Basic, Research : Functional Analysis of Sodium Taurocholate Cotransporting Polypeptide (Ntcp) as a Cellular Receptor for Entry of Hepatits B Virus

Background: Although many antiviral targets against hepatitis B virus (HBV) have been developed, the host receptor of HBV has not been documented until recently, when one report showed that sodium taurocholate cotransporting polypeptide (NTCP) has affinity for HBsAg and may work as a receptor for HBV entry in HepG2 cells. This study aimed to confirm whether NTCP expression could affect the HBV entry in Huh7 cell line, and whether HBV infection affect the transcription of NTCP. Methods: Huh-BAT cells, Huh7-derived cell line in which human NTCP under the control of CMV promoter was transfected, were used in our experiments. Supernatant of HepAD38 cells cultured in tetracycline-free medium was concentrated using polyethylene gycol (PEG) for the generation of infectious HBV particles. After adding HBV concentrates on Huh-7 and Huh-BAT cells, supernatants were collected at 24 and 48 hours. NTCP expression and HBV levels in the supernatant or cytosol of HBV treated cells were measured by real-time PCR. NTCP promoters were cloned in pGL3 plasmid and co-transfected with pHY92 plasmid encoding 1.1x HBV genome in HepG2 cells, and the promoter activity was assessed by luciferase assay. Results: NTCP expression level was negligible in Huh7 cells, whereas the expression was readily detected in Huh-BAT cells. At 24 hours after PEG-HBV treatment, HBV level of Huh-BAT supernatant was significantly higher compared to Huh-7 supernatant. However, the difference between supernatant HBV levels of 2 cell line was not significant at 48 hours. Cytosolic HBV concentration was slightly higher in HBV treated Huh- BAT cells than in Huh-7 cells on day 2. The promoter activity of NTCP was neither enhanced nor interrupted by replication of HBV. Conclusions: NTCP enhanced the binding of HBV onto Huh- BAT cells, and may enhance the entry of HBV by receptor-mediated endocytosis. Replication of HBV does not affect the transcription of NTCP. Dynamics of entry and release mechanism of HBV via NTCP in human HCC cells should be eluci dated in the future research.