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Journal of Virology, May 1999, p. 3778-3788, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Activation of Caspases and p53 by Bovine
Herpesvirus 1 Infection Results in Programmed Cell Death and
Efficient Virus Release
Laxminarayana R.
Devireddy and
Clinton J.
Jones*
Department of Veterinary and Biomedical
Sciences, Center for Biotechnology, University of
Nebraska
Lincoln, Lincoln, Nebraska 68583-0905
Received 12 October 1998/Accepted 15 January 1999
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ABSTRACT |
Programmed cell death (PCD), or apoptosis, is initiated in response
to various stimuli, including virus infection. Bovine herpesvirus 1 (BHV-1) induces PCD in peripheral blood mononuclear cells at the
G0/G1 phase of the cell cycle (E. Hanon, S. Hoornaert, F. Dequiedt, A. Vanderplasschen, J. Lyaku, L. Willems, and
P.-P. Pastoret, Virology 232:351-358, 1997). However, penetration of virus particles is not required for PCD (E. Hanon, G. Meyer, A. Vanderplasschen, C. Dessy-Doize, E. Thiry, and P. P. Pastoret, J. Virol. 72:7638-7641, 1998). The mechanism by which BHV-1
induces PCD in peripheral blood mononuclear cells is not understood,
nor is it clear whether nonlymphoid cells undergo PCD following
infection. This study demonstrates that infection of bovine kidney
(MDBK) cells with BHV-1 leads to PCD, as judged by terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, DNA
laddering, and chromatin condensation. p53 appears to be important in
this process, because p53 levels and promoter activity increased after
infection. Expression of proteins that are stimulated by p53
(p21Waf1 and Bax) is also activated after infection.
Cleavage of Bcl-xL, a protein that inhibits PCD, occurred
after infection, suggesting that caspases (interleukin-1
-converting
enzyme-like proteases) were activated. Other caspase substrates
[poly(ADP-ribose) polymerase and actin] are also cleaved during the
late stages of infection. Inhibition of caspase activity delayed
cytotoxic activity and virus release but increased the overall virus
yield. Taken together, these results indicate that nonlymphoid cells
undergo PCD near the end of productive infection and further suggest
that caspases enhance virus release.
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INTRODUCTION |
Bovine herpesvirus 1 (BHV-1) belongs
to the Alphaherpesvirinae subfamily and is a significant
viral pathogen of cattle (reviewed in reference 39).
Acute infection leads to conjunctivitis, tracheitis, and upper
respiratory tract infections and can induce a complex upper respiratory
infection called shipping fever. Although BHV-1 is not the sole
infectious agent associated with shipping fever, it initiates the
disorder by immunosuppressing infected cattle (reviewed in reference
84). Consequently, secondary bacterial infections
can cause pneumonia. Like other members of the
Alphaherpesvirinae subfamily, BHV-1 establishes a
latent infection in sensory ganglionic neurons (reviewed in reference
39). Viral DNA persists in sensory neurons for the
lifetimes of infected cattle but can periodically reactivate. Infection
of permissive cells by BHV-1 leads to rapid cell death and virus
spread. Viral gene expression is temporally regulated by two
immediate-early (IE) transcription units (75, 77, 92, 93).
IE gene expression is stimulated by a virion component (bTIF) which
interacts with a cellular transcription factor (Oct-1), and
subsequently this protein complex binds TAATGARAT motifs present in IE
promoters (52). It is generally agreed that tissue-specific
factors mediate latency and pathogenesis by influencing viral gene expression.
There are two types of cell death, necrosis and programmed cell death
(PCD) (apoptosis), and they differ in their morphological and
biochemical characteristics (reviewed in reference
48). PCD occurs during embryogenesis, tissue
differentiation, aging, or tumor regression (reviewed in reference
86). PCD can be triggered by a variety of stimuli,
including viral infection (reviewed in reference
79), growth factor withdrawal (26), and
DNA damage (reviewed in reference 25). Cells exhibit
nuclear fragmentation, chromatin condensation, cleavage of DNA into
nucleosomal oligomers, cell shrinkage, and appearance of apoptotic
bodies which are engulfed by surrounding cells (reviewed in references
91 and 95).
The tumor suppressor protein p53 monitors cell cycle checkpoints
(40), senses DNA damage (42), assembles DNA
repair machinery (89), modulates gene amplification
(96), and activates PCD (78, 97). The ability of
p53 to induce PCD is crucial for tumor suppressor function (15,
28, 47, 81, 97). However, whether p53's ability to activate gene
expression is required for PCD is controversial (6, 70, 88).
A p53-inducible gene product, p21Waf1, a component of
p53-dependent G1 arrest, is dispensable for p53-dependent PCD but is required for cell cycle arrest (reviewed in references 1, 17, 22, 41, and 49). A link
between p53 induction and activation of interleukin-1-converting
enzyme-like proteases, or caspases, following DNA damage has also been
established (9, 45, 71). p53-independent mechanisms of PCD
also exist, and they usually lead to caspase activation.
Many viruses induce PCD when cultured cells are infected (reviewed in
references 67, 79, and 82a).
