Inhibition of internal ribosomal entry site–directed translation of HCV by recombinant IFN-α correlates with a reduced La protein

Inhibition of Internal Ribosomal Entry Site–Directed Translation of HCV by Recombinant IFN-␣ Correlates With a Reduced La Protein Takeo Shimazaki, Masao Honda, Shuichi Kaneko, and Kenichi Kobayashi Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5ⴕ-nontranslated region (5ⴕNTR). We investigated the effect of interferon alfa (IFN-␣) on the IRES-directed translation of HCV, using two stably transformed cell lines, RCF-1 and RCF-26, of Huh7 cells derived from human hepatocellular carcinoma that express dicistronic reporter proteins, Renilla luciferase (RL) and firefly luciferase (FL), separated by HCV-IRES. After the administration of IFN-␣ or poly(I)-poly(C), HCV-IRES– directed translation was inhibited in a dose-dependent manner. The relative HCV-IRES activity (F/L) decreased to 60% at 5,000 IU/mL of IFN-␣ and 45% at 40 ␮g/mL of poly(I)-poly(C). Thus, IFN-␣ or poly(I)-poly(C) inhibited HCVIRES– directed translation more efficiently than a cellular cap– dependent translation. 2ⴕ,5ⴕ– oligoadenylate synthetase (2ⴕ,5ⴕAS) protein level in cells analyzed significantly increased after the administration of IFN-␣, but not upon poly(I)-poly(C). Overexpression of doublestranded RNA-activated protein kinase (PKR) gene did not mimic the selective inhibition of HCV-IRES– directed translation in the transformant cells, suggesting that neither the 2ⴕ,5ⴕAS nor the PKR system are involved in this selective inhibition. Interestingly, the expression of the autoantigen, La, which has been reported to enhance HCV-IRES– directed translation, was significantly reduced after the administration of IFN-␣ and poly(I)-poly(C) in a dose-dependent manner. Transient expression of La protein completely restored the selective inhibition of HCV-IRES– directed translation by IFN-␣ and poly(I)-poly(C). These findings suggested a new antiviral mechanism induced by IFN-␣ in that IFN-␣ or poly(I)-poly(C) selectively inhibited HCV-IRES– directed translation compared with the eukaryotic cap-dependent translation through the reduction of La protein. (HEPATOLOGY 2002;35:199-208.)

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epatitis C virus (HCV), a positive-strand, enveloped RNA virus, is classified within the genus Hepacivirus of the family Flaviviridae.1 HCV infects the human liver, leading to the development of chronic hepatitis, cirrhosis, and, in some instances, hepatocellular carcinoma.2-4 The treatment of chronic hepatitis C aims to eliminate viremia, and currently, the antiviral agent interferon alfa (IFN-␣) is commonly used worldwide.5-7 It is known that the antiviral action mechanisms of IFN-␣ chiefly include the transcriptional activation of 2⬘,5⬘– oligoadeny-

Abbreviations: HCV, hepatitis C virus; IFN-␣, interferon alfa; 2⬘,5⬘AS, 2⬘,5⬘– oligoadenylate synthetase; 2⬘,5⬘A, 2⬘,5⬘– oligoadenylate; PKR, RNA-activated protein kinase; IRES, internal ribosome entry site; 5⬘NTR, 5⬘nontranslated region; PTB, polypyrimidine tract binding protein; RL, Renilla luciferase; FL, firefly luciferase; CMV, cytomegalovirus; MTS, tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PBS, phosphatebuffered saline. From the First Department of Internal Medicine, Kanazawa University, Kanazawa, Japan. Received March 21, 2001; accepted October 4, 2001. Address reprint requests to: Masao Honda, M.D., Ph.D., First Department of Internal Medicine, Kanazawa University, Kanazawa, Japan. E-mail: [email protected]. kanazawa-u.ac.jp; fax: (81) 76-234-4250. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3501-0027$35.00/0 doi:10.1053/jhep.2002.30202

