title: NTCP-Reconstituted In Vitro HBV Infection System.
abstract: Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for hepatitis B virus (HBV). Expressing human NTCP in human hepatoma HepG2 cells (HepG2-NTCP) renders these cells susceptible for HBV infection. The HepG2-NTCP stably transfected cell line provides a much-needed and easily accessible platform for studying the virus. HepG2-NTCP cells could also be used to identify chemicals targeting key steps of the virus life cycle including HBV covalent closed circular (ccc) DNA, and enable the development of novel antivirals against the infection.Many factors may contribute to the efficiency of HBV infection on HepG2-NTCP cells, with clonal differences among cell line isolates, the source of viral inoculum, and infection medium among the most critical ones. Here, we provide detailed protocols for efficient HBV infection of HepG2-NTCP cells in culture; generation and selection of single cell clones of HepG2-NTCP; production of infectious HBV virion stock through DNA transfection of recombinant plasmid that enables studying primary clinical HBV isolates; and assessing the infection with immunostaining of HBV antigens and Southern blot analysis of HBV cccDNA.
authors.affiliation: National Institute of Biological Sciences, Beijing, 102206, China.
authors.forename: Yinyan
authors.initials: Y
authors.lastname: Sun
authors.affiliation: National Institute of Biological Sciences, Beijing, 102206, China.
authors.forename: Yonghe
authors.initials: Y
authors.lastname: Qi
authors.affiliation: National Institute of Biological Sciences, Beijing, 102206, China.|Graduate Program in School of Life Sciences, Peking University, Beijing, 100871, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Peng
authors.affiliation: National Institute of Biological Sciences, Beijing, 102206, China. liwenhui@nibs.ac.cn.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Li
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D004270:DNA, Circular; D004279:DNA, Viral; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D056945:Hep G2 Cells; D006509:Hepatitis B / Q000235:genetics / Q000378:metabolism* / Q000821:virology*; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006513:Hepatitis B e Antigens / Q000276:immunology; D006515:Hepatitis B virus / Q000502:physiology*; D006801:Humans; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D029363:Organic Anion Transporters, Sodium-Dependent; D012367:RNA, Viral; D011991:Receptors, Virus; D027981:Symporters; C071908:sodium-bile acid cotransporter
keywords: Antivirals; Cell culture; HBV; NTCP; Receptor; Viral infection; cccDNA