Premature PCD of infected cells reduces burst size and thus prevents
spread. Members of the Alphaherpesvirinae subfamily, e.g.,
herpes simplex virus type 1 (HSV-1), varicella-zoster virus, or BHV-1,
induce PCD after infection of cultured cells (24, 30-32, 43,
72). BHV-1-induced PCD occurs at the
G0/G1 phase of the cell cycle in cultured
peripheral blood mononuclear cells (PBMC) (24, 31). Since
inactivated BHV-1 induced PCD of PBMC (30, 32), it is
possible that PCD was a result of PBMC activation (90) and
not a direct result of infection. Furthermore, it is not clear whether
PCD occurs when fibroblasts or epithelial cells are infected with
BHV-1.
In this study, we analyzed BHV-1-induced PCD in Madin-Darby bovine
kidney (MDBK) cells. Following infection of MDBK cells, PCD occurred
during the late stages of infection. In contrast to the case for PBMC,
PCD did not occur when virus preparations were inactivated by Psoralen
or heat. Virus-induced PCD appeared to occur in a p53-dependent manner.
Processing and activation of caspase 2 and cleavage of poly(ADP-ribose)
polymerase (PARP), actin, and Bcl-xL occurred after
infection, suggesting that caspases were activated. Inhibition of
caspase activity increased virus yield but delayed or inhibited virus release.
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MATERIALS AND METHODS |
Cells and virus.
MDBK cells or CV-1 cells (African green
monkey kidney cells; American Type Culture Collection, Rockville, Md.)
were grown in Earle's modified Eagle's medium supplemented with 10%
fetal calf serum. The Cooper strain of BHV-1 was obtained from the
National Veterinary Services Laboratory, Animal and Plant Health
Inspection Services (Ames, Iowa). MDBK cells were infected with 5 PFU
of BHV-1/cell.
BHV-1 was inactivated by photochemical treatment as described
previously (30). Briefly, Psoralen (catalogue no. 8399;
Sigma) was dissolved in dimethyl sulfoxide (DMSO) to a final
concentration of 200 µg/ml. This stock was added to the viral
suspension (107 50% tissue culture infective doses/ml) to
a final concentration of 1 µg/ml. This viral suspension was then
irradiated with 254-nm-wavelength UV (UVP Products, San Gabriel,
Calif.). Alternatively, virus was inactivated by boiling the viral
suspension for 10 min.
Antibodies and plasmids.
Antibodies for p53 (sc-99 and
sc-100), Bcl-2 (sc-783), Bax (sc-526), p21Waf1/Cip1
(sc-528), Bcl-xL (sc-1690), actin (sc-1616), and caspase 2 (sc-626) were purchased from Santa Cruz Biotechnology (Santa Cruz,
Calif.). PARP antibodies (SA252) were purchased from BioMol (Plymouth
Meeting, Pa.).
A chloramphenicol acetyltransferase (CAT) plasmid containing the murine
p53 promoter, p0.7CAT (p53CAT), was provided by V.
Rotter (University
of South Carolina). Plasmid pA10CAT contains
the simian virus 40 early
promoter and was obtained from B. Howard
(National Institutes of
Health). The plasmid containing the intact
human
p21
Waf1/Cip promoter, pWWPCAT, was obtained from B. Vogelstein (Johns Hopkins
University). pSV2-
-gal was purchased from
Clontech, Palo Alto,
Calif.
TUNEL assay.
The terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling
(TUNEL) technique detects endonucleolytically cleaved DNA by the
addition of labeled dUTP to DNA ends by terminal transferase. MDBK
cells grown on coverslips were infected with 5 PFU/cell and stained
with an in situ cell death detection kit (catalogue no. 1684 809;
Boehringer Mannheim) according to the manufacturer's instructions.
Hoechst 33342 staining.
Monolayers of MDBK cells grown on
glass coverslips were infected with BHV-1 at 5 PFU/cell. At various
times postinfection (p.i.), cells were washed with phosphate-buffered
saline (PBS), fixed with ice-cold methanol-acetone (1:1, vol/vol) for
10 min at 
20°C, and then washed once with PBS. Nuclei were stained
with Hoechst 33342 (catalogue no. B-2261; Sigma) (0.5 µg/ml in PBS) for 10 min in the dark, washed once with PBS, and mounted on slides in
glycerol-citric acid phosphate buffer (9:1, vol/vol), pH 4.1. Cells
were visualized under UV with a fluorescence microscope (Microphot;
Nikon, Mellville, N.Y.).
Analysis of high-molecular-weight DNA after infection.
Monolayers of MDBK cells were grown in 100-mm-diameter plates and then
infected with 5 PFU of BHV-1 per cell. DNA from uninfected or infected
cells was extracted (43). Five micrograms of each DNA sample
was electrophoresed on a 1.5% agarose gel containing 0.1 µg of
ethidium bromide per ml. The DNA was visualized under UV light,
and the sizes of the respective amplified products were estimated
by comparing the mobilities with a 100-bp ladder (catalogue no.
15628-019; GIBCO-BRL).
Western blot analysis.
Extract from infected or uninfected
cells was prepared as described previously (33).
Approximately 20 µg of extract was resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
onto Immobilon (Millipore) membranes according to the manufacturer's
instructions. Membranes were probed with the indicated antibodies, and
Western blots were developed by chemiluminescence (ECL+ plus, RPN 2132;
Amersham). The sizes of the proteins on SDS-PAGE were estimated by
using Amersham molecular weight markers (catalogue no. RPN 756).
