Reovirus-Induced Apoptosis Requires Activation of Transcription Factor NF-kappa B

Abstract

Reovirus infection induces apoptosis in cultured cells and in vivo. To identify host cell factors that mediate this response, we investigated whether reovirus infection alters the activation state of the transcription factor nuclear factor kappa B (NF-kappaB). As determined in electrophoretic mobility shift assays, reovirus infection of HeLa cells leads to nuclear translocation of NF-kappaB complexes containing Rel family members p50 and p65. Reovirus-induced activation of NF-kappaB DNA-binding activity correlated with the onset of NF-kappaB-directed transcription in reporter gene assays. Three independent lines of evidence indicate that this functional form of NF-kappaB is required for reovirus-induced apoptosis. First, treatment of reovirus-infected HeLa cells with a proteasome inhibitor prevents NF-kappaB activation following infection and substantially diminishes reovirus-induced apoptosis. Second, transient expression of a dominant-negative form of IkappaB that constitutively represses NF-kappaB activation significantly reduces levels of apoptosis triggered by reovirus infection. Third, mutant cell lines deficient for either the p50 or p65 subunits of NF-kappaB are resistant to reovirus-induced apoptosis compared with cells expressing an intact NF-kappaB signaling pathway. These findings indicate that NF-kappaB plays a significant role in the mechanism by which reovirus induces apoptosis in susceptible host cells.

Full text

JOURNAL OF VIROLOGY,
0022-538X/00/$04.00⫹0
Apr. 2000, p. 2981–2989 Vol. 74, No. 7
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Reovirus-Induced Apoptosis Requires Activation of
Transcription Factor NF-␬B
JODI L. CONNOLLY,
1,2
STEVEN E. RODGERS,
1,2
PENNY CLARKE,
3
DEAN W. BALLARD,
1
LAWRENCE D. KERR,
1,4
KENNETH L. TYLER,
3,5,6,7,8
AND TERENCE S. DERMODY
1,2,9
*
Departments of Microbiology and Immunology,
1
Cell Biology,
4
and Pediatrics
9
and Elizabeth B. Lamb Center for
Pediatric Research,
2
Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Departments of
Neurology,
3
Medicine,
5
Microbiology,
6
and Immunology,
7
University of Colorado Health Sciences Center,
and Neurology Service, Denver Veterans Affairs Medical Center,
8
Denver, Colorado 80220
Received 5 October 1999/Accepted 29 December 1999
Reovirus infection induces apoptosis in cultured cells and in vivo. To identify host cell factors that mediate
this response, we investigated whether reovirus infection alters the activation state of the transcription factor
nuclear factor kappa B (NF-␬B). As determined in electrophoretic mobility shift assays, reovirus infection of
HeLa cells leads to nuclear translocation of NF-␬B complexes containing Rel family members p50 and p65.
Reovirus-induced activation of NF-␬B DNA-binding activity correlated with the onset of NF-␬B-directed
transcription in reporter gene assays. Three independent lines of evidence indicate that this functional form
of NF-␬B is required for reovirus-induced apoptosis. First, treatment of reovirus-infected HeLa cells with a
proteasome inhibitor prevents NF-␬B activation following infection and substantially diminishes reovirus-
induced apoptosis. Second, transient expression of a dominant-negative form of I␬B that constitutively
represses NF-␬B activation significantly reduces levels of apoptosis triggered by reovirus infection. Third,
mutant cell lines deficient for either the p50 or p65 subunits of NF-␬B are resistant to reovirus-induced
apoptosis compared with cells expressing an intact NF-␬B signaling pathway. These findings indicate that
NF-␬B plays a significant role in the mechanism by which reovirus induces apoptosis in susceptible host cells.
Many viruses are capable of inducing programmed cell
death, which results in apoptosis of infected cells (43, 45, 52,
60). Apoptotic cell death is characterized by cell shrinkage,
membrane blebbing, condensation of nuclear chromatin, and
activation of endogenous endonucleases. These changes occur
according to developmental programs or in response to certain
environmental stimuli (2, 43, 52, 71). In some cases, apoptosis
triggered by virus infection appears to serve as a host defense
mechanism to limit viral replication or spread. This defense
mechanism is mediated either directly by self-destruction of
the host cell prior to completion of viral replication or indi-
rectly through recognition of the infected cell by cytotoxic T
lymphocytes (43, 52). In other cases, induction of apoptosis
may enhance viral infection by facilitating virus spread or al-
lowing the virus to evade host inflammatory or immune re-
sponses (20, 43, 60). For some viruses, cellular factors operant
during apoptosis may function to increase the production of
viral progeny (45, 52).
Mammalian reoviruses have served as useful models for
studies of viral pathogenesis. Reoviruses are nonenveloped
icosahedral viruses with a genome consisting of 10 double-
stranded RNA gene segments (reviewed in reference 41). Af-
ter infection of newborn mice, reoviruses are highly virulent,
inducing injury to a variety of host organs including the central
nervous system, heart, and liver (reviewed in reference 62). In
both cultured cells (46, 63) and the murine central nervous
system (42) and heart (R. DeBiasi, B. Sherry, and K. Tyler,
Abstr. Am. Soc. Virol. 18th Annu. Meet., abstr. 52-1, p. 152,
1999), reoviruses induce the morphological and biochemical
features of apoptosis.
Insight into the mechanisms by which reoviruses trigger
apoptosis has emerged from studies of viral prototype strains
that vary in their capacity to elicit this cellular response. Reo-
virus strains type 3 Abney and type 3 Dearing (T3D) induce
apoptosis in cultured cells to a substantially greater extent than
does strain type 1 Lang (46, 63). Differences in the capacity of
these strains to induce apoptosis are determined by the viral S1
gene (46, 63), which encodes two proteins, attachment protein
␴1 and nonstructural protein ␴1s (25, 31, 50). Reovirus ␴1s-
null mutant T3C84-MA induces apoptosis with an efficiency
equivalent to its ␴1s-expressing parental strain, T3C84 (47),
which indicates that the ␴1 protein is the S1 gene product
responsible for mediating differences in the efficiency with
which reovirus strains induce apoptosis. Therefore, these stud-
ies suggest that apoptosis induced by reovirus is triggered by a
signaling pathway initiated by early steps in the virus replica-
tion cycle.
