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Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Department of Oncology, School of Medicine, Chiba University, Chiba
Department of Gastroenterology, Graduate School of Medicine, Okayama University, Okayama, Japan
Background.
We evaluated the association between variations in hepatitis C virus (HCV) core protein and hepatitis severity in patients with chronic HCV infection who achieved remission without viral eradication and had a biochemical response to interferon (IFN) therapy, to evaluate the effect of HCV core sequence in the absence of the influence of host factors.
Methods.
Using serum from 10 patients with a biochemical response and 10 patients with no response, we measured serum levels of interleukin (IL)1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-, and tumor necrosis factor before and after IFN therapy. Expression vectors with the core region were transfected into Huh7 cells, and cytokine induction was evaluated by reporter assay.
Results.
In biochemical responders, only IL-8 levels decreased after IFN therapy (P = .04). Changes in the C-terminal hydrophobic region were observed more frequently in biochemical responders. Activation of the IL-8 promoter by HCV core protein was significantly decreased in biochemical responders after IFN therapy (P = .04). When 69 C-terminal amino acids from before IFN therapy were replaced with those from after IFN therapy in 3 biochemical responders, their ability to transactivate IL-8 decreased.
Conclusions.
Differences in amino acids in the HCV core protein correlates with hepatitis activity through the modulation of IL-8 induction in HCV-infected patients.
Hepatitis C virus (HCV) infection causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma [1]. Although the pathogenesis of HCV is not fully understood, previous studies have reported that inflammatory cytokines (e.g., interleukin [IL]2, IL-6, IL-8, and IL-12) are involved [25]. Elevated IL-8 production is related to histological inflammation and severity of liver fibrosis [6].
The HCV genome codes an 3000 aa polyprotein cleaved into 10 structural or nonstructural proteins. The HCV core protein (aa 1191) is a highly conserved nucleocapsid protein [7] that modulates the production of several cytokines [3, 4, 8]. The HCV core protein up-regulates the IL-8 promoter by activating the NF-B signaling pathway [8, 9]. On the basis of the amino acid content and hydrophobicity pattern, the core protein is separated into 3 domains. Domains 2 (aa 123174) and 3 (aa 175191) are highly hydrophobic. C-terminal truncation of the HCV core protein (aa 152191) does not induce IL-8 production through the NF-B pathway by a change of subcellular localization from the cytoplasm to the nucleus [8, 9].
Interferon (IFN) has been used to treat chronic HCV infection by aiming at eradication of the virus. The latest antiviral therapy, pegylated IFN with ribavirin, has been shown to result in a sustained virological response in up to 46% of patients with HCV genotype 1 infection [10]. In patients with high baseline levels of HCV RNA, the viral eradication rate was 41%. This finding indicates that, as a whole, more than one-half of patients cannot achieve a sustained virological response. However, nearly 10% of patients showed sustained normalization of serum alanine aminotransferase (ALT) levels during HCV viremia (a so-called biochemical response) and showed slower progression to cirrhosis and hepatocellular carcinoma [1, 11, 12]. By analyzing biochemical responders, we might evaluate the viral factors that affect the activity of hepatitis in the absence of the influence of host factors. In the present study, we used serum from biochemical responders to analyze whether the properties of the HCV core protein, a viral factor, correlate with the activity of hepatitis.
PATIENTS, MATERIALS, AND METHODS
Patients.
We evaluated patients chronically infected with HCV genotype 1b who had received 6 months of IFN- therapy at the University of Tokyo or the Chiba University hospital between 1992 and 1997. Before IFN therapy, all of the patients had serum ALT levels that were 2-fold higher than the upper limit of normal (36 IU/L). A "biochemical response" was defined as the continuous normalization of ALT levels without viral eradication 6 months after IFN therapy. A persistently high ALT level with viremia was designated as a "nonresponse." We analyzed 12 biochemical responders (6 men and 6 women) with HCV genotype 1b infection. As control patients, we also evaluated 12 randomly selected nonresponders (6 men and 6 women) with HCV genotype 1b infection. Serum samples were obtained from all patients before and 6 months after the cessation of IFN therapy. All serum samples were stored at -80°C in a freezer immediately after they were obtained. Written, informed consent for the analyses was obtained from all patients.