RNA PCR analysis.
Total RNA from uninfected or infected
cells was extracted by using RNAgents (total RNA isolation system from
Promega; catalogue no. Z5110) according to the manufacturer's
instructions. Three micrograms of total RNA was reverse transcribed by
using a random primer (Invitrogen) with reverse transcriptase from
GIBCO-BRL according to the manufacturer's instructions. The
first-strand cDNA was amplified by using the primers described in Table
1. The cDNA was amplified in a Hybaid
thermal cycler under the following conditions: 95°C for 1 min, 60°C
for 1 min, 72°C for 2 min, and 72°C for 7 min to allow for final
extension. Amplified products were resolved on 2% agarose gels
(NuSieve). The sizes of the amplified products were estimated by using
X174 replicative DNA cleaved with HaeIII DNA (New England
Biolabs).
Immunoprecipitation.
Uninfected or infected MDBK cells were
rinsed twice with methionine-free Dulbecco modified Eagle medium (DMEM)
(GIBCO-BRL) and incubated in methionine-free DMEM at 37°C for 30 min.
The cells were then incubated (37°C for 4 h) with 200 µCi of [35S]methionine (Amersham) in 2 ml of
methionine-free DMEM supplemented with 1% dialyzed fetal bovine serum.
The labeled cells were then washed with ice-cold PBS. One milliliter of
lysis buffer (50 mM Tris-HCl [pH 7.3], 75 mM NaCl, 0.5% Nonidet
P-40, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM leupeptin, 1 mM
apopain) was added directly to the dish and rocked for 30 min at 4°C.
Cells were scraped into a microcentrifuge tube and centrifuged at
10,000 × g for 10 min at 4°C.
Cell lysate (500 µl) prepared from metabolically labeled cells was
precleared with normal mouse serum for 30 min, followed
by
precipitation with a p53 or actin antibody overnight at 4°C.
Twenty
microliters of protein A-agarose beads (Santa Cruz) was
incubated for
30 min at 4°C, and the beads were subsequently washed
three times
(each) with 1 ml of PBS containing 1, 0.5, or 0.05%
Triton X-100. The
beads were then suspended in 50 µl of 2× SDS
loading buffer and
boiled for 10 min. Proteins were separated
by SDS-PAGE and visualized
by fluorography with En
3Hance
(Amersham).
Extraction of PARP.
Infected or uninfected cells were washed
twice with ice-cold PBS and then once with ice-cold buffer A (100 mM
Tris-HCl [pH 7.4], 10 mM MgSO4, 500 mM sucrose, 10 mM
PMSF, 0.5 µg of leupeptin per ml, 0.75 µg of pepstatin per ml, and
5 µg of antipain per ml). Cells were permeabilized by incubation on
ice for 20 min with buffer B (100 mM Tris-HCl [pH 7.4], 10 mM
MgSO4, 500 mM sucrose, 10 mM PMSF, 1% Nonidet P-40; 0.25 µg of leupeptin per ml, 0.35 µg of pepstatin per ml, and 50 µg of
antipain per ml). PARP was extracted from permeabilized cells by
incubation with cold buffer C (200 mM K2HPO4,
100 mM Tris-HCl [pH 7.4], 10 mM MgSO4, 500 mM sucrose, 10 mM PMSF, 0.5 µg of leupeptin per ml, 0.75 µg of pepstatin per ml,
and 5 µg of antipain per ml) on ice for 20 min. The extract was
centrifuged at 2,000 × g for 10 min at 4°C, and the
supernatant was collected and mixed with 4 volumes of urea loading
buffer (62.5 mM Tris-HCl [pH 6.8], 6 M urea, 10% glycerol, 2% SDS,
0.00125% bromophenol blue, and 5% -mercaptoethanol).
CAT assay.
MDBK cells grown in 60-mm-diameter dishes were
transfected with 1 µg each of p0.7CAT or pWWPCAT and pSV2--gal
with Superfect reagent (Qiagen) according to the manufacturer's
instructions. Cells were infected with BHV-1 (5 PFU/cell) at
24 h after transfection. Cells were harvested at 24, 36, or
48 h p.i., and CAT assays were performed as previously described
(19). The amounts of extract used to measure CAT activity
were adjusted based on -galactosidase (-gal) activity. -gal
activity was measured by using
ortho-nitrophenyl--D-galactopyranoside (ONPG)
(Sigma) as a substrate in colorimetric assays (57).
In vitro reconstitution of PCD. (i) Preparation of cell
extracts.
Uninfected or infected MDBK cells were washed once with
PBS and then with cell extract buffer [50 mM
piperazine-N,N'-bis(2-ethanesulfonic acid)
(PIPES) (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM
MgCl2, 1 mM dithiothreitol (DTT), 10 µM
cytochalasin B, and 1 mM PMSF]. The cells were harvested,
swelled by treatment with an equal volume of cell extract buffer on ice
for 20 min, and then lysed in a Dounce homogenizer (Wheaton) by 20 strokes with a type B pestle. The homogenate was centrifuged at
4°C for 20 min at 14,000 rpm in a Beckman (Palo Alto, Calif.) Avanti
30 centrifuge, and the supernatant was stored at 70°C.
(ii) Preparation of nuclei.
