From the Division of Infectious Diseases and
Immunology, Saint Louis University, St. Louis, Missouri 63110 and
§ Cytokine Research Laboratory, Department of Molecular
Oncology, University of Texas M. D. Anderson Cancer Center,
Houston, Texas 77030
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ABSTRACT |
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Hepatitis C virus (HCV) putative core protein has
displayed many intriguing biological properties. Since tumor necrosis
factor (TNF) plays an important role in controlling viral infection, in
this study the effect of the core protein was investigated on the
TNF- induced apoptosis of human breast carcinoma cells (MCF7). HCV
core protein when expressed inhibited TNF-
-induced apoptotic cell
death unlike the control MCF7 cells, as determined by cell viability
and DNA fragmentation analysis. Additionally, HCV core protein blocked
the TNF-induced proteolytic cleavage of the death substrate
poly(ADP-ribose) polymerase from its native 116-kDa protein to the
characteristic 85-kDa polypeptide. Results from this study suggest that
the HCV core protein plays a role in the inhibition of TNF-
-mediated
cell death. Thus, the ability of core protein to inhibit the
TNF-mediated apoptotic signaling pathway may provide a selective
advantage for HCV replication, allowing for evasion of host antiviral
defense mechanisms.
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INTRODUCTION |
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Hepatitis C virus (HCV)1
is an important cause of morbidity and mortality worldwide, causing a
spectrum of liver disease ranging from an asymptomatic carrier state to
end-stage liver disease. The most important feature of persistent HCV
infection is the development of chronic hepatitis in half of the
infected individuals and the potential for disease progression to
hepatocellular carcinoma (1). Unfortunately, a number of important
issues related to HCV-mediated disease progression is unknown at this
time. An HCV genome contains a linear, positive-strand RNA molecule of
~9,500 nucleotides encoding a single polyprotein precursor of
~3,000 amino acids (2). The polyprotein is cleaved by both host and viral proteases (3, 4) to generate three putative structural proteins
(core, E1, and E2) and at least six nonstructural proteins (NS2, NS3,
NS4A, NS4B, NS5A, and NS5B). The genomic region encoding the putative
core protein is located between amino acids 1-191. HCV core protein
may be the fundamental unit for the encapsidation of genomic RNA to
facilitate virus morphogenesis. However, in vitro studies
suggest that the HCV core protein has many additional biological
properties. The core protein transactivates the human c-myc
proto-oncogene and unrelated viral promoters and suppresses c-fos, p53, and human immunodeficiency virus type 1 long
terminal repeat promoter activities (5-7). HCV core protein transforms primary rat embryo fibroblasts in association with a cooperative oncogene to a tumorigenic phenotype (8), interacts with the lymphotoxin- receptor to possibly modulate immune function (9), and
associates with apolipoprotein II for a potential role on lipid
metabolism (10). A recent study (11) suggests that missense mutations
in the clustering variable region of the hydrophilic domain (residues
39-76) of the core gene may be involved in the pathogenesis of chronic
HCV infection during hepatocellular carcinogenesis.
Viral infections may often induce an apoptotic response as a defense
mechanism in host cells, and many viruses encode proteins that inhibit
this mechanism (12). Alterations in cell survival contribute to the
pathogenesis of a number of human diseases including viral oncogenesis
(13). Tumor necrosis factor (TNF-) is a major inflammatory cytokine,
secreted primarily by activated macrophages and T lymphocytes, which is
thought to limit infections by a variety of microorganisms (14, 15).
