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Mol Med. 2006 Jan–Mar; 12(1-3): 47–53. doi: 10.2119/2006-00020.Wu. | PMCID: PMC1514550 |
Copyright 2006, The Feinstein Institute for Medical Research Effect of Hepatitis C Virus Core Protein on the Molecular Profiling of
Human B Lymphocytes Chuan-ging Wu,1 Anuradha Budhu,2 Sheng Chen,3 Xiaoling Zhou,4 Nicholas C. Popescu,4 Kristoffer Valerie,5 and Xin Wei Wang2 1 Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA 2 Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, MD, USA 3 Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, MD, USA 4 Laboratory of Experimental Carcinogenesis, National Cancer Institute, NIH, Bethesda, MD, USA 5 Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA, USA Received March 15, 2006; Accepted April 5, 2006. Hepatitis C virus (HCV) core protein features many intriguing properties
and plays a pivotal role in cellular immunity, cell growth, apoptosis, cell
transformation, and eventually in tumor development. However, the
role of B cells, the primary players in the humoral immune response, during
HCV infection is largely unknown. To explore the molecular effects
of HCV core on human B cells, we conducted gene expression profiling
of serial RNA samples from B cells that were infected with adenovirus
harboring full-length HCV core protein and β-galactosidase
as a reference using a microarray platform containing 22,149 human
oligo probes. The entire experiment was performed in duplicate in B lymphocytes
that were isolated from two individual donors and incubated
for up to 3 days after infection with adenovirus expressing HCV core protein
to identify dynamic gene expression patterns. Differential expression
of representative genes was validated by quantitative RT-PCR. We
found that HCV core significantly inhibited B-lymphocyte apoptosis. We
showed a dramatic downregulation of MHC class II molecules in B cells
expressing HCV core, whereas the expression of immunoglobulin genes
was not significantly altered. Moreover, genes associated with leukemia
and B-lymphoma were consistently upregulated by HCV core. In contrast, downregulation
of caspase-1 and caspase-4 was found to be associated
with core’s ability to prevent B-lymphocyte apoptosis. In summary, we
have identified several clusters of genes that are differentially
expressed in human B lymphocytes expressing HCV core, suggesting
a potential impairment of antigen processing and presentation, which
may provide more insights into HCV infection in B lymphocytes. Hepatitis C virus (HCV), with 1.8% prevalence of infection in the
United States and 170 million worldwide
( 1), is a major cause of cirrhosis and potentially leads to hepatocellular
carcinoma (HCC). The incidence of HCC is increasing in North America, Europe, and
Japan, largely because of the high rates of chronic HCV
infection
( 2). HCV replicates in T lymphocytes and suppresses T-cell proliferation
and cytokine production
( 3). For instance, in patients with chronic HCV infection, the frequencies
of antiviral CTLs are relatively low
( 4), and the proliferative response of HCV-specific CD8 + T cells is impaired
( 5). In addition, the production of Th1-type cytokines (i.e., IL-2 and IFN-γ ) is
dramatically suppressed in peripheral T cells of chronic
HCV patients
( 6, 7). These observations suggest that HCV chronic infection may be the result, at
least in part, of an inability to mount effective T-lymphocyte
responses, indicating that HCV gene products might be involved in modulating
or suppressing host immune responses. In comparison, the evidence
of HCV infection in B cells both in vivo and in vitro has been controversial; recent
studies have shown that HCV can infect and replicate
in B cells from HCV-infected patients
( 8), which suggests the direct pathological effect of HCV on B cells. HCV infection is associated with B-cell lymphoproliferative disorders, including
mixed cryoglobulinemia, usually a benign condition, and overt
B-cell lymphoma