late synthetase (2⬘,5⬘AS) that catalyzes 2⬘,5⬘-oligoadenylate (2⬘,5⬘A) synthesis,8 thereby activating the endonuclease, RNase L, which degrades viral and cellular RNA (2⬘,5⬘A/RNase L system), and also activating the double-stranded RNA-activated protein kinase (PKR),9,10 which phosphorylates the initiation factor elF2 and leads to the inhibition of translation.11,12 However, because IFN is used in various intracellular signal transduction pathways, we speculate that mechanisms of antiviral action by IFN-␣ other than that involving the 2⬘,5⬘A/RNase L system and PKR may exist. It is known that the translation of the HCV-RNA genome is initiated by a highly structured RNA segment,13,14 the internal ribosome entry site (IRES) that occupies most of the 5⬘ nontranslated (5⬘NTR) RNA.15-17 The IRES activity is highly dependent on both the primary sequence of this segment and its ability to form complex secondary and tertiary RNA structures.18-22 A number of in vitro studies have suggested that several cellular proteins, including both conventional translation initiation factors such as eukaryotic initiation factor 3 (eIF3)23,24 and noncanonical translation initiation factors such as the nuclear La protein25-29or polypyrimidine tract binding protein (PTB),30-32 may stimulate HCV translation. We previously reported that HCV translation is regulated in a cell-cycle– dependent manner and that cellular proteins that vary in abundance during the cell cycle may be involved in this process,33 but to date, the mechanism by which HCV translation is regulated in vivo is not well understood. 199

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Fig. 1. Organization of the transcriptional unit of plasmid pRL-HL.33 pRL-HL contains a dicistronic CMV transcriptional cassette in which an upstream RL gene and a downstream FL gene are separated by the complete 5⬘NTR and 66-nucleotide core sequence of HCV (nucleotides 1-407, strain 1b) placed within the intercistronic space. CMV, cytomegalovirus promoter; T7, Bacteriophage T7 RNA polymerase promoter; BGH-pA, bovine growth hormone polyadenylation signal.

In human hepatocytes chronically infected with HCV, antiviral signaling transduced by various cytokines, including endogenous IFN, plays an important role in eliminating infected viruses. It is important to understand the molecular mechanism of these antiviral systems in terms of viral replication to develop new antiviral drugs and improve the efficacy of IFN therapy. In this study, we investigated the effect of IFN-␣ on HCV-IRES– directed translation, using two stably transformed cell lines, RCF-1 and RCF-26, derived from human hepatoma cell line Huh-7 cells, which constitutively express dicistronic viral RNA transcripts containing sequences encoding 2 reporter proteins (Renilla luciferase [RL] and firefly luciferase [FL]) separated by a functional HCV IRES.

Materials and Methods Plasmids. The PKR expression vector, pEGFP-PKR, which encodes fusion protein of EGFP and PKR, was kindly provided by Takizawa et al.40 The La expression vector was constructed by excising the Eco RI and Xho I fragments from pGEX-La (kindly provided by Nomoto et al.13) and cloning into the pCR 3.1 (Invitrogen, San Diego, CA) under the cytomegalovirus (CMV) promoter. The constructed plasmid was designated pCMV-La. Cell Lines and DNA Transfection. RCF-1 and RCF-26 cell lines, which constitutively express dicistronic RNA transcripts containing sequences encoding 2 reporter proteins, RL and FL, separated by a functional HCV IRES (Fig. 1),33 were cultured in Dulbecco’s modified Eagle medium (Gibco BRL, Gaithersburg, MD), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 400 ␮g/mL of geneticin (active compound). Cells were cultured in a 5% CO2 incubator at 37°C and were transfected with 0.5 to 2.0 ␮g of plasmid DNA using FuGENE 6 (Roche Molecular Biochemicals, Basel, Switzerland) according to the manufacturer’s instructions. After 24- to 48-hour transfection, cells were harvested and used for the reporter-gene assay or Western blotting. Reporter-Gene Assays. RCF-1 and RCF-26 cells were seeded on 35- or 60-mm dishes and grown to 70% to 80% confluence. Recombinant IFN-␣ (Scherring-Plough Corp., Osaka, Japan) was added to the culture at concentrations of 500, 1,000, and 5,000 international units (IU)/mL. Poly(I)-poly(C) (Amersham Pharmacia Biotech, Uppsala, Sweden) was added to the culture at concentrations of 5, 10, 20, 40, and 80 ␮g/mL, respectively. The cells were lysed in 1 mL of passive lysis buffer (25 mmol/L 1,2-diaminocyclohexane-N,N,N⬘N⬘-tetraacetic acid, 10% glycerol, and 1% Triton X-100) 12 to 16 hours after the addition of IFN-␣ or poly(I)-poly(C). Twenty-microliter aliquots of cell lysates were used to measure RL and FL activities in the DualLuciferase Reporter Assay System (Promega, Madison, WI).

