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Journal of Virology, October 2008, p. 9639-9646, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00351-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The DNA Damage Sensors Ataxia-Telangiectasia Mutated Kinase and Checkpoint Kinase 2 Are Required for Hepatitis C Virus RNA Replication{triangledown}

Yasuo Ariumi,1 Misao Kuroki,1 Hiromichi Dansako,1 Ken-Ichi Abe,1 Masanori Ikeda,1 Takaji Wakita,2 and Nobuyuki Kato1*

Department of Molecular Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558, Japan,1 Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan2

Received 18 February 2008/ Accepted 18 July 2008


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ABSTRACT
 
Cellular responses to DNA damage are crucial for maintaining genome integrity, virus infection, and preventing the development of cancer. Hepatitis C virus (HCV) infection and the expression of the HCV nonstructural protein NS3 and core protein have been proposed as factors involved in the induction of double-stranded DNA breaks and enhancement of the mutation frequency of cellular genes. Since DNA damage sensors, such as the ataxia-telangiectasia mutated kinase (ATM), ATM- and Rad3-related kinase (ATR), poly(ADP-ribose) polymerase 1 (PARP-1), and checkpoint kinase 2 (Chk2), play central roles in the response to genotoxic stress, we hypothesized that these sensors might affect HCV replication. To test this hypothesis, we examined the level of HCV RNA in HuH-7-derived cells stably expressing short hairpin RNA targeted to ATM, ATR, PARP-1, or Chk2. Consequently, we found that replication of both genome-length HCV RNA (HCV-O, genotype 1b) and the subgenomic replicon RNA were notably suppressed in ATM- or Chk2-knockdown cells. In addition, the RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were suppressed in these knockdown cells after inoculation of the cell culture-generated HCV. Consistent with these observations, ATM kinase inhibitor could suppress the HCV RNA replication. Furthermore, we observed that HCV NS3-NS4A interacted with ATM and that HCV NS5B interacted with both ATM and Chk2. Taken together, these results suggest that the ATM signaling pathway is critical for HCV RNA replication and may represent a novel target for the clinical treatment of patients with chronic hepatitis C.


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INTRODUCTION
 
Hepatitis C virus (HCV) infection frequently causes chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma. HCV infection has now become a serious health problem, with at least 170 million people currently infected worldwide (28). HCV is an enveloped virus with a positive single-stranded 9.6-kb RNA genome, which encodes a large polyprotein precursor of approximately 3,000 amino acid residues. This polyprotein is cleaved by a combination of the host and viral proteases into at least 10 proteins in the following order: core, envelope 1 (E1), E2, p7, nonstructural 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (12, 13, 27).

Studies have shown that various viruses with distinct replication strategies—including the DNA viruses Epstein-Barr virus, herpes simplex virus 1, adenovirus, and simian virus 40 and the retrovirus human immunodeficiency virus type 1 (HIV-1)—can activate DNA damage response pathways and utilize these damage responses to facilitate their own viral reproduction and promote the survival of infected cells (2, 16, 17). In the case of HCV, it has been proposed that HCV infection causes double-stranded DNA (dsDNA) breaks and enhances the mutation frequency of cellular genes and that these effects are mediated by nitric oxide (18, 19). In addition, the HCV core, E1, and NS3 proteins have been suggested to be potent reactive oxygen species inducers, leading to DNA damage (19). Furthermore, we previously demonstrated that HCV NS5B-expressing PH5CH8 immortalized human hepatocyte cells were susceptible to DNA damage in the form of dsDNA breaks (23). Thus, HCV seems to be associated with the dsDNA damage response pathways.

Since the DNA damage sensors, such as ataxia-telangiectasia mutated kinase (ATM), ATM- and Rad3-related kinase (ATR), poly(ADP-ribose) polymerase 1 (PARP-1), and checkpoint kinase 2 (Chk2; a direct downstream target of ATM), play central roles in response to genotoxic stress (10), we hypothesized that these sensors might affect HCV replication.

To investigate the possible involvement of these cellular factors in HCV replication, we examined the level of HCV RNA in cells rendered defective for DNA damage sensors by RNA interference or by pharmacological inhibition.


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MATERIALS AND METHODS
 
Cell culture. 293FT cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS). The HuH-7-derived O cells harboring a replicative genome-length HCV RNA and the HuH-7-derived sO cells harboring the subgenomic replicon RNA of HCV-O were cultured in DMEM with 10% FBS and G418 (300 µg/ml geneticin; Invitrogen) as described previously (11, 14). Oc and sOc cells, which were created by eliminating HCV RNA from O cells and sO cells by interferon (IFN) treatment (11, 14), respectively, were also cultured in DMEM with 10% FBS.

