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Journal of Virology, September 2000, p. 8744-8750, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Activation of the Interferon-Inducible 2'-5'-Oligoadenylate Synthetase Gene by Hepatitis C Virus Core Protein

Atsushi Naganuma,1,2,3 Akito Nozaki,1 Torahiko Tanaka,1 Kazuo Sugiyama,1 Hitoshi Takagi,3 Masatomo Mori,3 Kunitada Shimotohno,4 and Nobuyuki Kato1,2,*

Virology and Glycobiology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045,1 Department of Molecular Biology, Institute of Cellular and Molecular Biology, Okayama University School of Medicine, Okayama 700-8558,2 The First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511,3 and Laboratory of Human Tumor Viruses, Department of Viral Oncology, The Institute for Virus Research, Kyoto University, Kyoto 606-8937,4 Japan

Received 3 February 2000/Accepted 23 June 2000


    ABSTRACT
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The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2'-5'-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2'-5'-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2'-5'-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2'-5'-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2'-5'-OAS gene.


    TEXT
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Abstract
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Hepatitis C virus (HCV) is a major causative agent of non-B chronic hepatitis and has been implicated in the etiology of hepatocellular carcinoma (7, 22, 33, 38). HCV is an enveloped positive single-stranded RNA (9.6-kb) virus belonging to the Flaviviridae (18, 44). The HCV genome shows remarkable sequence variation, and to date at least six major HCV genotypes, which have been further grouped into more than 50 subtypes, have been identified (4).

The HCV RNA genome encodes a polyprotein precursor of about 3,000 amino acid (aa) residues, and this precursor protein is cleaved by the host and viral proteases to generate at least 10 proteins: the core, envelope 1 (E1), E2, p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (13, 14, 29). These HCV proteins not only function in viral replication but also affect a variety of cellular functions (3, 6, 10, 11, 20, 24, 25, 27, 32, 34, 35, 36, 41, 45, 46, 48). All HCV proteins used in these studies to date were derived from cDNA clones which were obtained from sera of patients with hepatitis C. However, HCV shows typical quasispecies in human blood (26), and most circulating HCV has a defective viral genome (12, 26). In addition, nonhuman cells or nonhepatocyte cells were frequently used in these studies, although the main target of HCV is a human normal hepatocyte cell. Therefore, the functions of HCV proteins found in these studies may not reflect the functions in HCV-replicating human hepatocytes.

We recently developed HCV culture systems which could support HCV replication by using nonneoplastic human hepatocyte PH5CH1, PH5CH7, and PH5CH8 cells (16), which were cloned from PH5CH cells (immortalized by simian virus 40 large T antigen), and human T-cell lines MT-2A, MT-2B, MT-2C, MT-2D, and MT-2E (30), which were cloned from MT-2 cells (immortalized by human T-cell leukemia virus type 1 infection). A study of the dynamics of HCV populations during culture using three PH5CH clones (PH5CH1, PH5CH7, and PH5CH8) and three MT-2 clones (MT-2A, MT-2B, and MT-2C) found that the amino acid sequences of HCV proteins obtained from the infected cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection (19).

