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Departments of Pediatrics and Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, University of California, Los Angeles
Hepatitis C virus (HCV) core protein plays a significant role in the alteration of cellular gene expression. We expressed HCV core protein using a tetracycline-inducible expression system in HeLa cell lines. Profiles of gene expression in cells expressing the HCV core protein were compared with those in control cells by use of microarray analysis. Cells expressing the HCV core protein showed 86 down-regulated and 41 up-regulated genes, compared with control cells. One gene affected was cyclooxygenase 2 (COX-2). Levels of both COX-2 RNA and the Cox-2 protein were significantly inhibited after the expression of HCV core protein in HeLa cells. Similar results were obtained in hepatoma cells and in a functional assay that measured the production of the Cox-2 protein in response to a mitogenic stimulus. The inhibition of the Cox-2 protein could serve as a means of muting the cellular inflammatory response during HCV infection. Correlation of these findings with analysis of clinical specimens from chronically infected patients should lend further significance to the down-regulation of the inflammatory response via Cox-2.
Since its discovery in 1989 as the major cause of non-A, non-B hepatitis, hepatitis C virus (HCV) has proven to be a growing public health concern [1]. More than 4 million people in the United States are positive for HCV antibody, and transmission of the virus can be traced, in most cases, to contact with contaminated blood or tissue, injection drug use, vertical acquisition, and, very infrequently, sexual activity [2].
HCV is an enveloped, positive-sense, single-stranded RNA virus of 9600 nt in length. It consists of 1 long open-reading frame (ORF) that encodes the structural (core, E1, and E2) and nonstructural proteins (p7, NS2, NS3, NS4A+B, and NS5A+B). The ORF is flanked by a 5 untranslated region (UTR) and a 3 UTR [3]. An internal ribosome entry site (IRES) located within the 5 UTR allows the synthesis of a polyprotein that is processed by both cellular and virus-encoded proteases to yield mature viral structural and nonstructural proteins.
Despite its persistence in infected patients, viral replication has proven to be very difficult to sustain in an in vitro cell culture system. Cell lines have been identified that are permissive of HCV replication, and chimpanzees and trimera mice, which are SCID mice that are given concurrent transplants of bone marrow and human liver fragments, are, at present, the only animals available for the study of HCV infection [46]. Recent advances have been made in designing repliconsunits of self-replicating RNA that contain a partial or the full-length sequence of HCV RNA [79]. These models are limited, however, because they contain foreign selection markers and because they do not produce detectable virus particles, so they cannot completely represent a native infection.
The HCV core protein is highly conserved between different genotypes of HCV. Numerous studies have shown that the HCV core protein is a major cause of most of the pathogenic features associated with HCV infection. The HCV core protein has been shown to cause hepatic steatosis and hepatocellular carcinoma in transgenic mice [10, 11], suppress the promoter of the p53 tumor suppressor gene, and bind various receptors in the tumor necrosis factor family [12, 13]. The HCV core protein has been shown to immortalize primary hepatocytes in culture and also suppress or induce Janus kinase/signal transducer and activator of transcription (STAT) pathway signaling in response to interleukin (IL)6 or interferon (IFN) [14, 15]. A microarray analysis of Huh-7 cells expressing the HCV core protein showed altered response to oxidative stress [16]. In some clinical studies, high serum levels of the HCV core protein have been correlated with a more severe histologic grade on liver biopsy specimens, and serum levels of the HCV core protein are highly correlated with the response to IFN-based antiviral therapy [1719]. Few studies, however, have described anti-inflammatory phenomena associated with the HCV core protein. In one study, expression of the HCV core protein was associated with the activation of human monocytes, increased production of the anti-inflammatory cytokine IL-10, and inhibition of dendritic-cell differentiation [20].