( 9, 10). Epidemiological studies suggest that HCV infection may play a direct
role in the genesis of B-cell lymphoproliferative disorders, and clonal
B lymphocytes are frequently detected in the blood and liver of patients
with chronic HCV infection
( 11). Furthermore, antiviral treatment for HCV is associated with the regression
of B lymphoma
( 12, 13). Nevertheless, how HCV induces B-cell lymphoproliferative disorders and
whether HCV core plays any role in B-cell immunity is still unclear. Among the viral proteins encoded by HCV, core protein is the first to be
synthesized during the early phases of HCV infection. It can influence
cellular immunity, cell growth, apoptosis, cell transformation, and
eventually tumor development (hepatocellular carcinoma and possibly B
lymphoma)
( 14). Furthermore, whether HCV core protein plays any role in gene deregulation
in B cells and whether HCV core exerts any molecular effects on
apoptosis, immunoglobulin production, or antigen-processing need to be
addressed. In this study, we performed a systematic analysis for gene expression profiling
in primary human B lymphocytes overexpressing HCV core protein
from an adenoviral transfection construct. A total of 22,000 human expression
features were analyzed with a modified version of GoMiner, a
recently developed quantitative and statistical gene ontology software
package with a builtin control on false discovery rate (FDR)
( 15). This approach identified significant changes in gene expression in several
ontology categories, including apoptosis, leukemia/lymphoma, antigen
processing, and antigen presenting. These results suggest that HCV
core protein may play a role in B-cell escape from apoptosis and thus
contribute to the development of B-cell lymphoma, and may impair antigen
processing and presentation during HCV infection in B cells. Construction of Flag-HCV Core Protein Because the availability of antibodies is limited, the flag-tagged HCV
protein was generated from PCR amplification of the appropriate regions
of pCV-J416S, an infectious cDNA clone of HCV genotype 1b, followed
by restriction digesting and subcloning into pCMV-TAG. The Flag-tagged
construct was inserted into the adenoviral expression vector pZERO-TG (designated
as Ade-core).Adenovirus generation and purification were
conducted at the Massey Cancer Center Virus Vector Shared Resource, Virginia
Commonwealth University (Richmond, VA, USA). Likewise, the β -galactosidase
and HCV-NS3 (an HCV nonstructural protein) were generated
in adenovirus to be used as control constructs (designated as
Ade-β -gal and Ade-NS3, respectively). Viral amplification was
conducted by the Gene Therapy Center Virus Vector Core Facility, University
of North Carolina (Chapel Hill, NC, USA) as previously described
( 16). Human B-Cell Preparation and Viral Infection Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized
blood obtained from normal non-atopic donors (approved and provided
by The Department of Transfusion Medicine at NIH) by density-gradient
centrifugation on Ficoll Hypaque (Amersham Pharmacia Biotech). Cells
were then washed and resuspended in RPMI 1640 containing 10% heat-inactivated
fetal calf serum (JRH Biosciences), 2 mM l-glutamine, 50 μ g/mL streptomycin, and 100 units/mL penicillin (complete
medium). Purified B-cell populations were isolated from PBMCs
using a positive magnetic sorting system (Miltenyi Biotec, Auburn, CA, USA) with
magnetic beads conjugated to CD19 according to the manufacturer’s
instructions. The purity of B cells was detected with
FACS analysis using anti-CD20 antibody (Miltenyi Biotec). Cell viability
assessment of freshly isolated primary B cells from donors was examined
by Trypan blue dye exclusion. Twenty million B cells were infected
with viral stocks of Ade-core, Ade-NS3, or Ade-β -gal at 50 times
multiplicity of infection (MOI). Total RNA was extracted 24, 48, and 72 h
after viral infection using Trizol (Invitrogen, Carlsbad, CA) according
to the manufacturer’s instructions. The entire experiment