TNF-induced apoptosis requires the activation of one or more of the
interleukin-1
converting enzymes (ICEs), which function in the
apoptotic response. ICE is a cysteine protease that catalyzes the
proteolytic processing of the protein-inflammatory cytokine
interleukin-1
from an inactive precursor form to the active mature
form. The 116-kDa DNA repair enzyme poly(ADP-ribose) polymerase (PARP)
has been shown to be proteolytically processed to a signature 85-kDa
fragment from an aspartate-specific cleavage by an ICE-like protease,
which appears to be distinct from ICE. Importantly, PARP cleavage has been associated with a variety of apoptotic responses, including TNF-mediated cell death. In cultured cells infected with different DNA
or RNA viruses, TNF may act to inhibit virus replication or induce
apoptosis. Some viruses, in turn, have evolved strategies to block the
antiviral effects of TNF. Recently, several viral proteins have been
reported to interfere with the TNF-mediated signaling pathway leading
to apoptosis. For example, CrmA (a cowpox virus protein) or the
baculovirus protein p35 directly or indirectly inhibit ICE-like
protease and presumably preclude TNF function (16, 17). On the other
hand, the adenovirus E3-10.4K/14.5K protein complex inhibits
apoptosis by inhibiting TNF-induced translocation of cPLA2 from the
cytosol to membrane (18). Equine herpesvirus type 2 E8 protein and
molluscum contagiosum virus MC159 protein also block cytokine-mediated
apoptosis, probably by interfering with the ability of FLICE/caspase-8
(19, 20).
In this study, we examined whether HCV (genotype 1a) core protein has a
role in TNF--mediated cell death by utilizing a TNF sensitive MCF7
cell line. Our result suggests that the core protein inhibits the onset
of TNF-mediated apoptosis and the associated PARP cleavage.
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Transfections-- The TNF-sensitive MCF7 breast carcinoma cell line (16) was kindly provided by Dr. V. M. Dixit (University of Michigan Medical School, Ann Arbor). The plasmid pBabe core, containing the entire HCV-1a core genomic region, has been described previously (21). The MCF7 cell line was transfected with pBabe core plasmid DNA using LipofectAMINETM (Life Technologies, Inc.), and stable clones were selected using puromycin as described previously (21). The vector DNA-transfected MCF7 cell line as a positive control (MCF7-V), a pool of HCV core stable transfectants (Core-pool), and three individual clones (Core-7, Core-10, and Core-12) as the experimental cells were grown for further studies.
RNA Analysis--
Total RNA was isolated using an acid-phenol
extraction method (22) from the control and experimental cells.
Approximately 2 µg of RNA from each cell line was used for a reverse
transcription polymerase chain reaction. A reverse transcriptase
reaction was performed using random primers and avian myeloblastosis
virus reverse transcriptase at 45 °C for 30 min. Subsequently,
cDNA was amplified by polymerase chain reaction using synthetic
oligonucleotide primers (sense 5-CGTAGACCGGGATCCTGAGCACGAA-3
and
antisense 5
-GAAGCGGGTCTAGAGCAAGCAAGA-3
) at 94 °C for denaturing,
60 °C for annealing, and 72 °C for extension for 30 cycles. The
amplified products were analyzed by agarose gel electrophoresis and
followed by ethidium bromide staining.
Assessment of TNF-cytotoxicity and Apoptosis--
To determine
the level of TNF-induced toxicity in HCV core-transfected MCF7 cells,
various doses of TNF- were initially used to determine parent MCF7
cell death in a dose-dependent manner. To further examine
TNF-associated cell death, approximately 5 × 104
cells were exposed to 15 ng/ml TNF (3.75 × 107
units/mg, Promega) for 18 h in the absence of cyclohexamide and incubated an additional 48 h in TNF-free medium. Surviving cells were trypsinized and collected for counting by trypan blue exclusion. The sensitivity of MCF7 cells to TNF in the absence of cyclohexamide was also determined by the modified tetrazolium salt (MTT) assay as
described earlier (23). Briefly, cells (3 × 103/well)
were incubated for the indicated time frame in the presence or absence
of different concentrations of TNF in a final volume of 0.2 ml for
72 h. Analysis of cell viability was carried out using the
addition of 0.02 ml of a 5 mg/ml solution of MTT. After 2 h of
incubation at 37 °C, 0.1 ml of extraction buffer (20% SDS and 50%
dimethyl formamide, pH 4.7) was added. After incubation overnight at
37 °C, optical density was measured at 590 nm using a 96-well
multiscanner autoreader (Dynatech MR 5000, Dynatech Laboratories,
Chantilly, VA) with the extraction buffer serving as a blank. In a
different experiment, to examine the nature of TNF-induced cell death
in the transfected clones and control cells, floating and adherent
cells were collected after TNF treatment for an indicated period of
time. Cells were lysed, and genomic DNA was isolated as described
previously (21) for analysis by agarose gel electrophoresis.