In Vitro Translation. Plasmid pRL-HL DNA was linearized by digestion with Apa I (Fig. 1). Capped RNA was synthesized using T7 Cap-Scribe (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Transcripts were treated with RQ1 DNase (Promega) for 15 minutes at 37°C, extracted with phenolchloroform, and precipitated with ethanol/7.5 mol/L ammonium acetate. Before in vitro translation, the micrococcal nuclease–treated rabbit reticulocyte lysates (Promega) were incubated at 30°C for 30 minutes in the presence of various concentrations of poly(I)-poly(C) (0.1 ␮g/mL to 1,000 ␮g/mL) to activate endogenous PKR.34 Onehalf microgram of capped-synthetic template RNA was added to the poly(I)-poly(C)–treated reticulocyte lysates and incubated for an additional 30 minutes at 30°C. Two-microliter aliquots of lysates were used for the measurement of each luciferase activity. Evaluation of Viable Cells. Cells were seeded on 24-well plates at a density of 2 ⫻ 104 cells per well. The number of viable cells was determined using an MTS (tetrazolium compound (3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt) assay (Promega). Evaluation of the Reporter-Gene Transcripts. For the evaluation of reporter-gene transcripts, we used the cDNA microarray technique and the microarray slide including RL and FL genes.35 In the microarray analysis, mRNAs from reference and test samples were labeled with different fluorescent probes and competitively hybridized to the target cDNA spotted on the slide, which enabled relative changes in the expression of the test samples and reference samples to be compared. Two micrograms of mRNAs isolated from untreated cells and cells treated with IFN-␣ or poly(I)poly(C) was labeled with fluorescent probes, Cy3 (for untreated cells) and Cy5 (for treated cells), respectively. Each labeled mRNA was competitively hybridized to the target cDNA spotted on the slide. Fluorescence intensity in each spot was measured as described previously.35 The relative fluorescence intensities of Cy5 compared with those of Cy3 (Cy3:Cy5 ratio) of the Renilla and luciferase gene reflect changes in the expression of each reporter gene induced by the IFN-␣ or poly(I)-poly(C) treatment. Enzyme-Linked Immunosorbent Assay. The amount of albumin secreted from cells into the culture media during the 12- to 16-hour period after the addition of IFN-␣ or poly(I)-poly(C) was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Nephrat II, Exocell Inc., Philadelphia, PA). Indirect Immunofluoresence Staining. After 12- to 16-hour treatment of 5,000 IU/mL of IFN-␣ or 80 ␮g/mL of poly(I)poly(C), cells were fixed in cold acetone-methanol and subjected to indirect immunofluorescence staining. The fixed cells were incubated with rabbit anti-human albumin antibodies (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-PKR (Santa Cruz

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Biotechnology, Santa Cruz, CA), mouse anti-La (SW5; kindly provided by Dr. Prujin),36 or rabbit anti-PTB antibodies (kindly provided by Dr. Koide),37 diluted 1:250 at 37°C for 1 hour. After washing with phosphate-buffered saline (PBS), the same samples were reacted with fluorescein isothiocyanate–labeled anti-mouse IgG or anti-rabbit IgG at 37°C for 1 hour. Fluorescence was visualized under a confocal laser scanning microscope (Carl Zeiss Inc.). 2ⴕ-5ⴕAS Measurement. 2⬘-5⬘AS was radioimmunochemically assessed using an assay kit (Eiken Immunochemical Laboratory, Tokyo, Japan) in RCF-26 cells at 12 to 16 hours after the addition of IFN-␣ or poly(I)-poly(C). Western Blotting. To analyze the expression of La, albumin, and PKR, RCF-26 cells seeded in a 10-cm dish were grown to subconfluence and washed twice with PBS. Cells were lysed in a radioimmunoprecipitation assay buffer. Cell lysates were collected

Fig. 3. Inhibition of the cap-dependent and HCV-IRES– directed translation by poly(I)-poly(C). (A) Relative values for luciferase activities in cells 12 hours after poly(I)-poly(C) treatment at various concentrations compared with those obtained in the nontreated cells. (■), FL activity, (䊐), RL activity. (B) Changes in HCV-IRES– directed FL activities (F) relative to cap-dependent RL activities (R) in cells treated with poly(I)-poly(C) at various concentrations. F/R; relative ratio of FL activity to RL activity. F/R was defined as 1 in the nontreated condition. *P ⬍ .01.

Fig. 2. Inhibition of the cap-dependent and HCV-IRES– directed translation by IFN-␣. (A) Relative values for luciferase activities in cells 12 hours after IFN-␣ treatment at various concentrations compared with those obtained in the nontreated cells. (䊐), RL activity reflects cap-dependent translation; (■), FL activity reflects HCV-IRES– directed translation. Both luciferase activities were normalized to control (no treatment) values. (B) Changes in HCV-IRES– directed FL activities (F) relative to cap-dependent RL activities (R) in cells treated with IFN-␣ at various concentrations. F/R; relative ratio of FL activity to RL activity. F/R was defined as 1 in the control condition. *P ⬍ .01.