All patients tested positive for serum anti-HCV antibodies (by second-generation EIA; Ortho Diagnostics). HCV genotyping was performed as described elsewhere [13]. Serum HCV load was measured by use of the Amplicor HCV assay (Roche). All patients tested negative for serum hepatitis B surface antigen, antinuclear antibody, and antimitochondrial antibody and had no history of alcohol abuse or hepatotoxic drug intake. None of the patients had received alternative therapies, including glycyrrhizin, which possibly lowers aminotransferase levels. Liver-biopsy samples were obtained from all patients immediately before IFN therapy, and the absence of cirrhosis was confirmed according to the international classification system [14].
More than 6 months after the cessation of IFN therapy, the ALT levels of biochemical responders 1, 2, and 3 were again elevated. We obtained further serum samples from these patients and from biochemical responders 8 and 9, whose ALT levels did not again become elevated after the cessation of IFN therapy. The amino acid sequence of the HCV core protein was determined for each sample.
Serum concentrations of inflammatory cytokines.
Concentrations of IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-, and tumor necrosis factor (TNF) in serum obtained before and after IFN therapy were assayed by use of an ELISA kit (Quantine; R&D Systems).
RNA extraction, cDNA synthesis, and polymerase chain reaction (PCR).
RNA was extracted from the serum of each patient before and after IFN therapy by use of the SepaGeneRVR kit (Sanko Junyaku), in accordance with the manufacturer's instructions. To amplify a region of the HCV core protein, reverse-transcription PCR was performed as described elsewhere [15], by use of the following primers (the nucleotide positions in HCV-J [16] are shown in parentheses): sense (nt -42 to -37 from the transcription start site), 5-GGTGCTTGCGAGTGCC-3; antisense (nt 626645), 5-GCCTCATACACAATACTTGA-3. Each amplified product was used as a template for a second PCR with nested primers: sense (nt -22 to -4), 5-GAGGTCTCGTAGACCGTGC-3; antisense (nt 606624), 5-TTGGAGCAGTCGTTCGTGA-3.
Sequence determination.
The PCR products were purified by use of Qiaquick columns (Qiagen), in accordance with the manufacturer's instructions. Sequencing was performed directly from each purified PCR product by use of an automated DNA sequencer (Applied Biosystems) [17]. The sequencing primers were as follows: forward (nt 232254), 5-TCAGCCCGGGTACCCTTGGCCCC-3; reverse (nt 357378), 5-GTATCGATGACCTTACCCA-3.
The consensus amino acid sequence of the core protein of HCV genotype 1b (HCV 1b consensus) was determined by aligning the amino acid sequences of 99 HCV genotype 1b strains deposited in GenBank (accession numbers AB016785, AB049087101, AF05424750, AF139594, AF16504564, AF176573, AF20775274, AF208024, AJ000009, AJ1329967, AJ238799800, D10750, D10934, D11168, D11355, D13558, D14484, D30613, D45172, D504805, D63857, D85516, D89815, D89872, D90208, L02836, M58335, M84754, M96362, S62220, U01214, U16362, U45476, and X61596) and by identifying the most commonly observed amino acid at each position. The amino acid sequences were deduced and aligned with the HCV 1b consensus. Sequences from samples obtained both before and after IFN therapy were compared for each patient.
Cell culture.
Human hepatoma cells (Huh7) were obtained from the Riken cell bank. Cells were grown in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO2 atmosphere.
HCV core proteinexpressing plasmids.
With use of primers specific to each template sequence (nt 120 and 555573) with 5 hemagglutinin (HA: YPYDVPDYA) tags and 5 and 3 att DNA recombination sites, the HCV core region (nt 1573, aa 1191) was amplified with Platinum Pfx DNA polymerase (Invitrogen). The PCR products were cloned into pCXN2 (provided by J. Miyazaki, Osaka University, Osaka, Japan), a mammalian expression vector with a -actin promoter and cytomegalovirus enhancer [18], by use of the GATEWAY cloning system (Invitrogen) (pCXN2 core). The plasmids were purified by use of the Endfree plasmid kit (Qiagen) and were sequenced to confirm the core genes. At least 5 clones were sequenced for each serum sample, and results confirmed that the direct sequencing was consistent with the predominant clones in each sample.
