Down-Regulation of Cell Membrane Localized NTCP Expression in Proliferating Hepatocytes Prevents Hepatitis B Virus Infection

Background: Hepatocyte proliferation could result in the loss of covalently closed circular DNA (cccDNA) and the emergence of cccDNA-cleared nascent hepatocytes, which appear refractory to hepatitis B virus (HBV) reinfection with unknown mechanism(s). This study aims to uncover the underlying molecular mechanism(s) and its related clinical significance on HBV reinfection resistance of proliferating hepatocytes.   Methods: The transcriptional inhibition of Sodium taurocholate cotransporting polypeptide (NTCP) expression in proliferating hepatocytes and impact of NTCP protein levels on HBV infection were assayed both in vivo and in vitro. The clinical significance of NTCP down-regulation was explored in a cohort of 68 HBeAg-positive chronic hepatitis B (CHB) patients. Findings: Arresting HepG2-NTCP-tet cells in G0/G1 phase obviously up-regulated NTCP cell membrane expression and thus facilitated HBV infection in vitro. In liver-humanized mice, HBV reinfection occurred only after most hepatocytes had stopped proliferating and cell membrane expression of NTCP returned to normal. In patients, lower NTCP protein expression was correlated well with higher levels of hepatocyte proliferation and less HBsAg expression in HBV-related focal nodular hyperplasia (FNH) tissues. Clinically, significantly lower NTCP protein expression was correlated with more active hepatocyte proliferation in CHB patients with severe active necroinflammation and better antiviral treatment outcome. Mechanistically, p53, cyclin D1, S-phase kinase associated protein 2 and interleukin-6 could transcriptionally down-regulate NTCP expression.   Interpretation: Within the milieu of hepatocyte proliferation, lower NTCP expression level renders nascent hepatocytes resistant to HBV reinfection. This may accelerate virus clearance during immune-mediated cell death and compensatory proliferation of survival hepatocytes.   Funding Statement: This study was supported by grants from National Science and Technology Major Project of the Ministry of Science and Technology of China (2017ZX10202203, 2017ZX10302201, 2017ZX10202202), the National Natural Science Foundation of China (81672013) and the German Research Foundation (DFG, Collaborative Research Centre SFB-841: A5). Among them, the German Research Foundation (DFG, Collaborative Research Centre SFB-841: A5) supported the studies involved liver-humanized mice. The National Science and Technology Major Project of the Ministry of Science and Technology of China (2017ZX10202203, 2017ZX10302201, 2017ZX10202202), and the National Natural Science Foundation of China (81672013) supported the evolving of the other experiments. Declaration of Interests: All authors have no conflict of interest to disclose. Ethics Approval Statement: The animal experiments involving humanized mice were approved by the City of Hamburg, Germany (G118/16), conducted in accordance with the European Communities Council Directive (86/EEC). The other animals involved in the study were approved by the Bioethics Committees of Peking University, the reference number is LA2018181. The clinical data involved in the study was approved by the Bioethics Committees of Peking University at February 27th, 2018.