Confluent monolayers of CV-1
cells were washed with PBS, and nuclei were prepared (35).
The nuclei were suspended in storage buffer (10 mM PIPES [pH 7.4], 80 mM KCl, 20 mM NaCl, 250 mM sucrose, 5 mM EGTA, 1 mM DTT, 0.5 mM
spermidine, 0.2 mM spermine, 1 mM PMSF, and 50% glycerol) and stored
in aliquots at 70°C.
(iii) Cell-free apoptosis.
Cell extract (75 µg) was
incubated with 2 × 105 nuclei in 10 mM HEPES (pH
7.4)-50 mM NaCl-2 mM MgCl2-5 mM EGTA-1 mM DTT and incubated at 37°C for 1 h. The caspase inhibitor Z-VAD-FMK (25 µM) was added to extracts prior to incubation.
(iv) Hoechst staining of nuclei.
Four or five microliters of
the cell-free apoptosis reaction mixture was stained with 2 µl of 10 µM Hoechst 33342 (Sigma) in formalin on a glass slide for 2 to 3 min
in the dark. The slides were washed for 2 min with water, air dried,
and baked for 60 min at 50°C. Slides were mounted with 0.5 µg of
Canada balsam (catalogue no. C-1795; Sigma) per ml and observed under
UV light with a fluorescence microscope (Diaphot; Nikon).
Effect of caspase inhibitors on virus release and yield.
The
following caspase inhibitors were purchased from Calbiochem (La Jolla,
Calif.) and used for these studies: caspase 3 inhibitor II (Z-DEVD-FMK;
catalogue no. 264155), caspase 1 inhibitor I (Ac-YVAD-CHO; catalogue no. 400010), and caspase inhibitor I (Z-VAD-FMK;
catalogue no. 627610).
For the virus production assay, 25 µM Z-VAD-FMK was added at 6 and
24 h p.i., due to the short half-life of the peptide. Cells
and
medium were harvested at 48 h p.i. Nearly all cells which
were
infected but not treated with Z-VAD-FMK were rounded and
detached from
the plate at 48 h p.i. For total virus yield, cells
plus medium
were frozen and thawed three times to release virus
particles. The
yield of infectious virus was determined by estimating
the 50% end
points (
68).
For the virus release assay, cells and medium were separated by
centrifugation (1,000 rpm for 3 min) in a CR412 centrifuge
(Jouan, St.
Herblain Cedex, France). The resulting cell pellet
was frozen, thawed
three times, and suspended in 1 ml of DMEM.
Infectious virus yields in
the medium or in the cell pellet were
determined separately. The
percent virus release was calculated
by dividing the amount of
infectious virus released into the medium
by the total virus
yield.
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RESULTS |
BHV-1 induces PCD in cultured MDBK cells.
Although BHV-1
induces PCD in lymphoid cells (31, 32), it is not clear
whether infection of permissive fibroblasts or cells of epithelial
origin leads to PCD. The most commonly used markers of PCD are (i)
fragmentation of chromosomal DNA into oligonucleosomal fragments, which
can be detected by TUNEL assays or by agarose gel electrophoresis, and
(ii) chromatin condensation, which is identified by DNA staining
agents, Hoechst stain, or DAPI (4',6-diamidino-2-phenylindole), for
example (29). In addition, cell viability was assessed by trypan blue exclusion.
TUNEL-positive infected cells, but no TUNEL-positive mock-infected
cells, were readily detected at 48 h p.i. (Fig.
1). Condensed
chromatin, as judged by
Hoechst 33342 staining, was also detected
in many infected cells but
not in mock-infected cells (Fig.
1).
In mock-infected cells, chromatin
was diffusely stained, whereas
staining in infected cells was punctate
and marginated. Chromatin
condensation was observed in 3 to 5% of
infected cells at 24 h
p.i. and increased at 36 and 48 h p.i.
Since previous studies
demonstrated that inactivated BHV-1 initiates
PCD in PBMC (
30),
we tested whether inactivated virus could
induce PCD in MDBK cells.
In contrast to the case for PBMC, we were
unable to detect higher
levels of PCD after inactivated virus was added
to MDBK cells
(Fig.
2). Regardless of
whether the virus was inactivated by Psoralen
cross-linking or boiling,
chromatin condensation was not readily
detected at 48 h p.i.
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FIG. 1.
BHV-1 induces PCD in MDBK cells. MDBK cells were
infected with BHV-1 for 48 h. Mock-infected cells served as a
control. Hoechst 33342 staining was observed under UV light.
Magnifications, ×800 for Hoechst staining and ×200 for TUNEL assay.
Arrows point to TUNEL-positive cells or to nuclei that contain
condensed chromatin.
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FIG. 2.
Inactivated BHV-1 does not induce chromatin condensation
in MDBK cells. BHV-1 was treated with Psoralen or was heat inactivated
as described in Materials and Methods (magnification, ×400). As a
positive control, a similar amount of untreated BHV-1 was used to
infect cultures of MDBK cells (magnification, ×800). At 48 h
p.i., Hoechst 33342 staining was performed and the cells were observed
under UV light. Arrows point to a nucleus that contains condensed
chromatin.
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High-molecular-weight DNA prepared from cells infected for 48 h
was degraded and exhibited the characteristic laddering observed
after
PCD (Fig.
