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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.

SUBMITTER: Okuyama-Dobashi K

PROVIDER: S-EPMC4655410 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide.

Okuyama-Dobashi Kaori K Kasai Hirotake H Tanaka Tomohisa T Yamashita Atsuya A Yasumoto Jun J Chen Wenjia W Okamoto Toru T Maekawa Shinya S Watashi Koichi K Wakita Takaji T Ryo Akihide A Suzuki Tetsuro T Matsuura Yoshiharu Y Enomoto Nobuyuki N Moriishi Kohji K

Scientific reports 20151123

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-adh ...[more]

PMID: 26592202

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Project description:UnlabelledCell culture (cc)-derived hepatitis B virus (HBV) can infect differentiated HepaRG cells, but efficient infection requires addition of polyethylene glycol (PEG) during inoculation. Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor enabled ccHBV infection of NTCP reconstituted HepG2 cells, although very little hepatitis B surface antigen (HBsAg) is produced. We found infection by patient serum-derived HBV (sHBV), which required purification of viral particles through ultracentrifugation or PEG precipitation, was PEG independent and much more efficient in HepaRG cells than in HepG2/NTCP cells. In contrast to hepatitis B e antigen (HBeAg), HBsAg was not a reliable marker of productive sHBV infection at early time points. A low HBsAg/HBeAg ratio by ccHBV-infected HepG2/NTCP cells was attributable to dimethyl sulfoxide (DMSO) in culture medium, NTCP overexpression, and HBV genotype D. HepG2/NTCP cells released more viral antigens than HepG2 cells after HBV genome delivery by adeno-associated virus, and stable expression of NTCP in a ccHBV producing cell line increased viral mRNAs, proteins, replicative DNA, and covalently closed circular DNA. NTCP protein expression in HepG2/NTCP cells, despite being driven by the cytomegalovirus promoter, was markedly increased by DMSO treatment. This at least partly explains ability of DMSO to promote ccHBV infection in such cell lines. In conclusion, NTCP appeared inefficient to mediate infection by serum-derived HBV. It could promote HBV RNA transcription while inhibiting HBsAg secretion. Efficient PEG-independent sHBV infection of HepaRG cells permits comparative studies of diverse clinical HBV isolates and will help identify additional factors on virion surface promoting attachment to hepatocytes.ImportanceCurrently in vitro infection with hepatitis B virus (HBV) depends on cell culture-derived HBV inoculated in the presence of polyethylene glycol. We found patient serum-derived HBV could efficiently infect differentiated HepaRG cells independent of polyethylene glycol, which represents a more physiological infection system. Serum-derived HBV has poor infectivity in HepG2 cells reconstituted with sodium taurocholate cotransporting polypeptide (NTCP), the currently accepted HBV receptor. Moreover, HepG2/NTCP cells secreted very little hepatitis B surface antigen after infection with cell culture-derived HBV, which was attributed to NTCP overexpression, genotype D virus, and dimethyl sulfoxide added to culture medium. NTCP could promote HBV RNA transcription, protein expression, and DNA replication in HepG2 cells stably transfected with HBV DNA, while dimethyl sulfoxide could increase NTCP protein level despite transcriptional control by a cytomegalovirus promoter. Therefore, this study revealed several unusual features of NTCP as an HBV receptor and established conditions for efficient serum virus infection in vitro.

Project description:Hepatitis B virus (HBV) may directly infect human podocytes (HPCs). However, the mechanism of direct infection is unclear. We found that HPCs express sodium taurocholate cotransporting polypeptide (NTCP), a specific receptor for HBV entry into hepatocytes. Thus, we investigated whether NTCP mediates HBV infection and damage in HPCs and further clarified the specific mechanism. We constructed shRNA-NTCP1,2, shRNA-NC, WT-NTCP, and MUT-NTCP and transfected them into HPCs. HPCs were infected with HBV, and HBV infection markers were detected by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (RT-qPCR). The functional changes in HPCs were detected by Transwell migration and scratch assays, apoptosis was evaluated by flow cytometry (FCM), and podocytoskeletal proteins (nephrin, CD2AP, and synaptopodin) were determined by western blotting (WB). Compared with the control HPCs, HPCs infected with HBV showed increased levels of HBV infection markers and apoptosis along with decreased podocytoskeletal protein expressions, cell vitality, proliferation, and migration. Compared with the HPCs infected with HBV, the HPCs transfected with HBV + shRNA-NTCP, and HBV + MUT-NTCP showed decreased levels of HBV infection markers and apoptosis along with increased podocytoskeletal protein expressions, cell vitality, proliferation, and migration; the opposite effects were observed in the HPCs transfected with HBV + WT-NTCP. Overall, the changes to NTCP affected the susceptibility of HPCs to HBV and modulated HPC damage and repair. NTCP can mediate direct HBV infection and damage human podocytes, and the NTCP 157-165 locus is the main site of HBV entry. The findings provide a new target and theoretical basis for HBV-associated glomerulonephritis.ImportanceThis study identified for the first time that sodium taurocholate cotransporting polypeptide (NTCP) can mediate HBV direct infection and damage to human podocytes, and the NTCP157-165 locus is the main HBV entry site. The findings provide theoretical support for the pathogenesis of direct infection of HBV with kidney tissue. The findings provide a new target and theoretical basis for the treatment of HBV-related glomerulonephritis (HBV-GN). Blocking NTCP is a new target for the treatment of HBV-GN. We found that tacrolimus, a calcineurin inhibitor that blocks NTCP, can effectively treat HBV-GN. This study also provides a theoretical basis for the effective and safe treatment of immunosuppressant tacrolimus for HBV-GN.

Dataset Information

Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide.

Publications

Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide.

Similar Datasets

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