RNA interference. Oligonucleotides with the following sense and antisense sequences were used for the cloning of short hairpin RNA (shRNA)-encoding sequences targeted to Chk2 in lentiviral vector: 5'-GATCCCCGGGGGAGAGCTGTTTGACATTCAAGAGATGTCAAACAGCTCTCCCCCTTTTTGGAAA-3' (sense) and 5'-AGCTTTTCCAAAAAGGGGGAGAGCTGTTTGACATCTCTTGAATGTCAAACAGCTCTCCCCCGGG-3' (antisense). The oligonucleotides above were annealed and subcloned into the BglII-HindIII site, downstream from an RNA polymerase III promoter of pSUPER (5), generating pSUPER-Chk2i. To construct pLV-Chk2i, the BamHI-SalI fragments of the pSUPER-Chk2i were subcloned into the BamHI-SalI site of pRDI292, an HIV-1-derived self-inactivating lentiviral vector containing a puromycin resistance marker allowing for the selection of transduced cells (4). pLV-ATMi, pLV-ATRi, and pLV-PARP-1i were constructed as described previously (1).

Lentiviral vector production. The vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1-based vector system has been described previously (24). The lentiviral vector particles were produced by transient transfection of the second-generation packaging construct pCMV-{Delta}R8.91 (30) and the VSV-G envelope plasmid pMDG2 as well as the lentiviral vector into 293FT cells with FuGene6 (Roche Diagnostics, Mannheim, Germany).

Quantitative reverse transcription-PCR analysis. Quantitative reverse transcription-PCR analysis for HCV RNA was performed by real-time LightCycler PCR as described previously (11).

Western blot analysis. Cells were lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 4 mM EDTA, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Supernatants from these lysates were subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting analysis using anti-ATM (2C1; GTX70103 [GeneTex, San Antonio, TX]), anti-ATR (GTX70133; GeneTex), anti-Chk2 (NT; ProSci, Poway, CA), anti-Chk2 (DCS-273; Medical and Biological Laboratories, Nagoya, Japan), anti-phospho-Chk2 (Thr68) (Cell Signaling, Danvers, MA), anti-PARP-1 (C-2-10; Calbiochem, Merck Biosciences, Darmstadt, Germany), anti-hemagglutinin (HA) (HA-7; Sigma, St. Louis, MO), anti-core protein (CP-9 and CP-11; Institute of Immunology, Tokyo, Japan), anti-NS3 and anti-NS5B (no. 14; a generous gift from M. Kohara, the Tokyo Metropolitan Institute of Medical Science, Japan), anti-NS5A (no. 8926; a generous gift from A. Takamizawa, The Research Foundation for Microbial Diseases of Osaka University, Japan), and anti-β-actin (Sigma) Antibodies.

Immunofluorescence and confocal microscopic analysis. Cells were fixed in 3.5% formaldehyde in phosphate-buffered saline (PBS) and permeabilized in 0.1% NP-40 in PBS at room temperature. Cells were incubated with anti-ATM antibody (5C2; GTX70107 [GeneTex] or PM026 [MBL]), anti-HA antibody (3F10), anti-NS5B antibody and/or anti-NS3 antibody at a 1:300 dilution in PBS containing 3% bovine serum albumin at 37°C for 30 min. Cells were then stained with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA) or anti-Cy3-conjugated anti-mouse antibody (Jackson ImmunoResearch) at a 1:300 dilution in PBS containing bovine serum albumin at 37°C for 30 min. Following extensive washing in PBS, cells were mounted on slides using a mounting medium of 90% glycerin-10% PBS with 0.01% p-phenylenediamine added to reduce fading. Samples were viewed under a confocal laser-scanning microscope (LSM510; Zeiss, Jena, Germany).

Immunoprecipitation. Cells were lysed in buffer containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, 10 mM NaF, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Lysates were precleared with 30 µl of protein G-Sepharose (GE Healthcare Biosciences, Uppsala, Sweden). Precleared supernatants were incubated with 5 µg of anti-HA antibody (3F10; Roche), 10 µl of anti-NS5B antibody, 5 µg of anti-Chk2 antibody (DCS-273; MBL), 5 µg of anti-FLAG antibody (M2; Sigma), or 5 µg of anti-ATM antibody (2C1) (GTX70103; GeneTex) at 4°C for 1 h. Following absorption of the precipitates on 30 µl of protein G-Sepharose resin for 1 h, the resin was washed four times with 700 µl of lysis buffer. Proteins were eluted by boiling the resin for 5 min in 2x Laemmli sample buffer. The proteins were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting analysis using anti-ATM, anti-Chk2, anti-HCV core protein (CP-9 and CP-11 mixture), anti-NS5A, anti-NS5B, anti-HA (HA-7; Sigma), or anti-NS3 antibody.