3A). Degraded DNA was not
readily detected in
mock-infected cells or in cells infected for
24 h. As expected,
the percentage of viable cells decreased as a
function of time
as determined by using trypan blue to monitor viable
cells (Fig.
3B). Taken together, these studies indicated that PCD
occurred
in MDBK cells during the late stages of infection.
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FIG. 3.
Infection induces DNA degradation and reduces cell
viability. (A) Agarose gel electrophoresis of DNA extracted from MDBK
cells at 24, 36, or 48 h p.i. Lane 0, DNA prepared from
mock-infected cells; lane M, 100-bp ladder. Numbers on the left
indicate base pairs. (B) Measurement of cell viability as judged by
trypan blue staining. Data from one of two experiments is shown.
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Examination of p53 after infection.
The p53 protein inhibits
cell cycle progression after DNA damage, thus facilitating DNA repair
(69), or it can induce PCD if extensive DNA damage occurs
(97). During p53-dependent PCD, p53 levels generally
increase (reviewed in reference 2). Although steady-state levels of p53 protein were not dramatically induced after
infection (Fig. 4A), higher levels of
35S-labeled p53 protein were present at 24 or 36 h
p.i. (Fig. 4B). The antibody used for immunoprecipitation recognizes
wild-type p53 but not mutated forms, suggesting that MDBK cells contain wild-type p53. Compared to actin RNA levels, p53 RNA levels were slightly higher at 24 h p.i. (Fig. 4C). At 36 h p.i., a
sixfold increase in p53 promoter activity (p53CAT) was observed (Fig. 4D). In contrast, the simian virus 40 early promoter (pA10CAT) was not
stimulated during the late stages of infection.
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FIG. 4.
Analysis of p53 after infection. MDBK cells were
infected with BHV-1 for the indicated times (hours p.i.). Mock-infected
cells (lanes M) served as a control. (A and B) Western blot analysis of
p53 and actin in infected cells (A) and fluorography of
35S-labeled p53 and actin (B). Immunoprecipitation was
carried out with an antibody that specifically recognizes p53 or actin.
Arrows on the right of each panel indicate the position of p53 or
actin. The numbers to the left of each panel represent the positions of
protein markers (in kilodaltons). (C) RNA PCR analysis with primers
specific for p53 or -actin. Three micrograms of total RNA was
reverse transcribed with Superscript reverse transcriptase, and the
cDNA was amplified by PCR. The arrows on the right represent the
positions of the amplified cDNA. Numbers on the left are the positions
of X174 DNA digested with HaeIII (in base pairs). (D) A
mouse p53 promoter construct (p53CAT) or pA10CAT was cotransfected with
pSV2--gal into MDBK cells with Superfect transfection reagent from
Qiagen. Twenty-four hours after transfection, the cells were infected
with 5 PFU/cell. Cells were harvested at the indicated times p.i., and
CAT activity was measured. The amount of extract used to measure CAT
activity was adjusted based on -gal activity in the respective
samples. Ac-CM, acetylated chloramphenicol.
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Overexposure of p53 Western blots revealed a band that migrated with an
apparent molecular mass of 40 kDa at 48 h p.i. (Fig.
5). The 40-kDa band was detected in
several different experiments.
Following DNA damage, p53 binds
specifically to damaged DNA and
is cleaved into 40- and 50-kDa products
(p40 or p50, respectively)
(
61). Cleavage of p53 occurs at
the C terminus by autoproteolysis
and is important for p53-dependent
PCD. In summary, these studies
demonstrated that infection increased
p53 protein levels slightly
and that p53 was cleaved.
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FIG. 5.
Cleavage of p53 after infection. Western blot analysis
of p53 with extracts prepared from mock-infected (lane M) or infected
(24, 36, or 48 h p.i.) cells is shown. The blots were probed with
an antibody that detects the N terminus of p53 (Pab 240). Cleaved
products are designated p50 and p40. The numbers to the left represent
the positions of protein markers (in kilodaltons).
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Examination of Bax, Bcl-2, and p21Waf1/Cip1 after
infection.
Several genes that regulate the cell cycle or apoptosis
are transactivated by p53. These include the genes encoding
p21Waf1/Cip1 (21), GADD45 (40), Mdm2
(62), cyclin G (60), and Bax (53).
Since p53 protein expression increased after infection, the levels of
the p21 and Bax proteins were examined by Western blot analysis. Bax
and p21Waf1/Cip1 levels increased as a function of time
after infection (Fig. 6A). Following
infection, the p21Waf1/Cip1 promoter (pWWPCAT) was
activated more than 30-fold (Fig. 6B), which agreed with an increase in
p21Waf1/Cip1 protein levels.
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FIG. 6.
Analysis of p21Waf1/Cip1 and Bax after
infection. (A) Whole-cell lysate was prepared from mock-infected cells
(lane M) or infected cells (24, 36, or 48 h p.i.), and Western
blot analysis was performed with an antibody which specifically
recognizes Bax or p21. Arrows indicate the positions of the respective
proteins. The numbers to the left represent the positions of protein
markers (in kilodaltons). (B) A human p21Waf1 promoter
construct (pWWPCAT) was cotransfected with pSV2--gal into MDBK cells
with Superfect transfection reagent from Qiagen. Twenty-four hours
after transfection, cells were infected with 5 PFU/cell. Cells were
harvested at the indicated times p.i., and CAT activity was measured.