To investigate whether HCV proteins affect certain signal transduction pathways, we made several expression vectors using HCV cDNA clones obtained from HCV-infected cells. The plasmids used in this study were constructed to produce several proteins under the control of the cytomegalovirus immediate-early promoter fused to the Moloney murine leukemia virus long terminal repeat at the TATA box in the 5'-U3 region and named the pCXbsr (a kind gift of T. Akagi, Osaka Bioscience Institute) series. Two core expression vectors [pCXbsr/core(M) and pCXbsr/core(P)] were constructed by inserting the core-encoding regions of two cDNA clones (no. 15-2 and no. 16-6 in reference 19) derived from HCV-infected MT-2C and PH5CH7 cells, respectively, into pCXbsr. The amino acid sequence of core(M) was identical to the consensus sequences from the original inoculum 1B-2 and was the same as the consensus sequence of the HCV 1b genotype (19). Although only 1 aa [aa 70, Arg for core(M) and Gln for core(P)] differed between core(M) and core(P) (19), the amino acid sequence of core(M) differed at 2 and 8 aa from HCV-J (18) and HCV-K (6) strains, respectively. The plasmid vectors expressing E1 and E2 [pCXbsr/E1(P) and pCXbsr/E2(P)] were also constructed from a cDNA (no. 16-6 in reference 19) clone derived from HCV-infected PH5CH7 cells. The NS5A expression vectors [pCXbsr/NS5A(M) and pCXbsr/NS5A(P)] were constructed from two cDNA clones which were obtained from HCV-infected MT-2C and PH5CH7 cells, respectively. The amino acid sequence of NS5A from HCV-infected cells converged with the consensus amino acid sequence of NS5A from the original inoculum 1B-2, as previously shown for the other regions (19). Only 1 aa [aa 413, Tyr for NS5A(M) and Cys for NS5A(P)] differed between NS5A(M) and NS5A(P) (A. Naganuma et al., unpublished data). The amino acid sequence of NS5A(M) was different from that of NS5A of the HCV-J strain (18) at 28 aa positions. All constructed plasmids were checked by nucleotide sequence analysis.

We confirmed the transient expression of HCV proteins from these vectors in PH5CH8 cells. As shown in Fig. 1, core(M) and core(P) proteins derived from pCXbsr/core(M) and pCXbsr/core(P), respectively, were 21 kDa in size. E1(P) and E2(P) proteins were approximately 35 and 60 kDa in size, respectively. NS5A(M) and NS5A(P) proteins derived from pCXbsr/NS5A(M) and pCXbsr/NS5A(P), respectively, were both 58 kDa in size. The sizes of these proteins were the same as those in the previous reports (14, 28).


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  		FIG. 1.   Western blot analysis of HCV proteins expressed in PH5CH8 cells. Expression plasmids pCXbsr/core(M) (lane 1), pCXbsr/core(P) (lane 2), pCXbsr/E1(P) (lane 4), pCXbsr/E2(P) (lane 6), pCXbsr/NS5A(M) (lane 8), and pCXbsr/NS5A(P) (lane 9) were transfected using FuGENE 6 transfection reagent (Boehringer Mannheim) into PH5CH8 cells. The proteins expressed in these cells were analyzed on an immunoblot using the region-specific antibodies as described previously (14, 17); proteins were resolved by electrophoresis on a sodium dodecyl sulfate-15% (lanes 1 to 3), -12% (lanes 4 and 5), -7.5% (lanes 6 and 7), and -10% (lanes 8 to 10) polyacrylamide gel. The lysate of cells transfected with expression vector pCXbsr was analyzed as a negative control (lanes 3, 5, 7, and 10). Products (21 kDa for core, 35 kDa for E1, 60 kDa for E2, and 58 kDa for NS5A) that were specifically detected by region-specific antibodies are indicated by arrows. M and P indicate HCV proteins derived from MT-2C and PH5CH7 cells, respectively. C, negative control.

