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Previous Article | Next Article 
J Virol, July 1998, p. 5654-5660, Vol. 72, No. 7
Laboratory of Genetics, The Salk Institute,
San Diego, California 92186-5800,1 and
Department of Medicine, University of California, San Diego,
California 92093-06132
Received 25 November 1997/Accepted 23 March 1998
Apoptosis is a central host defense mechanism to eliminate
virus-infected cells. Activation of NF- Programmed cell death, or apoptosis,
is a fundamental biochemical process that plays an essential role in
normal development and tissue homeostasis (15, 34).
Apoptosis is also utilized by the host to defend against invading
microbes. In particular, the relationship between viruses and apoptosis
has been well established, and the subject has been reviewed (37,
44, 46). Viruses can trigger apoptosis through recognition by
cytotoxic T cells, by viral disruption of cellular metabolism and cell
cycle regulation, and by induction of proinflammatory cytokines such as
tumor necrosis factor (TNF) (44). Perhaps the best evidence
demonstrating the importance of apoptosis in controlling viral
replication are the many different strategies that viruses have evolved
to avoid host programmed cell death. Virus-encoded antiapoptotic
proteins include Bcl-2 homologs, like adenovirus E1B, that indirectly
inhibit activation of caspases (2, 9, 10, 14, 35), cowpox
virus CrmA and baculovirus p35, which are direct inhibitors of the
caspases (13, 36, 55), and a new class of proteins called
viral FLICE-inhibitory proteins (v-FLIPs) encoded by gammaherpesviruses
and poxvirus (8, 47). Infection of cells with mutant viruses
lacking these genes often results in premature apoptotic death and
altered yields of progeny virus, indicating that antiapoptotic proteins
are necessary for efficient virus replication (11, 23, 33).
In addition, suppression of apoptotic cell death is important for viral
persistence (26) and may be required for the establishment
of certain latent infections (22). Therefore, a better
understanding of the signaling pathways important for apoptosis of
virally infected cells will provide further insight into virus
replication and persistence and facilitate development of novel drug
targets.
NF- In their initial reporting of mice with targeted disruption of the
NF- Accordingly, we present data showing that loss of p50 signaling during
picornavirus infection can attenuate the virulence of EMCV infection in
vivo during the acute phase of viral replication through an
IFN- Cell and cell culture.
The cells used in these studies were
L929 cells (24) (a kind gift from S. A. Huber,
University of Vermont, Burlington) and primary S129 wild-type, p50
Virus and viral assays.
EMCV was isolated from murine heart
and was a kind gift from S. A. Huber (24). Infected
murine hearts were aseptically harvested and homogenized in
high-glucose Dulbecco's modified Eagle medium supplemented with 2%
heat-inactivated fetal bovine serum; after three cycles of
freeze-thawing for release of intracellular virus, the supernatant was
used for plaque-forming assay, with titers determined on L929 cells
(24). For in vitro studies, EMCV was passaged through L929
cells and MEF were infected at a multiplicity of infection of 5. After
30 min of incubation and three washing steps to remove residual virus,
new medium was added to the cells; at the time points indicated,
supernatants were assayed for viral progeny.
Mice.
Wild-type S129, IFNRI Annexin V assay.
At various time points after infection,
cells were harvested by trypsinization, pelleted, resuspended in
Annexin V buffer (29), washed, and then resuspended in
Annexin V buffer containing 1 µg of Annexin V-fluorescein
isothiocyanate (Bender Med Systems, Vienna, Austria) per ml. After 30 min of incubation at room temperature, the cells were immediately
analyzed with FACScan (Becton Dickinson, San Jose, Calif.) and LYSIS II
software.
Assays for DNA fragmentation.
To detect host cell DNA
degradation, total cellular DNA was collected by using a Puregene DNA
isolation kit (Gentra Systems, Minneapolis, Minn.), and 3 µg of DNA
per lane was run on a 1.5% agarose gel. The gel was stained with
ethidium bromide and photographed. To detect DNA strand breaks in situ,
cells were grown on Lab-Tek two-chamber slides (Nalgene, Naperville,
Ill.), and the TUNEL (terminal deoxynucleotidyltransferase-mediated
dUTP nick end labeling) assay was performed by using an Apotag Plus kit
(Oncor, Gaithersburg, Md.), with the following modifications
(32): prior to the terminal deoxytidyltransferase reaction,
endogenous alkaline phosphatase was quenched with levamisole (Vector
Laboratories, Burlingame, Calif.), and the detection system was changed
to alkaline phosphatase, using anti-digoxigenin-alkaline phosphatase
Fab fragments (Boehringer Mannheim, Indianapolis, Ind.) diluted 1:500
in phosphate-buffered saline containing 3% bovine serum albumin
(Sigma) and Vector Red (Vector) as a substrate.