by pelleting cell debris, and the concentration of protein was quantified using a dye-binding assay (Bio-Rad, Hercules, CA). Eighty micrograms of cell lysate was electrophoresed in a sodium dodecyl sulfate–12.5% polyacrylamide gel and electrotransferred to a nitrocellulose membrane. After blocking with PBST (PBS with 0.3% Tween20) containing 5% skim milk for 1 hour, the membranes were reacted with rabbit anti-human albumin antibodies, mouse anti-human La protein antibodies (SW5), or mouse anti-PKR antibodies diluted 1:250. After washing with PBST, membranes were reacted with horseradish peroxidase– conjugated anti-mouse IgG or anti-rabbit IgG antibodies diluted 1:3,000. Membranes were washed again, and then visualized by an ECL kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Statistical Analysis. All data are expressed as means ⫾ SEM. The significance was tested by 1-way ANOVA using Bonferroni’s method.

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Fig. 5. The changes in the 2⬘,5⬘AS expression level in RCF-26 cells after being treated with IFN-␣ or poly(I)-poly(C) at various concentrations. Fig. 4. Evaluation of apoptotic cell death by the MTS assay. Twelve hours after RCF-26 cells were treated with IFN-␣ or poly(I)-poly(C), viable cells were assessed. The viable cell number in the treated culture was normalized to that in the nontreated control culture. NS, not significant.

Results Inhibition of HCV-IRES–Directed Translation by Recombinant IFN-␣. First, to investigate how IFN-␣ inhibits HCVIRES– directed translation, we added various concentrations of IFN-␣ to a culture of RCF-1 or RCF-26 cells and the translation levels were assessed. RCF-1 and RCF-26 constitutively express dicistronic mRNA consisting of the RL gene, the complete 5⬘NTR and the N terminal 66-nucleotide core sequence of HCV, and the FL gene (Fig. 1). RL in the first cistron is translated in a capdirected translation mechanism common to eukaryotes, and the second-cistron FL fused with the N terminal core sequence is translated in a HCV-IRES– dependent manner. As shown in Fig. 2A, the administration of 500, 1,000, or 5,000 IU/mL of IFN-␣ to the RCF-26 culture inhibited FL activity (reflecting HCV-IRES– directed translation) in a dose-dependent manner, resulting in a decrease to 35% of its activity before administration of 5,000 IU/mL IFN-␣. Likewise, RL activity (reflecting cap-dependent translation) was decreased in response to IFN-␣. Interestingly, HCV-IRES– directed translation was relatively more inhibited than cap-dependent translation. The administration of 5,000 IU/mL of IFN-␣ reduced the relative IRES-directed translation activity, and the ratio of IRES-directed translation (firefly activity) to cap-dependent translation (RL activity) was decreased to 60% of the preadministration level (Fig. 2B), demonstrating that IFN-␣ preferentially inhibited IRES-directed translation. Similar results were obtained with RCF-1 cells (data not shown).

Inhibition of HCV-IRES–Directed Translation by poly(I)poly(C). The replication of the Flavivirus genome involves the synthesis of negative-strand RNA complementary to positivestrand viral RNA in forming double-stranded RNA. The synthesis of negative-strand RNA has also been reported for HCV during replication.38,39 Double-stranded RNA is regarded as an activator of various bioactive substances involved in viral replication including double-stranded PKR.11,40 Thus, we investigated HCV-IRES– directed translation capacity in the stably transformed cells after the addition of poly(I)-poly(C) to mimic the physiology of HCVinfected cells with respect to the control of viral replication. As shown in Fig. 3, poly(I)-poly(C) suppressed FL activity, representing HCV-IRES– directed translation capacity in a dose-dependent manner, and to 45% of the preaddition level at 40 ␮g/mL of poly(I)-poly(C) (Fig. 3A). On the other hand, RL activity, representing cap-dependent translation capacity, remained at 84% of the preaddition level even after the addition of 80 ␮g/mL of poly(I)-poly(C). Relative values of IRES-directed translation com-

Table 1. Cy5:Cy3 Ratio After IFN and Poly(I)-Poly(C) Administration The Cy5/Cy3 ratio after IFN administration The Cy5/Cy3 ratio after poly(I)-poly(C) administration

RL

FL

1.15 ⫾ 0.065

1.08 ⫾ 0.10

1.13 ⫾ 0.04

1.07 ⫾ 0.016

Fig. 6. Expression of PKR in RCF-26 cells treated with IFN-␣ and poly(I)poly(C). Western blotting of PKR in RCF-26 cells 12 hours after being treated with IFN-␣ or poly(I)-poly(C).