We first examined whether core or NS5A protein affects several signal transduction pathways using an in vivo cis reporting assay system. The expression of the firefly luciferase gene, which is controlled by a synthetic promoter that contains tandem repeats of cyclic AMP response element or serum response element or binding sites for NF-kappa B, AP-1, or p53, was monitored in the presence or absence of core or NS5A protein in PH5CH8 cells. In this study, to accurately evaluate the firefly luciferase activities, we used a dual-luciferase reporter assay system (Promega) using pRL-CMV, which expressed Renilla luciferase under the control of the cytomegalovirus promoter, as an internal control reporter. The results revealed that no signal transduction pathways (NF-kappa B, AP-1, cyclic AMP response element, serum response element, and p53) were significantly affected by core or NS5A protein production in the cells (data not shown). During the course of these experiments, we found that core protein affected the interferon (IFN)-stimulated response element (ISRE) pathway. Analysis of the effects of HCV proteins against ISRE using reporter plasmids p2'-5'OAS(-159)-Luci (2), which contains the -159-to-+82 region of the 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene, and pGBP(-216)-Luci (23), which contains the -216 to +19 region of the human guanylate-binding protein GBP-1 gene, showed that both core(M) and core(P) specifically activated the human 2'-5'-OAS gene promoter in PH5CH8 cells (Fig. 2), although the activation of the human GBP-1 gene promoter by core was weaker than that of the 2'-5'-OAS gene promoter (data not shown). The 2'-5'-OAS gene promoter was not activated by E1(P), E2(P), NS5A(M), or NS5A(P) proteins (Fig. 2). From these results, we focused on the activation of the 2'-5'-OAS gene promoter by core protein. First, we checked whether PH5CH8 cells respond to alpha IFN (IFN-alpha ), using a p2'-5'OAS(-159)-Luci reporter plasmid. IFN-alpha at 500 U/ml gave approximately sixfold enhancement of luciferase activity (Fig. 2), indicating that PH5CH8 cells showed a good response to IFN-alpha . Treatment with IFN-alpha up to 1,000 U/ml led to no growth or morphological changes in PH5CH8 cells (16). Since the maximum effect of IFN-alpha in PH5CH8 cells was observed from 6 to 12 h after treatment (data not shown), we chose 6 h after IFN-alpha treatment as the time point for the measurement of luciferase activity. As shown in Fig. 2, we found that both core(M) and core(P) proteins further enhanced 2'-5'-OAS gene promoter activity, while E1(P), E2(P), NS5A(M), or NS5A(P) showed no enhancement of activity. The activation potentials of core(M) and core(P) proteins were equivalent, indicating that the amino acid difference at position 70 did not affect the activation of the 2'-5'-OAS gene promoter.


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  		FIG. 2.   Effects of HCV protein production on human 2'-5'-OAS gene promoter activity in PH5CH8 cells treated with and without IFN-alpha . A total of 1.5 × 105 cells was seeded in a six-well plate 24 h before transfection. Then 0.5 µg of p2'-5'OAS(-159)-Luci, 2 µg of various expression plasmids (for the production of core, E1, E2, and NS5A), and 5 ng of pRL-CMV (internal control) were transfected into PH5CH8 cells with FuGENE 6. At 42 h posttransfection, some cells were treated with IFN-alpha (Sigma; 500 IU/ml) for 6 h as indicated. A whole-cell lysate was prepared and assayed for firefly and Renilla (internal control) luciferase activities according to the manufacturer's protocol for the dual-luciferase assay (Promega). The relative luciferase activity was normalized to the activity of Renilla luciferase. The data represent the means of the normalized luciferase activities of triplicates in at least three repeated experiments. (M) and (P) indicate that the HCV proteins are from MT-2C and PH5CH7 cells, respectively.

To examine whether activation of the 2'-5'-OAS gene promoter by core protein occurs in other human hepatocyte cell lines, HepG2 and PLC/PRF/5 cells were used in the same experiment as PH5CH8 cells. As shown in Fig. 3, HepG2 and PLC/PRF/5 cells showed results similar to those obtained with PH5CH8 cells. In both cell lines, core(P) protein, but not NS5A(P) protein, activated the 2'-5'-OAS gene promoter and further enhanced the promoter activity in the presence of IFN-alpha , although the level of activation by core protein in these cells was slightly lower than that in PH5CH8 cells (Fig. 2 and 3). This difference might be due to the different properties of these cell lines: HepG2 and PLC/PRF/5 cells were derived from hepatocellular carcinoma, while PH5CH8 cells (16) were derived from nonneoplastic hepatocytes. Therefore, the 2'-5'-OAS gene promoter is activated by core protein in human cultured hepatocytes.


