Scientific Reports 5, Article number: 17047 (2015)
doi:10.1038/srep17047
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Hepatitis B | Hepatitis B virus
Received:
30 July 2015
Accepted:
07 October 2015
Published online:
23 November 2015
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.
Introduction
Hepatitis B virus (HBV) chronically infects approximately 3.4% of the world’s population and is a major factor for hepatocellular carcinoma following liver cirrhosis1. Interferon-alpha or nucleot(s)ide analogue inhibitors against the viral reverse transcriptase are currently approved for therapy for hepatitis B patients; however, these therapies are not necessarily effective for all such patients due to side effects and the emergence of escape mutant virus2. Thus, the development of new antiviral drugs that target several factors is still needed to prevent the liver diseases caused by HBV infection. Reliable and inexpensive cell culture systems and animal models of HBV infection are required in investigations of the underlying infection mechanism and pathogenesis of HBV. Although primary human hepatocytes (PHH), primary Tupaia belangeri hepatocytes (PTH), and the HepaRG cell line3 have been used as ex vivo HBV infection systems, these are typically employed under limited conditions, are expensive, and have difficulties maintaining stable susceptibility to HBV infection.
The HBV nucleocapsid is enveloped by a lipid bilayer enclosed within glycoproteins: the large (L), middle (M), and small (S) proteins of the HBV surface antigen (HBs)4. The L protein consists of preS1 and preS2 domains and the S protein, while the M protein consists of the preS2 domain and the S protein4. The S protein of the HBV virion has been shown initially, but weakly, to attach to heparan sulfate proteoglycans on hepatocytes5,6. Infection by HBV or hepatitis D virus (HDV) was previously reported to be neutralized by the antibody reacting to the preS1 region7 or by the myristoylated or acylated synthetic peptide composed of 47 N-terminal amino acids of the preS1 domain8,9,10, suggesting that the preS1 domain of the L protein is responsible for binding to the putative entry receptor(s). The sodium taurocholate cotransporting polypeptide (NTCP) was recently identified as a functional receptor for HBV and HDV because the myristoylated N-terminal region of the preS1 domain bound to NTCP and expression of NTCP rendered the HepG2 cell line susceptible to HBV infection11. The N-terminally myristoylated synthetic peptide corresponding to the region spanning from amino acid residue (aa) 2 to 48 of preS1 has been shown to interact with NTCP with high affinity11. The region spanning from aa 157 to 165 of NTCP was responsible for HBV infection and preS1 binding, while the region from aa 84 to 87 was for HBV infection but not preS1 binding11,12,13,14, suggesting that the region from aa 84 to 87 plays a role in a post-attachment step. Differences in these regions may determine host specificity for a member of the family Hepadnaviridae. Previous studies also suggested that the expression of NTCP provides HBV infectivity in the HepG2 cell line11,15,16,17. In the reported models, HBV could infect NTCP-expressing hepatoma cell lines under adherent monolayer-cell conditions11,15,16,17. However, NTCP-expressing HepG2 cells showed susceptibility to HBV infection compared with the parent cell line HepG2, but its infectivity was not high, which was indicated in the review process11. Schulze et al. reported that treatment with EGTA increased HBV infectivity in HepaRG cells18, suggesting that loosening of cell-cell junctions may promote HBV infectivity. Several reports suggest that NTCP is mainly expressed at the basolateral membrane of hepatocytes19,20,21. Thus, we hypothesized that the sufficient disruption of cell-cell junctions would expose NTCP to HBV virions in the medium, thereby promoting infectivity.
In the present study, we found lateral expression of NTCP in HepG2 cells transfected with the human SLC10A1 gene (NTCP gene), investigated the effect of non-adherent cell conditions on HBV infection, and then established a novel cell culture system for NTCP-dependent HBV infection. We also examined the effects of several compounds on HBV infectivity by using our culture system. 作者: StephenW 时间: 2015-11-24 16:58