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pared with those of cap-dependent translation decreased to 64% and 45% of the preaddition level, after the addition of 5 ␮g/mL and 40 ␮g/mL of poly(I)-poly(C), respectively (P ⬍ .001). Poly(I)poly(C) caused a more significant inhibition of HCV-IRES– directed translation than IFN-␣. These results indicate that IFN-␣ or poly(I)-poly(C) more preferentially inhibited HCV-IRES– directed translation than cap-dependent translation. Similar results were obtained with RCF-1 cells (data not shown). Evaluation of Apoptosis. To investigate whether the decreases in RL and FL activity reflected decreases in viable cell counts as a result of apoptosis, we examined changes in the number of viable cells using an MTS assay. As shown in Fig. 4, the MTS assay did not reveal any significant changes in the number of viable cells following IFN-␣ or poly(I)-poly(C) administration. These findings indicate that the decreases in RL and FL activity were caused by the inhibitory effects of IFN-␣ and poly(I)-poly(C) on translations of viral messages. Cellular Levels of mRNA Encoding the 2 Reporters, RL and FL. We investigated changes in the expressed levels of mRNA encoding in the reporter genes in cultured cells after administration of IFN-␣ or poly(I)-poly(C), using a cDNA microarray prepared in our laboratory.35 The microarray chip was spotted with serial dilutions of the RL and FL genes, and mRNAs were isolated from cells before and after IFN-␣ or poly(I)-poly(C) administration and conjugated with the fluorescent dyes, Cy3 or Cy5, respectively, followed by competitive hybridization with DNA spots. IFN-␣– or poly(I)-poly(C)–induced changes in the amount of RNA expression were represented by the relative fluorescence intensities of Cy5 compared with those of Cy3 (Cy5:Cy3 ratio). The Cy5:Cy3 ratio after IFN administration was 1.15 ⫾ 0.065 for RL and 1.08 ⫾ 0.10 for FL. The Cy5:Cy3 ratio after poly(I)-poly(C) administration was 1.13 ⫾ 0.04 for RL and 1.07 ⫾ 0.016 for FL. These results indicate that IFN or poly(I)-poly(C) administration did not affect the expression levels of the reporter genes (Table 1).

Fig. 7. Translational activity in RCF-26 cells transfected with PKR expressive vector. Mock and PKR represent cells transfected with control vector and vectors expressing PKR, respectively. (■), FL activity, (䊐), RL activity. Relative values for the translational activities in PKR-transfected cells were compared with those in mock-transfected cells for both luciferase activities.

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Fig. 8. Expressions of La protein (upper panel), PTB (second panel), albumin (third panel), and PKR (lower panel) in RCF-26 cells treated with IFN-␣ or poly(I)-poly(C) detected by indirect immunofluorescence staining.

2ⴕ,5ⴕAS Activity. To elucidate the mechanism of inhibition by IFN-␣ or poly(I)-poly(C) of HCV-IRES– directed translation, we analyzed bioactive substances potentially induced by these compounds. 2⬘-5⬘AS has been reported to be activated in the presence of double-stranded RNA,41 and to accelerate the synthesis of 2⬘5⬘A, activate RNase L, and degrade viral RNA and “cellular” RNA (not just viral RNA might be degraded) (2⬘,5⬘AS/RNase L system). 2⬘,5⬘AS in RCF-26 cells showed a significant dose-dependent increase after IFN-␣ administration, but did not show significant changes after poly(I)-poly(C) administration (Fig. 5). Therefore, we speculated that the specific inhibition of HCV-IRES– directed translation by both IFN-␣ and poly(I)-poly(C) observed in RCF-26 cells was not mediated by the 2⬘5⬘AS/RNase L system. Double-Stranded PKR. Next, we investigated the expression of PKR after IFN-␣ or poly(I)-poly(C) administration. Western blotting (Fig. 6) and indirect immunofluoresence staining showed a slight increase in the expression of PKR after IFN-␣ or poly(I)poly(C) administration. To confirm whether an excessive expression of PKR is involved in the specific inhibition of HCV-IRES– directed translation, a PKR expression vector40 was transiently transfected into RCF-26 cells, and these cells, which have an excessive expression of PKR, were used for the evaluation of translation capacity. We found that cap-dependent translation capacity markedly decreased, while HCV-IRES– directed translation was less effected (Fig. 7). These findings suggest that the specific inhibition of HCV-IRES– directed translation by IFN-␣ or poly(I)-

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Fig. 9. Western blotting of La protein and albumin in RCF-26 cells. Twelve hours after being treated with IFN-␣ or poly(I)-poly(C) at various concentrations, cells were harvested and the expressions of La protein and albumin were evaluated. The expression ratios of La protein to albumin assessed by NIH image are shown under the gel images.