To analyze the effect of domains 2 and 3 of the core protein on IL-8 transactivation in biochemical responders 1, 2, and 3, we constructed chimeric clones that expressed fusion HCV core proteins with domain 1 from the core sequence isolated before IFN therapy and domains 2 and 3 from the core sequence isolated after IFN therapy. Briefly, each pCXN2 core was digested by use of ClaI (which recognizes 367372 bp of the HCV core protein) and HindIII (which recognizes a site in pCXN2). In each biochemical responder, a DNA fragment containing the 122 N-terminal amino acids of the HCV core protein (domain 1) isolated from serum obtained before IFN therapy and a DNA fragment containing the 69 C-terminal amino acids of the HCV core protein (domains 2 and 3) isolated from serum obtained after IFN therapy were ligated to each other by use of the DNA ligation kit (version 1; Takara Biomedical).
Aiming to evaluate the effects of each amino acid change in domains 2 and 3 of the core protein on IL-8 gene induction, we used site-directed mutagenesis via a quick-change site-directed mutagenesis kit (Stratagene). We used the core protein subcloned from serum from biochemical responder 2 before IFN therapy as a template, because amino acid changes before and after IFN therapy were observed only in domains 2 and 3 in biochemical responder 2. We prepared the following primers for site-directed mutagenesis: 5-GCGCATGGCGTCTGGGTTCTAGAGG-3 (nt 454-478, W156R), 5-CAGGGAATTTGCCCGGTTGCTCTTTTTC-3 (nt 497-524, T170P), and 5-GTCCTGTTTGACCATCCCAGCTTCCGC-3 (nt 546-572, T187I). Introduced mutations were verified by sequencing.
Intracellular signaling pathway reporter plasmids.
The effect of each HCV core protein on IL-8 gene expression was evaluated by use of the Photinus pyralis (firefly) luciferase reporter plasmid that encoded the 5 flanking region of the IL-8 gene spanning -133 to +44 bp (pIL-8-133 wild-type Luc) [19].
Transfection.
Approximately 4 × 105 cells/well were plated in 6-well tissue-culture plates (Iwaki Glass) for 24 h before transfection. To examine the effect of each cloned HCV core protein on intracellular signaling pathways, Huh7 cells were transfected with a total of 0.4 g of plasmid DNA consisting of 0.19 g of firefly luciferase reporter plasmid, 0.01 g of pRL-TK (Promega), a control plasmid expressing the Renilla reniformis (seapansy) luciferase driven by the herpes simplex thymidine kinase promoter, and 0.2 g of pCXN2 or pCXN2 core, by use of Effectine transfection reagent (Qiagen). As a positive control, pFCmitogen-activated protein kinase/extracellular signalregulated kinase kinase (MEKK; Stratagene), which constitutively expresses active MEKK 1 (aa 360672) driven by a cytomegalovirus promoter, was added to the transfection complexes that contained pCXN2. Transfection efficiency was monitored by cotransfection with pRL-TK.
Expression of HCV core proteins.
The expression of each HCV core protein was examined by the ECL-Plus Western blotting detection system (Amersham Pharmacia Biotech), by use of extracts of Huh7 cells transiently transfected with HCV core proteinexpression plasmids. Initially, HCV core protein expression was confirmed by use of antiHCV core protein monoclonal antibody 515S (provided by M. Kohara, Tokyo Metropolitan Institute of Medical Science, Japan). Subsequently, anti-HA polyclonal antibody (Santa Cruz Biotechnology) was used to confirm that, for each patient, the amount of expressed protein did not differ between the 2 core-expression plasmids cloned from serum obtained before and after IFN therapy. Indirect immunofluorescent staining was performed to evaluate the subcellular localization of the HCV core protein, as described elsewhere 9.
Luciferase assay.
Cells were harvested 48 h after transfection by use of the PicaGene dual seapansy system (Toyo Ink) with a luminometer (Lumat LB9507; EG&G Berthold). Firefly luciferase activity and seapansy luciferase activity were measured in relative light units in triplicate. Firefly luciferase activity was then normalized against seapansy luciferase activity.
Statistics.
Statistical significance was evaluated by Student's t test, the 2 test, and Fisher's exact test. Two-tailed P < .05 was considered to be statistically significant.
RESULTS
Patients' clinical data.
Table 1 summarizes the clinical data on biochemical responders and nonresponders (12 each). There were no significant differences between biochemical responders and nonresponders in sex (P = .68), age (P = .26), ALT values before IFN therapy (P = .11), amounts of IFN received (P = .56), and level of hepatic fibrosis (P = .24). One biochemical responder and 3 nonresponders had F3 fibrosis (P = .58). Serum viral loads before and after IFN therapy were not significantly different in each group (for biochemical responders, P = .17; for nonresponders, P = .46).