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RESULTS
 
ATM and Chk2 are required for HCV RNA replication. To determine the potential role of DNA damage sensors in HCV replication, we first used lentiviral vector-mediated RNA interference to stably knockdown ATM, ATR, PARP-1 (1), or Chk2 in the following human hepatoma HuH-7-derived cell lines: O cells harboring a replicative genome-length HCV RNA (HCV-O, genotype 1b) (11), Oc cells derived from O cells (created by eliminating genome-length HCV RNA from O cells by IFN treatment) (11), sO cells harboring the subgenomic replicon of HCV-O (14), or RSc cells that cell culture-generated HCV (HCVcc) (JFH1, genotype 2a) (29) could infect and effectively replicate (3). To express shRNAs targeted to ATM, ATR, PARP-1 (1), or Chk2, we used a VSV-G-pseudotyped HIV-1-based vector system (24). We used puromycin-resistant pooled cells 10 days after the lentiviral transduction in all experiments. Western blot analysis of the lysates demonstrated very effective knockdown of ATM, ATR, Chk2, and PARP-1 in Oc cells (Fig. 1A). The effective knockdown of ATM, ATR, Chk2, or PARP-1 in O cells or sO cells was also confirmed by Western blot analysis (data not shown). In this context, the efficiency of colony formation (ECF) in ATM- or Chk2-, but not ATR- or PARP-1-, knockdown Oc cells transfected with the genome-length HCV-O RNA with an adapted mutation at amino acid position 1609 in the NS3 helicase region (ON/C-5B K1609E RNA) (11) was notably reduced compared with the control cells (Fig. 1B) even though Chk2-knockdown cells had a slightly faster growth rate than the control cells (Fig. 1C), suggesting that both ATM and Chk2 are crucial for HCV RNA replication. To further confirm this observation, we quantitatively examined the level of HCV RNA in the O cell- or sO cell-derived knockdown cells. Consequently, we found that replication of both genome-length HCV RNA (HCV-O) and its subgenomic replicon RNA (sO) were notably suppressed in ATM- or Chk2-knockdown cells but not in ATR- or PARP-1-knockdown cells (Fig. 1D and E). Consistent with this finding, the expression levels of core and NS5B proteins were also significantly decreased in the cell lysates of ATM- or Chk2-knockdown O cells (Fig. 1F). We next examined the replication level of HCV-JFH1 in ATM-, ATR-, Chk2-, or PARP-1-knockdown RSc cells (Fig. 2A). The results revealed that RNA replication of HCV-JFH1 and release of core protein into the culture supernatants were suppressed in only ATM- or Chk2-knockdown RSc cells after inoculation with HCVcc (Fig. 2B and C). Interestingly, the release of core protein into the culture supernatant was also significantly suppressed in ATR-knockdown RSc cells, while HCV RNA replication was slightly suppressed in these cells (Fig. 2B and C), suggesting that ATR participates in the production of HCV virion.


Figure 1
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  		FIG. 1. The ATM signaling pathway is required for HCV RNA replication. (A) Inhibition of ATM, ATR, Chk2, or PARP-1 expression by shRNA-producing lentiviral vectors. The results of the Western blot analysis of cellular lysates with anti-ATM, anti-ATR, anti-Chk2, anti-PARP-1, or anti-β-actin antibody in Oc cells expressing shRNA targeted to ATM (ATMi), ATR (ATRi), Chk2 (Chk2i), or PARP-1 (PARP-1i) as well as in Oc cells transduced with a control lentiviral vector (Con) are shown. (B) ECF in ATM-, ATR-, Chk2-, or PARP-1-knockdown cells. In vitro transcribed ON/C-5B K1609E RNA (2 µg) was transfected into the ATM-, ATR-, Chk2, or PARP-1-knockdown Oc cells or the Oc cells transduced with a control lentiviral vector (Con). G418-resistant colonies were stained with Coomassie brilliant blue at 3 weeks after electroporation of RNA. Experiments were done in duplicate, and a representative result is shown. (C) The cell growth curve of ATM (ATMi), ATR (ATRi), Chk2 (Chk2i), or PARP-1 (PARP-1i)-knockdown Oc cells or the Oc cells transduced with a control lentiviral vector (Con). Results from three independent experiments are shown. (D) The level of genome-length HCV-O RNA was monitored by real-time LightCycler PCR (Roche). Experiments were done in triplicate, and columns represent the mean percentage of HCV RNA. (E) The level of subgenomic replicon (sO cells) RNA was monitored by real-time LightCycler PCR. Results from three independent experiments are shown as described in panel D. (F) The HCV core or NS5B protein expression level in ATM-, ATR-, Chk2-, or PARP-1-knockdown cells. The results of Western blot analysis of cellular lysates with anti-HCV core protein, anti-HCV NS5B, or anti-β-actin antibody in O cells expressing shRNA targeted to ATM (ATMi), ATR (ATRi), Chk2 (Chk2i), or PARP-1 (PARP-1i) as well as in O cells transduced with a control lentiviral vector (Con) are shown.


Figure 2
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  		FIG. 2. ATM affects HCV infection. (A) Inhibition of ATM, ATR, Chk2, or PARP-1 expression by shRNA-producing lentiviral vectors. The results of Western blot analysis of cellular lysates with anti-ATM, anti-ATR, anti-PARP-1, anti-Chk2, or anti-β-actin antibody in RSc cured cells expressing shRNA targeted to ATM (ATMi), ATR (ATRi), Chk2 (Chk2i), or PARP-1 (PARP-1i) as well as in RSc cells transduced with a control lentiviral vector (Con) are shown. (B) The level of genome-length HCV (JFH1) RNA was monitored by real-time LightCycler PCR after inoculation of the HCVcc. Results from three independent experiments are shown as described in the legend of Fig. 1D. (C) The levels of the core protein in the culture supernatants were determined by enzyme-linked immunosorbent assay (Mitsubishi Kagaku Bio-Clinical Laboratories). Experiments were done in triplicate, and columns represent the mean core protein levels.