In the present study, we demonstrate that a number of cellular genes are differentially regulated by the HCV core protein. Among those genes down-regulated is the one that encodes the inducible cyclooxygenase (Cox)2 protein. Enzymes in the Cox family are involved in the first step of prostaglandin synthesis by their conversion of arachidonic acid to a transient intermediary in the pathway, prostaglandin G2 [21]. There are 3 known isoforms of the enzyme: Cox-1, which is constitutively expressed; Cox-2, which is expressed in response to inflammatory or mitogenic stimuli; and Cox-3, which is a constitutively expressed splice variant of Cox-1 present in neural tissue [22]. Both Cox-1 and Cox-2 contribute to homeostasis and to inflammatory pathways [23, 24]. The COX-2 gene is 8 kb, containing 10 exons located on chromosome 1, and its promoter contains a TATA box as well as binding sites for a number of transcription factors [21, 23, 25]. Cox-2 shares 60% of its protein sequence with Cox-1 [21]. Drugs that preferentially inhibit 1 or both of these enzymes have been prescribed for many years as anti-inflammatory agents. Our results demonstrating the possible anti-inflammatory activities of the HCV core protein could help explain, at least in part, how the virus causes chronic infection over many years without causing many symptoms.
MATERIALS AND METHODS
Cloning of the gene encoding the HCV core protein.
The gene encoding the HCV core protein was amplified from the infectious HCV type 1a clone (pCV-H77C) [5, 26] by use of gene-specific oligonucleotide primers (table 1) containing SacII and NotI endonuclease restriction sequences. The gel-purified product of the polymerase chain reaction (PCR) and the pTRE2 vector (Clontech) were digested at 37°C for 2 h in SacII and NotI. The digested products were ligated at a product-to-vector ratio of 10 : 1 by use of T4 DNA Ligase (Promega) at 25°C for 1 h. A 5-L sample from the ligation was used to transform DH5 subcloning-competent Escherichia coli cells (Life Technologies). Plasmid DNA was isolated, by use of QIAprep Spin Miniprep kit (Qiagen), from a number of colonies grown in ampicillin-containing Luria-Bertani medium and was restriction digested to screen for fragment insertion. The proper cloning of the gene encoding the HCV core protein was confirmed by sequence analysis.
Transfection, screening, and analysis of pTET-On cell lines.
HeLa cells were transfected with the pTET-On promoter vector (Clontech) by use of Effectene reagent (Qiagen). Transfectants were selected by use of G418-containing medium at a concentration of 900 g/mL. Surviving colonies were transfected with pTRE-Luc luciferase vector (Clontech) and were incubated overnight in medium and various concentrations of tetracycline. Cell lysates were assayed for luciferase activity by use of a Luciferase Assay kit (Promega). Two colonies with a low background expression and a high inducible expression of pTET-On cell lines were selected for transfection with pTRE-HCV core protein vector. These transfections were performed in combination with a transfection with hygromycin-resistance plasmid (pTK-Hyg; Clontech), and 1 g DNA of each vector was used. Two days after transfection, cells were incubated in medium containing 50 g/mL of hygromycin (Clontech), and the medium was changed every 48 h until colonies appeared. The subcultured colonies were lysed, and the DNA was analyzed by PCR for the presence of the gene encoding the HCV core protein. A colony of cells that survived selection but did not harbor the gene sequence encoding the HCV core protein was used as a negative control.
Western blot analysis.
Lysates (30 L) from cells grown in medium containing 10 g/mL of tetracycline were electrophoresed and then were transferred to a nitrocellulose membrane. The membrane was incubated with primary monoclonal mouse antiHCV core protein antibody (Austral Biologicals) and secondary goat horseradish-peroxideconjugated anti-mouse antibody (Roche Biologicals). The HCV core protein was detected by chemiluminescence (Pierce Biologicals).
For Western blot analysis, monoclonal antibodies were used for the initial incubation (antihuman Cox-2 antibody; Cayman Chemical), and species-specific horseradish-peroxideconjugated secondary antibodies were used for the subsequent staining. Membranes were stripped of antibodies by incubation for 30 min in a -mercaptoethanolcontaining buffer before being probed again.
RNA isolation and microarray analysis.