Microscopy and immunocytochemistry.
EMCV-infected cells were
identified with murine monoclonal antibody 10D3 (18) at a
1:400 dilution in phosphate-buffered saline-3% bovine serum albumin
(a kind gift from Ann C. Palmenberg, University of Wisconsin, Madison).
Antibody 10D3 was originally raised against mengovirus 3D polymerase
but exhibits strong cross-reactivity against the 3D polymerase of EMCV
(18). Bound antibody was detected with polyclonal
fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G
at a 1:100 dilution (Jackson Immunoresearch Laboratories, West Grove,
Pa.). Nuclear morphology was visualized with Hoechst 33342 dye (Sigma)
at 1 µg/ml for 2 min. Finally, slides were mounted with Vectashield
mounting medium (Vector) and imaged in an MRC 1024 confocal microscope
(Bio-Rad, Hercules, Calif.) equipped with Lasersharp software.
Deficiency of the p50 subunit of NF-
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
NF-
B-Mediated Inhibition of Apoptosis Is
Required for Encephalomyocarditis Virus Virulence: a Mechanism of
Resistance in p50 Knockout Mice
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B suppresses apoptosis following some types of stimulation in vitro. To test the physiological importance of this pathway in vivo, we studied murine
encephalomyocarditis virus (EMCV) infection in mice and cell lines
defective in NF-B1 (p50) signaling. As previously reported, we find
that all p50 knockout (p50 
/) mice survive an EMCV infection that
readily kills normal mice. By introducing the p50 mutation into
interferon (IFN) type I receptor knockout (IFNRI /) mice, we find
that this resistance is not mediated by IFN-
as previously thought. While no IFNRI / mice survive, the double-knockout mice survive 60% of the time. The survival is tightly linked to the animals' ability to clear the virus from the heart in vivo. Using murine embryonic fibroblasts (MEF) derived from wild-type, p50 /, and p65
/ embryos, we found that NF-B is not required for the
replication cycle of EMCV. However, during these experiments we
observed that p50 / and p65 / MEF infected with EMCV
undergo enhanced, premature cytotoxicity. Upon examination of this cell
death, we found that EMCV infection induced both plasma membrane and
nuclear changes typical of apoptosis in all cell lines. These apoptotic
processes occurred in an accelerated and pronounced way in the
NF-B-defective cells, as soon as 6 h after infection, when
virus is beginning to be released. Previously, only the RelA (p65)
subunit of NF-B has been shown to play a role in suppressing
apoptosis. In our studies, we find that p50 is equally important in
suppressing apoptosis during EMCV infection. Additionally, we show that
suppression of apoptosis by NF-B1 is required for EMCV virulence in
vivo. The attenuation in p50 / mice can be explained by rapid
apoptosis of infected cells which allows host phagocytes to clear
infected cells before the viral burst leading to a reduction of the
viral burden and survival of the mice.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B is a transcription factor central to immune and inflammatory
responses as well as viral replication (3, 4, 49, 52). Viral
infection or cell stimulation by proinflammatory cytokines like TNF-
allow rapid nuclear translocation of NF-B through degradation of
IB inhibitory cytoplasmic retention proteins. A dominant negative
IB that lacks both constitutive (5, 42) and inducible
(12, 49) phosphorylation sites is capable of eliminating
NF-B activity in transfected cells and rendering the cells highly
susceptible to TNF--induced apoptosis (50). Similar
results were obtained by stimulating p65 / mouse embryo fibroblasts (MEF) with TNF- (6). These experiments
demonstrate that activation of NF-B inhibits some forms of
apoptosis. In its absence, the cell dies. Less is known regarding the
role of p50 in NF-B-mediated inhibition of apoptosis. At present,
the importance of the NF-B signaling pathway in vivo is still in question.
B1 subunit, p50 (p50 / mice), Sha et al. discovered that
while these mice are more susceptible to certain bacterial pathogens,
they are resistant to murine encephalomyocarditis virus (EMCV)
infections; which cause myocarditis and dilated cardiomyopathy and
which kill normal healthy mice (43, 53). They suggested that
the resistance may be caused by elevated beta interferon (IFN-)
levels in the mice. While IFN- signaling is clearly important for
control of picornavirus replication, another possibility to explain the
inhibition of virulence following EMCV infection of p50 / mice is
that p50 / cells undergo premature apoptosis during viral
infection. Recent data suggests that under certain conditions and in
certain cell types infection with picornaviruses can activate markers
of apoptosis (25, 48). Since NF-B is activated with
picornavirus infection (56), we hypothesized that the
attenuation of EMCV virulence in p50 / mice is related to early
induction of markers of apoptosis and cell death from the lack of
NF-B signaling and can therefore occur independent of type I IFN
(IFN-/) signaling. Premature activation of apoptosis in infected
cells in vivo may limit viral replication through phagocytosis of
infected apoptotic cells (19, 21, 31).