poly(C) administration is not involved in the conventional antiviral 2⬘5⬘AS/RNase L or PKR system. Assessment of the Expression of La Protein That Stimulates HCV-IRES–Directed Translation. Currently, several host proteins such as La protein and PTB are considered to be involved in the control of efficacy of HCV-IRES– directed translation by binding to IRES.25,26,28,42 La protein is a transcription factor for RNA polymerase III and has been reported to stimulate the translation of viral genes by binding to the 5⬘NTR in several viruses such as polio,43 rubella,44 and HCV.25 We therefore investigated the possible change in the cellular levels of La protein after IFN-␣ or poly(I)-poly(C) administration. The fluorescent antibody method using monoclonal anti-La antibody (SW5)36 revealed that La proteins were mainly localized in the nucleoli. Strong staining was observed mainly in the nucleoli pre- and post-IFN treatment. (Fig. 8). IFN-␣ or poly(I)-poly(C) administration caused a marked decrease in the fluorescence levels of La protein (Fig. 8). In contrast, neither administration caused any detectable changes in the expression levels of albumin or PTB in cells (Fig. 8). In addition, ELISA did not reveal any significant changes in the amount of albumin secreted into culture fluid (data not shown). Western blot analysis showed a marked decrease in La protein levels after IFN-␣ or poly(I)-poly(C) administration in a dose-dependent manner, but not as much as La in the expression levels of albumin as a control (Fig. 9). The relative expression ratio of La to albumin decreased from 0.139 to 0.069 by 5,000IU/mL IFN-␣ and 0.139 to 0.021 by 80-␮g/mL poly(I)-poly(C) administration, respectively (Fig. 9). Because marked enhancement of HCV-IRES– directed translation by La protein has been reported,25,28 the present findings strongly suggest that the specific inhibition of HCV-IRES– directed translation after IFN-␣ or poly(I)-poly(C) administration is caused by the specific decrease in La protein. To further confirm these findings, transient transfection of the La expression vector, pCMV-La, to RCF-26 was performed. The expression level of La protein was monitored by Western blotting in every assay. The HCV-IRES– directed translation was significantly increased by overexpression of La protein almost in a dose-dependent manner (Fig. 10). The administration of 80 ␮g/␮L of poly(I)-poly(C) and 5,000 IU/mL of IFN-␣ decreased HCV-IRES– directed translation specifically (Fig. 11); however, overexpression of La protein (1 ␮g and 2 ␮g of transfected DNA) restored this inhibition completely (Fig. 11). The Western blotting results showed that the transfection of RCF-26 cells with 0.5 ␮g of pCMV-La did not rescue the reduction of La protein by IFN-␣ or poly(I)-poly(C), whereas 1.0 ␮g of pCMV-La restored the La protein level com-

pletely, which correlates well with the results of the reporter-gene assay (Fig. 12). These data strongly support the notion that La protein is directly involved in the specific inhibition of HCVIRES– directed translation by IFN-␣ or poly(I)-poly(C). Further supporting data were obtained from an in vitro translation system using a rabbit reticulocyte lysate in which La protein may not be present. Only a trace amount of La protein was detected in the rabbit reticulocyte lysates (Fig. 13A). Analysis of translation after adding poly(I)-poly(C) using capped RNA showed that poly(I)poly(C) administration reduced RL and FL activity to the same degree, without the specific inhibition of HCV-IRES– directed translation as observed in cultured cells (Fig. 13B, 13C).

Fig. 10. Overexpression of La protein in RCF-26 cells and its effect on cap-dependent and HCV-IRES– directed translation. (A) Various amounts of the La expression vector, pCMV-La, were transfected into RCF-26 cells, and the reporter-gene assay was performed 24 hours after transfection. The La protein level was assessed by Western blotting in every assay (Fig. 12). (䊐), RL activity reflects cap-dependent translation. (■), FL activity reflects HCVIRES– directed translation. Both luciferase activities were normalized to control (no treatment) values. La 0.5, 0.5 ␮g; La 1.0, 1.0 ␮g; La 2.0, 2.0 ␮g; La 4.0, 4.0 ␮g of pCMV-La, respectively. (B) Changes in HCV-IRES– directed FL activities (F) relative to cap-dependent RL activities (R) in cells transfected with pCMV-La. F/R; relative ratio of FL activity to RL activity. F/R was defined as 1 in the nontreated condition. *P ⬍ .01.