Total cellular RNA was isolated from 5 × 106 cells by use of TRIzol reagent (Life Technologies) and was suspended in 40 L of nuclease-free water. A 20-L sample was digested in RNase-free DNase (Promega) at 37°C for 1 h, was purified using the RNeasy Mini kit (Qiagen), and was analyzed on a 1% agarose gel, to confirm purity. The samples were submitted to the DNA Microarray Core facility at the University of California, Los Angeles, for hybridization and scanning by use of a standardized protocol. Commercial oligonucleotide microarray chips (HuFL 35) were obtained from Affymetrix. Data were analyzed with Microarray Suite software (version 4.0.1; Affymetrix). A number of statistical approaches for the designation of reproducibly altered genes have been proposed [27]. We included in our list only those genes that met the quality-control criteria for a good clone spot and whose expression were altered by a factor of 2 in 3 independent experiments. A 2-fold cutoff value has been frequently used to select differentially expressed genes, and both theoretical and empirical calculations have supported the use of 2-fold changes as a robust and somewhat conservative cutoff value [28, 29]. Statistical analysis was performed by applying the 1- or 2-class (unpaired) response type of Significance Analysis of Microarrays (SAM; version 1.20) [30]. The number of permutations was set to 24 (3-array sets) or to 300 (10-array sets). The identity of SAM-significant spots was obtained through the SOURCE database [31].
Reverse transcription (RT)PCR.
One microgram of total purified RNA was used as a template for RT-PCR that used custom primers (table 2) and the SuperScript One-Step RT-PCR system (Invitrogen). The RT-PCR conditions were as follows: RT at 45°C for 10 min and then at 95°C for 2 min, denaturation at 95°C, annealing at 55°C for -actin primers and at 60°C63°C for custom primers, and extension at 72°C for 35 cycles. Samples that did not produce a band that was visible by agarose gel electrophoresis after the initial round of RT-PCR were subjected to 25 cycles of an additional PCR that used Platinum Taq (Invitrogen) and the same gene-specific primers that were used in the first reaction.
Cox-2 induction.
Cells induced in the absence and in the presence of tetracycline were placed in medium containing 0.5% fetal bovine serum for 16 h, to control for the background expression of Cox-2. Cells were then washed and incubated in the following conditions: no tetracycline, tetracycline alone (baseline), tetracycline plus dimethyl sulfoxide (DMSO) at a dilution of 1 : 2000, and tetracycline plus 12-O-tetradecanoylphorbol 13-acetate (TPA) dissolved in DMSO at a dilution of 1 : 2000 (final concentration, 50 ng/mL). After 6 h, cell lysates were prepared using lysis buffer that contained protease inhibitor.
Image quantitation and comparison.
All comparisons of agarose gels from RT-PCR and Western blot analysis were performed by scanning the images and analyzing the band density by use of Image Quant software. The reported relative fold change is based on the comparison of the resulting band densities.
RESULTS
Cellular genes regulated by the HCV core protein.
Microarray analysis was used to examine the regulation of cellular genes by the HCV core protein, as described in Materials and Methods. Of 7129 genes examined, 86 were found to be down-regulated, and 41 were found to be up-regulated. The genes that were differentially regulated in statistically relevant amounts by the expression of the HCV core protein are given in table 2. The down-regulated genes fell into 3 general categories of activity: apoptosis/cell growth, immune response/cell recognition, and transcriptional/translational control. A significant number of the up-regulated genes were transcription factors and/or genes that encode proteins, which are involved in the regulation of transcription.
HCV core protein and expression of COX-2 RNA in both HeLa and Huh-7 cells.
When we used gene-specific primers for COX-2 to examine levels of RNA, no detectable product was observed after 1 round of RT-PCR (figure 3A). After subsequent rounds of RT-PCR with the same specific primers and 10 L of the product of the original RT-PCR, a 900-bp band was detected (figure 3B). The relative difference between the intensity levels of the bands generated by the control cells and by the cells expressing the HCV core protein was 45 fold. Omission of RT during the first round of PCR resulted in no detection of the 900-bp band after a round of RT-PCR (data not shown). These results, therefore, confirmed the results of our microarray.