In contrast, highly efficient knockdown of PARP-1 had no observable effects on the ECF (Fig. 1B), HCV RNA replication (Fig. 1D and E and 2B), or core protein expression in the cell lysate or in the supernatant (Fig. 1F and 2C), suggesting that our finding was not due to a nonspecific event. Thus, we have demonstrated for the first time that DNA damage sensors, ATM and Chk2, are required for HCV RNA replication.

ATM kinase activity in HCV RNA-replicating cells. Although it has been proposed that HCV causes dsDNA breaks (18, 19), little is known about whether HCV activates or inhibits the ATM-dependent damage response pathway. In this regard, it is worth noting that we observed weak but significant Chk2 phosphorylation at threonine 68, the specific marker for ATM activation (20, 21), in the HCV RNA-replicating cells (O and sO cells) but not in the HCV-negative Oc and sOc cells (created by eliminating replicon RNA from sO cells by IFN treatment) (Fig. 3A), suggesting that the persistent HCV RNA replication stimulated the ATM-dependent DNA damage response. Furthermore, a 2-h treatment with 100 nM adriamycin, a dsDNA break inducer, markedly induced Chk2 phosphorylation in Oc, O, and sO cells (Fig. 3A). Importantly, Chk2 phosphorylation was not inhibited even in the HCV RNA-replicating cells (O and sO cells) (Fig. 3A), suggesting that the persistent HCV RNA replication and the HCV proteins are not able to suppress the ATM-dependent DNA damage response. To examine whether such a DNA damage response activates HCV RNA replication, we quantified the level of HCV RNA in the O cells treated with 100 nM adriamycin for 24 h. The results show that HCV RNA replication was increased (approximately 1.3-fold) after treatment with adriamycin (Fig. 3B), suggesting that the DNA damage response activates HCV RNA replication.


Figure 3
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  		FIG. 3. ATM-dependent DNA damage response in HCV RNA-replicating cells. (A) Stimulation of Chk2 phosphorylation in the HCV RNA-replicating cells. The Oc, O, or sO cells were treated with 100 nM adriamycin (Sigma) for 2 h. The results of Western blot analysis of cellular lysates with anti-phospho-Chk2 (Thr68) (P-Chk2 T68), anti-Chk2, or anti-core protein antibody are shown. (B) Effect of adriamycin on HCV RNA replication. The O cells were treated with 100 nM adriamycin for 24 h. The level of genome-length HCV-O RNA was monitored by real-time LightCycler PCR. Results from three independent experiments are shown as described in the legend of Fig. 1D. DMSO, dimethyl sulfoxide. (C) Effect of ATM kinase inhibitor on Chk2 phosphorylation. The sO or O cells were pretreated with 10 µM ATM kinase inhibitor (KU-55933) (Calbiochem) for 2 h, followed by treatment with 100 nM adriamycin for 2 h. The results of Western blot analysis of cellular lysates with anti-phospho-Chk2 (Thr68) or anti-Chk2 antibody are shown.

Suppression of HCV RNA replication by a small-molecule inhibitor of the ATM kinase. We next examined the effect of a specific small-molecule inhibitor of the ATM kinase (2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one [KU-55933]) (16) on HCV RNA replication. As expected, the ATM kinase inhibitor effectively inhibited Chk2 phosphorylation after adriamycin treatment in both sO and O cells (Fig. 3C). In this context, the ATM kinase inhibitor could efficiently suppress genome-length HCV RNA replication with an in vitro 50% effective concentration (EC50) of approximately 2 µM at 72 h after treatment with adriamycin (Fig. 4A). Although this ATM kinase inhibitor did not affect cell viability at 24 h after the treatment, there was a slight decrease in the cell viability at 72 h after treatment (Fig. 4B). Thus, this or other ATM kinase inhibitors may be useful for the clinical treatment of patients with chronic hepatitis C.


Figure 4
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  		FIG. 4. Suppression of HCV RNA replication by ATM kinase inhibitor. (A) The level of genome-length HCV-O RNA was monitored by real-time LightCycler PCR after treatment with the indicated concentration of ATM kinase inhibitor for 72 h. Results from three independent experiments are shown as described in the legend of Fig. 1D. (B) Cell viabilities after treatment with the indicated concentration of ATM kinase inhibitor for 24 h or 72 h are shown.

Interaction of HCV NS3-4A with ATM. Since HCV NS3 has been proposed to be a viral factor involved in the induction of dsDNA breaks (18, 19), we first examined the subcellular localization of NS3-NS4A ([NS3-4A] 1B-1 or HCV-O strain) and ATM by confocal laser scanning microscopy. In most of the observed cells, ATM partially colocalized with NS3-4A in the perinuclear region and in dispersed points throughout the cytoplasm (Fig. 5A). In particular, we observed prominent colocalization of ATM with NS3-4A in some cells (Fig. 5A). Next, using anti-FLAG and anti-ATM antibodies, we immunoprecipitated lysates from 293FT cells in which FLAG-tagged ATM and either NS3-4A (HCV-O) or NS3 (HCV-O) were overexpressed and then performed immunoblotting analysis using either anti-ATM or anti-NS3 antibody to determine whether ATM binds to NS3-4A or NS3. The results revealed that ATM preferentially bound to NS3-4A over NS3 alone (Fig. 6A). Similarly, we found that ATM bound to NS3-4A using the O cell lysates (Fig. 6B), while HA-tagged Chk2 did not bind to NS3-4A in immunoprecipitation analysis using lysates from 293FT cells in which NS3-4A and HA-tagged Chk2 were overexpressed (Fig. 6C). Although NS3-4A has protease activity, ATM was not cleaved by the NS3-4A protease (Fig. 6D). Taking these results together, we conclude that ATM is able to interact with NS3-4A.