The nuclear factor kappa B (NF-␬B) family of transcription
factors plays a key role in the regulation of cell growth and
survival. The prototypical form of NF-␬B exists as a hetero-
dimer of proteins p50 and p65 (RelA) (4, 27). In quiescent
cells, NF-␬B is sequestered in the cytoplasm by the I␬B family
of inhibitory proteins (3, 66). Following exposure of cells to a
variety of stimuli (including tumor necrosis factor alpha [TNF-
␣], interleukin-1, and lipopolysaccharide), activation of NF-␬B
is accomplished by a mechanism involving site-specific phos-
phorylation, ubiquitination, and proteasomal degradation of
I␬B (11, 12, 17, 61). Release of I␬B reveals a nuclear localiza-
tion signal on NF-␬B, which allows NF-␬B to translocate to the
nucleus (7), where it serves as a transcriptional regulator (re-
viewed in references 38 and 66). In systems in which NF-␬Bis
activated during induction of apoptosis, NF-␬B can either pre-
vent (6, 35, 65, 69) or potentiate (1, 29, 32, 34) apoptosis.
We conducted experiments to investigate the role of NF-␬B
in reovirus-induced apoptosis. We show that NF-␬B complexes
* Corresponding author. Mailing address: Lamb Center for Pediat-
ric Research, D7235 MCN, Vanderbilt University School of Medicine,
Nashville, TN 37232. Phone: (615) 343-9943. Fax: (615) 343-9723.
E-mail: terry.dermody@mcmail.vanderbilt.edu.
2981

are activated in HeLa cells in response to reovirus infection
and that these complexes contain both p50 and p65. Using two
independent methods to block NF-␬B activation following re-
ovirus infection, we demonstrate that reovirus-induced apopto-
sis requires NF-␬B. Furthermore, reovirus-induced apoptosis
is inhibited in cells deficient in expression of either p50 or p65.
These results provide strong evidence that reovirus activates
NF-␬B and that NF-␬B activation is required for apoptosis
induced by reovirus infection.
MATERIALS AND METHODS
Cells and viruses. Murine L929 (L) cells were maintained as previously de-
scribed (47). p50⫹/⫹, p50⫺/⫺, p65⫹/⫹, and p65⫺/⫺ immortalized fibroblasts
were obtained from David Baltimore, California Institute of Technology, Pasa-
dena, Calif. HeLa, p50⫹/⫹, p50⫺/⫺, p65⫹/⫹, and p65⫺/⫺ cells were grown in
Dulbecco’s modified Eagle’s medium (Gibco BRL, Gaithersburg, Md.) supple-
mented to contain 10% fetal bovine serum (Intergen, Purchase, N.Y.), 2 mM
L-glutamine, 100 U of penicillin per ml, 100 ␮g of streptomycin per ml (Sigma
Chemical Co., St. Louis, Mo.), and 0.25 ␮g of amphotericin B per ml (Irvine
Scientific, Santa Ana, Calif.).
Reovirus strain T3D is a laboratory stock. Purified virion preparations were
made as previously described using second-passage L-cell lysate stocks of twice-
plaque-purified reovirus (26). Concentrations of virions in purified preparations
were determined from the equivalence 1 optical density at 260 nm unit ⫽ 2.1 ⫻
10
12
virions per ml (54).
Quantitation of reovirus growth. Cells grown in 24-well tissue culture plates
(Costar, Cambridge, Mass.) were infected with T3D at a multiplicity of infection
(MOI) of 1 PFU per cell. After viral adsorption for 1 h, the inoculum was
removed, 1.0 ml of fresh medium was added, and the cells were incubated at 37°C
for various intervals. After incubation, cells and culture medium were frozen
(⫺70°C) and thawed twice, and viral titers in cell lysates were determined by
plaque assay using L-cell monolayers (67).
Quantitation of apoptosis by acridine orange staining. Cells grown in 24-well
tissue culture plates were treated with 20 ng of human recombinant TNF-␣
(Sigma) per ml or infected with T3D at an MOI of 100 PFU per cell. This MOI
was chosen to produce a synchronous infection and to ensure maximum levels of
apoptosis. The percentage of apoptotic cells was determined using acridine
orange staining as previously described (23, 46, 63). The cell culture medium was
removed, and the cells were incubated with trypsin-EDTA (Irvine Scientific).
The cell culture medium and trypsinized cells were combined and centrifuged.
The cell pellet was resuspended in 200 ␮l of phosphate-buffered saline and
stained using 10 ␮l of a solution containing 100 ␮g of acridine orange (Sigma)
per ml and 100 ␮g of ethidium bromide (Sigma) per ml. For each experiment,
200 to 300 cells were counted and the percentage of cells exhibiting condensed
chromatin was determined by epi-illumination fluorescence microscopy using a
fluorescein filter set (Photomicroscope III; Zeiss, Oberko¨chen, Germany).
EMSA. Cells grown in 75-cm
2
tissue culture flasks (Costar) were either treated
with 20 ng of TNF-␣ per ml or adsorbed with T3D at an MOI of 100 PFU per
cell. After incubation at 37°C for various intervals, nuclear extracts were pre-
pared by washing cells in phosphate-buffered saline and incubating them in
hypotonic lysis buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl
2
, 0.5
mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, protease inhibitor
cocktail [Boehringer Mannheim, Indianapolis, Ind.]) at 4°C for 15 min. Then
1/20 volume of 10% Nonidet P-40 was added to the cell lysate, and the sample
was vortexed for 10 s and centrifuged at 10,000 ⫻ g for 5 min. The nuclear pellet
was washed once in hypotonic buffer, resuspended in high-salt buffer (25%
glycerol, 20 mM HEPES [pH 7.9], 0.42 M NaCl, 1.5 mM MgCl
2
, 0.2 mM EDTA,
0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, protease inhibitor
cocktail), and incubated at 4°C for 2 to 3 h. Samples were centrifuged at 10,000 ⫻
g for 10 min, and the supernatant was used as the nuclear extract.
