Analysis of Naturally Occurring Mutations in Reverse Transcriptase Region of Hepatitis B Virus Genotype C2 Polymerase

Abstract

Hepatitis B virus (HBV) is a family of hepatotropic DNA viruses, and replicates its DNA genome through an RNA intermediate during the viral life cycle using reverse transcriptase (RT). Mutations within RT probably affect the replication capacity of HBV, which may in turn alter the antigenicity and generation of drug resistance. HBV is classified into eight genotypes, type A-H, according to the differences in the genome sequence. Patients infected with HBV genotype C are associated with more aggressive liver diseases and poorer response to antiviral therapy. Full-length HBV RT sequences were analyzed from 131 treatment-naïve Korean patients infected with HBV genotype C2. The patients had two distinct clinical statuses: chronic hepatitis (CH) and hepatocellular carcinoma (HCC). To analyze the patterns and the frequencies of mutations in the RT region, a nested polymerase chain reaction (nested-PCR) based sequencing protocol was used. The mutation frequency was significantly (p<0.05) higher in the HCC patients and the HBV envelope antigen (HBeAg) negative patients. Notably the A-B interdomain region had a significantly (p<0.05) higher mutation frequency than that of the other regions, and it overlaps the A-determinant of the HBV surface antigen (HBsAg) which is the region involved in vaccine escape. The patients with three type of mutations (rt80, rt139, and rt204) were significantly (p<0.05) relative to the HCC and the severe liver disease progression. In addition, among 71 out of 131 patients who had received the follow-up antiviral treatment, those with the previously unknown mutation (rt55) and the well known YMDD mutation (rt204) significantly (p<0.05) showed no response to the antiviral treatment. In order to determine how the mutants affected the HBV replication, this study confirmed with in vitro transfection systems, using full-genome HBV constructs with the site-directed mutation (H55Q, L80I, N139K, and M204I), quantifying of HBV DNA and HBsAg levels, and treatment of antiviral agents. In conclusion, naturally occurring RT mutations might be indicators for the prediction of the liver disease progression and antiviral treatment response of patients with HBV C2 infection.