/-independent mechanism. This was done by showing that mice
deficient in type I IFN receptor (IFNRI) signaling (IFNRI / mice)
are extremely sensitive to EMCV infection, while p50 / IFNRI /
double-knockout mice exhibit a significant resistance to EMCV
infection. Additionally, we show that p50 is not required for viral
replication and that EMCV infection of cells that lack p50 results in
accelerated cell death with activation of markers of apoptosis. This
finding suggests that NF-B signaling is required for inhibition of
apoptosis and of premature cell death and that these mechanisms are
required for unrestricted viral replication in vivo and for EMCV
virulence.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/, and p65 / MEF (7, 43) (kind gifts from D. Baltimore). The cells were cultured in Dulbecco's modified Eagle
medium with high glucose (4,500 mg/liter) supplemented with 10%
heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 2 mM
L-glutamine, streptomycin (100 mg/ml), and penicillin G
(100 U/ml) (Irvine Scientific, Santa Ana, Calif.) at 37°C and 5%
CO2 under humidified conditions. Cells were routinely
subcultured every 2 to 3 days.
/ (30), and p50
/ (43) mice were maintained in a germ-free environment
and housed in microisolators during the challenges. Double-knockout
mice lacking both the IFNRI and the p50 subunit of NF-B (p50 /
IFNRI / mice) were generated, and the genotype of the offspring was
verified by PCR. At 4 to 6 weeks of age, mice were challenged with an
intraperitoneal (i.p.) injection of EMCV (103 PFU) as
previously described (41, 43). These mice were monitored daily for the survival study or sacrificed 2 days later for the measurements of viral titers.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B in vivo can limit
the early phase of EMCV replication and confer resistance by an
IFNRI-independent signaling mechanism.
A previous report
demonstrates that p50 / mice are highly resistant to EMCV infection
(43). The precise mechanism for this resistance to infection
has not been thoroughly established. To determine in vivo
whether the attenuated virulence in EMCV-infected mice might be
secondary to more rapid induction of apoptosis during the early
phase of viral replication, through an IFNRI-independent mechanism,
we bred p50 / mice with IFNRI / mice. The
resulting double-knockout animals were infected with EMCV, and the
mortality data from these experiments are shown in Fig.
1A. As expected, the p50 / mice had
no significant mortality upon an i.p. injection with
103 PFU of EMCV, while the IFNRI / mice had
100% mortality by day 3 postinfection. Surprisingly, the p50 /
IFNRI / double-knockout mice survived 60% of the time, while the
wild-type mice had only a 20% survival rate by day 5. Mice
heterozygous for the p50 knockout and homozygous for the IFNRI knockout
(p50 +/ IFNRI / mice) had an intermediate mortality (data not
shown), demonstrating a gene dose effect.
View larger version (13K):
[in a new window]
FIG. 1.
Survival curves and viral titers in the hearts of mice
infected with EMCV. (A) Wild-type (wt), p50 /, IFNRI /, and
double-knockout (p50 / IFNRI /) mice, 4 to 6 weeks
old, were challenged with an i.p. injection of 103 PFU of
EMCV. Survival was monitored at daily intervals. Ten mice were infected
in each group. (B) Viral titers in the hearts of mice at day 2 after
infection with EMCV. Viral titers were determined as detailed in
Materials and Methods. Titers are expressed as PFU per milligram of
cardiac tissue. Data points from each heart are shown.
/ mice is related to
the ability of the virus to replicate in vivo (Fig. 1B). This time
point is prior to activation of a significant cellular immune response
and is a measure of the initial phase of viral replication
(57). Wild-type mice consistently had titers of about
105 to 106 PFU per mg of cardiac tissue. The
p50 / mice could generate only significantly lower titers, with 2 to 3 orders of magnitude fewer PFU/milligram of cardiac tissue, and in
some cases we were unable to recover any virus from the hearts of these
animals. The p50 / IFNRI / mice had slightly higher titers than
the p50 / animals, which were still significantly lower than those found in the wild-type mice.
