The DNA damage sensors ataxia-telangiectasia mutated kinase and checkpoint kinase 2 are required for hepatitis C virus RNA replication

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.

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.

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.

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.

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.

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.

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.