Serum cytokine levels.
Serum levels of IL-8 significantly decreased after IFN therapy in biochemical responders (P = .04), whereas those in nonresponders did not (P = .67) (table 2). Serum levels of TNF- had a tendency to increase after IFN therapy in biochemical responders (P = .08). There were no significant changes in the levels of other cytokines after IFN therapy in either group.
Amino acid sequence of the HCV core protein.
The number of amino acid changes was greater in biochemical responders than that in nonresponders (figure 1): the median (range) was 2.5 (15) in biochemical responders and 0.5 (03) in nonresponders (P < .01). In domain 3, the difference in the number of amino acid changes was significant (P = .02), whereas the differences in domains 1 and 2 were not significant (P = .15 and P = .63, respectively). There was no common amino acid substitution.
Detection of transiently expressed HCV core protein.
HCV core protein cDNA sequences obtained from 6 patients in each of the groups (biochemical responders 16 and nonresponders 15 and 9) were cloned into pCXN2. The expression of each HCV core protein was examined by Western blotting with extracts of transiently transfected Huh7 cells (data not shown). Anti-HA polyclonal antibodies were used to detect HCV core protein.
The core protein was mainly located in the cytoplasm in all clones. For all patients, there was no marked difference in the subcellular localization of the HCV core protein before and after IFN therapy (data not shown).
Activation of the IL-8 promoter by the HCV core protein.
Activation of the IL-8 promoter by HCV core protein was compared before and after IFN therapy (figure 2). In biochemical responders, this activation was reduced after IFN therapy by 27% (median, 73%; 25th percentile, 61%; 75th percentile, 93%) (P = .04), whereas no significant differences were observed in nonresponders (median, 112%; 25th percentile, 98%; 75th percentile, 124%) (P = .17).
Effect of HCV core domains 2 and 3 on IL-8 inducibility.
Our previous results showed that truncation of HCV core protein domains 2 and 3 eliminated IL-8 transactivation through the NF-B pathway [8]. In the present study, we have evaluated the effects that the postIFN therapy changes to domains 2 and 3 have on IL-8 transactivation for biochemical responders 1, 2, and 3. To do this, we constructed plasmids encoding chimeric HCV core proteins consisting of domain 1 sequences from serum obtained before IFN therapy and sequences of domains 2 and 3 from serum obtained after IFN therapy. Subsequently, a luciferase assay was performed to evaluate the ability of these chimeric proteins to induce IL-8 transcription. For the 3 patients examined, the luciferase activities were reduced by changing the 69 C-terminal amino acids of the HCV core protein (figure 3), which suggests that domains 2 and 3 play important roles in the induction of IL-8 transcription.
Effect of each amino acid change in HCV core protein domains 2 and 3 on IL-8 inducibility.
In biochemical responder 2, 3 amino acid changes were observed in domains 2 and 3 after IFN therapy at positions aa 156 (argininetryptophan), 170 (prolinethreonine), and 187 (isoleucinethreonine). To evaluate the effect of each amino acid change on the ability of the core protein to induce IL-8, we used 2 pCXN2-core plasmids carrying core protein cDNA from serum from biochemical responder 2 before and after IFN therapy (BR2before IFN and BR2after IFN). When BR2after IFN was used as a template, the 3 amino acids that were different from in the BR2before IFN core protein were individually mutated back to the amino acids found in the BR2before IFN core protein. This involved the preparation of 3 plasmids (BR2-W156R, BR2-T170P, and BR2-T187I). None of the individual mutants were able to transactivate the IL-8 promoter to the same degree as did the BR2before IFN core protein, which suggests that all 3 amino acid changes are important in altering IL-8 induction (figure 4).
DISCUSSION
More-frequent amino acid changes were observed after IFN therapy in domains 2 and 3 of the HCV core protein in biochemical responders than in nonresponders. These changes in the amino acid sequences of the HCV core protein altered its ability to induce the production of IL-8. In addition, in biochemical responders, serum IL-8 levels were significantly decreased after IFN therapy, which indicates that IL-8 is associated with the activity of hepatitis. These results suggest that the sequence of the C-terminal hydrophobic region of the HCV core protein affects the induction of IL-8 and that it may be a viral factor associated with the activity of hepatitis. Although further confirmation is required, the follow-up sequencing data for biochemical responders 1, 2, 3, 8, and 9 support our conclusions.