Figure 5
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  		FIG. 5. Subcellular localization of ATM and Chk2 in HCV NS3-4A- or NS5B-expressing cells. (A) ATM partially colocalized with HCV NS3-4A. 293FT cells cotransfected with 300 ng of pCX4bsr/NS3-4A (1B-1) (8) or pCX4bsr/NS3-4A (O) (8) and 300 ng of pcDNA3-FLAG-ATMwt (6) were examined by confocal laser scanning microscopy. Cells were stained with anti-NS3 and anti-ATM (5C2) antibodies and then visualized with FITC (NS3) or Cy3 (ATM). (B) ATM partially colocalized with HCV NS5B. 293FT cells cotransfected with 300 ng of pCX4bsr/NS5B (1B-1) (23) and 300 ng of pcDNA3-FLAG-ATMwt (6). Cells were stained with anti-NS5B (no. 14) and anti-ATM (PM026) antibodies and then visualized with FITC (ATM) or Cy3 (NS5B). (C) Chk2 partially colocalized with HCV NS5B. 293FT cells cotransfected with 300 ng of pCX4bsr/NS5B (1B-1) (23) and 300 ng of pcDNA3-HA-Chk2wt (20, 21). Cells were stained with anti-NS5B and anti-HA (3F10) antibodies and then visualized with FITC (HA-Chk2) or Cy3 (NS5B). Images were visualized using confocal laser scanning microscopy (LSM510; Carl Zeiss). The right panels exhibit two-color overlay images (Merged). Colocalization is shown in yellow.


Figure 6
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  		FIG. 6. Interaction of HCV NS3-4A and NS5B with the ATM signaling pathway. (A and B) ATM bound to HCV NS3-4A. (A) 293FT cells were transfected with 4 µg of pCX4bsr/NS3-4A (O), 4 µg of pCX4bsr/NS3 (O), or 4 µg of pcDNA3-FLAG-ATMwt. The cell lysates of expressed FLAG-ATM were mixed with lysates expressing either NS3-4A or NS3. The cell lysates were immunoprecipitated with both anti-FLAG (M2) and anti-ATM (2C1) antibodies, followed by immunoblotting analysis using either anti-ATM (2C1) or anti-HCV NS3 antibody. The results of Western blot analysis of 1/10 of the cellular lysates with anti-ATM or anti-NS3 antibody are also shown. (B) 293FT cells were cotransfected with 4 µg of pcDNA3-FLAG-ATMwt and/or 4 µg of pCX4bsr/NS3-4A (O). The cell lysates of expressed FLAG-ATM alone were mixed with the O cell lysates. Immunoprecipitation and Western blot analysis were performed as described in panel A. (C) Chk2 did not bind to NS3-4A. 293FT cells were cotransfected with 4 µg of pcDNA3-HA-Chk2wt and/or 4 µg of pCX4bsr/NS3-4A (O). The cell lysates of expressed HA-Chk2 alone were mixed with the O cell lysates. The cell lysates were immunoprecipitated with anti-HA antibody (3F10), followed by Western blot analysis using either anti-HA (HA-7) or anti-HCV NS3 antibody. The results of Western blot analysis of 1/10 of the cellular lysates with anti-HA or anti-NS3 antibody are also shown. (D) ATM was not cleaved by HCV NS3-4A protease. 293FT cells were cotransfected with 4 µg of pCX4bsr/NS3-4A (O) and/or 4 µg of pcDNA3-FLAG-ATMwt. The results of Western blot analysis of cellular lysates with anti-ATM or anti-NS3 antibody are shown. (E to G) ATM bound to HCV NS5B. (E) The lysates of O or Oc cells were immunoprecipitated with anti-ATM antibody (2C1), followed by immunoblotting analysis using either anti-ATM or anti-HCV NS5B antibody (no. 14). The results of Western blot analysis of 1/10 of the cellular lysates with anti-NS5B antibody are also shown. (F) 293FT cells were cotransfected with 4 µg of pCX4bsr/NS5B (1B-1) and 4 µg of pcDNA3-FLAG-ATMwt. The cell lysates were immunoprecipitated with both anti-FLAG and anti-ATM antibodies, followed by immunoblotting analysis using either anti-ATM or anti-HCV NS5B antibody. (G) Western Blot analysis was performed with anti-NS5B antibody, reusing the same blotted membrane that was used for panel B. (H to J) Chk2 bound to HCV NS5B. (H) 293FT cells were cotransfected with 4 µg of pcDNA3-HA-Chk2wt. The cell lysates of expressed HA-Chk2 were mixed with the O cell lysates and were immunoprecipitated with anti-HA antibody (3F10), followed by immunoblotting analysis using anti-HCV NS5B, anti-HCV NS5A (no. 8926), anti-HCV core protein (CP-9 and CP-11 mixture), or anti-HA (HA-7) antibody. The results of Western blot analysis of 1/10 of the cellular lysates with the same antibodies are also shown. (I) The lysates of O cells were immunoprecipitated with anti-NS5B or anti-Chk2 antibody (DCS-273), followed by immunoblotting analysis using anti-HCV NS5B antibody. The result of Western blot analysis of 1/10 of the cellular lysates with anti-NS5B antibody is also shown. (J) 293FT cells were cotransfected with 4 µg of pCX4bsr/NS5B (1B-1) and 4 µg of pcDNA3-HA-Chk2wt. The cell lysates were immunoprecipitated with anti-HA antibody (3F10), followed by immunoblotting analysis using either anti-HA (HA-7) or anti-HCV NS5B antibody. IP, immunoprecipitation; WB, Western blotting; IgG, immunoglobulin G.

