Background: HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence. Methods: The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane. Results: We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells. Conclusions: LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity. Export

Hepatitis B virus (HBV) specifically infects hepatocytes and has a complex life cycle owing to the stabilization and pooling of covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. We previously reported that the suppression of dedicator of cytokinesis 11 (DOCK11) decreases cccDNA and HBV-DNA levels and identified it as a new HBV therapeutic target. The DOCK11-associated gene, Wnt/β-catenin signaling regulator tankyrase (TNKS), was identified using in vitro methods; however, its function in the HBV life cycle remains unknown. Here, we used various inhibitors, antagonists, and short-hairpin RNA treatments related to TNKS signaling in HBV-infected hepatocytes. The role of TNKS-related Wnt/β-catenin signaling in the HBV life cycle was evaluated using immunoprecipitation assays with DOCK11 and bulk RNA sequencing methods. TNKS and Wnt/β-catenin signaling inhibitors significantly repressed cccDNA and HBV-DNA levels. Conversely, certain Wnt/β-catenin signaling agonists enhanced the HBV life cycle. DOCK11 directly binds to β-catenin to regulate HBV using its nuclear transport system. SKL2001, normally used as a Wnt/β-catenin signaling agonist, strongly reduced cccDNA in HBV-infected hepatocytes and in combination with entecavir predominantly eradicated HBV without cytotoxicity. Therefore, DOCK11 and other Wnt/β-catenin signaling molecules may be therapeutic targets to prevent persistent HBV infection.

We developed a novel hepatitis B virus (HBV) infection-monitoring system using a luminescent, 11-amino acid reporter (HiBiT). We performed high-throughput antiviral screening using this system to identify anti-HBV compounds. After the infection of primary human hepatocytes with the recombinant virus HiBiT-HBV, which contains HiBiT at its preS1, 1262 compounds were tested in a first screening using extracellular HiBiT activity as an indicator of viral infection. Following a second screening, we focused on the compound skimmianine, which showed a potent antiviral effect. When skimmianine was added at the same time as HiBiT-HBV infection, skimmianine inhibited HiBiT activity with EC₅₀ of 0.36 pM, CC₅₀ of 1.67 μM and a selectivity index (CC₅₀:EC₅₀ ratio) of 5,100,000. When skimmianine was added 72 h after HiBiT-HBV infection, the EC₅₀, CC₅₀ and selectivity index were 0.19 μM, 1.87 μM and 8.79, respectively. Time-lapse fluorescence imaging analysis using another recombinant virus, ReAsH-TC155HBV, with the insertion of tetra-cysteine within viral capsid, revealed that skimmianine inhibited the accumulation of the capsid into hepatocytes. Furthermore, skimmianine did not inhibit either attachment or internalization. These results imply that skimmianine inhibits the retrograde trafficking of the virus after internalization. This study demonstrates the usefulness of the recombinant virus, HiBiT-HBV, for high-throughput screening to identify anti-HBV compounds.

Hepatitis B virus (HBV) specifically infects hepatocytes and has a complex life cycle owing to the stabilization and pooling of covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. We previously reported that the suppression of dedicator of cytokinesis 11 (DOCK11) decreases cccDNA and HBV-DNA levels and identified it as a new HBV therapeutic target. The DOCK11-associated gene, Wnt/β-catenin signaling regulator tankyrase (TNKS), was identified using in vitro methods; however, its function in the HBV life cycle remains unknown. Here, we used various inhibitors, antagonists, and short-hairpin RNA treatments related to TNKS signaling in HBV-infected hepatocytes. The role of TNKS-related Wnt/β-catenin signaling in the HBV life cycle was evaluated using immunoprecipitation assays with DOCK11 and bulk RNA sequencing methods. TNKS and Wnt/β-catenin signaling inhibitors significantly repressed cccDNA and HBV-DNA levels. Conversely, certain Wnt/β-catenin signaling agonists enhanced the HBV life cycle. DOCK11 directly binds to β-catenin to regulate HBV using its nuclear transport system. SKL2001, normally used as a Wnt/β-catenin signaling agonist, strongly reduced cccDNA expression in HBV-infected hepatocytes and in combination with entecavir predominantly eradicated HBV without cytotoxicity. Therefore, DOCK11 and other Wnt/β-catenin signaling molecules may be therapeutic targets to prevent persistent HBV infection.