspase inhibitors function downstream of Bcl-2
(10). (The effect of Bcl-xL overexpression on
viral replication in mouse brains is currently under study.) Thus, it remains possible that viral persistence in mouse brains is facilitated not only by the presence of endogenous apoptosis inhibitors but also by
a reduction in virus replication.
We thank Emily Cheng for the GST-CrmA plasmid, Ellen and Jim
Strauss for plasmids C481G and 12V, and Yukio Shirako for advice with
the transcleavage assays. We thank Lesia Dropulic, Jennifer Lewis, and
Diane Griffin for helpful suggestions and protocols and Shifa Zou for
excellent technical assistance.
This work was supported by NIH grants NS34175 (J.M.H.) and AI40246
(B.L.) and by a James S. McDonnell Foundation Scholar award (B.L.).
A.R. is a Pew Scholar in the Biomedical Sciences. V.E.N. is funded in
part by the Consejo Nacional de Investigaciones Cientificas de
Venezuela. R.J.C. is a Postdoctoral Fellow of the American Cancer
Society.
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Abstract
Introduction
, nerve growth factor withdrawal, and extracellular matrix
disruption but does not inhibit cell death induced by DNA-damaging agents or staurosporine. However, both the CrmA-dependent and CrmA-independent pathways lead to activation of caspase 3, a
more-downstream protease in the cascade (13, 42). Because
CrmA does not inhibit caspase 3 effectively, CrmA may be working to
protect cells by affecting upstream proteases. However, the lack of
specific inhibitors that can definitively distinguish individual
caspases makes the task of delineating the cell type- or
stimulus-specific death pathways difficult. Nevertheless, we found that
the Sindbis virus-induced death pathway is sensitive to both p35
(43a) and CrmA. Thus, Sindbis virus-induced apoptosis may
share upstream components of the death pathway mediated by Fas, TNF,
and nerve growth factor withdrawal that do not involve CrmA-resistant
proteases (Fig. 8). However, consistent with our study on Sindbis
virus, transfected CrmA and zVAD-FMK treatment do not block cell death
indefinitely, which has been assumed to mean that these inhibitors are
unable to stave off all of the intracellular caspases. Nevertheless, this observation leaves open the possibility that caspase-independent pathways are also operational in Sindbis virus-infected cells (Fig. 8).