was conducted in duplicate using B lymphocytes derived from
two donors. Apoptotic Cell Death Assay To quantify apoptosis, cultured B cells were double-stained with Annexin
V-FITC conjugate and propidium iodide (PI) using TACS Annexin V Kits
from Oncogene (San Diego, CA, USA), according to the step-by-step protocol
as provided by the manufacturer, and immediately analyzed using
a FACSCalibur flow cytometer (Becton Dickinson). Briefly, 0.5 million
B cells were incubated in annexin binding buffer (10 mM HEPES, pH 7.4, 150 mM
NaCl, 5 mM KCl, 1 mM MgCl2, and 2 mM CaCl2) and stained with annexin V-FITC for 10 min on ice in the dark. Apoptotic
cells were counted on a flow cytometer using a dual filter set for
FITC and PI. Early apoptotic cells were positive for annexin V-FITC conjugate
but did not stain with PI because their membranes were still
intact. Late-stage apoptotic cells or dead cells that had damaged permeable
plasma membranes stained concurrently with annexin V-FITC conjugate
and PI. For data analysis, FlowJo software (Tree Star) was used. Western Blot After RNA isolation, protein was isolated according to the manufacturer’s
protocol (Invitrogene). Protein concentration was determined
by the Bradford Assay according to the manufacturer’s protocol (Bio-Rad, Hercules, CA, USA). An identical amount of total protein
was loaded in each lane of a 16% PAGE gel, followed by transferring
and blocking in 5% nonfat dry milk. Blots were probed with
antiactin followed by incubation with horseradish peroxidase–conjugated
secondary antibody. Membranes were stripped and reported
with anti-HA antibody (Roche, Indianapolis, IN, USA) to detect HCV-core
expression. Antibody-antigen complexes were detected by enhanced chemiluminescence
according to the manufacturer’s protocol (Amersham, Piscataway, NJ, USA). Oligo microarray Human operon oligonucleotide chips (v. 2.1, Qiagen Human Array-Ready Oligo
Set) were generated by the National Cancer Institute (Bethesda, MD, USA) microarray
facility at the Advanced Technology Center. The oligo
array platform contains 22,149 70-mer probes. Detailed hybridization, quality
control, data acquisition, and filtering were performed as previously
described
( 17). In a pilot experiment, we had conducted both forward and reverse labeling
of Cy3/Cy5 and Cy5/Cy3 dye as a standard procedure. Scatterplot
analysis of the ratio of gene expression in forward and reverse labeling
of control samples was shown to be highly correlated and ensured dye
labeling specificity. For each experiment, fluorescent probes were therefore
prepared by an indirect labeling approach of a reference RNA
from B cells infected with Ade-β -gal (Cy5), and B cells infected
with Ade-core were labeled with Cy3. All microarray data analyses
were performed in duplicate in B cells from two donors. TaqMan Analyses Total RNA prepared from B cells was used to perform microarray analyses
and to monitor cellular RNA by quantitative reverse transcription PCR (qRT-PCR). PCR
primers and probes (Assays on Demand) were purchased (Applied
Biosystems, Foster City, CA, USA), and qRT-PCR reactions were
performed according to the manufacturer’s instructions. For each
time point of gene detection, an average of four replicate reactions
was calculated. Human 18S RNA labeled with VIC reporter dye was used
as an endogenous control for normalization in validation assays. Reactions
were performed with the ABI PRISM 7700 Sequence Detector System (Applied
Biosystems). Analysis and Statistics The percentage of apoptotic B cells after Ade-core, Ade-NS3, and Ade-β -gal
infection was compared and analyzed with Pearson χ 2 test. For microarray quantification, data processing and cluster analyses
were conducted as described ( http://nciarray.nci.nih.gov). A class comparison based on univariate F tests at a significance level of P < 0.005 was used to find genes differentially expressed among various
time points. Significant genes were included in the GoMiner analysis, developed
by Zeeberg et al.