The pathogenesis of HCV infection involves complex interactions between host and viral factors [20]. As a viral factor, HCV genotype affects the response to IFN therapy [10]. Although the host's immune system may play a key role, individual differences among patients make it difficult to understand the mechanism of hepatitis. In the present study, we compared viral factors obtained from individual patients at different time points, to minimize the influence of the diversity of immune responses. For this purpose, we considered the use of biochemical responders to be optimal. In addition, because samples were obtained 6 months after the cessation of IFN therapy, its effect on the host immune response can be disregarded.
Our previous results suggested that the HCV core protein interacts with TNF receptorassociated molecules at its C-terminal hydrophobic region and that the core protein up-regulates IL-8 transcription through the activation of NF-Bassociated signals. Thus, the HCV core protein may be associated with the pathogenesis of HCV infection [8, 9]. A previous study of clinical samples showed an association between deletion of the C-terminal region of the HCV core protein and the severity of hepatitis [21]. In the cases described here, neither a deletion nor a change in subcellular localization was observed. However, amino acid changes in the C-terminal region of the HCV core protein were clearly observed more frequently in biochemical responders than in nonresponders. We propose that some amino acid changes are sufficient to modulate the affinity of the core protein for TNF receptorrelated signaling molecules and that these changes lead to differences in the ability of the core protein to induce IL-8 production, despite no major changes being observed in subcellular localization. Viral loads did not change after IFN therapy, which suggests that these amino acid changes did not affect the function of the HCV core protein as a nucleocapsid protein.
Of 12 biochemical responders, 4 (4, 5, 11, and 12) showed no decrease in serum IL-8 levels. None of these showed amino acid changes in domain 3 after IFN therapy, whereas 7 of the remaining 8 patients did show some amino acid changes. This observation might support our conclusion that the amino acid changes in domain 3 are associated with the inducibility of IL-8 transcription by the HCV core protein. However, in these 4 patients, we have to consider the possibility that other mechanisms played a role in achieving the biochemical response. Also, it might be possible that relatively low ALT levels before IFN therapy in biochemical responders 4, 5, and 12 were related to the lack of significant decrease in serum IL-8 levels.
The levels of TNF- in biochemical responders were significantly higher than those in nonresponders, both before (P = .049) and after (P = .041) IFN therapy. This finding might allow us to speculate that the TNF-mediated NF-B signaling pathway is interrupted to some extent in biochemical responders. We could not see any statistically significant difference in levels of other cytokines.
Several studies that have used cell lines have reported that the HCV core protein induces the expression of IL-2 [3], IL-8, IL-12, and nitric oxide [4]. Some clinical evidence has suggested that the induction of IL-8 plays a major role in the pathogenesis of chronic HCV infection [5, 6]. By analyzing biochemical responders, we have revealed that IL-8 is associated with the activity of hepatitis. Recently, the HCV nonstructural 5A protein has been reported to induce IL-8 transcription [22]. Thus, changes in the amino acid sequence of NS5A could possibly alter the induction of IL-8 and hepatitis activity. This indicates that chronic HCV infection may potentially involve factors other than the HCV core protein.
Recent clinical trials that have used IFN for the treatment of chronic HCV infection have shown that this disease remains intractable, with overall virological response rates of nearly 50%, and a risk of hepatic cancer also exists [10]. Recent cohort studies have shown that the status of the biochemical response does not persist and that a reactivation of hepatitis may occur in some patients [23]. These patients are still at high risk for cirrhosis and hepatocellular carcinoma. The results of retreatment with IFN with and without an additional arm have not been satisfactory [24]. Achieving a persistent biochemical response would have a great impact on the treatment strategy for intractable chronic HCV infection, especially with genotype 1. Our results may provide clues to potential therapeutic targets. In conclusion, our results show that amino acid changes in the C terminus of the HCV core protein correlate with its ability to induce IL-8 gene transcription and that this may be associated with hepatitis activity in patients with HCV genotype 1b infection.
Acknowledgments
We thank Dr. J. Miyazaki, for providing us with plasmid; Dr. M. Kohara, for providing antihepatitis C virus core antibodies; M. Tsubouchi, for technical assistance; and Y. Otori, for secretarial assistance.
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