Nuclear extracts were assayed for NF-␬B activation by an electrophoretic
mobility shift assay (EMSA) using a
32
P-labeled oligonucleotide consisting of the
NF-␬B consensus binding sequence (Santa Cruz Biotechnology, Santa Cruz,
Calif.). Nuclear extracts (5 to 10 ␮g of total protein) were incubated at 4°C for
20 min with a binding-reaction buffer containing 2 ␮g of poly(dI-dC) (Sigma) in
20 mM HEPES (pH 7.9)–60 mM KCl–1 mM EDTA–1 mM dithiothreitol–5%
glycerol. Radiolabeled NF-␬B consensus oligonucleotide (0.1 to 1.0 ng) was
added, and the mixture was incubated at room temperature for 20 min. For
competition experiments, a 10-fold excess of unlabeled consensus oligonucleo-
tide or of an oligonucleotide containing a point mutation in the NF-␬B consensus
site (Santa Cruz Biotechnology) was added to reaction mixtures. For supershift
experiments, 1 ␮l of rabbit polyclonal antiserum raised against either human p50
or p65 (Santa Cruz Biotechnology) or a control antibody raised against reovirus
nonstructural protein ␴1s (47) was added to binding-reaction mixtures and in-
cubated at 4°C for 30 min prior to the addition of radiolabeled oligonucleotide.
Nucleoprotein complexes were subjected to electrophoresis on native 5% poly-
acrylamide gels at 180 V, dried under vacuum, and exposed to Biomax MR film
(Kodak, Rochester, N.Y.).
Luciferase gene reporter assay. The NF-␬B-dependent luciferase reporter
construct was a gift from Lucy Ghoda. The construct is composed of pGL2-Basic
(Promega, Madison, Wis.) and three NF-␬B-binding sites from the major histo-
compatibility complex class I promoter. HeLa cells (1.5 ⫻ 10
5
) in six-well tissue
culture plates (Costar) were incubated for 24 h and then transfected with 10 ␮g
of the luciferase reporter construct and 2 ␮g of a cytomegalovirus (CMV)–␤-
galactosidase reporter construct (Clontech, Palo Alto, Calif.) using Lipo-
fectAMINE (Gibco BRL). After an additional 24-h incubation, cells were either
mock infected or infected with T3D at an MOI of 100 PFU per cell and
incubated at 37°C for various intervals. The cells were resuspended in 1 ml of
sonication buffer (91 mM dithiothreitol, 0.91 M K
2
HPO
4
[pH 7.8]), centrifuged
at 2,000 ⫻ g for 10 min, and resuspended in 100 ␮l of sonication buffer. The cells
were then vortexed, frozen (⫺20°C) and thawed three times, and centrifuged at
14,000 ⫻ g for 10 min. Samples (10 ␮l) were assessed for luciferase activity after
addition of 350 ␮l of luciferase assay buffer (85 mM dithiothreitol, 0.85 M
K
2
HPO
4
[pH 7.8], 50 mM ATP, 15 mM MgSO
4
) by determining the optical
density in a luminometer (Monolight 2010; Analytical Luminescence Laborato-
ry). Samples were assayed for ␤-galactosidase activity using standard procedures
(49) to normalize for transfection efficiency.
Proteasome inhibitor treatment. The proteasome inhibitor Z-L
3
VS was ob-
tained from Hidde Ploegh (10). HeLa cells were incubated at 37°C for 1 h with
medium containing 5 ␮M Z-L
3
VS. TNF-␣ at 20 ng per ml was added, and the
cells were incubated at 37°C for 18 h, or the medium was removed and the cells
were adsorbed at 4°C for 1 h with T3D at an MOI of 100 PFU per cell in gelatin
saline containing 5 ␮M Z-L
3
VS. Following adsorption, medium containing the
proteasome inhibitor was added and the cells were incubated at 37°C for various
intervals. Cells were harvested for EMSA or acridine orange staining assays.
Transient transfection of HeLa cells. The coding sequence of FLAG epitope-
tagged human I␬B␣ lacking amino acids 1 to 36 (I␬B␣-⌬N) (11) was inserted
into the multiple-cloning site of pHook-2 (Invitrogen, Carlsbad, Calif.) to gen-
erate pHook-2/I␬B␣-⌬N. HeLa cells (7 ⫻ 10
5
) in 60-mm dishes (Corning, Corn-
ing, N.Y.) were incubated at 37°C for 24 h and then transfected with either 5 ␮g
of pHook-2/lacZ (Invitrogen) or 5 ␮g of pHook-2/I␬B␣-⌬N using Lipo-
fectAMINE PLUS reagent (Gibco BRL). Transfected cells were selected using
Capture-Tec magnetic beads (Invitrogen) 24 h following infection and plated in
24-well plates for use in acridine orange staining assays.
Statistical analysis. Acridine orange staining data were tested using paramet-
ric statistical analysis with a two-sample t test. Statistical analysis was performed
using Minitab statistical software (Addison-Wesley, Reading, Mass.).
RESULTS
Reovirus replicates efficiently and induces apoptosis in
HeLa cells. To determine whether reovirus is capable of pro-
ductively infecting HeLa cells, reovirus strain T3D was ad-
sorbed to cells at an MOI of 1 PFU per cell and viral yields
were determined 24 and 48 h after infection (Fig. 1A). T3D
replicated efficiently in HeLa cells, producing yields of approx-
imately 800 and 8,000 progeny virions per input 24 and 48 h
following infection, respectively. To determine whether reovi-
rus induces apoptosis of HeLa cells, T3D was adsorbed to cells
at an MOI of 100 PFU per cell and apoptosis was assessed by
acridine orange staining 24 and 48 h after infection (Fig. 1B).
In previous work, we showed that cell death detected using
acridine orange staining of infected L cells and Madin-Darby
canine kidney (MDCK) epithelial cells correlates with ultra-
structural changes characteristic of apoptosis and formation of
oligonucleosome-length DNA ladders (46, 63). T3D infection
of HeLa cells induced chromatin condensation and the mor-
phological changes of apoptosis in approximately 70% of cells
24 h after infection and 80% of cells 48 h after infection. These
results indicate that reovirus grows efficiently in HeLa cells and
induces the death of these cells by apoptosis.
NF-␬B is activated by reovirus. To determine whether
NF-␬B is activated following reovirus infection of HeLa cells,
we used EMSAs to detect NF-␬B in nuclear extracts prepared
from reovirus-infected cells. HeLa cells were either mock in-
fected or infected with reovirus strain T3D, and nuclear ex-
tracts were prepared at various times after viral adsorption.
Extracts were incubated with a
32
P-labeled oligonucleotide
consisting of the NF-␬B consensus binding sequence and re-
solved in a nondenaturing polyacrylamide gel (Fig. 2A). Fol-
lowing infection with reovirus, proteins capable of shifting the
radiolabeled oligonucleotide to a higher relative molecular
2982 CONNOLLY ET AL. J. VIROL.

mass were increased in nuclear extracts. NF-␬B activation was
first detected at 4 h postinfection, peaked at 10 h postinfection,
and was diminishing by 12 h postinfection. Activated com-
plexes could not be detected in mock-infected cultures at any
time point (data not shown). As assessed by EMSA, NF-␬B
was activated in L cells and MDCK cells with similar kinetics
following reovirus infection (data not shown).
