title: Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide.
abstract: Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection.
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Yamanashi, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Okuyama-Dobashi
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Yamanashi, Japan.
authors.forename: Hirotake
authors.initials: H
authors.lastname: Kasai
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Yamanashi, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Yamanashi, Japan.
authors.forename: Atsuya
authors.initials: A
authors.lastname: Yamashita
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Yamanashi, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Yasumoto
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Yamanashi, Japan.
authors.forename: Wenjia
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Molecular Virology, Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Okamoto
authors.affiliation: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Yamanashi 409-3898, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Maekawa
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Watashi
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.affiliation: Department of Molecular Biodefense Research, Yokohama City University Graduate School of Medicine Kanagawa 236-0004, Japan.
authors.forename: Akihide
authors.initials: A
authors.lastname: Ryo
authors.affiliation: Department of Infectious Diseases, Hamamatsu University School of Medicine, Shizuoka, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Molecular Virology, Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Matsuura
authors.affiliation: First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Yamanashi 409-3898, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Enomoto
authors.affiliation: Department of Microbiology, Faculty of Medicine, University of Yamanashi, Japan.
authors.forename: Kohji
authors.initials: K
authors.lastname: Moriishi
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002018:Bufanolides / Q000494:pharmacology; D002448:Cell Adhesion; D004074:Digitoxin / Q000494:pharmacology; D004077:Digoxin / Q000494:pharmacology; D015870:Gene Expression; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000187:drug effects / Q000502:physiology; D057166:High-Throughput Screening Assays; D006801:Humans; D029363:Organic Anion Transporters, Sodium-Dependent / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010793:Phthalazines / Q000494:pharmacology; D011442:Proscillaridin / Q000494:pharmacology*; D011991:Receptors, Virus / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D019821:Simvastatin / Q000494:pharmacology; D013328:Strophanthins / Q000494:pharmacology; D027981:Symporters / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D014759:Viral Envelope Proteins / Q000494:pharmacology; D053586:Virus Internalization / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D002018:Bufanolides; C096362:L protein, hepatitis B virus; D029363:Organic Anion Transporters, Sodium-Dependent; D010793:Phthalazines; D011991:Receptors, Virus; D013328:Strophanthins; D027981:Symporters; D014759:Viral Envelope Proteins; C071908:sodium-bile acid cotransporter; D004077:Digoxin; D019821:Simvastatin; D004074:Digitoxin; C000505:convallatoxin; D011442:Proscillaridin; C022777:bufalin; C020976:azelastine