The amount of extract used to measure CAT activity was adjusted based
on -gal activity in the respective samples. Ac-CM, acetylated
chloramphenicol.
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In contrast to that of Bax and p21
Waf1/Cip1, expression of
the antiapoptotic protein Bcl-2 is negatively regulated by p53
(
54). Bcl-2
protein levels in infected cells were slightly
lower at 36 or
48 h p.i. than those in mock-infected cells (Fig.
7B). To confirm
that Bcl-2 expression was
repressed after infection, RNA PCR analysis
was performed with total
RNA prepared from mock-infected cells
or cells infected with BHV-1. A
268-bp fragment corresponding
to the Bcl-2 amplified product was
detected in mock-infected cells
but was not readily detected at 24, 36, or 48 h p.i. (Fig.
7A).
As expected, actin RNA and protein levels
were not altered dramatically.
These studies demonstrated that Bax and
p21
Waf1/Cip1 were induced after infection but that Bcl-2
levels decreased.
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FIG. 7.
Analysis of Bcl-2 after infection. (A) RNA PCR analysis
of Bcl-2 or actin mRNA in mock-infected cells (lane M) or infected
cells (24, 36, or 48 h p.i.). Three micrograms total RNA was
reverse transcribed with Superscript reverse-transcriptase, and the
cDNA was amplified by PCR. Numbers on the left are the positions of
X174 DNA digested with HaeIII (base pairs). (B)
Whole-cell lysate was prepared from mock-infected cells (lane M) or
infected cells (24, 36, or 48 h p.i.), and Western blot analysis
was performed with an antibody which specifically recognized Bcl-2 or
actin. The arrows indicate the position of Bcl-2 or actin. The numbers
to the left represent the positions of protein markers (in
kilodaltons).
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Proteins that are cleaved during PCD undergo proteolysis after
infection.
Caspases are cysteine proteases that play a pivotal
role in PCD (65). Caspases 2, 3, and 7 are activated by
cleavage during p53-dependent PCD (9, 45, 71). To test
whether infection led to cleavage of caspase 2, an antibody that
recognizes intact caspase 2 and its 12-kDa cleavage product was
utilized. At 36 or 48 h p.i., an increase in the 12-kDa cleavage
product was detected (Fig. 8).
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FIG. 8.
Analysis of caspase 2 after infection. Whole-cell lysate
was prepared from mock-infected cells (lane M) or infected cells (24, 36, or 48 h p.i.), samples were electrophoresed in a 12.5% gel,
and Western blot analysis was performed with an antibody which
specifically recognizes caspase 2. The arrow at 12 kDa indicates the
cleaved form of caspase 2, and the one at 51 kDa shows the location of
intact caspase 2. Molecular mass markers are indicated on the left and
are expressed as kilodaltons.
|
|
To determine whether other caspase substrates were cleaved following
infection, Western blot analysis was performed with antibodies
directed
against Bcl-x
L, actin, or PARP. The Bcl-x
L gene
encodes
two distinct proteins as a result of alternative splicing, a
long
form (Bcl-x
L) and a short form (Bcl-x
S
(
4). Bcl-x
L inhibits
apoptosis, but
Bcl-x
S antagonizes the antiapoptotic action of
Bcl-x
L and Bcl-2 (
51). Bcl-x
L is
cleaved during PCD by caspases
(
12). Actin is cleaved by
caspase 3, and this appears to occur
after cleavage of PARP
(
50). At 36 or 48 h p.i., cleavage of
PARP was detected
(Fig.
9A). Cleavage of Bcl-x
L
and actin was
detected at 48 h p.i. (Fig.
9B and C). Thus,
cleavage of caspase
2, PARP, actin, and Bcl-x
L occurred
during the late stages of
infection, suggesting that the caspase
cascade was activated.
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|
FIG. 9.
Cleavage of PARP, Bcl-xL, and actin after
infection. Whole-cell lysate was prepared from mock-infected cells
(lanes M) or infected cells (24, 36, or 48 h p.i.), and Western
blot analysis was performed with an antibody which specifically
recognized PARP (A), Bcl-xL (B), or -actin (C). The
arrows indicate the positions of the respective cleaved proteins, and
the ovals indicate the intact proteins.
|
|
Inhibition of caspases delays BHV-1-induced cytopathogenic effects
but enhances total virus yield.
To determine if caspases play a
role during infection, MDBK cells were treated with caspase inhibitors
at 6 h p.i., and the degree of cytopathology was monitored by
light microscopy. For these studies, three different classes of caspase
inhibitors were tested: (i) Z-VAD-FMK, which inhibits caspase 1-like
proteases and prevents apoptosis mediated by a variety of stimuli
(7, 80); (ii) Ac-YVAD-CHO, which inhibits
interleukin-1-converting enzyme (83); and (iii)
Z-DEVD-FMK, which inhibits caspase 3-like enzymes (59).