To confirm the specificity of NF-␬B DNA-binding activity in
these experiments, HeLa cells were either mock infected or
infected with T3D at an MOI of 100 PFU per cell and nuclear
extracts were prepared 10 h after adsorption. Nuclear extracts
were incubated with a
32
P-labeled NF-␬B consensus oligonu-
cleotide in the presence of a 10-fold excess of either unlabeled
consensus oligonucleotide or unlabeled mutant oligonucleo-
tide (Fig. 2B). The mutant oligonucleotide consists of the
NF-␬B consensus sequence with a single point mutation that
abolishes NF-␬B binding. Binding of the radiolabeled probe
was competed with unlabeled consensus oligonucleotide but
not with mutant oligonucleotide. We conclude that the gel shift
activity detected following reovirus infection is specific for se-
quences that are bound by NF-␬B.
To identify NF-␬B family members present in complexes
activated following reovirus infection, nuclear extracts were
prepared from mock-infected cells and cells infected with T3D
10 h after viral adsorption. The nuclear extracts were incubated
with a p50-specific antiserum, a p65-specific antiserum, or both
antisera prior to addition of the
32
P-labeled NF-␬B consensus
oligonucleotide (Fig. 2C). The addition of anti-p50 or anti-p65
antiserum or both antisera resulted in bands of higher relative
molecular mass, indicating that both p50 and p65 are present
in complexes activated following reovirus infection. These
findings indicate that reovirus infection of HeLa cells results in
nuclear translocation of NF-␬B complexes and that these com-
plexes contain NF-␬B family members p50 and p65.
To determine whether NF-␬B is capable of stimulating tran-
scription following reovirus infection, we used a reporter gene
construct containing NF-␬B-binding sites to direct the expres-
sion of luciferase. Following transfection of HeLa cells with
FIG. 1. (A) Growth of reovirus in HeLa cells. Cells (1 ⫻ 10
5
) were infected
with reovirus strain T3D at an MOI of 1 PFU per cell. After adsorption for 1 h,
the inoculum was removed, fresh medium was added, and the cells were incu-
bated at 37°C for 0, 24, or 48 h. The cells were frozen and thawed twice, and viral
titers were determined by a plaque assay. The results are expressed as the mean
viral yields, calculated by dividing the viral titer at 24 or 48 h by the viral titer at
0 h, for three independent experiments. Error bars indicate standard error of the
mean. (B) Apoptosis induced by reovirus infection of HeLa cells. Cells (5 ⫻ 10
4
)
were either mock infected or infected with reovirus strain T3D at an MOI of 100
PFU per cell. After adsorption for 1 h, the cells were incubated at 37°C for 24 or
48 h and stained with acridine orange. The results are expressed as the mean
percentage of cells undergoing apoptosis in three independent experiments.
Error bars indicate standard error of the mean.
FIG. 2. (A) Time course of NF-␬B gel shift activity in nuclear extracts pre-
pared from reovirus-infected HeLa cells. Cells (5 ⫻ 10
6
) were either mock
infected or infected with T3D at an MOI of 100 PFU per cell and incubated at
37°C for the times shown. Uninfected cells also were treated with 20 ng of TNF-␣
per ml for 1 h. Nuclear extracts were prepared and incubated with a
32
P-labeled
oligonucleotide consisting of the NF-␬B consensus binding sequence. Incubation
mixtures were resolved by acrylamide gel electrophoresis, dried, and exposed to
film. NF-␬B-containing complexes are indicated. (B) Specificity of NF-␬B gel
shift activity. Nuclear extracts were prepared as in panel A 10 h after viral
adsorption. Extracts were incubated with
32
P-labeled NF-␬B consensus oligonu-
cleotide alone (lanes N), a 10-fold excess of unlabeled consensus probe (lanes C),
or a 10-fold excess of unlabeled mutant probe consisting of the NF-␬B consensus
sequence with a point mutation that abolishes NF-␬B binding (lanes M). NF-
␬B-containing complexes are indicated. (C) Identification of NF-␬B family mem-
bers activated by reovirus infection. Nuclear extracts were prepared as in panel
A 10 h after viral adsorption. Extracts were incubated with no antibody, a control
antibody (Ab), p50-specific antiserum, p65-specific antiserum, or both p50- and
p65-specific antisera. NF-␬B complexes not shifted by antibody and supershifted
complexes containing p50 or p65 are indicated.
VOL. 74, 2000 REOVIRUS-INDUCED APOPTOSIS REQUIRES NF-␬B 2983

this construct, cells were either mock infected or infected with
T3D. After incubation for various intervals, cell extracts were
prepared and assayed for luciferase activity (Fig. 3). T3D in-
fection was associated with substantial NF-␬B-dependent lu-
ciferase expression in comparison to mock-infected cells. Lu-
ciferase activity in infected cells was first detected between 6
and 12 h postinfection and was greatest 18 h postinfection,
after which it declined and was nearly undetectable by 48 h
postinfection. Transfection efficiencies were normalized by
cotransfection of a ␤-galactosidase reporter construct driven
by the CMV promoter. ␤-Galactosidase expression from the
CMV reporter was not altered by reovirus infection (data not
shown). These results indicate that reovirus infection of HeLa
cells activates NF-␬B-dependent transcription, which is consis-
tent with the results obtained from biochemical experiments.
Proteasome inhibitor treatment inhibits reovirus-induced
apoptosis. To determine the role of NF-␬B activation in reo-
virus-induced apoptosis, HeLa cells were treated with Z-L
3
VS,
a synthetic inhibitor of proteasome function (10). Previous
studies of other proteasome inhibitors have demonstrated that
NF-␬B activation induced by a variety of stimuli, including
TNF-␣, lipopolysaccharide, and phorbol esters, is blocked by
treatment of cells with inhibitors of proteasome catalytic ac-
tivity (36, 44). Since degradation of I␬B and the subsequent
release of NF-␬B require proteasome activity (17), proteasome
inhibitors lead to sequestration of NF-␬B in the cytoplasm. To
determine whether Z-L
3
VS is capable of blocking NF-␬B ac-
tivation following reovirus infection, cells were cultured in the
absence or presence of 5 ␮M Z-L
3
VS and then either mock
infected or infected with T3D at an MOI of 100 PFU per cell.