Interaction of HCV NS5B with ATM and Chk2. We next examined the subcellular localization of ATM and/or Chk2 in HCV NS5B-expressing cells by confocal laser scanning microscopy since we previously demonstrated that HCV NS5B-expressing PH5CH8 immortalized human hepatocyte cells were susceptible to DNA damage in the form of dsDNA breaks (23). ATM partially colocalized with NS5B in dispersed points throughout the cytoplasm (Fig. 5B), similar to the subcellular localization of HCV NS3-4A and ATM. Furthermore, Chk2 also partially colocalized with NS5B in the perinuclear region and in dispersed points in the nucleus (Fig. 5C). To determine whether endogenous ATM binds to NS5B, lysates from Oc or O cells were immunoprecipitated with anti-ATM antibody, and then immunoblotting analysis using either anti-ATM or anti-NS5B antibody was performed. The results revealed that endogenous ATM bound to endogenous NS5B (Fig. 6E). Furthermore, we confirmed that ATM bound to NS5B in immunoprecipitation analysis using lysates from 293FT cells, in which NS5B (1B-1 strain) and FLAG-tagged ATM were overexpressed (Fig. 6F). Similarly, we confirmed that FLAG-tagged ATM bound to NS5B derived from O cell lysates in immunoprecipitation analysis using lysates from 293FT cells in which FLAG-tagged ATM was overexpressed (Fig. 6G). Finally, to determine which HCV protein binds to Chk2, the 293FT cell lysates of overexpressed HA-Chk2 were mixed with the O cell lysates and were immunoprecipitated with anti-HA antibody, followed by Western blot analysis using anti-HCV NS5B, anti-HCV NS5A, anti-HCV core protein, or anti-HA antibody. Consistent with the immunofluorescence result that Chk2 partially colocalized with NS5B (Fig. 5C), we observed that HA-tagged Chk2 bound to NS5B (Fig. 6H). Importantly, we found that endogenous Chk2 bound to endogenous NS5B derived from O cells (Fig. 6I). In addition, HA-tagged Chk2 bound to NS5B in immunoprecipitation analysis using lysates from 293FT cells in which NS5B (1B-1 strain) and HA-tagged Chk2 were overexpressed (Fig. 6J). Thus, Chk2 also interacted with NS5B as well as ATM. Taking these results together, we conclude that HCV targets ATM and Chk2 DNA damage sensors and that the ATM signaling pathway is required for HCV RNA replication.


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DISCUSSION
 
ATM has been implicated as a target of most DNA viruses, harboring their genomes in the form of dsDNA which can activate or inhibit the ATM signaling pathway (17). In this study, we have demonstrated for the first time that the ATM signaling pathway is required for HCV RNA replication even though HCV does not have a dsDNA genome, unlike DNA viruses. In this regard, Machida et al. previously proposed that HCV infection and the expression of HCV NS3 and core protein induced dsDNA breaks (18, 19). Furthermore, NS3 has DNA helicase activity by which it unwinds dsDNA, suggesting that NS3 affects host dsDNA (22, 25). Thus, HCV infection might trigger the activation of ATM without a dsDNA genome. In fact, we observed weak but significant phosphorylation of Chk2 at threonine 68, the specific marker for ATM activation, in the HCV RNA-replicating cells (O and sO cells) but not in the HCV-negative Oc and sOc cells (Fig. 3A), suggesting that the ATM-dependent DNA damage response is constantly stimulated in persistent HCV RNA-replicating cells. Furthermore, we demonstrated that ATM preferentially bound to NS3-4A over NS3 alone (Fig. 5B) and that ATM partially colocalized with NS3-4A in the perinuclear region, where HCV is known to form a replication complex and replicate itself, and in dispersed points throughout the cytoplasm (Fig. 5A), indicating the interaction of ATM with NS3-4A. Interestingly, Lai et al. very recently reported that NS3-4A impaired DNA repair and enhanced sensitivity to ionizing radiation through interaction with ATM (15). However, we observed an equivalent level of Chk2 phosphorylation at threonine 68, a direct downstream target of ATM (20, 21), in both HCV RNA-replicating cells (O cells) and HCV-negative cells (Oc cells) after treatment with adriamycin (Fig. 3A), suggesting that Chk2 phosphorylation by ATM is not impaired by HCV RNA replication. In this regard, Gaspar and Shenk also showed that human cytomegalovirus could inhibit a DNA damage response by mislocalizing ATM and phosphorylated Chk2 at threonine 68 to a cytoplasmic virus assembly zone, indicating that human cytomegalovirus blocked at the level of Chk2 (9). On the other hand, dsDNA triggers IFN immune defenses through retinoic acid-induced gene I, the mitochondrial antiviral signaling protein, or the DNA-dependent activator of IFN-regulatory factor (7, 26); and NS3-4A protease, which is known to cleave the mitochondrial antiviral signaling protein, can block it (26), suggesting that interaction of NS3-4A with ATM is partially involved in such a common antiviral signaling pathway. On the other hand, we previously demonstrated that HCV NS5B-expressing PH5CH8 immortalized human hepatocyte cells were susceptible to DNA damage in the form of dsDNA breaks (23). In this regard, we have found that HCV NS5B could bind to both ATM and Chk2 (Fig. 5B and C and 6E to J). Together, these results indicate that HCV might hijack ATM and Chk2 and utilize ATM and Chk2 for HCV RNA replication, thereby resulting in impairment of DNA repair, enhancement of mutation frequency, and development of hepatocellular carcinoma.