Since adherence of infected cells to the tissue culture dish is a
reliable marker for viability of BHV-1-infected MDBK cells, we used
this as a means to estimate what role, if any, caspase inhibitors
played during infection. Relative to untreated infected cultures or
cultures which were infected and treated with DMSO, more cells were
attached to the plate when cultures were treated with Z-VAD-FMK at
48 h p.i. (Fig. 10). Although
Z-DEVD-FMK or Ac-YVAD-CHO treatment increased the number of cells
attached to plastic after infection, the effect was not as dramatic as that with Z-VAD-FMK. Furthermore, addition of all three caspase inhibitors did not dramatically enhance the protective effect. If
cultures were incubated for longer periods of time, all cells were
eventually released from the substrate and were not viable (20). The simplest explanation of this study was that
caspases were not absolutely required for virus infection.
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FIG. 10.
Z-VAD-FMK delays cell death after infection. The
indicated caspase inhibitor (25 µM) was added at 6 and 24 h p.i.
Representative areas of the cultures were photographed (magnification,
×200) at 48 h p.i. Since the caspase inhibitors were suspended in
DMSO, a control experiment was performed to test the effects of DMSO on
PCD.
|
|
To test whether inhibition of caspases by Z-VAD-FMK treatment had an
effect on virus release, the amount of virus released
from infected
cells was measured in cultures treated with Z-VAD-FMK
and compared to
that for untreated cultures. Since Z-VAD-FMK has
a short half-life
(
14) and the medium obtained from infected
cells was diluted
more than 1,000-fold for measuring virus, it
was unlikely that residual
levels of Z-VAD-FMK were active or
affected the study. When cultures
were subjected to three cycles
of freeze-thawing (
70 to 37°C) to
estimate the total amount of
virus in infected cells, a twofold
increase in virus titers was
observed following treatment with
Z-VAD-FMK (Fig.
11B). Twofold-less
virus was present in the medium that was removed at 48 h p.i.
when
cells were treated with Z-VAD-FMK (Fig.
11A). In summary,
these studies
suggested that Z-VAD-FMK treatment enhanced the
amount of total
infectious virus but reduced or delayed virus
release.
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FIG. 11.
Z-VAD-FMK enhances virus yield but reduces virus
release at 48 h p.i. Z-VAD-FMK (25 µM) was added to cultures 6 and 24 h p.i. Cells were removed from the dish at 48 h p.i.
(A) Cell-free virus release was measured by collecting the medium from
infected cells at 48 h p.i. after removal of cells by
centrifugation. (B) Cells plus culture medium were subjected to three
freeze-thaw cycles (70 to 37°C), and cells were removed by
centrifugation. The amount of infectious virus was measured as
described in Materials and Methods. Data are the means and standard
deviations from three independent experiments. TCID50, 50%
tissue culture infective dose.
|
|
In vitro reconstitution of PCD.
Experiments were performed to
confirm the importance of caspases during virus-induced PCD and to
prove that the components necessary to initiate PCD of healthy nuclei
were present in infected cells. Several studies have demonstrated that
cell extracts prepared from cells undergoing PCD mimic the
intracellular environment of a dying cell and thus can induce nuclear
changes which are observed during PCD (chromatin condensation or DNA
laddering, for example) (34, 44, 58). Cell extract was
prepared from infected cells (24, 36, or 48 h p.i.) and added to
nuclei prepared from uninfected cells, and alterations in nuclear DNA
were monitored by Hoechst 33342 staining. Nuclei from CV-1 cells were
used because these cells have a low frequency of PCD when actively
growing and because CV-1 cells are not permissive for BHV-1 infection. Incubation of healthy CV-1 nuclei with extract prepared from
BHV-1-infected cells (24, 36, or 48 h p.i.) induced chromatin
condensation (Fig. 12). A
higher percentage of nuclei contained condensed chromatin when the
nuclei were incubated with extracts prepared from cells infected for 48 versus 24 h p.i. The same extract prepared from mock-infected
cells did not efficiently induce chromatin condensation. Incubation of
the extract prepared from infected cells (48 h p.i.) with Z-VAD-FMK
inhibited chromatin condensation. These studies indicated that extract
prepared from infected cells can induce chromatin condensation of
nuclei prepared from nonpermissive cells and that active caspases play
a role during this process.
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|
FIG. 12.
Extracts prepared from infected cells induce chromatin
condensation. Nuclei prepared from CV-1 cells (2 × 105) were incubated with 75 µg of extract prepared from
mock-infected or infected cells. For caspase inhibition, 25 µM
Z-VAD-FMK was added prior to addition of nuclei. Nuclei were incubated
with the respective cell extracts for 2 h at 37°C. Nuclei were
stained with Hoechst 33342 as described in Materials and Methods, and
the samples were observed under a UV microscope. Magnification,
×400.
|
|
| |
DISCUSSION |
Although PCD occurred in MDBK cells after infection,
inactivated virus did not efficiently induce PCD (Fig. 2). This
finding is in contrast to those from previous studies using PBMC
(30-32) or activated CD4+ T cells
(24). In general, lymphoid cells are prone to PCD, whereas fibroblasts or epithelial cells are more resistant
(90), suggesting that inactivated virus can induce PCD in
cells which easily undergo PCD. It is also possible that novel
virus-host interactions may be important for inactivated virus to
initiate PCD in lymphocytes. For example, a novel member of the tumor
necrosis factor (TNF) NGF receptor family (HVEM) (56)
mediates HSV-1 entry into activated T cells. Conversely, entry of HSV-1
and BHV-1 into epithelial or other nonlymphoid cells is mediated by an
unrelated membrane glycoprotein which resembles the poliovirus
receptor (27). If BHV-1 entry into PBMC is mediated by
binding of a viral glycoprotein to a TNF-like receptor, this
interaction could lead to PCD in the absence of active infection,
because binding of ligand to TNF receptors can initiate PCD (reviewed
in references 26, 82a, and 91).