Nuclear extracts were prepared 10 h following infection and
used in an EMSA (Fig. 4A). Treatment of HeLa cells with
Z-L
3
VS abolished NF-␬B activation following reovirus infec-
tion, which confirms that inhibition of proteasome function
blocks nuclear translocation of NF-␬B.
To determine the effect of blockade of NF-␬B activation on
reovirus-induced apoptosis, HeLa cells were cultured in the
absence or presence of 5 ␮M Z-L
3
VS and then either mock
infected or infected with T3D at an MOI of 100 PFU per cell.
Apoptosis was assessed using acridine orange staining 18 h
after infection (Fig. 4B). This time point was chosen because
more prolonged incubation with the proteasome inhibitor re-
sulted in cytotoxicity. Approximately 20% of reovirus-infected
cells cultured in the absence of the proteasome inhibitor were
apoptotic 18 h after infection; however, only 6% of infected
cells cultured in the presence of the proteasome inhibitor were
apoptotic (P ⫽ 0.005). The level of apoptosis detected in in-
fected cells cultured with Z-L
3
VS did not significantly differ
from that observed in uninfected cells cultured with the pro-
teasome inhibitor (P ⫽ 0.25). As a control for the blockade of
NF-␬B using Z-L
3
VS, cells also were treated with TNF-␣ (Fig.
4C), which has been shown to increase the levels of apoptosis
in cells lacking functional NF-␬B (6, 35, 65, 69). Apoptosis was
increased in TNF-␣-treated cells cultured in the presence of
Z-L
3
VS, suggesting that alterations in apoptosis induction me-
diated by the proteasome inhibitor are due to blockade of
FIG. 3. NF-␬B-dependent luciferase expression in reovirus-infected HeLa
cells. Cells (1.5 ⫻ 10
5
) were transfected with 10 ␮g of a luciferase reporter
construct containing NF-␬B binding sites. After 24 h, the cells were either mock
infected or infected with T3D at an MOI of 100 PFU per cell and incubated at
37°C for the times shown. Cell extracts were prepared, and luciferase activity was
determined. The results are expressed as the mean luciferase units in two inde-
pendent experiments. Error bars indicate standard error of the mean.
FIG. 4. (A) NF-␬B gel shift activity in reovirus-infected cells cultured in the
presence of Z-L
3
VS. Cells (5 ⫻ 10
6
) were either mock infected or infected with
T3D at an MOI of 100 PFU per cell and cultured in the absence or presence of
5 ␮M Z-L
3
VS. After incubation at 37°C for 10 h, nuclear extracts were prepared
and incubated with a
32
P-labeled oligonucleotide consisting of the NF-␬B con-
sensus sequence. Incubation mixtures were resolved by acrylamide gel electro-
phoresis, dried, and exposed to film. NF-␬B-containing complexes are indicated.
(B) Quantitation of apoptosis in reovirus-infected HeLa cells cultured in the
presence of Z-L
3
VS. Cells (5 ⫻ 10
4
) were either mock infected or infected with
T3D at an MOI of 100 PFU per cell and cultured in the absence or presence of
5 ␮M Z-L
3
VS. After incubation at 37°C for 18 h, the cells were stained with
acridine orange. (C) Quantitation of apoptosis in TNF-␣-treated HeLa cells
cultured in the presence of Z-L
3
VS. Cells (5 ⫻ 10
4
) were either untreated or
treated with 20 ng of TNF-␣ per ml and cultured in the absence or presence of
5 ␮M Z-L
3
VS. After incubation at 37°C for 18 h, the cells were stained with
acridine orange. The results of the experiments in panels B and C are expressed
as the mean percentage of cells undergoing apoptosis in three independent
experiments. Error bars indicate standard error of the mean.
2984 CONNOLLY ET AL. J. VIROL.

NF-␬B. These results provide evidence that interference with
signal-dependent degradation of I␬B prevents reovirus-in-
duced apoptosis.
Expression of a transdominant inhibitor of NF-␬B inhibits
reovirus-induced apoptosis. To exclude the possibility that ex-
posure of cells to a proteasome inhibitor results in the block-
ade of apoptosis by inhibiting viral replication or by exerting
other nonspecific effects, we tested whether transient transfec-
tion of a transdominant inhibitor of NF-␬B, I␬B␣-⌬N, alters
reovirus-induced apoptosis. HeLa cells were transfected with
the pHook-2 plasmid containing human I␬B␣-⌬N appended at
the amino terminus with a FLAG epitope. I␬B␣-⌬Nisa36-
amino-acid amino-terminal truncation of I␬B␣ that lacks the
two serine residues required for I␬B␣ degradation (12, 51, 61).
I␬B␣-⌬N cannot be targeted for proteasome-mediated degra-
dation and thus functions as a trans-dominant inhibitor of
NF-␬B. The pHook-2 plasmid allows the selection of trans-
fected cells from a population of cells by virtue of coexpression
of a cell surface marker that allows subsequent isolation using
magnetic beads (19). Cells selected following transfection of
either pHook-2/I␬B␣-⌬N or a control plasmid, pHook-2/lacZ,
were either mock infected or infected with T3D at an MOI of
100 PFU per cell. Apoptosis was assessed using acridine or-
ange staining 24 h after infection (Fig. 5A). Approximately
40% of pHook-2/lacZ-transfected cells were apoptotic follow-
ing infection with reovirus. In sharp contrast, only 15% of
pHook-2/I␬B␣-⌬N-transfected cells were apoptotic following
reovirus infection (P ⫽ 0.002). This low level of apoptosis in
the pHook-2/I␬B␣-⌬N-transfected cells was similar to the level
of apoptosis in mock-infected cultures (approximately 10%).
The percentage of apoptotic cells in mock-infected cultures
was higher than routinely observed for untransfected cells,
which is probably due to transfection and selection conditions.
