Hepatitis C virus infection activates the immunologic (type II) isoform of nitric oxide synthase and thereby enhances DNA damage and mutations of cellular genes

Abstract

Hepatitis C virus (HCV) infection causes hepatitis, hepatocellular carcinoma, and B-cell lymphomas in a significant number of patients. Previously we have shown that HCV infection causes double-stranded DNA breaks and enhances the mutation frequency of cellular genes, including proto-oncogenes and immunoglobulin genes. To determine the mechanisms, we studied in vitro HCV infection of cell culture. Here we report that HCV infection activated the immunologic (type II) isoform of nitric oxide (NO) synthase (NOS), i.e., inducible NOS (iNOS), thereby inducing NO, which in turn induced DNA breaks and enhanced the mutation frequencies of cellular genes. Treatment of HCV-infected cells with NOS inhibitors or small interfering RNA specific for iNOS abolished most of these effects. Expression of the core protein or nonstructural protein 3 (NS3), but not the other viral proteins, in B cells or hepatocytes induced iNOS and DNA breaks, which could be blocked by NOS inhibitors. The core protein also enhanced the mutation frequency of cellular genes in hepatocytes derived from HCV core transgenic mice compared with that in control mice. The iNOS promoter was activated more than fivefold in HCV-infected cells, as revealed by a luciferase reporter assay driven by the iNOS promoter. Similarly, the core and NS3 proteins also induced the same effects. Therefore, we conclude that HCV infection can stimulate the production of NO through activation of the gene for iNOS by the viral core and NS3 proteins. NO causes DNA breaks and enhances DNA mutation. This sequence of events provides a mechanism for HCV pathogenesis and oncogenesis.

FIG. 1.

Detection and kinetic analysis of iNOS mRNA expression. (A) cDNA from Raji cells infected with HCV and control cells was serially diluted (0-, 5-, and 25-fold) and used for PCR amplification to detect iNOS mRNA (32 PCR cycles). β-Actin mRNA levels (22 PCR cycles) were used as a control. These experiments were repeated three times with similar results. (B) Kinetics of iNOS expression in HCV-infected Raji cells were followed from day 2 up to day 28 postinfection (p.i.). HCV RNA was detected by RT-PCR assay. (C) NO3−-NO2− production in cell culture medium in the presence or absence of 1400W or L-NMMA. For a positive control, cells were treated with a mixture of cytokines (Cyt) (interleukin-1β at 0.5 ng/ml; gamma interferon at 100 U/ml, and tumor necrosis factor alpha at 10 ng/ml) or 0.3 mM SNAP, releasing endogenous and exogenous NO, respectively. HCV (−), cells treated with UV-inactivated HCV.

FIG. 2.

HCV infection induces NO-dependent DNA damage. (A and B) DSBs in HCV-infected and uninfected Raji (A) or JT (B) cells in the presence or absence of NOS inhibitors were detected by LM-PCR and separated by gel electrophoresis as previously described (15, 31). A control PCR assay was done with β-actin. (C and D) The region-specific DSBs were detected with p53- and V_H-specific primers (15). (E) HCV RNA was detected by RT-PCR assay. (F) Control PCR: amplification of the V_H gene of genomic DNA as an internal control.

FIG. 3.

iNOS-specific siRNA lowers HCV-induced DSBs. (A and B) Effect of siRNA on levels of iNOS protein (A) and RNA (B) in HCV-infected cells. (A) Western blot analysis shows iNOS protein expression at day 8 postinfection in HCV-infected cells. A Western blot assay of β-actin was used as a loading control. HCV RNA was detected by RT-PCR. (B) iNOS and β-actin mRNAs in serial dilutions from Raji cells transfected with iNOS or control siRNA. (C) DSBs in cells transfected with iNOS or control siRNA. DSBs were examined at day 8 postinfection.

FIG. 4.

Core or NS3 protein induces NO-dependent DNA damage. (A) Raji cells were transfected with various expression plasmids. Expression of HCV proteins was determined by Western blot analysis with specific antibodies. V, vector-transfected cells. (B) iNOS mRNA expression in cells transfected with viral proteins was determined by a semiquantitative RT-PCR assay in Raji cell expressing various HCV proteins. Serial dilutions (0-, 5-, and 25-fold) of each cellular cDNA were used. (C) DSB formation was determined by LM-PCR in Raji cells expressing various HCV proteins. Vector, vector control; SNAP, Raji cells treated with the NO donor SNAP; Cytokines, Raji cells treated with a cytokine mixture. (D) DSBs in Raji cells transfected with various HCV proteins in the presence or absence of NOS inhibitors (1400W and L-NMMA). Vec, vector.

FIG. 5.

DSBs in hepatocytes expressing various HCV proteins. DSBs were determined by LM-PCR assay. (A) Core protein-expressing (Core) or neomycin-resistant (Neo) HepG2 cells. For some of the experiments indicated, the iNOS inhibitors 1400W and L-NMMA were added to the cell culture 5 days before the cells were harvested. (B) Huh7 cells transiently expressing NS3 or the vector plasmid. Cells were examined 2 days after transfection. (C) Huh7 cells with the HCV replicon. (D) NO_X concentration in serum from HCV core transgenic (Tg) mice and littermates. (E) DSB formation in hepatocytes from HCV core transgenic mice. In panels D and E, each symbol and each lane represents an individual animal.

FIG. 6.

Assays of iNOS promoter activity in HCV-infected cells and in cells transfected with the core or NS3 protein. (Top two panels) Mock-infected or HCV-infected Raji cells were transfected with the indicated luciferase reporter constructs containing different fractions of the iNOS promoter at 8 days postinfection of HCV. Luciferase activity was determined from the cell lysates at 2 days posttransfection. (Bottom three panels) Uninfected Raji cells were transfected with the vector or the core- or NS3-expressing plasmid and one of the luciferase reporter controls as described on the left. Luciferase activity was assayed at 2 days posttransfection. Data are the mean values of three experiments with assays done in triplicate, expressed as the luciferase activity (relative light units [RLU]) normalized for total protein content. Positions of NF-κB and AP-1 binding sites in the promoter are depicted.

FIG. 7.

Postulated mechanism of HCV-induced DNA damage.