From these data, we conclude that IFN- and - are important for
protection against EMCV, but the attenuated virulence conferred by
targeted disruption of p50 is independent of IFN-/ signaling. These data also demonstrate that EMCV can replicate in p50 / mice;
however, in these animals the virus is unable to reach titers that are
associated with mortality.
NF-B is not required for the replication cycle of EMCV.
One
potential explanation for the in vivo results for p50 / mice is
that p50 is required for efficient EMCV replication. To test this
possibility, we determined in vitro the kinetics of EMCV replication in
infected MEF derived from wild-type and p50 / mice. Wild-type, p50
/, and p65 / cells were able to support productive viral
replication to comparable levels (Fig. 2), as were cells expressing a dominant
negative IB (data not shown). The viral burst occurred between 6 and 12 h postinfection. These results demonstrate that EMCV can
establish a productive infection independent of NF-B signaling.
|
NF-B-deficient cells die prematurely with EMCV infection.
Recently it has been shown that mammalian cells require p65 to induce
an antiapoptotic response following TNF- stimulation (6, 28,
50, 52). Therefore, we hypothesized that the altered virulence
observed in p50 / mice might be related to the ability of the virus
to induce cell death, thus limiting viral replication in vivo.
/ and p65 / MEF showed
evidence of cell death (Fig. 3B), similar to results for cells
expressing a dominant negative IB (Fig. 3C).
|
EMCV infection induces markers of apoptosis in MEF.
Since
picornaviruses can activate both lytic and apoptotic features of cell
death (25, 40, 48), we sought to demonstrate that EMCV
infection could activate markers of apoptosis. To this end, we
investigated nuclear hallmarks of apoptosis in EMCV-infected MEF.
Morphologically, nuclei of infected cells from wild-type and p50 /
MEF displayed nuclear shrinkage with chromatin condensation and
margination as well as formation of apoptotic bodies with DNA strand
breaks, as evidenced by in situ TUNEL staining 12 h postinfection
(Fig. 4A to F). Quantitative flow
cytometry of TUNEL-stained cells demonstrated that 70% of infected
cells became TUNEL positive at 12 h postinfection, compared to
less than 10% of uninfected cells (data not shown). Furthermore, EMCV
infection led to degradation of host cell genomic DNA, as shown by gel
electrophoresis (Fig. 4G). There was degradation of genomic DNA in
EMCV-infected p50 / MEF before it was observed in infected
wild-type MEF. Significant degradation of genomic DNA does not occur in
wild-type MEF until 18 h postinfection. From these results, we
conclude that EMCV infection of MEF induces an apoptotic phenotype with
nuclear changes typical of apoptosis and DNA degradation that occurs
earlier in cells that lack p50.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
During infection, many viruses, including picornaviruses, induce a
cytopathic effect ultimately leading to host cell death and release of
viral progeny (40). In principal, an infected cell can die
like any other cell, of apoptosis or lysis or a combination of these
two pathways (16). Picornavirus infection often results in
lysis of the host cell (40, 48). However, under
nonpermissive conditions (i.e., infection with a guanidine-sensitive
virus variant in the presence of guanidine), poliovirus has been
reported to induce apoptosis of the host cell (48), and
recently coxsackievirus B3 infection of cultured cardiac myocytes has
been shown to induce markers of apoptosis (25). However,
little is known regarding the role of apoptotic mechanisms during
infection with picornaviruses. Our data indicate that NF-B signaling
is required to prevent premature death of infected cells and is
required for in vivo virulence by an IFN-/-independent mechanism.
Given the important antiviral properties of IFN-/ we anticipated
that mice that lacked the IFNRI would have early and consistent mortality by days 2 to 3 after EMCV infection, a time point that precedes activation of the cellular immune response (39,
57). Based on the fact that loss of NF-B leads to accelerated
apoptosis (6, 7, 50), we hypothesized that if disruption of
p50 increased the rate of apoptotic cell death in infected mice that lacked the IFNRI, there would be an improvement in survival of the
infected mice in spite of loss of IFNRI signaling. If such were the
case, EMCV titers would be lower in infected mice during the early
phase of viral replication. Our results are consistent with this
hypothesis, demonstrating a substantial improvement in survival in
IFNRI / mice that also lacked p50 and showing that survival from
the EMCV challenge was linked to the animals' ability to limit viral
replication.
To identify the mechanism(s) responsible for these in vivo findings, we
studied p50-deficient MEF in culture. EMCV is able to establish a
productive viral infection in MEF lacking the p50 subunit of NF-B
that is similar to that which occurs in wild-type MEF, indicating that
the p50 subunit of NF-B is not required for EMCV infectivity and
replication.