As a control, transfected cells also were treated with TNF-␣
(Fig. 5B), which has been shown to increase levels of apoptosis
in cells lacking p65 (6) and in cells expressing mutant forms of
I␬B␣ (65, 69). Levels of apoptosis were increased following
TNF-␣ treatment of cells transfected with pHook-2/I␬B␣-⌬N
in comparison to cells transfected with pHook-2/lacZ. There-
fore, it is likely that transfection with pHook-2/I␬B␣-⌬N effec-
tively blocks NF-␬B activation. Stable expression of mutant
forms of I␬B in L cells and MDCK cells also inhibits both
NF-␬B activation and apoptosis following reovirus infection
(data not shown). These results demonstrate that expression of
an NF-␬B trans-dominant inhibitor blocks reovirus-induced
apoptosis and further supports the hypothesis that NF-␬B ac-
tivation is required for apoptosis induced by reovirus infection.
Reovirus-induced apoptosis is inhibited in cell lines defi-
cient for p50 or p65. Since both p50 and p65 are present in
NF-␬B complexes activated following reovirus infection, we
performed experiments to specifically determine whether p50
or p65 is required for reovirus-induced apoptosis. Immortal-
ized embryonic fibroblasts containing a null mutation in the
gene encoding either the p50 or p65 subunit of NF-␬B were
infected with reovirus and assayed for NF-␬B activation and
apoptosis induction. To determine whether NF-␬B complexes
are activated following reovirus infection of the null cell
lines, the p50⫺/⫺ and p65⫺/⫺ cell lines and their respective
p50⫹/⫹ and p65⫹/⫹ littermate control cell lines were either
mock infected or infected with T3D at an MOI of 100 PFU
per cell. Nuclear extracts were prepared 6 h (p50⫹/⫹ and
p50⫺/⫺) or 8 h (p65⫹/⫹ and p65⫺/⫺) following infection and
used in EMSAs (Fig. 6A and 7A). The results demonstrate
that NF-␬B complexes are not activated in the p50⫺/⫺ and
p65⫺/⫺ cell lines following infection. This result was antici-
pated based on the biochemical results with HeLa cells, which
demonstrated that p50 and p65 are the primary constituents of
NF-␬B complexes activated by reovirus.
To determine whether reovirus is capable of inducing apo-
ptosis in the mutant cell lines, p50⫹/⫹, p50⫺/⫺, p65⫹/⫹, and
p65⫺/⫺ cells were either mock infected or infected with T3D
at an MOI of 100 PFU per cell. Apoptosis was assessed using
acridine orange staining 48 h after infection (Fig. 6B and 7B).
Reovirus infection of both p50⫹/⫹ and p65⫹/⫹ cell lines
resulted in apoptosis of approximately 25% of cells. However,
only 5% of p50⫺/⫺ cells and 1% of p65⫺/⫺ cells were apo-
ptotic following infection. Although apoptosis was not abol-
ished in p50-deficient cells, the levels of apoptosis were signif-
icantly reduced in comparison to those in p50-expressing
control cells (P ⫽ 0.013). This result suggests that p50 serves as
an enhancer of reovirus-induced apoptosis but is not absolutely
required for this effect. Reovirus infection of p65-deficient
cells resulted in levels of apoptosis indistinguishable from
those of mock-infected cells (P ⫽ 0.78), which indicates a strict
requirement for p65 in the signaling pathway that results in
apoptosis following reovirus infection. Differences in p65⫹/⫹
and p65⫺/⫺ cell apoptosis induced by reovirus were highly
statistically significant (P ⫽ 0.01).
A previous study of the p50⫺/⫺ and p65⫺/⫺ cell lines
demonstrated that p65 but not p50 is required to inhibit apo-
ptosis induced by TNF-␣ (6), the opposite effect observed with
reovirus infection. To confirm that p65⫺/⫺ cells but not
p50⫺/⫺ cells are more sensitive to TNF-␣-induced cell death,
FIG. 5. Quantitation of apoptosis in HeLa cells transiently expressing I␬B␣-
⌬N. Cells (1 ⫻ 10
6
) were either transfected with 5 ␮g of pHook-2/I␬B␣-⌬Nor
5 ␮g of pHook-2/lacZ. After 24 h, transfected cells were isolated using Capture-
Tec magnetic beads and plated in 24-well plates. (A) Transfected cells (2 ⫻ 10
4
)
were either mock infected or infected with T3D at an MOI of 100 PFU per cell.
After incubation at 37°C for 24 h, the cells were stained with acridine orange. (B)
Transfected cells (2 ⫻ 10
4
) were either not treated or treated with 20 ng of
TNF-␣ per ml. After incubation at 37°C for 24 h, the cells were stained with
acridine orange. The results of the experiments in both panels are expressed as
the mean percentage of cells undergoing apoptosis in three independent exper-
iments. Error bars indicate standard error of the mean.
VOL. 74, 2000 REOVIRUS-INDUCED APOPTOSIS REQUIRES NF-␬B 2985

the null and control cell lines were either not treated or treated
with TNF-␣ (Fig. 6C and 7C). TNF-␣ treatment induced
apoptosis of p65⫺/⫺ cells but did not alter the viability of
p50⫺/⫺ or control cell lines. These results provide strong
genetic evidence that both p50 and p65 are critical for medi-
ating the apoptotic response triggered by reovirus infection
and support the idea that reovirus and TNF-␣ engage NF-␬B
in fundamentally different ways to influence stimulus-induced
cell death.
Growth of reovirus is diminished in cell lines deficient for
p50 and p65. For some viruses, induction of apoptosis may lead
to the activation of cellular signaling molecules required to
render a cell fully permissive for virus replication. Apoptosis
may also facilitate virus release and dissemination from in-
fected cells, resulting in an increase in viral progeny. To de-
termine whether NF-␬B family members are required for max-
imal viral replication in cultured cells, yields of reovirus were
determined after viral growth in the p50⫺/⫺ and p65⫺/⫺ cell
lines and their respective p50⫹/⫹ and p65⫹/⫹ littermate con-
trol cell lines (Fig. 8). Cells were infected with T3D at an MOI
of 1 PFU per cell, and viral yields were determined 24 and 48 h
after infection. T3D replicated efficiently in all four cell lines;
however, viral yields in the control cell lines were two- to
fivefold greater, after 24 or 48 h of viral growth, than were the
yields in their respective null cell lines. In p50⫹/⫹ cells, T3D
produced yields of approximately 1,300 and 9,100 progeny
virions per input 24 and 48 h following infection, respectively.
