Amino acid substitutions in codons 9-11 of hepatitis C virus core protein lead to the synthesis of a short core protein product

Background: Previous in vitro experiments have indicated that if the ninth codon of the hepatitis C virus (HCV) core gene is mutated from arginine to lysine, a short 16-kDa (P16) instead of a 21-kDa (P21) core protein will be produced. In this study, we aimed to investigate whether similar mutations existed in patients with chronic HCV infection and whether such mutations led to the expression of P16. Methods: The core gene was isolated from patients' sera by reverse transcription-polymerase chain reaction and sequenced. Results: Three of 10 patients with hepatocellular carcinoma were found to have mutant viruses with missense mutations at codons 9-11: arginine-to-glycine mutation at codon 9 (case 1); lysine-to-glutamine mutation at codon 10 (case 5); and lysine-to-asparagine/threonine-to-alanine double mutations at codons 10 and 11 (case 8). Site-directed mutagenesis and in vitro translation experiments revealed that P16 was expressed by all three mutants. Using gel-purified P21 and P16 proteins obtained from transformed Escherichia coli, the serum titres of anti-P21 and anti-P16 were assayed. Unequal titres of anti-P16 and anti-P21 were found in only cases 1, 5 and 8. A rabbit antibody directed against P16 but not P21 was thus generated for immunohistochemical analysis. P16 was detected in the nuclei of hepatocytes in the peri-hepatoma tissue of a single case (case 1). Conclusions: These data indicate that missense mutations at codons 9-11 can occur during chronic HCV infection, which results in the expression of P16 core protein. (C) 2000 Blackwell Science Asia Pty Ltd.