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  		FIG. 3.   Effects of core or NS5A production on human 2'-5'-OAS gene promoter activity in HepG2 and PLC/PRF/5 cells treated with or without IFN-alpha . Using HepG2 or PLC/PRF/5 cells, DNA transfection, IFN-alpha treatment, and dual-luciferase assay were carried out as indicated in the Fig. 2 legend. (P) indicates that core and NS5A are from PH5CH7 cells.

To examine whether the dose of core protein affects the level of activation of the 2'-5'-OAS gene promoter, p2'-5'OAS(-159)-Luci (0.5 µg), pRL-CMV (5 ng), and pCXbsr/core(P) (0.5 to 4 µg) were cotransfected following the dual-luciferase reporter assay procedure into PH5CH8 cells. To maintain the efficiency of transfection, up to 4 µg of pCXbsr instead of pCXbsr/core(P) was used as effector plasmid DNA. As shown in Fig. 4, activation of the 2'-5'-OAS gene promoter by core(P) protein was clearly observed in a dose-dependent manner regardless of IFN-alpha treatment. Similar results were also obtained using core(M) protein (data not shown).


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  		FIG. 4.   Dose dependency of core on the activation of the 2'-5'-OAS gene promoter. PH5CH8 cells were transfected with various doses (0.5 to 4 µg) of pCXbsr/core(P) in addition to p2'-5'OAS(-159)-Luci (2 µg) and pRL-CMV (5 ng). IFN-alpha treatment and dual-luciferase assay were carried out as indicated in the Fig. 2 legend.

To confirm that core protein activates the 2'-5'-OAS gene promoter, we examined the effect of blocking core protein production by using pCXbsr/core(P)R, which expresses antisense RNA complementary to core-encoding RNA. Cotransfection with p2'-5'OAS(-159)-Luci (0.5 µg), pRL-CMV (5 ng), pCXbsr/core(P) (2 µg), and/or pCXbsr/core(P)R (2 µg) following the dual-luciferase reporter assay was performed in PH5CH8 cells. As shown in Fig. 5A, the expression of pCXbsr/core(P)R partially suppressed the activation by core protein regardless of IFN-alpha treatment. By Western blot analysis, we confirmed that the amount of core protein was decreased by the expression of pCXbsr/core(P)R (Fig. 5B). The level of suppression by antisense RNA was correlated with the ratio of pCXbsr/core(P) and pCXbsr/core(P)R used (data not shown). To exclude the possibility that the RNA corresponding to core-encoding sequence activates the 2'-5'-OAS gene promoter, we examined the effect of pCXbsr/core(P)Delta P, which is produced by the mutation of the AUG initiation codon to a GUG codon. The expression of pCXbsr/core(P)Delta P showed no enhancement of the promoter activity (data not shown). These results suggest that the activation of the 2'-5'-OAS gene promoter is due to the production of core protein in PH5CH8 cells.


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  		FIG. 5.   Effects of antisense RNA complementary to core-encoding RNA. pCXbsr/core(P)R, which expresses antisense RNA complementary to core-encoding RNA, was made by inserting the PCR product (reverse direction) into the pCXbsr. PH5CH8 cells were transfected with pCXbsr/core(P) (2 µg) and/or pCXbsr/core(P)R (2 µg), which expresses antisense RNA complementary to core-encoding RNA, in addition to p2'-5'OAS(-159)-Luci (2 µg) and pRL-CMV (5 ng). To avoid the squelching effect of the promoter, the amount of plasmid DNA was maintained with pCXbsr (2 µg) instead of pCXbsr/core(P)R. IFN-alpha treatment was carried out as indicated in the Fig. 2 legend. (A) Suppression of core activity on 2'-5'-OAS gene promoter by antisense RNA complementary to core-encoding RNA. The dual-luciferase assay was carried out as indicated in the Fig. 2 legend. (B) Western blot analysis of HCV core and beta -actin expressed in PH5CH8 cells. Core and beta -actin expressed in the cells were detected on an immunoblot using the anticore antibody (upper portion) and anti-beta -actin antibody (Santa Cruz; lower portion); proteins were resolved by electrophoresis on a sodium dodecyl sulfate-15% polyacrylamide gel. The lysate of cells transfected with expression vector pCXbsr/core(P) without (lane 1) or with (lane 2) pCXbsr/core(P)R was used for detection of core protein. The lysate of cells transfected with expression vector pCXbsr was used as a control (lane 3).