To analyze the kinetics of gene regulation in cells expressing the HCV core protein, we performed a time-course experiment using RT-PCR and gene-specific primers for COX-2. Samples taken at 0 h, 24 h, and 96 h after induction were analyzed (figure 3C). In this experiment, very low levels of COX-2 RNA were detected at 0 h after induction, but there was no significant difference between levels of COX-2 RNA in cells expressing the HCV core protein and in control cells (lanes 1 and 2). At 48 h and 96 h after induction, COX-2 RNA was expressed in significant amounts (lanes 3 and 5). In contrast, the level of COX-2 RNA remained at control levels in cells expressing the HCV core protein until 96 h after induction (lanes 4 and 6). These results suggest that the level of COX-2 RNA is drastically affected in cells expressing the HCV core protein.
To examine whether the HCV core protein is able to down-regulate the expression of COX-2 RNA in a liver-derived cell line, Huh-7 cells were transiently transfected with either a plasmid expressing the HCV core protein or a control plasmid lacking the HCV core proteinencoding sequence. The intensity of the band generated when the COX-2specific primer was used was drastically reduced (78 fold) in cells transfected with the plasmid expressing the HCV core protein, compared with that in cells transfected with the control plasmid (figure 3D, lanes 1 and 2). These results suggest that the expression of COX-2 RNA is down-regulated by the HCV core protein in both HeLa and Huh-7 cells.
Levels of HCV core protein and Cox-2 protein.
The level of Cox-2 protein was examined by Western blot by use of a Cox-2specific antibody. The blot containing cellular proteins was stained for Cox-2 protein, the HCV core protein, and -actin successively, using monoclonal antibodies (figure 4A). Compared with that in control cells, the expression of the Cox-2 protein was reduced by 34-fold in cells expressing the HCV core protein (figure 4A, lanes 3 and 4). The HCV core protein was present only in experimental cell lysates. Lysates from a murine colon carcinoma cell line that constitutively expresses Cox-2 (lane 2) was used as a positive control.
HCV core protein and induction of Cox-2 expression.
A TPA induction assay was used to examine the effect of the HCV core protein on the inducible expression of Cox-2. The cells were incubated for 16 h in medium containing 0.5% serum, to minimize the background expression of Cox-2. DMSO was used as a control, because the TPA used for Cox-2 induction was dissolved in DMSO. Control cells at baseline and those treated with DMSO showed an approximately equivalent, moderate level of Cox-2 expression by Western blot analysis (figure 4B, lanes 1 and 3).
The control cells incubated with TPA for 6 h showed strong induction of Cox-2 expression (an increase of 7-fold, compared with that of control cells at baseline). Cells expressing the HCV core protein showed a marked decrease in the expression of Cox-2 on all levels and showed very little induced expression after incubation with TPA (figure 4B, lanes 5 and 6). In fact, in cells expressing the HCV core protein, the level of Cox-2 after incubation with TPA was below the baseline level (figure 4B, lanes 1 and 6). Thus, the HCV core protein almost completely mutes the inducible expression of Cox-2 by inflammatory stimuli. Taken together, these results suggest that the expression of both COX-2 RNA and Cox-2 protein is significantly down-regulated by the HCV core protein.
DISCUSSION
The results of our microarray showed that many genes were affected by the expression of the HCV core protein in HeLa cells. These generally fell into 3 broad categories of activity: apoptosis/cell growth, immune response/cell recognition, and transcriptional/translational control. These results generally correspond with those of a previous study that examined the effect of the HCV core protein in Huh-7 cells [16]. The similarity of results found when cell lines of different origins were used indicates that the HCV core protein exerts similar effects on different cell types. In addition, several translational factors were identified in our analysis, most notably eukaryotic translation initiation factor (eIF)2 and eIF-5, which is consistent with data published elsewhere that the HCV core protein down-regulates cap-dependent, cap-independent, and IRES-mediated translation [35].
