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Abstract
Hepatitis C virus (HCV)-induced hepatic stress is associated with increased oxidative DNA damage and has been implicated in hepatic inflammation. However, HCV infection and replication are uneven and vary among individual hepatocytes. To investigate the effect of the viral load on host DNA damage, we used an Enhanced Yellow Fluorescent Protein gene (EYFP)-tagged HCV virus to distinguish between HCV intracellular high viral load (HVL) cells and low viral load (LVL) cells. The cell sorting efficiency was confirmed by the high expression of the HCV polyprotein. We found DNA damage γ-H2AX foci in the HVL population. Comet assays demonstrated that HVL was related to the extent of the DNA strand breaks. Surprisingly, the DNA qPCR arrays and western blotting showed that the damage-related genes GPX2, MRE11, phospho-ATM, and OGG1 were significantly up-regulated in LVL cells but inversely down-regulated or consistently expressed in HVL cells. The colony survival assay to examine the repair abilities of these cells in response to irradiation showed that the LVL cells were more resistant to irradiation and had an increased ability to repair radiation-induced damage. This study found that intracellular viral loads drove cellular DNA damage levels but suppressed damage-related gene expression. However, the increase in damage-related gene expression in the LVL cells may be affected by ROS from the HVL cells. These findings provide new insights into the distinct DNA damage and repair responses resulting from different viral loads in HCV-infected cells.
Highlights
Hepatitis C virus (HCV) replicates in the cytoplasm and results in a chronic infection that may cause chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) [1]
The cell population with the lowest 20% Enhanced Yellow Fluorescent Protein gene (EYFP) fluorescent intensity was gated as the low viral load (LVL) cells, and the cell population with the highest 20% EYFP fluorescent intensity was gated as the high viral load (HVL) cells (S1 Fig)
The sorting efficiencies for the LVL and HVL cells were 94% and 89%, respectively. q-PCR confirmed that the HVL cells expressed significantly higher HCV-NS5B mRNA levels than the LVL cells and unsorted JFH1-EYFP infected control cells (P < 0.001) (Fig 1A)
Summary
Hepatitis C virus (HCV) replicates in the cytoplasm and results in a chronic infection that may cause chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) [1]. The associations between viral and oxidative DNA damage responses are particular and increasing scientific interest. Viral replication within a host cell requires a large amount of exogenous genetic material, including DNA fragments and atypical structures. It is generally accepted that HCV viral replication induces DNA damage stress and activates DNA damage signal pathways that lead to apoptosis as part of the host cell immune surveillance defense. Sustained oxidative damage may contribute to the development of virus-associated HCC [10]; determining whether HCV perturbs this process and the involved mechanisms requires further investigation
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