( 15), which was performed using a modified program with a built-in control
of false discovery rate at P < 0.05. Generation and Expression of Adenovirus Harboring HCV Core Protein and β -Galactosidase Because human B cells have very low proliferation capacity, adenoviral
vectors harboring HCV core protein, HCV NS3, and β -galactosidase
were generated to ensure high efficiency of transgene expression in
primary B cells. To optimize infection conditions, B cells were infected
with Ade-core, Ade-NS3, and Ade-β -gal at MOIs ranging from 5 to 100 for 24 h. An
MOI of 50 resulted in efficient expression of
HCV core encoded genes as determined by Western blot at 48 h as shown
in , and the same viral dose was used in further experiments for up to 72 h. The
entire experiment was conducted in duplicate in B cells isolated
from two normal donors. B-cell purity reached more than 90% as
detected by FACS analysis using anti-CD20 antibody (data not shown). Decreased Apoptotic Cells in HCV Core–Infected B Cells During 3 days of viral infection with Ade-core, Ade-NS3, and Ade-β -gal
in primary B cells, increased apoptotic cells were observed in
a time-dependent manner as seen in . The percentage of apoptotic cells, especially of the control cells carrying
Ade-β -gal, began to rise from 27.9% on day 1 to 44.6% on
day 2 and 58.6% on day 3. Likewise, the increase
of apoptotic cells harboring HCV-NS3 was similar to the control
cells, with up to 64.9% of apoptosis on day 3. In comparison, the
cell death of those cells carrying Ade-core was only 27% on
day 2 and 35.1% on day 3. The difference of cell death between
B cells infected with Ade-β -gal or Ade-NS3 and those with
Ade-core became statistically significant (P < 0.01 and P < 0.001) on days 2 and 3, respectively. Differentially Expressed Genes Microarray was conducted and analyzed with serial RNA samples from infected
B cells isolated from two donors. The B cells were infected with
Ade-core and Ade-β -gal over 3 days. As shown in , 216 genes were differentially expressed, with a univariate P value of < 0.005 and with a greater than 2-fold ratio changes in at
least two arrays. Among them, most (90%) genes appeared to be
either upregulated (genes shown in red) or downregulated (genes shown
in green) on the first 2 days of infection with Ade-core versus Ade-β -gal. These
include the cluster of genes related to myeloid/lymphoid
leukemia, B lymphoma, transcription regulation, apoptosis, and
potential modulators of immune cell activities. Validation of Microarray Data To validate the microarray data, six representative genes were chosen to
verify the gene expression levels and patterns using qRT-PCR (). The quantitations of gene expression were highly reproducible among
the duplicate experiments throughout 3 days of viral infection. As shown
in , when CASP4 was downregulated in microarray analysis from day 1 to day 3 of
the experiment, the duplicate qRT-PCR after being standardized with 18S
RNA showed the exact same patterns. Likewise, when MLLT3 was upregulated
as detected by microarray during viral infection up to day 3, qRT-PCR
revealed the identical pattern. The same was true with four
other genes tested using qRT-PCR despite some discrepancy in terms of
fold of gene expression level compared with microarray analysis (). The overall correlation of gene expression for each of the six genes
after normalization with 18S RNA between microarray and qRT-PCR was highly
significant (R2 = 0.86, P < 0.01). Identification of the Gene Clusters Affected by Core Protein Genes related to leukemia or lymphoma Among the most differentially expressed genes, several of them related
to myeloid/lymphoid leukemia and B lymphoma, such as MLLT3, BAL, and BMI1, exhibited
the greatest enhancement. The microarray analysis showed
that MLLT3 expression increased at the first day of experiment and continued
to increase up to more than 10 fold. The expression pattern was
confirmed by duplicate quantitative RT-PCR. Similarly, BAL, a B-lymphoma
related gene, started going up on the second day of infection as
detected by both microarray and quantitative RT-PCR. Immunoglobulin genes Because HCV is frequently associated with mixed cryoglobulinemia, it was
noteworthy to see if any changes in immunoglobulin genes occur during
early viral infection with HCV core protein. We examined the entire
immunoglobulin gene families, and found that none of them showed any significant
changes in transcription levels throughout the 3 days of Ade-core
infection in duplicate experiments (data not shown). Apoptosis genes To pursue the cause of decreased cell death during Ad-HCV core infection
in B cells, we examined the transcriptional levels of all apoptosis-related
genes. Among the genes down-regulated by HCV core protein, CASP4 was
shown to be suppressed more than 2 fold as early as the first day, and
sustained through day 3. CASP1 showed a gene expression pattern