To identify the region responsible for the activation of the 2'-5'-OAS gene promoter, we carried out deletion analysis of the core protein coding region using our reporter assay system. First, we constructed three carboxyl-truncated forms of core(P) containing aa 1 to 102, 1 to 152, and 1 to 173, respectively. However, surprisingly, these truncated forms of core protein were unstable in PH5CH8 cells, because only very weak signals were obtained from these expression vectors by Western blot analysis (data not shown), although full-length core protein was observed in Western blot analysis (Fig. 1 and 6B). This observation was not consistent with the recent report (47) concerning similar truncated forms of core protein expressed in rabbit kidney cells, suggesting that the stability of the core protein differs with cell types and that the carboxyl portion of the core protein is involved in intracellular stability. We therefore then constructed four plasmid vectors expressing internal core deletion mutants [core(P)Delta 38-43, core(P)Delta 61-80, coreDelta 101-120, and coreDelta 140-159], by PCR with pCXbsr/core(P) as a template (Fig. 6A). Western blot analysis indicated that these core mutants were stably expressed in PH5CH8 cells (Fig. 6B). The low mobility of core(P)Delta 140-159 (lane 6, Fig. 6B) might be due to the lack of processing at aa 173 for the production of mature core protein (173 aa) (47). PH5CH8 cells were cotransfected with the plasmid vectors expressing these core mutants, p2'-5'OAS(-159)-Luci and pRL-CMV, and the dual-luciferase reporter assay was performed. In this experiment, PH5CH8 cells were treated with IFN-alpha for 6 h at 42 h after transfection. As shown in Fig. 6C, results revealed that all of the deletion mutations of core protein reduced the activation of the 2'-5'-OAS gene promoter by full-size core protein, although the core(P)Delta 140-159 mutant showed a slightly stronger effect than did other mutants. This result suggests that most regions of core protein are required for the activation of the 2'-5'-OAS gene promoter.


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  		FIG. 6.   Deletion analysis of HCV core protein. (A) Schematic presentation of core deletion mutants. The expression plasmids for internally deleted core protein were made by inserting PCR products into pCXbsr. aa 38 to 43 encode a putative nuclear localization signal (43). (B) Western blot analysis of core and beta -actin expressed in PH5CH8 cells. Core and beta -actin were detected on an immunoblot using the anticore antibody (upper portion) and anti-beta -actin antibody (lower portion). The lysate of cells transfected with expression vector, pCXbsr/core(P) (lane 2), pCXbsr/core(P)Delta 38-43 (lane 3), pCXbsr/core(P)Delta 61-80 (lane 4), pCXbsr/core(P)Delta 101-120 (lane 5), or pCXbsr/core(P)Delta 140-159 (lane 6) was loaded. The lysate of cells transfected with expression vector pCXbsr was used as a control (lane 1). (C) Effects of core deletion mutants on human 2'-5'-OAS gene promoter activity in PH5CH8 cells treated with and without IFN-alpha . DNA transfection, IFN-alpha treatment, and dual-luciferase assay were carried out as indicated in the Fig. 2 legend except for the amount of effector plasmid. To maintain the level of core protein, 1.5 µg of pCXbsr/core(P), 3.75 µg of pCXbsr/core(P)Delta 38-43, 1.5 µg of pCXbsr/core(P)Delta 61-80, 4.5 µg of pCXbsr/core(P)Delta 101-120, 4.5 µg of pCXbsr/core(P)Delta 140-159, and 4.5 µg of pCXbsr (control) were transfected, and the total amount of plasmid DNA was adjusted to 4.5 µg by the addition of pCXbsr.