However, in p50⫺/⫺ cells, the yields of T3D were reduced to
approximately 350 and 3,900 progeny per input 24 and 48 h
following infection, respectively. Similarly, in p65⫹/⫹ cells,
FIG. 6. (A) NF-␬B gel shift activity in reovirus-infected p50⫹/⫹ and p50⫺/⫺
immortalized fibroblast cells. Cells (5 ⫻ 10
6
) were either mock infected or
infected with T3D at an MOI of 100 PFU per cell. After incubation at 37°C for
6 h, nuclear extracts were prepared and incubated with a
32
P-labeled DNA probe
consisting of the NF-␬B consensus sequence. Incubation mixtures were resolved
by acrylamide gel electrophoresis, dried, and exposed to film. NF-␬B-containing
complexes are indicated. (B) Quantitation of apoptosis in reovirus-infected
p50⫹/⫹ and p50⫺/⫺ cells. Cells (2.5 ⫻ 10
4
) were either mock infected or
infected with T3D at an MOI of 100 PFU per cell. After incubation at 37°C for
48 h, the cells were stained with acridine orange. (C) Quantitation of apoptosis
in TNF-␣-treated p50⫹/⫹ and p50⫺/⫺ cells. Cells (2.5 ⫻ 10
4
) were either
untreated or treated with 20 ng of TNF-␣ per ml. After incubation at 37°C for
24 h, the cells were stained with acridine orange. The results of the experiments
in panels B and C are expressed as the mean percentage of cells undergoing
apoptosis in three independent experiments. Error bars indicate standard error
of the mean.
FIG. 7. (A) NF-␬B gel shift activity in reovirus-infected p65⫹/⫹ and p65⫺/⫺
immortalized fibroblast cells. Cells (5 ⫻ 10
6
) were either mock infected or
infected with T3D at an MOI of 100 PFU per cell. After incubation at 37°C for
8 h, nuclear extracts were prepared and incubated with a
32
P-labeled DNA probe
consisting of the NF-␬B consensus sequence. Incubation mixtures were resolved
by acrylamide gel electrophoresis, dried, and exposed to film. NF-␬B-containing
complexes are indicated. (B) Quantitation of apoptosis in reovirus-infected
p65⫹/⫹ and p65⫺/⫺ cells. Cells (2.5 ⫻ 10
4
) were either mock infected or
infected with T3D at an MOI of 100 PFU per cell. After incubation at 37°C for
48 h, the cells were stained with acridine orange. (C) Quantitation of apoptosis
in TNF-␣-treated p65⫹/⫹ and p65⫺/⫺ cells. Cells (2.5 ⫻ 10
4
) were either
untreated or treated with 20 ng of TNF-␣ per ml. After incubation at 37°C for
24 h, the cells were stained with acridine orange. The results of the experiments
in panels B and C are expressed as the mean percentage of cells undergoing
apoptosis in three independent experiments. Error bars indicate standard error
of the mean.
2986 CONNOLLY ET AL. J. VIROL.

T3D produced yields of approximately 1,700 and 57,500 prog-
eny per input 24 and 48 h following infection, respectively.
However, in p65⫺/⫺ cells, the yields of T3D were reduced to
approximately 500 and 12,900 progeny per input 24 and 48 h
following infection, respectively. Similar two- to fivefold reduc-
tions in viral yields were observed in the p50⫺/⫺ and p65⫺/⫺
cell lines relative to the control cell lines when cells were
infected at an MOI of 100 PFU per cell (data not shown).
These results suggest that expression of p50 and p65 confers a
modest viral growth advantage. The capacity of p50⫹/⫹ and
p65⫹/⫹ cells to undergo apoptosis following reovirus infection
may directly enhance viral replication, or expression of p50 and
p65 may allow a more permissive cellular environment to
achieve maximal viral growth.
DISCUSSION
Mammalian reoviruses have served as a useful experimental
system for studies of viral pathogenesis, and studies of these
viruses have provided important insights into how viruses in-
teract with host cells (68). In this study, we demonstrate that
NF-␬B is activated following infection of cultured cells with
reovirus. This conclusion is supported by two lines of evidence.
First, reovirus infection of HeLa cells and immortalized cell
lines derived from murine embryonic fibroblasts leads to nu-
clear translocation of NF-␬B complexes containing the p50 and
p65 subunits. Second, reovirus infection of HeLa cells induces
NF-␬B-directed expression of a luciferase reporter gene. Max-
imal luciferase activity follows the peak of NF-␬B gel shift
activity, as would be expected to allow NF-␬B-directed gene
expression. Thus, reovirus infection is capable of functional
activation of NF-␬B.
Our results using proteasome inhibitor Z-L
3
VS and tran-
sient expression of mutant forms of I␬B␣ suggest that reovirus-
induced activation of NF-␬B involves targeted degradation of
I␬B␣ by the 26S proteasome. However, the precise mechanism
by which reovirus activates NF-␬B remains unknown. We con-
sider it unlikely that NF-␬B activation is triggered solely by
attachment of the virus to its cognate cellular receptor, because
peak NF-␬B activity follows reovirus adsorption by several
hours. Activation of NF-␬B in response to physiologic recep-
tor-ligand interactions occurs with more rapid kinetics (66).
Therefore, we suspect that reovirus transcription or translation
is a prerequisite for access to the host NF-␬B pathway, which
is more consistent with the delayed response observed in our
studies. It is also possible that reovirus infection induces a
soluble factor that mediates activation of NF-␬B, which also
would account for the delay in NF-␬B activation.
Prior studies have firmly established that NF-␬B activation
can be achieved by a broad spectrum of biochemical inducing
cues, resulting in either enhancement (1, 29, 32) or inhibition
(6, 8, 65) of programmed cell death (reviewed in reference 55).
Using a proteasome inhibitor and a trans-dominant inhibitor of
NF-␬B, we demonstrated that interference with the NF-␬B
pathway leads to inhibition of reovirus-induced apoptosis.
These findings strongly suggest that NF-␬B enhances apoptosis
in response to reovirus infection. In support of this contention,
cell lines deficient for either p50 or p65, the primary constitu-
ents of NF-␬B complexes activated by reovirus, are signifi-
cantly more resistant to reovirus-induced apoptosis than are
control cell lines. In fact, fibroblasts deficient in p65 expression
do not undergo apoptosis in response to reovirus infection over
an observation period of 48 h. These results provide compel-
ling evidence that NF-␬B plays an essential role in the mech-
anism by which reovirus triggers an apoptotic program in in-
fected cells.