similar to that of CASP4, especially in the first 2 days. In contrast, changes
in other apoptosis genes including CASP2, CASP3, CASP7, CASP8, and
CASP10, as well as BCL2, BAD, and Granzyme B, were not detected (data
not shown). Transcription regulator genes Among the early response genes, several transcription elements were identified
in the first day of HCV core infection. NF.BIA, a nuclear factor
of κlight peptide inhibitor gene, and TBP, a TATA box binding
protein, appeared to be upregulated by more than 4 fold (), whereas other genes, such as STAT1, were down-regulated at the beginning
of HCV core infection (Data not shown). GoMiner Data Analysis With the advent of GoMiner, a resource for biological interpretation of
genomic and proteomic data, we were able to explore and display the major
gene family of MHC class I molecules, and especially MHC class II
molecules among significant genes detected in microarray. As shown in , all MHC class II molecules including HLA-DMA, HLA-DMB, HLA-DQB1, and
HLA-DRA, together with CD74, an MHC class II molecule related gene, were
found to be dramatically suppressed on the first day of Ade-core infection
even though some B cell–specific molecules like CD79B
were somewhat upregulated. As two of six representative genes chosen for
validation with quantitative RT-PCR, HLA-DRA and CD74 appeared to be
as much as 10-fold down regulated in both microarray and qRT-PCR analyses
on day 1 (). High-throughput genomic studies have been employed to investigate gene
profiling associated with hepatitis virus infection, thereby providing
more insights into the molecular mechanism of viral infection
( 16, 18). More recently, a number of microarray analyses have been performed in
chimpanzees and in patients infected with HCV virus in an attempt to
identify specific gene expression profiling and potential markers
( 10, 19). To our present knowledge, however, the role of HCV, and in particular
HCV core protein, in regulation of gene expression in human primary
B cells has not been studied by microarray analysis. To systematically
examine the molecular effects of HCV core protein on regulating host
gene expression in primary human B cells, we have performed microarray
and GoMiner analyses for gene expression profiling in the human B lymphocytes
that were infected with adenovirus harboring HCV core protein
in a time-dependent manner using oligo microarray covering nearly 22,000 human
oligonucleotides. Infection of primary human B cells with an adenoviral vector expressing
HCV core protein significantly reduced B-cell death in a time-dependent
manner compared with control vectors expressing β -gal or HCV-NS3, an
essential HCV nonstructural helicase protein for viral replication, suggesting
that HCV core protein may specifically rescue B lymphocytes
from cell death. To identify candidate B-cell genes that might
be involved in core-mediated inhibition of apoptosis, we conducted
microarray analyses with serial RNA samples from infected B cells isolated
from two donors. Under stringent selection criteria, 216 differentially
expressed genes were clustered based on significance ( P < 0.005) and greater than 2-fold ratio changes in at least two arrays. Most
genes appeared to be either upregulated or downregulated after 2 days
of core expression, indicating that HCV core can modulate cellular
gene expression in host B cells. Among the genes downregulated by
HCV core protein, CASP4, a member of the CASP1 superfamily, was suppressed
more than 2 fold as early as the first day, and this suppression
was sustained through day 3 of the experiment. Similarly, CASP1 showed
the same pattern as CASP4, especially in the first 2 days. In comparison, changes
in other caspase genes including CASP2, CASP3, CASP7, CASP8, and
CASP10, as well as BCL2, BAD, and Granzyme B, were not detected. These
results suggest that HCV core may rescue B cells from apoptosis
possibly through suppression of CASP1 and CASP4. It is plausible
that modulation of apoptosis may involve binding of HCV core protein to
the intra-cellular signal transducing portion of death receptors and
displacement of signaling molecules. Hence, monitoring caspase activation
might provide a reliable tool to estimate the efficacy of HCV therapy, and
might open challenging therapeutic strategies in HCV infection
( 20). Whether these changes in gene expression are due to HCV core’s
transactivation of cellular promoters, including NF-κB and
AP-1, or due to its interaction with c-JNK and MAPK signaling as previously
indicated
( 21), still needs to be fully investigated. qRT-PCR was used to verify and quantify the microarray data of six representative
genes, chosen by their gene expression levels, patterns, and
biological functions. Quantification of gene expression levels and patterns
were highly reproducible among the duplicate experiments throughout
the 3 days of viral infection. The overall correlation of the six