Detection of HCV Core Protein in MCF7 Stable Transfectants and
PARP Cleavage Following TNF Treatment--
The preparation of cell
lysates and immunoblotting of TNF--treated cells was performed as
described earlier (16). Similar amounts of cellular proteins
transferred onto nitrocellulose membrane were incubated with a rabbit
polyclonal antiserum (1:1000 dilution) to the HCV core protein (kindly
provided by Michael M. C. Lai, University of Southern California,
Los Angeles) for 2 h at room temperature. An anti-rabbit
immunoglobulin coupled with horseradish peroxidase was used as the
second antibody for detection of HCV core protein by chemiluminescence
(ECL, Amersham). For detection of PARP cleavage following treatment of
cells with TNF-
, the anti-PARP monoclonal antibody (kindly provided
by S. Chatterjee and N. Berger, Case Western Reserve University, Ohio)
was used at a dilution of 1:500. A secondary antibody conjugate
(anti-mouse Ig/horseradish peroxidase, Amersham) was used at a dilution
of 1:10,000. The peroxidase signal was visualized by chemiluminescence (ECL). The molecular weight of HCV core protein bands in the immunoblot or PARP cleavage products was estimated from the migration of standard
protein molecular weight markers (Life Technologies, Inc.).
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RESULTS AND DISCUSSION |
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Transfection of MCF7 Cells with HCV Core Gene--
TNF-sensitive
MCF7 cells have been used earlier to study the role of viral proteins
in apoptotic cell death (16, 17, 19, 20). We selected the MCF7 cell
line as a model in our study due to its sensitivity to TNF-mediated
apoptosis. This cell line is amenable to viral proteins for regulation
of this important biological process. MCF7 cells stably transfected
with HCV core gene under the control of murine leukemia virus long
terminal repeat in the pBabe-puro plasmid (21) were analyzed for the level of TNF--mediated apoptosis indicated in the cell line. A pool
of stable transfectants and three individual colonies were selected
following treatment with puromycin and arbitrarily included in this
study. mRNA synthesis for the core protein in stable transfectants was characterized by reverse transcription polymerase chain reaction. HCV core gene-transfected pooled cells (Core-pool) and the three clones
exhibited core specific mRNA expression, and the results are shown
in Fig. 1, panel A. Stable
core transfectants of MCF7 cells displayed protein expression when
studied by immunoblot analysis, and the results are shown in Fig. 1,
panel B. Individual clones exhibited differences in the
level of protein expression, and the clone, Core-7, appeared to have
the maximum core protein expression among the cell lines included in
this study. A study to assess cell viability by the trypan blue
exclusion method suggested that HCV core protein expression in MCF7
cells did not have an apparent effect upon the growth rate as compared
with the vector-transfected control cells except clone 12. The reason
for higher growth of clone 12 is not clear at this time.
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HCV Core Protein Inhibits TNF--induced
Cytotoxicity--
Various doses of TNF-
were initially used to
determine the dose-dependent response of positive control
MCF7-V and core-transfected MCF7 for cell death. At 15 ng/ml
TNF-treated cells, 53% cell viability was observed in MCF7-V positive
control cells (Fig. 2). Under similar
conditions, HCV core transfection inhibits TNF-induced cytotoxicity to
a varying degree in selected clones and pooled stable transfectants.
The protection provided by HCV core from TNF-
-induced apoptosis
correlated with the level of protein expression as determined by
immunoblot assay. Thus, the expression of HCV core protein appeared to
inhibit TNF-
-induced death in MCF7 cells.