Since PCD was not observed until late after infection, the early events
of productive infection may inhibit PCD, as previously described for
HSV-1 (43). The possibility that BHV-1 encodes positive and
negative regulators of PCD supports the hypothesis that
cell-type-specific interactions are important during PCD.
The levels of newly synthesized p53 protein and promoter activity
increased after infection (Fig. 4), suggesting that p53 plays a role in
virus-induced PCD. Genes that are regulated by p53 (those for Bax,
p21Waf1/Cip1, and Bcl-2, for example) were also induced or
repressed in a p53-dependent fashion (Fig. 6 and 7). Cleavage of p53
occurs in response to DNA damage, generating p50 or p40
(55), and consequently alters the transcriptional regulatory
activity of p53 (11, 36, 37). HSV-1 and HSV-2 induce DNA
damage after infection (64, 74), suggesting that infection
by BHV-1 leads to DNA damage and p53 induction. E1A, E2F-1, or c-Myc
can also sensitize cells to undergo p53-dependent PCD (3, 7, 18,
26, 46, 66, 94). Myc-mediated PCD may have relevance to BHV-1
infection, because c-Myc levels are elevated after infection (20,
31). Despite the findings that link p53 to BHV-1-induced PCD,
p53-independent mechanisms may also be important. This hypothesis is
supported by the finding that p53 induction was modest compared to
induction by adenovirus infection (82) and that adenovirus
infection induces PCD by p53-dependent and -independent mechanisms
(82, 82a). A p53
/ primary bovine cell line
is not available, making it difficult to directly address this issue.
The importance of caspase activation during PCD is well established
(87). The finding that caspase 2 is cleaved after infection indicated that caspases are activated. A number of substrates for
caspases have been identified: protein kinases (8, 23), Rb
(38), Sp1 (63), Bcl-2 (12), ICAD
(73), Bcl-xL (16), and cytoskeletal
proteins (lamin B1 and actin) (5, 9, 50). Bcl-xL protects cells from p53-dependent PCD (51,
76), and cleavage of Bcl-xL inactivates this function
or induces PCD (16), suggesting that cleavage of
Bcl-xL after infection is important. Although cleavage of
PARP, Bcl-xL, and actin occurred after infection, cleavage
of PARP was detected earlier. In general, PARP cleavage is an early
event during PCD, while cytoplasmic proteins are necessary for
maintaining the structural integrity of the cell and thus are cleaved
later (25, 91).
PCD is an important host cell defense that limits viral infection.
Prevention of PCD enhances production of human immunodeficiency virus
(13), simian immunodeficiency virus (10), and
adenoviruses (14). Conversely, induction of PCD by specific
viral genes near the end of infection may promote cell-to-cell spread
and virus release and interfere with inflammatory responses. The
adenovirus E3 gene promotes virus release and enhances virus growth in
cultured cells by promoting cell death that is distinct from PCD
(85). The finding that Z-VAD-FMK enhanced total infectious
virus production at 48 h p.i. implied that prevention of PCD
enhanced virus yield. Since virus release was reduced at 48 h p.i.
when cells were treated with Z-VAD-FMK (Fig. 10), we hypothesized that
caspase activation or other events during PCD led to virus assembly or
release. Infection of cattle typically leads to upper respiratory tract
infections and transient immunosuppression, thus allowing opportunistic
bacterial infections (reviewed in reference 84). If
extensive lymphocyte PCD occurs after infection, immunosuppression
would occur. PCD of nonlymphoid cells in the upper respiratory tract
could also lead to virus spread and interfere with an inflammatory
response. To understand the role that PCD plays with respect to BHV-1
pathogenesis, it will be necessary to determine what cell types undergo
PCD in infected cattle and correlate these findings to symptoms
associated with infection.
| |
ACKNOWLEDGMENTS |
We are grateful to Kotlo Kumar (University of Illinois, Chicago)
for suggesting cell-free apoptosis and several experimental protocols
and to Harikrishna Nakshatri (Indiana University Medical Center,
Indianapolis) and Nagendra Prasad (University of California, San
Francisco) for invaluable suggestions. We thank Heidi Hoff and Judi
Wheeler for help with the microscopy. We also thank Marty Dickman and
Fernando Osorio for carefully reading the manuscript.
This work is supported by grants from the USDA (9702394 and 9802064)
and the Center for Biotechnology, University of NebraskaLincoln. L. Devireddy was supported in part by a fellowship from the Center for
Biotechnology, University of NebraskaLincoln.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Veterinary and Biomedical Sciences, Center for Biotechnology,
University of NebraskaLincoln, Fair St. at East Campus Loop, Lincoln,
NE 68583-0905. Phone: (402) 472-1890. Fax: (402) 472-9690. E-mail: cj{at}unlinfo.unl.edu.
| |
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Journal of Virology, May 1999, p. 3778-3788, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
