西辻 裕紀

ABSTRACT Hepatitis B virus (HBV) infects hepatocytes by using sodium taurocholate cotransporting polypeptide (NTCP) as a receptor. NTCP can render non-permissive hepatocyte cell lines such as HepG2 permissive to HBV infection. However, only a few reports investigate the NTCP function in HBV infection beyond virus binding. Here, we performed immunopurification followed by liquid chromatography-tandem mass spectrometry to identify ZO-2 (aka TJP2) as a novel NTCP-binding protein. Knockdown or knockout of ZO-2 in NTCP-transduced HepG2 cells decreased the amount of NTCP at the cell surface, leading to reductions in cellular attachment and infection by HBV. Incubation of cells with HBV surface molecules preS1 resulted in the dissociation of NTCP from ZO-2 and in the formation of NTCP-preS1-actin complexes that were internalized into the cell. Latrunculin A, an inhibitor of actin polymerization, suppressed the preS1 internalization into hepatocytes and HBV infection. In conclusion, ZO-2, together with actin, regulates the function of NTCP as a receptor of HBV, providing a new model for HBV-cell interactions and virus internalization. IMPORTANCE Although a number of candidates have been reported to bind to the hepatitis B virus (HBV) envelope, accumulating evidence indicates that NTCP is accepted as a functional receptor for HBV infection. Thus, NTCP is an attractive target for antiviral therapies. Here, we showed that ZO-2 interacts with NTCP. The silencing of ZO-2 decreased HBV infection, whereas ZO-1 and ZO-3 knockdown had no effect on HBV infection. Moreover, knockout of ZO-2 induced the downregulation of NTCP from the cell surface. This aberrant NTCP localization causes the reduction of the half-life of NTCP in ZO-2 knockout cells. PreS1 treatment or HBV infection disrupted the NTCP/ZO-2 complex through the dissociation of the actin-binding domain of ZO-2, leading to internalization of a newly formed preS1/NTCP/actin complex into the cell. The actin polymerization inhibitor latrunculin A suppressed HBV infection. These results suggest that ZO-2 regulates cell surface localization of NTCP.

Hepatitis B virus (HBV) infection remains a major global health challenge. While sodium taurocholate co-transporting polypeptide (NTCP) is the primary receptor for HBV entry, the molecular mechanisms regulating NTCP-mediated viral entry remain incompletely understood. Here, we identified CD46 as a crucial regulatory factor for NTCP membrane expression. We found that CD46 interacted with NTCP in cis at the plasma membrane through proximity-based labeling screening. The depletion of CD46 significantly reduced cell-surface NTCP levels and HBV infection in hepatocytes. Anti-CD46 monoclonal antibodies, particularly clone E4.3, inhibited HBV infection by triggering NTCP internalization from the plasma membrane to intracellular vesicles. The antiviral effect of CD46 antibodies was also confirmed in primary human hepatocytes. Our study reveals a previously unknown mechanism regulating NTCP-mediated HBV entry and suggests CD46 as a potential therapeutic target for HBV infection.

Hepatitis B virus (HBV) infection is a major global health burden worldwide despite the availability of an effective vaccine and effective anti-HBV drugs. The currently approved anti-HBV drugs-i.e., nucleos(t)ide analogues and pegylated interferon α-can effectively suppress HBV replication, but rarely achieve a functional cure. Accordingly, new anti-HBV agents targeting different aspects of the HBV life cycle are needed. In this study, we screened for anti-HBV agents using the recombinant HBV expressing NanoLuc (NL) reporter gene (HBV/NL) and our original synthetic heterocyclic compound library. As a result, we identified a synthetic bile acid derivative, SO-145, as a potential novel anti-HBV agent, and investigated its effects in several cellular models of HBV. Treatment of HepG2-NTCP-C4 cells with SO-145 suppressed their NL activity following infection with HBV/NL. SO-145 suppressed HBV replication in PXB-cells infected with HBV genotype D, but did not show any inhibitory effect on HBV replication in Hep38.7-Tet cells. These results suggest that SO-145 specifically inhibits the early phase of the HBV life cycle. In other experiments, SO-145 was also shown to inhibit hepatitis D virus infection. Immunofluorescence analysis using fluorescent-labeled preS1 peptide revealed that SO-145 does not inhibit the preS1 attachment to the NTCP, but does markedly inhibit the HBV/preS1 internalization. Moreover, SO-145 does not inhibit the bile acid uptake facilitated by NTCP. Further mechanistic analysis suggested that SO-145 interferes with the NTCP oligomerization. Taken together, these results suggest that SO-145 inhibits HBV entry into hepatocytes by interfering with the NTCP oligomerization.

BACKGROUND AND AIMS: HBV leads to severe liver diseases, such as cirrhosis and HCC. Identification of host factors that regulate HBV replication can provide new therapeutic targets. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV entry receptor has enabled the establishment of hepatic cell lines for analyzing HBV infection and propagation. Using this new system, studies aimed at identifying host factors that regulate HBV propagation have increased. APPROACH AND RESULTS: We established an HBV-based-reporter gene expression system that mimics HBV replication from transcription to virus egress. Using this approach, we screened 1827 Food and Drug Administration-approved compounds and identified glycogen synthase kinase 3 (GSK3)alpha/beta inhibitors, including AZD1080, CHIR-98014, CHIR-98021, BIO, and AZD2858, as anti-HBV compounds. These compounds suppressed HBeAg and HBsAg production in HBV-infected human primary hepatocytes. Proteome analysis revealed that GSK3alpha/beta phosphorylated forkhead box K1/2 (FOXK1/2)s. A double-knockout of FOXK1/2 in HBV-infected HepG2-NTCP cells reduced HBeAg and HBsAg production. The rescue of FOXK2 expression, but not FOXK1 expression, in FOXK1/2-double-knockout cells restored HBeAg and HBsAg production. Importantly, phosphorylation of FOXK2 at Ser 424 is required for GSK3alpha/beta-mediated HBeAg and HBsAg production. We observed the binding of FOXK2 to HBV DNA in HepG2-NTCP cells. CONCLUSIONS: Our recombinant HBV-based screening system enables the discovery of new targets. Using our approach, we identified GSK3 inhibitors as potential anti-HBV agents.