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Fig. 11. Reversal of the negative effects on HCV-IRES– directed translation during IFN-␣ and poly(I)-poly(C) treatment by a transient overexpression of La protein in RCF-26. RCF-26 cells were transfected with various amounts of pCMV-La (0.5 ␮g, 1.0 ␮g, 2.0 ␮g), 24 hours before administration of IFN-␣ or poly(I)-poly(C). After 24-hour transfection, RCF-26 cells were treated with IFN-␣ (5,000 IU/mL) or poly(I)-poly(C) (80 ␮g/mL) for 12 hours. (A) poly(I)-poly(C)&La protein. Relative values for luciferase activities in cells 12 hours after poly(I)-poly(C) treatment. (䊐), RL activity reflects cap-dependent translation. (■), FL activity reflects HCV-IRES– directed translation. Both luciferase activities were normalized to control (no treatment) values. (B) poly(I)-poly(C)&La protein. Changes in HCV-IRES– directed FL activities (F) relative to cap-dependent RL activities (R) in cells treated. F/R; relative ratio of FL activity to RL activity. F/R was defined as 1 in the control condition. *P ⬍ .01. (C) IFN and La protein. Relative values for luciferase activities in cells 12 hours after IFN-␣ treatment. Both luciferase activities were normalized to control (no treatment) values. (D) IFN and La protein. Changes in HCV-IRES– directed FL activities (F) relative to cap-dependent RL activities (R) in cells treated. F/R; relative ratio of FL activity to RL activity. F/R was defined as 1 in the control condition. IF5000, IFN-␣ 5,000 IU/mL; La 0.5, 0.5 ␮g; La 1.0, 1.0 ␮g; La 2.0, 2.0 ␮g of pCMV-La. P80, poly(I)-poly(C) 80 ␮g/mL.

Discussion HCV frequently causes persistent infection, resulting in the development of chronic hepatitis and cirrhosis45; hepatocellular carcinoma develops from these disease conditions. Although IFN-␣ is widely used as an effective agent in treating HCV,5-7 the underlying antiviral action mechanism has not been fully elucidated. Regarding the mechanism of the antiviral activity of IFN-␣ in general, it is known that IFN-␣ induces the transcriptional activation of the 2⬘,5⬘AS and subsequently activates the endonuclease RNase L (2⬘,5⬘A/RNase L system),8 resulting in the degradation of viral RNA. IFN also induces the expression of PKR that phosphorylates the initiation factor, eIF2, and results in the sup-

Fig. 12. Western blotting of La protein in RCF-26 cells transfected with pCMV-La. La1.0 and La2.0, 0.5, 1.0, and 2.0 ␮g of pCMV-La.

pression of translation.9,12 However, these cellular responses to IFN-␣ may not only lead to antiviral action, but may also lead to the inhibition of protein synthesis in host cells. Thus, the mechanisms by which IFN-␣ specifically inhibits viral replication are still not clear. In this study, we investigated the mechanism of IFN-␣ action on the translation activity of HCV using 2 Huh-7– derived cell lines, RCF-1 and RCF-26, in which the HCV-IRES– directed reporter gene was stably introduced, resulting in the constitutive expression of the reporter genes. More concretely, RCF-1 and RCF-26 constitutively express the RL gene translated by a capdependent mechanism, and a fusion gene of the N-terminal HCV core (66 nucleic acid residues) with FL translated by HCV-IRES

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Fig. 13. (A) Comparison of the amount of La protein in rabbit reticulocyte lysate and RCF-26 cells examined by Western blot analysis. La protein was below detectable levels in RRL (rabbit reticulocyte lysate). (B) Relative values for luciferase activities of each reporter protein translated in RRL programmed with poly(I)-poly(C) treatment compared with those obtained in nontreated RRL. (■), FL activity, (䊐), RL activity. (C) The HCV-IRES– directed activities (F) relative to the cap-dependent activities (R) in RRL programmed with poly(I)-poly(C) treatment at various concentrations. F/R ratio was defined as 1 in nontreated RRL. P ⬍ .01.

(nucleotides 1-407) in the dicistronic CMV transcriptional cassette (Fig. 1). The administration of IFN-␣ or poly(I)-poly(C) inhibited the HCV-IRES– directed translation in a dose-dependent manner. Interestingly, the HCV-IRES– directed translation was inhibited more significantly than the cap-dependent translation. The evaluation of apoptosis showed that IFN-␣ and poly(I)poly(C) did not cause any significant changes in the number of viable cells, and that they did not cause any significant changes in the level of RNA encoding reporters. It is difficult to completely rule out the possibility of a different stability of RNA encoding each reporter; however, cDNA microarray analysis using different reporter genes, RL and FL cDNA, showed no significant differences in the expression level of each reporter RNA (Table 1). To further investigate the mechanism of specific inhibition of this HCV-IRES– directed translation, we investigated the expression level of the 2⬘,5⬘A/RNase L system and PKR, representative responses to IFN-␣ action. The administration of IFN-␣ markedly increased 2⬘,5⬘AS in the RCF-1 and RCF-26 cells, whereas the administration of poly(I)-poly(C) caused only a slight increase in 2⬘,5⬘AS. Because the specific inhibitory effect of poly(I)-poly(C) on HCV-IRES– directed translation was more marked than that of IFN-␣, the specific inhibitory effect on HCV-IRES– directed translation may not involve the 2⬘,5⬘AS/RNase L system. The administration of IFN-␣ or poly(I)-poly(C) enhanced the expression of PKR (Fig. 6); however, under the excessive expression of PKR, the specific

