Analysis of HBV integration in mitochondrial DNA of HepAD38 cells by the high-throughput HBV integration sequencing and RNASeq approaches

Abstract: Introduction Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC) worldwide. The integration of HBV DNA into the host genome is one of the carcinogenic mechanisms of HBV. It has been recently demonstrated that mitochondrial DNA (mtDNA) can be a target of HBV, suggesting a new potential mechanism by which HBV integration may contribute to liver damage and HCC development. Aim (1) To conduct a deeper investigation of HBV integration in mtDNA of HepAD38 cell line, which derives from HepG2 cells and supports tetracycline (Tet)-off inducible HBV replication; (2) to evaluate mitochondrial function in HBV-replicating HepAD38 cells. Methods We used a high-throughput HBV integration sequencing (HBIS) approach and RNASeq to investigated HBV integration in mitochondria isolate from HepAD38 cells after Tet removal for 7 days. Moreover, at the same time point we analysed mitochondrial function of HepAD38 cells using the Seahorse XFp analyzer. Results After 7 days Tet removal, mean amounts of HBV DNA and HBV RNA in HepAD38 cells were 1.1 × 103 ± 6.0 × 102 and 1.0 × 10 ± 5.9 × 10−1 copies/cell, respectively; while mean amounts of HBV DNA and HBsAg in the cell supernatants were 2 × 106 ± 2.7 × 104 copies/mL and 4.1 × 103± 7.2 × 102 IU/mL, respectively. At the same time point, HBIS led us to detect a mean amount of 81± 11.9 HBV integration sites in mtDNA from HBV-replicating HepAD38. In particular, HBV integration sites were detected at high frequency in COX1, RNR2, and ND2 mitochondrial genes. In addition, RNASeq analysis ledus to detect large amount (mean±S.D.: 635± 143.7) of HBV-mitochondria chimeric transcripts in HBV-induced HepAD38 cells. These transcripts contained - at higher frequency - sequences corresponding COX1, RNR2, ND2, ND4, and ND6 mitochondrial genes. The analysis of mitochondrial function showed that, compared to HepAD38 cells without HBV replication and viral integration, HBV replicating HepAD38 cells had a 2-fold reduction of basal respiration, of ATPlinked respiration, and of maximal respiration. Conclusions (1) HBV may integrate into mtDNA of HBV replicating HepAD38 cells; (2) The site of HBV insertion into mtDNA may be transcriptionally active; (3) HBV replication and viral integration into mtDNA may induce mitochondrial dysfunction