Impact of amino acid substitutions in hepatitis C virus core region on the severe oxidative stress

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, leading to liver steatosis, fibrosis, and hepatocellular carcinoma (HCC). Despite the accumulation of clinical data showing the impact of amino acid substitutions at positions 70 (R70Q/H) and/or 91 (L91M) in the HCV core protein in progressive liver diseases, including HCC, the underlying mechanisms have not been elucidated. We analyzed 72 liver biopsy specimens from patients with chronic HCV genotype 1b (HCV-1b) infection prior to antiviral treatment. Levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor erythroid 2-related factor 2 (NRF2) in the nucleus were quantified using liver tissue immunohistochemistry. The effects of amino acid substitutions in the HCV core region on hepatocellular oxidative stress were investigated using wild-type or double-mutant (R70Q/H+L91M) HCV-1b core transfection and stable expression in human hepatoma HuH-7 cells. Overall, 24, 19, 11, and 18 patients had the wild-type, R70Q/H, L91M, and R70Q/H+L91M genotypes, respectively, in the HCV core. A significantly higher accumulation of hepatocellular 8-OHdG and a lower NRF2/8-OHdG ratio were observed in patients with R70Q/H+L91M than in those with the wild-type disease. Increased levels of intracellular superoxide and hydrogen peroxide in the cytoplasm and mitochondria, mRNA expression of enzymes generating oxidative stress, and nuclear expression of nicotinamide adenine dinucleotide phosphate oxidase 4 were augmented in cells treated with R70Q+L91M. HCV core proteins harboring either or both substitutions of R70Q/H or L91M enhanced hepatocellular oxidative stress in vivo and in vitro. These amino acid substitutions may affect HCC development by enhancing hepatic oxidative stress in patients with chronic HCV-1b infection.