In support of the hypothesis that EMCV infection of p50-deficient cells
leads to earlier cell death, we noted that p50 / MEF had
an accelerated cytopathic effect with premature cell death compared to
the wild-type cells. This was apparent at the microscopic level, with
detachment of cells from the plate, and was quantitated by flow
cytometric analysis of Annexin+ cells. Since death of
virally infected cells can be mediated by lysis and/or apoptosis, and
since cells which are initially apoptotic later progress to have
disrupted cell membranes, we quantitated the rate of total cell death
by using Annexin V. Annexin+ cells include cells in the
early stages of apoptosis with externalized phosphatidylserine
(29) and cells that are at a late stage in the apoptotic
process with disrupted cell membranes or have lysed secondary to viral
replication. Most importantly, this assay has been demonstrated to
reliably predict a cell's susceptibility to phagocytosis in vivo
(21).
In addition to the premature cell death that was observed in p50 /
MEF, it was clear that infected cells also had an increase of both
membrane and nuclear alterations consistent with apoptosis. The cell
death and induction of apoptosis in EMCV-infected p50 / mice are
very similar to that observed with mutant adenoviruses lacking the E1B
19-kDa protein. This protein is a homolog of the cellular
proto-oncoprotein Bcl-2 and suppresses apoptosis caused by the
adenovirus E1A protein (35). Infections with viruses carrying the E1B 19-kDa protein deletion lead to a phenotype
characterized by premature and enhanced cytopathic effect as well as
degradation of host cell genomic DNA (33). The DNA
degradation upon adenovirus infection can be observed as either a smear
or a ladder of chromosomal DNA, depending on multiple parameters such
as the multiplicity of infection, the host cell type used, and the time
course of the infection (54). Subsequent experiments with
adenovirus and adenovirus gene products have shown that both forms of
DNA degradation are due to apoptosis (35). It is also known
that in many model systems of apoptosis, oligonucleosomal DNA fragments
may be apparent only after more than 24 h following initiation of
apoptosis (1). Taken together, our results show that
EMCV-infected MEF exhibit nuclear changes typical of apoptosis, such as
chromatin alterations, DNA strand breaks, and degradation of the
genomic DNA. However, the cells do not reach the later stages of
apoptosis because of the simultaneous viral replication that lyses the
cells. While previous experiments have clearly demonstrated that p65 is
required to suppress apoptosis following stimulation with TNF-, this
is the first example of a stimulus that can induce accelerated cell death in p50-deficient cells.
The data for cultured MEF make a compelling argument that the
protective effect in the p50 / mice is secondary to accelerated cell death with activation of markers of apoptosis. Inhibition of
apoptosis is a well-recognized strategy by which viruses can evade one
component of the host defense system. The difference in the kinetics of
cell death and activation of apoptotic markers following infection may
be very relevant to viral production in vivo. It is known that
apoptotic cells in vivo are cleared by phagocytic cells in a way that
toxic cellular components (i.e., virus) are not released (19,
31). Reports in the literature have described at least five
separate membrane changes that may lead to recognition of apoptotic
cells by phagocytes (29). Of particular relevance to this
study are the convincing data for the involvement of phosphatidylserine
in the recognition of apoptotic cells by macrophages (20,
21). Thus, premature apoptosis with externalization of
phosphatidylserine in p50-deficient cells may allow clearance of
infected cells by host phagocytes before the virus is able to complete
its full replication cycle. In this way, apoptosis acts as a defense
mechanism used by the host to limit viral spread and to allow survival
of the mice upon picornavirus infection.
In summary, EMCV infection of MEF induces a cytopathic effect that
displays features of both apoptotic and lytic cell death. Thus, a
picornavirus infection can induce markers of apoptosis in the host
cell. The observed apoptotic events included plasma membrane and
nuclear changes characteristic of apoptosis. Cell death following
picornavirus infection is accelerated in cells lacking the p50 subunit
of NF-B. A likely explanation for our findings in vivo and in vitro
is that NF-B is required for the expression of yet unknown
antiapoptotic survival genes during EMCV infection. This may explain
the resistance to EMCV in the p50 / mice that occurs during early
phases of viral replication independent of IFNRI signaling. It will be
of great interest if future studies can provide us with a method to
capitalize on this mechanism as novel drug targets for the treatment of
viral heart disease.
| |
ACKNOWLEDGMENTS |
|---|
Edward M. Schwarz and Cornel Badorff contributed equally to this project.
We thank D. Baltimore, S. A. Huber, and A. C. Palmenberg for the kind provision of reagents used in this study. We are indebted to D. Young and D. Peterson for assistance with flow cytometry and confocal microscopy, respectively.