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HCV Core Protein Inhibits TNF--induced DNA
Fragmentation--
Endonucleolysis is considered a key biochemical
event of apoptosis, resulting in the cleavage of nuclear DNA into
oligonucleosome-sized fragments. DNA isolated from control MCF7 cells
following treatment with TNF-
(15 ng/ml) for 24 h exhibited a
typical oligonuclear fragmentation pattern on agarose gel
electrophoresis (Fig. 3), whereas DNA
isolated from core-transfected MCF7 cell clones treated with TNF-
inhibited DNA fragmentation. Similar inhibition of DNA fragmentation
was also observed following TNF-
treatment of core gene-transfected
pooled cells. Results from this study indicated that HCV core protein
inhibits TNF-
-induced fragmentation of cellular DNA.
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HCV Core Protein Inhibits TNF--induced Cleavage of
Poly(ADP-ribose) Polymerase Substrate--
Recent studies have pointed
to a role for the family of caspase proteases (ICE/ced-3 proteases) in
apoptosis, which act upstream of endonuclease (24). Proteases in
apoptosis came to the forefront with studies on the proteolysis of
PARP, initially described in cells induced to undergo apoptosis by
various chemotherapeutic agents including etoposide (25, 26). This
event was later determined to be catalyzed by a protease resembling
ICE. The human homolog of this protease has been cloned and is now
known (27, 28) as caspase-3 (CPP 32/yama/apopain). To determine whether the core protein has any effect on activation of caspase-3 following TNF-
treatment, PARP cleavage activity was examined. Analysis of the
integrity of the death substrate PARP in control MCF7-V cells showed
almost complete cleavage of the native 116-kDa PARP to the signature
85-kDa proteolytic fragment within 24 h of TNF-
treatment.
However, experimental MCF7 cells exhibited a low level of PARP
cleavage. The typical results from control MCF7-V cells and a
core-expressing experimental cell clone (Core-7) are shown in Fig.
4. Densitometric scanning of the
autoradiogram suggested an 88% PARP cleavage in MCF7-V cells after
24 h of incubation, whereas the Core-7 clone showed only 7% PARP
cleavage. The Core-10, Core-12, and Core-pooled cells showed a 16, 24, and 27% PARP cleavage, respectively. Use of cyclohexamide in this
experiment reduced the time of incubation from 24 to 3 h for onset
of PARP cleavage (data not shown). Results from this study indicated
that MCF7 cells stably transfected with HCV core gene inhibit
TNF-
-mediated PARP cleavage under our experimental conditions.
Inhibition of PARP cleavage has also been shown in cowpox virus CrmA
protein and baculovirus p35. However, CrmA is a serpin capable of
inhibiting ICE family members. Similar to p35, HCV core protein
possesses no homology to any known protease inhibitor.
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ACKNOWLEDGEMENTS |
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We thank Robert B. Belshe for helpful discussions, Michael Houghton for providing the HCV cDNA (Blue4/C5p-1), Vishva M. Dixit for providing the TNF-sensitive MCF7 cells, Michael M. C. Lai for providing the antiserum to HCV core protein, S. Chatterjee for providing monoclonal antibody to PARP and SuzAnn Price for preparation of the manuscript.
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FOOTNOTES |
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* This research was supported by funding from Saint Louis University, National Institutes of Health Grants CA52799 from NCI (to R. B. R.) and AI-45250 from NIAID (to R. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Div. of Infectious Diseases and Immunology, Saint Louis University Health Sciences Center, 3635 Vista Ave., FDT-8N, St. Louis, MO 63110-0250. Tel.: 314-577-8648. Fax: 314-771-3816.
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The abbreviations used are: HCV, hepatitis C
virus; PARP; poly(ADP-ribose) polymerase; TNF, tumor necrosis factor;
ICE, interleukin-1 converting enzyme; MTT, 3-(4,5-dimethyl
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide.
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REFERENCES |
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