Analysis of integrated hepatitis B virus DNA and flanking cellular sequence by inverse polymerase chain reaction

Abstract

Although hepatitis B virus (HBV) DNA has been detected in the human hepatoma cell line, HAGS 2.1, viral and cellular junction sequences have not been investigated fully. To facilitate the analysis of HBV DNA integration sites in HAGS 2.1 cells, a combination of conventional polymerase chain reaction (PCR) and inverse PCR (IPCR) was carried out to identify the junction between the viral and the cellular gene. The HBV integrant and its cellular counterpart sequence were cloned and analyzed. The sequencing data indicated that the breakpoints on the HBV integrant are at nucleotide 2111 of the C gene and nucleotide 1558 of the X gene. The length of the integrated HBV DNA in HAGS 2.1 was approximately 2.6 kb, which includes partial C, P, and X genes and an intact S gene. The cellular sequence flanking the integrated HBV gene was very similar to a human satellite III repetitive sequence with 43 and 56 of GGAAT repeats on the left- and right-hand side, respectively. Although the findings on the viral–cellular junction in HAGS 2.1 cells cannot explain the liver tumorigenesis, the current study shows that by choosing the nearest restriction site, which can be determined by conventional PCR rather than using a unique site within the integrated viral sequence to do IPCR, gives a higher successful rate for cloning and the subsequent analysis of the viral–cellular junctions.