This work was supported by grants from the Arthritis Foundation to E.M.S., grant Ba 1668/1-1 from the Deutsche Forschungsgemeinschaft to C.B., and grants from the American Heart Association (96-303A) and UCSD Biotechnology Star Project (S96-38) to K.U.K. I.M.V. is an American Cancer Society Professor of Molecular Biology and receives funding from an OIG award from the National Institutes of Health.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Medicine, 0613c, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0613. Phone: (619) 822-1363. Fax: (619) 822-1365. E-mail: kknowlton{at}ucsd.edu.
Present address: Immunology/Rheumatology Unit, University of
Rochester, Rochester, NY 14642.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. | Ankarcrona, M., J. M. Dypbukt, E. Bonfoco, B. Zhivotovsky, S. Orrenius, S. A. Lipton, and P. Nicotera. 1995. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15:961-973[Medline]. |
| 2. |
Armstrong, R. C.,
T. Aja,
J. Xiang,
S. Gaur,
J. F. Krebs,
K. Hoang,
X. Bai,
S. J. Korsmeyer,
D. S. Karanewsky,
L. C. Fritz, and K. J. Tomaselli.
1996.
Fas-induced activation of the cell death-related protease CPP32 Is Inhibited by Bcl-2 and by ICE family protease inhibitors.
J. Biol. Chem.
271:16850-16855 |
| 3. | Baeuerle, P. A., and T. Henkel. 1994. Function and activation of NF-kappa B in the immune system. Annu. Rev. Immunol. 12:141-179[Medline]. |
| 4. | Baldwin, A. S., Jr. 1996. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649-683[Medline]. |
| 5. |
Barroga, C. F.,
J. K. Stevenson,
E. M. Schwarz, and I. M. Verma.
1995.
Constitutive phosphorylation of I kappa B alpha by casein kinase II.
Proc. Natl. Acad. Sci. USA
92:7637-7641 |
| 6. |
Beg, A. A., and D. Baltimore.
1996.
An essential role for NF-kappaB in preventing TNF-alpha-induced cell death.
Science
274:782-784 |
| 7. | Beg, A. A., W. C. Sha, R. T. Bronson, S. Ghosh, and D. Baltimore. 1995. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 376:167-170[Medline]. |
| 8. |
Bertin, J.,
R. C. Armstrong,
S. Ottilie,
D. A. Martin,
Y. Wang,
S. Banks,
G. H. Wang,
T. G. Senkevich,
E. S. Alnemri,
B. Moss,
M. J. Lenardo,
K. J. Tomaselli, and J. I. Cohen.
1997.
Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis.
Proc. Natl. Acad. Sci. USA
94:1172-1176 |
| 9. | Boulakia, C. A., G. Chen, F. W. Ng, J. G. Teodoro, P. E. Branton, D. W. Nicholson, G. G. Poirier, and G. C. Shore. 1996. Bcl-2 and adenovirus E1B 19 kDa protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose)-polymerase. Oncogene 12:529-535[Medline]. |
| 10. | Boyd, J. M., S. Malstrom, T. Subramanian, L. K. Venkatesh, U. Schaeper, B. Elangovan, C. D'Sa-Eipper, and G. Chinnadurai. 1994. Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell 79:341-351[Medline]. |
| 11. | Brooks, M. A., A. N. Ali, P. C. Turner, and R. W. Moyer. 1995. A rabbitpox virus serpin gene controls host range by inhibiting apoptosis in restrictive cells. J. Virol. 69:7688-7698[Abstract]. |
| 12. |
Brown, K.,
S. Gerstberger,
L. Carlson,
G. Franzoso, and U. Siebenlist.
1995.
Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation.
Science
267:1485-1488 |
| 13. |
Bump, N. J.,
M. Hackett,
M. Hugunin,
S. Seshagiri,
K. Brady,
P. Chen,
C. Ferenz,
S. Franklin,
T. Ghayur,
P. Li, et al.
1995.
Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35.
Science
269:1885-1888 |
| 14. |
Chinnaiyan, A. M.,
K. Orth,
O. R. K. H. Duan,
G. G. Poirier, and V. M. Dixit.
1996.
Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3-like apoptotic proteases.
