Figure 3

NPD8716 inhibited HBV preS1 attachment to host hepatocytes. (A) HBV replication was evaluated by detecting nucleocapsid-associated HBV DNA in Hep38.7-Tet cells treated with or without the indicated compounds (200 μM NPD8716, 100 nM entecavir, 100 nM preS1 peptide) under tetracycline depletion for 6 days by southern blot analysis. rcDNA and ssDNA show HBV relaxed circular DNA and single strand DNA, respectively. (B) HBV preS1 attachment to HepG2-hNTCP-C4 cells were evaluated by treating cells with TAMRA-labeled preS1 peptide in the presence or absence of the indicated compounds (100 nM preS1 peptide, or 200 μM NPD8716) for 30 min. Red and blue signals show TAMRA-labeled preS1 peptide and the nucleus, respectively. NPD8716 was suggested to inhibit HBV preS1-mediated host cell attachment. (C) Hepatitis D virus (HDV) infection assay. HepG2-hNTCP-C4 cells were inoculated with HDV in the absence or presence of compounds (100 nM pre-S1 peptide or 200 μM NPD8716) as shown in Materials and Methods. HBV infection was evaluated with HDV RNA levels in the cells quantified by real-time RT-PCR analysis. (D) Hepatitis C virus (HCV) infection assay. HCV entry was evaluated by HCV pseudoparticle system as described in Methods upon treatment with or without 10 nM bafilomycin A1 or 200 μM NPD8716.