inhibition of HCV-IRES– directed translation was not observed. Unexpectedly, cap-dependent translation was more hammered by the overexpression of PKR in this study (Fig. 7). The reason for this result is not currently clear; however, it is possible that the minimum requirement of the initiation factor (eIF-2␣) for efficient translation may be different between cap-dependent translation and HCVIRES– directed translation in cells. The other possibility is that the PKR activation is inhibited by the HCV RNA for some reason. It is reported that small virus RNA such as adenovirus VA1 or EBER encoded by Epstein-Barr virus binds to PKR and inhibits its activity.46,47 Adenovirus VA1 and EBER form secondary or tertiary structures such as HCV IRES forms these structures. Thus, we speculated that the specific inhibitory effect of IFN-␣ or poly(I)-poly(C) on HCV-IRES– directed translation is not the result of protein synthesis inhibition mediated by the 2⬘5⬘A/RNase L system or PKR, and we investigated the possible roles of host cell factors in the present study that may have influenced HCV-IRES activity. La protein and PTB have been reported as host factors influencing HCV-IRES activity,25-32 and in particular, La protein has been reported to be very important for the translation activity of HCV.25,28 La protein is considered to enhance translation by binding to the stem-loop IV of the HCV-IRES structure, resulting in the unwinding of the stem loop,25 and the stability of a stem loop involving the initiator, AUG, controls the efficiency of the

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internal initiation of translation on HCV RNA.21 Thus, we focused on La protein and assessed changes after the administration of IFN-␣ or poly(I)-poly(C). Interestingly, IFN-␣ and poly(I)poly(C) markedly reduced the expression levels of La protein (Figs. 8 and 9), whereas they did not cause any significant changes in the albumin or PTB levels. Studies with immunostaining, Western blotting, and ELISA gave similar results. Importantly, overexpression of La protein in RCF-26 enhanced HCV-IRES– directed translation in a dose-dependent manner and completely restored the specific inhibition of HCV-IRES– directed translation by IFN-␣ and poly(I)-poly(C) administration completely. These results strongly suggested that the specific inhibitory effect of IFN-␣ and poly(I)-poly(C) on HCV-IRES– directed translation was the result of a decrease in La protein after administration. In this study, poly(I)-poly(C) treatment inhibited HCVIRES– directed translation more specifically than IFN-␣ (Figs. 2 and 3). This suggests that poly(I)-poly(C) itself may have some specific action on HCV-IRES– directed translation. As poly(I)poly(C) binds to La protein,48,49 it is possible that poly(I)-poly(C) sequester La protein from binding to HCV IRES, resulting in a decrease of HCV-IRES– directed translation. In addition to these results, an analysis with an in vitro translation system using rabbit reticulocyte lysates, which contained little La protein, showed that the addition of poly(I)-poly(C) inhibited both cap-dependent and HCV-IRES– directed translation to the same extent, without exerting a specific inhibitory effect on HCVIRES– directed translation. The mechanism of La protein decreasing after IFN-␣ or poly(I)-poly(C) administration is not clear. However, La protein has been reported to undergo phosphorylation and proteolytic cleavage in the early stage of apoptosis.50 Such a modification may induce a change in the stability of La protein, leading to a decrease in the expression level. In establishing an antiviral strategy, it is obviously important to elucidate the mechanisms involved in the viral replication, and various host factors including La protein are considered to participate in the control of viral replication. In cells infected with poliovirus, La protein was reported to undergo cleavage by protease 3C and migrate from the nucleus to the cytoplasm,51 suggesting a mechanism functioning in favor of viral replication. In this study, we first revealed the possibility that La protein expression is directly related to IFN-␣ or poly(I)-poly(C) action on HCV-IRES– directed translation. It is possible that La protein may be a target of IFN, and this could be a new antiviral mechanism of IFN. The establishment of a definite relationship between the expression of La protein and the amount of HCV in the liver tissue of patients with chronic hepatitis C remains a subject for future studies. Acknowledgment: The authors thank Dr. Prujin for supplying mouse anti-La antibody (SW5). We also thank Dr. Koide for supplying rabbit anti-polypyrimidine tract-binding protein antibodies (PTB), Dr.Takizawa for supplying the PKR expression vector, and Prof. Nomoto for supplying the La expression vector.

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