J. Biol. Chem.
271:4573-4576 |
| 15. | Cohen, J. J., R. C. Duke, V. A. Fadok, and K. S. Sellins. 1992. Apoptosis and programmed cell death in immunity. Annu. Rev. Immunol. 10:267-293[Medline]. |
| 16. | Darzynkiewicz, Z., G. Juan, X. Li, W. Gorczyca, T. Murakami, and F. Traganos. 1997. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1-20[Medline]. |
| 17. | DiDonato, J. A., M. Hayakawa, D. M. Rothwarf, E. Zandi, and M. Karin. 1997. A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB. Nature 388:548-554[Medline]. |
| 18. | Duque, H., and A. C. Palmenberg. 1996. Epitope mapping of monoclonal antibodies raised to recombinant mengo 3D polymerase. Virus Genes 13:159-168[Medline]. |
| 19. | Duvall, E., A. H. Wyllie, and R. G. Morris. 1985. Macrophage recognition of cells undergoing programmed cell death (apoptosis). Immunology 56:351-358[Medline]. |
| 20. | Fadok, V. A., J. S. Savill, C. Haslett, D. L. Bratton, D. E. Doherty, P. A. Campbell, and P. M. Henson. 1992. Different populations of macrophages use either the vitrionectin reseptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. J. Immunol. 149:4029-4035[Abstract]. |
| 21. | Fadok, V. A., D. R. Voelker, P. A. Campbell, J. J. Cohen, D. L. Bratton, and P. M. Henson. 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148:2207-2216[Abstract]. |
| 22. | Gregory, C. D., C. Dive, S. Henderson, C. A. Smith, G. T. Williams, J. Gordon, and A. B. Rickinson. 1991. Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis. Nature 349:612-614[Medline]. |
| 23. |
Hershberger, P. A.,
J. A. Dickson, and P. D. Friesen.
1992.
Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication.
J. Virol.
66:5525-5533 |
| 24. |
Huber, S. A.
1994.
VCAM-1 is a receptor for encephalomyocarditis virus on murine vascular endothelial cells.
J. Virol.
68:3453-3458 |
| 25. | Jeon, E.-S., Z. Sheng, and K. U. Knowlton. 1996. Coxsackievirus induces myocyte apoptosis. Circulation 94(Suppl. I):I-356. |
| 26. | 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]. |
| 27. | Liou, H. C., and D. Baltimore. 1993. Regulation of the NF-kappa B/rel transcription factor and I kappa B inhibitor system. Curr. Opin. Cell Biol. 5:477-487[Medline]. |
| 28. | 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-kappaB activation prevents cell death. Cell 87:565-576[Medline]. |
| 29. |
Martin, S. J.,
C. P. Reutelingsperger,
A. J. McGahon,
J. A. Rader,
R. C. van Schie,
D. M. LaFace, and D. R. Green.
1995.
Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.
J. Exp. Med.
182:1545-1556 |
| 30. |
Muller, U.,
U. Steinhoff,
L. F. Reis,
S. Hemmi,
J. Pavlovic,
R. M. Zinkernagel, and M. Aguet.
1994.
Functional role of type I and type II interferons in antiviral defense.
Science
264:1918-1921 |
| 31. |
Newman, S. L.,
J. E. Henson, and P. M. Henson.
1982.
Phagocytosis of senescent neutrophils by human monocyte derived macrophages and rabbit inflammatory macrophages.
J. Exp. Med.
156:430-442 |
| 32. | Perry, S. W., L. G. Epstein, and H. A. Gelbard. 1997. Simultaneous in situ detection of apoptosis and necrosis in monolayer cultures by TUNEL and trypan blue staining. BioTechniques 22:1102-1106. [Medline] |
| 33. |
Pilder, S.,
J. Logan, and T. Shenk.
1984.
Deletion of the gene encoding the adenovirus 5 early region 1b 21,000-molecular-weight polypeptide leads to degradation of viral and host cell DNA.
J. Virol.
52:664-671 |
| 34. | Raff, M. C. 1992. Social controls on cell survival and cell death. Nature 356:397-400[Medline]. |
| 35. |
Rao, L.,
M. Debbas,
P. Sabbatini,
D. Hockenbery,
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 |
| 36. | Ray, C. A., R. A. Black, S. R. Kronheim, T. A. Greenstreet, P. R. Sleath, G. S. Salvesen, and D. J. Pickup. 1992. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell 69:597-604[Medline]. |
| 37. | Razvi, E. S., and R. M. Welsh. 1995. Apoptosis in viral infections. Adv. Virus Res. 45:1-60[Medline]. |
| 38. | Regnier, C. H., H. Y. Song, X. Gao, D. V. Goeddel, Z. Cao, and M. Rothe. 1997. Identification and characterization of an IkappaB kinase. Cell 90:373-383[Medline]. |
| 39. | Rousseau, V., I. Cremer, E. Lauret, I. Riviere, M. Aguet, and E. De Maeyer. 1995. Antiviral activity of autocrine interferon-beta requires the presence of a functional interferon type I receptor. J. Interferon Cytokine Res. 15:785-789[Medline]. |
| 40. | Rueckert, R. R. 1996. Picornaviridae: the viruses and their replication, p. 609-654. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Virology, 3rd ed. Lippincott-Raven Publishers, Philadelphia, Pa. |
| 41. |
Schwarz, E. M.,
P. Krimpenfort,
A. Berns, and I. M. Verma.
1997.