Since the 2'-5'-OAS gene promoter is known to be regulated through the ISRE (nucleotides [nt] -101 to -88 of the 2'-5'-OAS gene promoter) (2), we examined whether the ISRE mediates the activation by core protein using two deletion mutants of the 2'-5'-OAS gene promoter (Delta A and Delta B in Fig. 7A). The regions of nt -159 to -109 and -108 to -87 were deleted in Delta A and Delta B, respectively. In the dual-luciferase assay, both the relative luciferase activity and the activation by core protein were almost abolished when Delta B was used. However, the activation by core protein still remained when Delta A was used. These results suggest that the ISRE mediates the activation by core protein. The possibility that the region of nt -159 to -109 also mediates the activation by core protein is not ruled out, because the luciferase activity decreased when Delta A was used. Further analysis is required to determine whether the ISRE is the only element regulating transcriptional activation by core protein in the 2'-5'-OAS gene promoter.


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  		FIG. 7.   Deletion analysis of the 2'-5'-OAS gene promoter. (A) Schematic presentation of deletion mutants of the 2'-5'-OAS gene promoter. Delta (-) indicates no deletion of the promoter, Delta A indicates the deletion of -159 to -108 of the 2'-5'-OAS gene promoter, and Delta B indicates the deletion of -108 to -86 (ISRE) of the 2'-5'-OAS gene promoter. (B) Activation of the 2'-5'-OAS gene promoter by core protein occurred through the ISRE. DNA transfection, IFN-alpha treatment, and dual-luciferase assay were carried out as indicated in the Fig. 2 legend.

To clarify whether the enhancement of luciferase activity by core protein occurs at the transcriptional level, we examined the expression of firefly luciferase mRNA by Northern blot analysis. The level of firefly luciferase mRNA (3.1 kb) was enhanced approximately fourfold by core protein in the absence of IFN-alpha and was further enhanced by core protein in the presence of IFN-alpha (Fig. 8A and B), indicating that the activation by core protein occurred at the transcriptional level. The level of enhancement of transcription was consistent with that of luciferase activity. Although our experiments clearly showed the transcriptional activation of the exogenous 2'-5'-OAS gene, it is important to clarify the effect of core protein on endogenous 2'-5'-OAS gene expression. The level of expression of the 2'-5'-OAS gene in PH5CH8 cells was examined by Northern blot analysis. Endogenous 2'-5'-OAS mRNA (1.8 kb) was also elevated by core protein in the absence of IFN-alpha and further enhanced by core protein in the presence of IFN-alpha (Fig. 8A and C). Although the level of 2'-5'-OAS mRNA in the presence of core protein was increased approximately twofold, it is estimated that the actual increase in mRNA would be about sixfold, because the efficiency of transfection with FuGENE 6 was consistently only about 20% (data not shown). Therefore, this result clarified that the activation of the endogenous 2'-5'-OAS gene by core protein also occurred at the transcriptional level.