In contrast to our finding that NF-␬B functions as a proapo-
ptotic factor during reovirus infection of cultured cells, NF-␬B
plays an antiapoptotic role in cells treated with TNF-␣ (6, 35,
65, 69) (Fig. 4 to 7). Engagement of the TNF receptor by
TNF-␣ induces protein-protein interactions that lead directly
to the activation of NF-␬B (30), which results in inhibition of
apoptosis (6, 35, 65, 69). Since activation of NF-␬B by reovirus
is not likely to occur directly following receptor ligation, it is
possible that the mechanism that promotes activation of
NF-␬B by reovirus explains its proapoptotic effects. Alterna-
tively, reovirus infection and TNF-␣ receptor engagement may
induce different auxiliary factors that influence the effects of
NF-␬B within a given cell type.
The requirement for NF-␬B activation in reovirus-induced
apoptosis suggests that NF-␬B functions to increase the ex-
pression of proapoptotic genes. Several genes encoding pro-
teins involved in mediating apoptosis induced by a variety of
stimuli are regulated by NF-␬B and contain NF-␬B response
elements in their promoters. Such NF-␬B-responsive proapo-
ptotic proteins include p53 (70), caspase-1 (14), and FasL (58).
Activation of NF-␬B following reovirus infection may induce
the expression of one or more of these genes or other proapo-
ptotic genes, which include death receptors and their ligands,
such as DR4, DR5, and TRAIL; effector or initiator caspases,
such as caspase-3 and caspase-9; and prodeath Bcl-2 family
members, such as Bax, Bik, and Bad. In a previous study, we
demonstrated that inhibitors of calpain, a calcium-dependent
papain-like cysteine protease, block reovirus-induced apopto-
FIG. 8. Growth of reovirus in p50⫺/⫺ and p65⫺/⫺ immortalized fibroblast
cells. p50⫹/⫹ and p50⫺/⫺ cells (A) or p65⫹/⫹ and p65⫺/⫺ cells (B) (2.5 ⫻ 10
4
cells per experiment) were infected with T3D at an MOI of 1 PFU per cell. After
adsorption for 1 h, the inoculum was removed, fresh medium was added, and the
cells were incubated at 37°C for 0, 24, or 48 h. The cells were frozen and thawed
twice, and viral titers were determined by a plaque assay. The results are pre-
sented as the mean viral yields (viral titer at 24 or 48 h divided by viral titer at
0 h) in three independent experiments. Error bars indicate standard error of the
mean.
VOL. 74, 2000 REOVIRUS-INDUCED APOPTOSIS REQUIRES NF-␬B 2987

sis (21). NF-␬B may function to upregulate the expression of
calpain activator proteins (22, 48, 53) or growth factors (15,
40), which have been shown to increase calpain activity. NF-␬B
also may be involved in regulating genes that control cellular
calcium flux, which is required for calpain activation (57). It is
also possible that NF-␬B induces the expression of transcrip-
tion factors that in turn augment the transcription of proapo-
ptotic genes not directly under the control of NF-␬B.
Why would reovirus activate NF-␬B? One possibility is that
induction of apoptosis of infected cells would reduce host
inflammatory responses, potentially leading to increased dis-
semination of the virus. Thus, viruses capable of this response
would have a clear selective advantage. Given the well-estab-
lished role of NF-␬B in signal-induced cell growth pathways
(reviewed in reference 66), a second possibility is that activa-
tion of NF-␬B produces a cellular environment that is more
permissive for reovirus replication. Reovirus yields are sub-
stantially higher in rapidly dividing or transformed cells (24, 56,
59), which suggests that cellular factors associated with cell
growth augment viral replication. Reovirus-induced NF-␬B ac-
tivation might lead to expression of growth-associated cellular
genes that promote more efficient viral nucleic acid or protein
synthesis, intracellular transport of viral proteins, or assembly
and release of progeny virions. In support of this idea, we
found that reovirus yields are decreased in both p50⫺/⫺ and
p65⫺/⫺ cells relative to control cells.
Other viruses induce NF-␬B activation (13, 34, 37, 64, 72),
and in some cases NF-␬B is required for maximal viral repli-
cation. For instance, the long terminal repeat of human im-
munodeficiency virus contains ␬B response elements, and ac-
tivation of NF-␬B directly stimulates viral gene expression (16,
18). The human T-cell leukemia virus Tax protein induces
NF-␬B activation (9, 72), which in turn induces the expression
of cellular genes that promote human T-cell leukemia virus
replication (5, 28, 33, 39). Thus, the NF-␬B signaling pathway
may be a common pathway by which viruses confer an opti-
mum environment to achieve their replication.
The results reported here establish that NF-␬B is activated
following reovirus infection and demonstrate that activation of
NF-␬B is required for reovirus-induced apoptosis. Most RNA-
containing viruses, such as reovirus, are thought to replicate
independently of the nucleus. Our results, however, clearly
show that infection with an RNA-containing cytoplasmic virus
triggers a signal transduction pathway involving nuclear com-
ponents, which leads to cellular gene expression. Activation of
this signaling pathway is critical for cell death caused by reo-
virus and probably contributes to reovirus-induced pathology
(42; DeBiasi et al., Abstr. Am. Soc. Virol. 18th Annu. Meet.
1999). Understanding the signaling pathways used by reovirus
to induce cellular gene expression and apoptosis will contrib-
ute important new information about mechanisms by which
viruses produce cell death and disease.
ACKNOWLEDGMENTS
We thank David Baltimore for the NF-␬B null and control cell lines
and Hidde Ploegh for the proteasome inhibitor. We are grateful to
Zhi-Liang Chu, David Scherer, and Alexander Hoffman for expert
advice. We thank Erik Barton, Jim Chappell, and Tibor Valyi-Nagy for
careful review of the manuscript.
This work was supported by Public Health Service awards T32
GM07347 from the National Institute of General Medical Studies for
the Vanderbilt Medical Scientist Training Program (S.E.R.), AI33839
from the National Institutes of Allergy and Infectious Diseases
(D.W.B.), AG14071 from the National Institute on Aging (K.L.T.),
and AI38296 from the National Institute of Allergy and Infectious
Diseases (J.L.C. and T.S.D.); Department of Veterans Affairs Merit
and REAP Awards (K.L.T.); U.S. Army grant DAMD17-98-1-8614
(K.L.T.); and the Elizabeth B. Lamb Center for Pediatric Research
(J.L.C. and T.S.D.). Additional support was provided by Public Health
Service awards CA68485 for the Vanderbilt Cancer Center and
DK20593 for the Vanderbilt Diabetes Research and Training Center.
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