The specific finding that COX-2 is down-regulated in the presence of the HCV core protein is intriguing. Certainly, the Cox-2 protein plays a significant role in the host immune response, in which its production is linked to exposure to catalase and peptidoglycan [36, 37]. Cox-2 has specifically been shown to play a role in the pathogenesis of certain viral infections [38], notable examples being the dependence on Cox-2 for replication of cytomegalovirus [39] and the induction of Cox-2 by primary human herpesvirus 6 infection [40]. However, Epstein-Barr virus (EBV), a herpesvirus that establishes latency in white blood cells, has been shown to inhibit both Cox-2 and prostaglandin E2 in monocytes [41]. Neither Cox-1 nor Cox-2 has previously been linked with HCV infection. Inhibition of the inducible form of the enzyme by the HCV core protein would disable a major mechanism that allows up-regulation of the inflammatory response. Analogous to the example of EBV, this could be a survival pathway that the virus has developed over time. This finding could explain how HCV can replicate to such high RNA copy levels without producing much inflammation in the liver. It may also help to explain why studies on the correlation between HCV loads and various antigen levels have not shown a direct correlation with disease severity in biopsy specimens. Unlike HCV, the herpesviruses are cytolytic and use the inflammatory response to their advantage.
The ability of various stimuli (e.g., cytokines) to induce transcription of the COX-2 gene likely occurs via the various cis-acting elements present within the 5 UTR of the COX-2 gene. These transcriptional elements include TATA box, AP2, STAT-1, STAT-3, nuclear factor (NF), NF-IL6, cAMP response element, and CCAAT enhancer binding protein transcription factors [42, 43, 44]. Down-regulation of 1 of these factors could negatively affect the expression of COX-2. The expression of COX-2 is also regulated posttranscriptionally and is determined by the presence of multiple copies of adelylate and uridylate (AU)rich elements within the 3 UTR of COX-2 mRNA [45]. The AU-rich elements of many protooncogene and cytokine mRNAs confer posttranscriptional control of expression by acting as mRNA instability determinants [46, 47]. Binding of cellular proteins to these elements presumably regulates the stability of these mRNAs. Future studies will determine whether the HCV core protein influences the stability of COX-2 mRNA.
It is believed that TPA activates the family of activator protein 1/mitogen-activated protein kinases that subsequently work at the promoter region of the COX-2 gene to increase expression [48]. Given that data published elsewhere have shown that the HCV core protein inhibits other cellular kinases [15], it is likely that it plays a role in the inhibition of the induction of Cox-2 by TPA via this mechanism. It is unlikely that the HCV core protein has any direct interaction with TPA.
We chose to use HeLa cells in the present study because HeLa cells could support the replication of the HCV subgenomic replicons [50], and microarray analysis in cells expressing the HCV core protein has already been reported in liver-derived cells [16, 49]. The focus of these studies was on markers of oxidative stress in response to the expression of the HCV core protein, and they did not mention COX-2 in their data or discussion. Our finding that COX-2 is down-regulated in HeLa cells is further supported by the replication of results in both Huh-7 (figure 3D) and HepG2 cell lines (data not shown).
The effect of the HCV core protein when it is expressed alone lacks the context of natural infection. The other structural and nonstructural proteins are absent, and one could argue that the phenomena observed were the result of artificially derived circumstances in the absence of viral replication and packaging. Correlation of these findings with analysis of clinical specimens from chronically infected patients should lend further significance to the down-regulation of the inflammatory response via COX-2.
To summarize, we may have uncovered a pathway by which HCV possibly evades the host inflammatory response via the down-regulation of COX-2. Future studies will be directed at determining the mechanisms by which the HCV core protein accomplishes this.
Acknowledgments
We thank the DNA Microarray Core facility at the University of California, Los Angeles; Zugen Chen, for assistance with the completion of this project; and Weimin Tsai and Kathy Takahashi, for their help in seeing this project to its completion.
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