Immunological defects in mice with a targeted disruption in Bcl-3.
Genes Dev.
11:187-197 |
| 42. |
Schwarz, E. M.,
D. Van Antwerp, and I. M. Verma.
1996.
Constitutive phosphorylation of IB by casein kinase II occurs preferentially at serine 293: requirement for degradation of free IB.
Mol. Cell. Biol.
16:3554-3559[Abstract].
|
| 43. | Sha, W. C., H. C. Liou, E. I. Tuomanen, and D. Baltimore. 1995. Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell 80:321-330[Medline]. |
| 44. | Shen, Y., and T. E. Shenk. 1995. Viruses and apoptosis. Curr. Opin. Genet. Dev. 5:105-11[Medline]. |
| 45. | Siebenlist, U., G. Franzoso, and K. Brown. 1994. Structure, regulation and function of NF-kappa B. Annu. Rev. Cell Biol. 10:405-455. |
| 46. | Teodoro, J. G., and P. E. Branton. 1997. Regulation of apoptosis by viral gene products. J. Virol. 71:1739-1746[Medline]. |
| 47. | Thome, M., P. Schneider, K. Hofmann, H. Fickenscher, E. Meinl, F. Neipel, C. Mattmann, K. Burns, J. L. Bodmer, M. Schroter, C. Scaffidi, P. H. Krammer, M. E. Peter, and J. Tschopp. 1997. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386:517-521[Medline]. |
| 48. | Tolskaya, E. A., L. I. Romanova, M. S. Kolesnikova, T. A. Ivannikova, E. A. Smirnova, N. T. Raikhlin, and V. I. Agol. 1995. Apoptosis-inducing and apoptosis-preventing functions of poliovirus. J. Virol. 69:1181-1189[Abstract]. |
| 49. | Traenckner, E. B., H. L. Pahl, T. Henkel, K. N. Schmidt, S. Wilk, and P. A. Baeuerle. 1995. Phosphorylation of human I kappa B-alpha on serines 32 and 36 controls I kappa B-alpha proteolysis and NF-kappa B activation in response to diverse stimuli. EMBO J. 14:2876-2883[Medline]. |
| 50. |
Van Antwerp, D. J.,
S. J. Martin,
T. Kafri,
D. R. Green, and I. M. Verma.
1996.
Suppression of TNF-alpha-induced apoptosis by NF-kappaB.
Science
274:787-789 |
| 51. |
Verma, I. M.,
J. K. Stevenson,
E. M. Schwarz,
D. Van Antwerp, and S. Miyamoto.
1995.
Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation.
Genes Dev.
9:2723-2735 |
| 52. |
Wang, C. Y.,
M. W. Mayo, and A. S. Baldwin, Jr.
1996.
TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB.
Science
274:784-787 |
| 53. |
Wang, W. Z.,
A. Matsumori,
T. Yamada,
T. Shioi,
I. Okada,
S. Matsui,
Y. Sato,
H. Suzuki,
K. Shiota, and S. Sasayama.
1997.
Beneficial effects of amlodipine in a murine model of congestive heart failure induced by viral myocarditis. A possible mechanism through inhibition of nitric oxide production.
Circulation
95:245-251 |
| 54. |
White, E., and B. Stillman.
1987.
Expression of adenovirus E1B mutant phenotypes is dependent on the host cell and on synthesis of E1A proteins.
J. Virol.
61:426-435 |
| 55. | Xue, D., and H. R. Horvitz. 1995. Inhibition of the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein. Nature 377:248-251[Medline]. |
| 56. | Zhu, Z., W. Tang, A. Ray, Y. Wu, O. Einarsson, M. L. Landry, J. Gwaltney, Jr., and J. A. Elias. 1996. Rhinovirus stimulation of interleukin-6 in vivo and in vitro. Evidence for nuclear factor kappa B-dependent transcriptional activation. J. Clin. Invest. 97:421-430[Medline]. |
| 57. | Zinkernagel, R. M. 1996. Immunology taught by viruses. Science 271:173-178[Abstract]. |
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) is EMCV infected,
stains positive for DNA strand breaks, and shows formation of an
apoptotic body. Similar results were obtained with p50