Finally, consistent with our finding that ATM was required for HCV RNA replication, an ATM kinase inhibitor efficiently suppressed genome-length HCV RNA replication at an EC50 of approximately 2 µM at 72 h after the treatment (Fig. 4A). Similarly, Lau et al. reported that the same ATM kinase inhibitor could suppress HIV-1 replication at an EC50 of approximately 2.3 µM (16). Importantly, the EC50 for HIV-1 replication is similar to that for HCV replication. Thus, this or other ATM kinase inhibitors may represent a novel approach for the clinical treatment of patients with chronic hepatitis C as well as AIDS patients.


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ACKNOWLEDGMENTS
 
We thank D. Trono, R. Agami, R. Iggo, M. Kastan, S. J. Elledge, M. Kohara, A. Takamizawa, and M. Hijikata for the VSV-G-pseudotyped HIV-1-based vector system (pCMVR8.91 and pMDG2) and for pSUPER, pRDI292, pcDNA3-FLAG-ATM, and pcDNA3-HA-Chk2, and for anti-NS3 antibody, anti-NS5B antibody, anti-NS5A antibody, and 293FT cells. We also thank A. Morishita and T. Nakamura for their technical assistance.

This work was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT); by a Grant-in-Aid for Research on Hepatitis from the Ministry of Health, Labor, and Welfare of Japan; by the Ichiro Kanehara Foundation; and by a Research Fellowship from the Japan Society for the Promotion of Science.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558, Japan. Phone: 81 86 235 7385. Fax: 81 86 235 7392. E-mail: nkato{at}md.okayama-u.ac.jp Back