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  		FIG. 8.   Transcriptional activation of exogenous and endogenous 2'-5'-OAS gene promoter by core. (A) Northern blot analysis of firefly luciferase mRNA and endogenous 2'-5'-OAS mRNA. PH5CH8 cells were transfected with 0.5 µg of p2'-5'OAS(-159)-Luci and 2 µg of pCXbsr (lanes 1 and 3) or pCXbsr/core(P) (lanes 2 and 4). At 42 h posttransfection, cells were treated with (lanes 3 and 4) or without (lanes 1 and 2) IFN-alpha (500 IU/ml). Total RNA was extracted from PH5CH8 cells using the ISOGEN extraction kit (Nippon Gene, Toyama, Japan). Ten micrograms of total RNA from the cells was used for the detection of firefly luciferase mRNA (3.1 kb) and endogenous 2'-5'-OAS mRNA (1.8 kb). Northern blotting and hybridization were performed as described previously (21). To indicate the quality of RNA, ethidium bromide-stained bands of 28S and 18S rRNAs are shown. (B) Quantification of firefly luciferase mRNA. The bands on Northern blots (A) were quantified using a BAS2000 Image analyzer. The measured values were normalized to the intensity of 28S rRNA shown in panel A. (C) Quantification of endogenous 2'-5'-OAS mRNA. Quantification was carried out as indicated for panel B.

The 2'-5'-OAS gene is generally induced by viral infection and IFN-alpha /beta and plays a major role in the antiviral activity of host cells, by activating RNase L to cleave viral RNA and thereby inhibit viral protein synthesis (1, 15). However, several viruses have mechanisms of resistance to this host cell antiviral activity. In herpes simplex virus-infected cells, 2'-5'-adenylate analog accumulates and interferes with the activation of RNase L (5). The human immunodeficiency virus type 1 Tat protein, human T-cell leukemia virus type 1 Rex protein, and vaccinia virus E3L gene product all inhibit 2'-5'-OAS activity (37, 39). In contrast, some viral RNA species including EBER1 RNA of Epstein-Barr virus, VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-response element RNA of human T-cell leukemia virus type 1 can act as 2'-5'-OAS activators (8, 31, 39, 40). Recently, the NS5A and E2 proteins have been shown to inactivate the IFN-induced double-stranded RNA-dependent protein kinase PKR (11, 45), suggesting a mechanism by which HCV can resist the antiviral effect of IFN and proliferate. In contrast, in this study we found that core protein activated the 2'-5'-OAS gene promoter. Taken together, these results suggest that core protein can activate the 2'-5'-OAS-RNase L pathway (to decrease virus dose) and that NS5A and E2 proteins can suppress the PKR pathway (to increase virus dose). These viral proteins are probably involved in the maintenance of a low steady state of virus in infected cells, enabling HCV to escape from the host immunosurveillance system and facilitating persistent viral infection.

To date, two reports have described elevated levels of 2'-5'-OAS in serum or peripheral blood mononuclear cells of patients with chronic hepatitis C compared with normal healthy controls (9, 42). This might reflect the activation of 2'-5'-OAS gene expression by core protein, although IFN induced by HCV infection should also function in the elevation of 2'-5'-OAS activity.

To directly clarify whether the activation of the 2'-5'-OAS-RNase L pathway by HCV core protein contributes to the degradation of HCV genomic RNA, further experiments using an efficient HCV replication system are required. We are currently developing an HCV replication system using an infectious HCV cDNA clone.

Nucleotide sequence accession number. The nucleotide sequence data reported in this article will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB036519 to AB036521.


    ACKNOWLEDGMENTS

We are grateful to M. Saito (Virology and Glycobiology Division, National Cancer Center Research Institute) for helpful suggestions and discussion. We thank T. Kobayashi and K. Hashimoto for helpful assistance. We thank T. Akagi (Osaka Bioscience Institute) and T. Matsuyama (Nagasaki University) for helpful suggestions.

A. Naganuma and A. Nozaki are recipients of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research, Japan. This work was supported by grants from Grants-in-Aid for Cancer Research and for the Second-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare and Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and the Organization for Pharmaceutical Safety and Research (OPSR).


    FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology, Institute of Cellular and Molecular Biology, Okayama University Medical School, Shikata-cho, 2-5-1, Okayama 700-8558, Japan. Phone: 81-86-235-7385. Fax: 81-86-235-7392. E-mail: nkato{at}med.okayama-u.ac.jp.


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Journal of Virology, September 2000, p. 8744-8750, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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