genes, standardized with 18S RNA, was highly significant between microarray
and qRT-PCR analyses ( R2 = 0.86, P < 0.01) despite some discrepancy in terms of fold of gene expression
level. These results demonstrated that microarray analyses performed
in this study using oligo chips made at NCI are valid, although microarray
is not adequately quantitative. This observation is also consistent
with similar studies from others
( 22). Several genes related to myeloid/lymphoid leukemia and B lymphoma were
among the most enhanced by expression of core protein. MLLT3 is involved
in translocations associated with both acute lymphoblastic and acute
myelogenous leukemia
( 23). BAL (B-aggressive lymphoma) is a novel risk-related gene in diffuse
large B-cell lymphomas that enhances cellular migration. A recent study
showed that stable BAL-over expressing B-cell lymphoma transfectants
had significantly higher rates of migration than vector-only transfectants, indicating
that the risk-related BAL gene promotes malignant B-cell
migration
( 24). Further elucidation of the deregulation of these genes in B cells as
an early molecular event following HCV infection, and the potential underlying
mechanism, may be important to the development of novel therapeutics
for HCV-associated B-cell lymphoma. HCV is frequently associated with mixed cryoglobulinemia
( 25, 26), raising the possibility that HCV core may influence the regulation of
immunoglobulin genes in B cells. Surprisingly, no member of the entire
immunoglobulin gene family showed significant transcriptional changes
throughout 3 days of Ade-core infection. These results suggest that
overproduction of immunoglobulin in HCV-related B-lymphocyte disorders
may require other viral HCV proteins that possibly contribute to the
regulation in vivo. Alternatively, other cell types, for example, T cells
or macrophages, may activate B cells and eventually lead to the overproduction
of immunoglobulin in patients. Moreover, most cryoglobulinemia
is related to B-lymphocyte proliferation, which we did not observe
in this study. GoMiner is a useful software package that organizes lists of up- and downregulated
genes for biological interpretation in the context of the
Gene Ontology
( 15). With the advent of GoMiner, we were able to explore and display the
transcriptional expression of the major gene family of MHC class I molecules, and
especially MHC class II molecules, from thousands of genes
detected in microarray. All MHC class II molecules including HLA-DMA, HLA-DMB, HLA-DQB1, and
HLA-DRA, together with CD74, an MHC class II molecule
related gene, were suppressed on the first day of Ade-core infection. The
data were verified by quantitative RT-PCR, and both HLA-DRA
and CD74 appeared to be as much as 10-fold downregulated in both microarray
and TaqMan RT-PCR analyses. Our recent FACS analysis indicates
that HCV core also downregulates HLA class II protein expression (unpublished
data). These results suggest that HCV core inhibits MHC class
II molecules, thereby impairing antigen-processing and presentation. Thus, it
is plausible that HCV core protein modulation of MHC class II
expression and antigen presentation to CD4 + T cells provides HCV with a means of avoiding early immune recognition. Our novel finding is in agreement with a recent study demonstrating reduced
expression of MHC class II molecules in human cytomegalovirus-infected
macrophage cultures from 90% of the donors
( 18). Human cytomegalovirus uses different mechanisms to decrease the expression
of MHC class II molecules on infected macrophages either independent
of or dependent on viral replication. Moreover, CMV-infected macrophages
exhibited a 66% to 90% reduced capacity of stimulating
an antigen-specific proliferative CD4 + T-cell response
( 27). It is generally accepted that genes within the major histocompatibility
complex play a central role in the development of the immune response
against HCV. MHC class II molecules may be important for viral clearance, because
particular alleles are associated with chronic HCV infections
in patients, and MHC class II molecules restrict HCV-specific
CTL responses
( 28, 29). Furthermore, inhibition of MHC class II molecules, and therefore inhibition
of antigen processing and presentation, may account for delayed
development of neutralizing antibodies against HCV, as observed both
in animal experiments and in HCV-infected patients
( 30, 31). Moreover, MHC class II molecules can influence T-cell function, because
HCV core binds directly to gC1qR on CD4 + and CD8 + T cells
( 32, 33). Future studies directed at the mechanism of suppression of MHC class
II molecules and regulation of T cell function by HCV core protein may
identify new approaches for therapeutic regulation of HCV infection. We would like to thank Drs. B. Golding, D. Scott, M-y.W. Yu, and J. Giovanelli
for their critical review and discussion. We also thank Drs. Jean
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