{triangledown} Published ahead of print on 30 July 2008. Back


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REFERENCES
 
    1
  1. Ariumi, Y., P. Turelli, M. Masutani, and D. Trono. 2005. DNA damage sensors ATM, ATR, DNA-PKcs, and PARP-1 are dispensable for human immunodeficiency virus type 1 integration. J. Virol. 79:2973-2978.[Abstract/Free Full Text]
    2. 2
  2. Ariumi, Y., and D. Trono. 2006. Ataxia-telangiectasia-mutated (ATM) protein can enhance human immunodeficiency virus type 1 replication by stimulating Rev. function. J. Virol. 80:2445-2452.[Abstract/Free Full Text]
    3. 3
  3. Ariumi, Y., M. Kuroki, K. Abe, H. Dansako, M. Ikeda, T. Wakita, and N. Kato. 2007. DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication. J. Virol. 81:13922-13926.[Abstract/Free Full Text]
    4. 4
  4. Bridge, A. J., S. Pebernard, A. Ducraux, A.-L. Nicoulaz, and R. Iggo. 2003. Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet. 34:263-264.[CrossRef][Medline]
    5. 5
  5. Brummelkamp, T. R., R. Bernard, and R. Agami. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550-553.[Abstract/Free Full Text]
    6. 6
  6. Canman, C. E., D.-S. Lim, K. A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E. Apella, M. B. Kastan, and J. D. Siliciano. 1998. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281:1677-1679.[Abstract/Free Full Text]
    7. 7
  7. Cheng, G., J. Zhong, J. Chung, and F. V. Chisari. 2007. Double-stranded DNA and double-stranded RNA induces a common antiviral signaling pathway in human cells. Proc. Natl. Acad. Sci. USA 104:9035-9040.[Abstract/Free Full Text]
    8. 8
  8. Dansako, H., M. Ikeda, and N. Kato. 2007. Limited suppression of the interferon-beta production by hepatitis C virus serine protease in cultured human hepatocytes. FEBS J. 274:4161-4176.[CrossRef][Medline]
    9. 9
  9. Gaspar, M., and T. Shenk. 2006. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins. Proc. Natl. Acad. Sci. USA 103:2821-2826.[Abstract/Free Full Text]
    10. 10
  10. Harper, J. W., and S. J. Elledge. 2007. The DNA damage response: ten years after. Mol. Cell 28:739-745.[Medline]
    11. 11
  11. Ikeda, M., K. Abe, H. Dansako, T. Nakamura, K. Naka, and N. Kato. 2005. Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system. Biochem. Biophys. Res. Commun. 329:1350-1359.[CrossRef][Medline]
    12. 12
  12. Kato, N., M. Hijikata, Y. Ootsuyama, M. Nakagawa, S. Ohkoshi, T. Sugimura, and K. Shimotohno. 1990. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc. Natl. Acad. Sci. USA 87:9524-9528.[Abstract/Free Full Text]
    13. 13
  13. Kato, N. 2001. Molecular virology of hepatitis C virus. Acta Med. Okayama 55:133-159.[Medline]
    14. 14
  14. Kato, N., K. Sugiyama, K. Namba, H. Dansako, T. Nakamura, M. Takami, K. Naka, A. Nozaki, and K. Shimotohno. 2003. Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in vitro. Biochem. Biophys. Res. Commun. 306:756-766.[CrossRef][Medline]
    15. 15
  15. Lai, C. K., K. S. Jeng, K. Machida, Y. S. Cheng, and M. M. Lai. 2008. Hepatitis C virus NS3/4A protein interacts with ATM, impairs DNA repair and enhances sensitivity to ionizing radiation. Virology 370:295-309.[Medline]
    16. 16
  16. Lau, A., K. M. Swinbank, P. S. Ahmed, D. L. Taylor, S. P. Jackson, G. C. Smith, and M. J. O'Connor. 2005. Suppression of HIV-1 infection by a small molecule inhibitor of the ATM kinase. Nat. Cell Biol. 7:493-500.[CrossRef][Medline]
    17. 17
  17. Lilley, C. E., R. A. Schwartz, and M. D. Weitzman. 2007. Using or abusing: viruses and the cellular DNA damage response. Trends Microbiol. 15:119-126.[CrossRef][Medline]
    18. 18
  18. Machida, K., K. T. Cheng, V. M. Sung, S. Shimodaira, K. L. Lindsay, A. M. Levine, M. Y. Lai, and M. M. Lai. 2004. Hepatitis C virus induces a mutator phenotype: enhanced mutations of immunoglobulin and protooncogenes. Proc. Natl. Acad. Sci. USA 101:4262-4267.[Abstract/Free Full Text]
    19. 19
  19. Machida, K., K. T. Cheng, V. M. Sung, K. J. Lee, A. M. Levine, and M. M. Lai. 2004. Hepatitis C virus infection activates the immunologic (type II) isoform of nitric oxide synthase and thereby enhances DNA damage and mutations of cellular genes. J. Virol. 78:8835-8843.[Abstract/Free Full Text]
    20. 20
  20. Matsuoka, S., M. Huang, and S. J. Elledge. 1998. Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282:1893-1897.[Abstract/Free Full Text]
    21. 21
  21. Matsuoka, S., G. Rotman, A. Ogawa, Y. Shiloh, K. Tamai, and S. J. Elledge. 2000. Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc. Natl. Acad. Sci. USA 97:10389-10394.[Abstract/Free Full Text]
    22. 22
  22. Myong, S., M. M. Bruno, A. M. Pyle, and T. Ha. 2007. Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science 317:513-516.[Abstract/Free Full Text]
    23. 23
  23. Naka, K., H. Dansako, N. Kobayashi, M. Ikeda, and N. Kato. 2006. Hepatitis C virus NS5B delays cell cycle progression by inducing interferon-β via Toll-like receptor 3 signaling pathway without replicating viral genomes. Virology 346:348-362.[CrossRef][Medline]
    24. 24
  24. Naldini, L., U. Blömer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, and D. Trono. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263-267.[Abstract]
    25. 25
  25. Pang, P. S., E. Jankowsky, P. J. Planet, and A. M. Pyle. 2002. The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J. 21:1168-1176.[CrossRef][Medline]
    26. 26
  26. Takaoka, A., Z. Wang, M. K. Choi, H. Yanai, H. Negishi, T. Ban, Y. Lu, M. Miyagishi, T. Kodama, K. Honda, Y. Ohba, and T. Taniguchi. 2007. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 448:501-505.[CrossRef][Medline]
    27. 27
  27. Tanaka, T., N. Kato, M. J. Cho, and K. Shimotohno. 1995. A novel sequence found at the 3' terminus of hepatitis C virus genome. Biochem. Biophys. Res. Commun. 215:744-749.[CrossRef][Medline]
    28. 28
  28. Thomas, D. L. 2000. Hepatitis C epidemiology. Curr. Top. Microbiol. Immunol. 242:25-41.[Medline]
    29. 29
  29. Wakita, T., T. Pietschmann, T. Kato, T. Date, M. Miyamoto, Z. Zhao, K. Murthy, A. Habermann, H. G. Kräusslich, M. Mizokami, R. Bartenschlager, and T. J. Liang. 2005. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11:791-796.[CrossRef][Medline]
    30. 30
  30. Zufferey, R., D. Nagy, R. J. Mandel, L. Naldini, and D. Trono. 1997. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15:871-875.[CrossRef][Medline]

Journal of Virology, October 2008, p. 9639-9646, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00351-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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