We then investigated the ability of NTCP to render nonsusceptible cells susceptible to viral infection. NTCP mRNA expression is low in human hepatocarcinoma cell lines that are not susceptible to HBV or HDV infection. The levels of NTCP mRNA in Huh-7 and HepG2 cells were about 10,000 times lower than that in primary human and Tupaia hepatocytes ( Figure 5A ). We first examined if NTCP expression renders Huh-7 susceptible to HDV infection. Human NTCP-transfected Huh-7 cells supported HDV infection with an efficiency comparable to that of PTHs; nearly 10% of cells were infected as shown by staining of the HDV delta antigen that mainly locates in cell nuclei, whereas Huh-7 cells transfected with a vector plasmid allowed no HDV infection ( Figure 5B ). Moreover, the infection could be blocked by known HBV entry inhibitors, such as pre-S1 lipopeptide and hepatitis B immune globulin (HBIG), demonstrating a genuine infection of HDV mediated by HBV envelope proteins on these cells ( Figure 5C ). HDV RNAs, including antigenomic RNA that is only produced during HDV replication, rapidly increased over time in the infected cells ( Figure 5D ). Moreover, the infection efficiency correlated with both the inoculation dose of HDV ( Figure 5E ) and the expression level of hNTCP ( Figure 5F ). HDV also infected HepG2 cells transiently transfected with hNTCP ( Figure 5 -figure supplement 1) as well as a cell line established by G418 selection of HepG2 cells after hNTCP transfection, which expresses hNTCP stably and could be readily stained by the FITC-pre-S1 peptide ( Figure More efficient HBV infection was achieved on stable HepG2-hNTCP cells with about 5-10% of the cells being infected as revealed by intracellular staining of HBsAg, whereas there was no HBV infection in the parental HepG2 cells ( Figure 6A ). HBV infection in the HepG2-hNTCP stable cells was further evidenced by the continuously increased production of HBeAg during the testing period. HBV entry inhibitors, in particular the Myr-59 peptide and 17B9, efficiently inhibited the infection ( Figure 6B ). The infection efficiency as evidenced by HBV total and 3.5 kb RNA levels correlated with the inoculation dose. Moreover, the formation of HBV covalently closed circular DNA (cccDNA), which is a replicative intermediate and transcriptional template for production of viral RNAs, was confirmed by Southern blot analysis ( Figure 6D ). To further demonstrate that the replicative intermediates of HBV were synthesized de novo in HepG2-hNTCP cells after infection, we performed additional time course experiments. HBV viral replicative intermediates, including cccDNA, the 3.5 kb HBV RNA, as well as the total HBV RNA in the HBV-infected HepG2-hNTCP cells were quantified at different time points. The cccDNA became detectable at 24 hr post-infection. It markedly increased at day 3 post-infection and maintained a relatively stable level for the rest of the time points examined, whereas the formation of HBV cccDNA was completely abolished if entry inhibitor Myr-59 was included with the initial virus inoculation ( Figure 6E) . Consistently, HBV RNA levels of the 3.5 kb transcript and the total HBV viruses AAV8-HBV and Lenti-VSV-G. For HDV and HBV, PTHs were infected at 500 and 100 genome equivalent copies per cell, respectively. The level of HDV viral RNAs in infected cells was quantified by qRT-PCR on 6 dpi. Strand-specific primers were used to differentiate the HDV genomic and antigenomic RNAs (see 'Materials and methods'). For VSV-G control virus infection, recombinant lentivirus pseudotyped by VSV-G carrying a luciferase reporter was inoculated to PTHs 3 days after siRNA transfection. The luciferase activity was assessed on 6 dpi. For HBV infection, the kinetics of secreted viral antigens HBsAg and HBeAg were measured by ELISA. The medium was changed every 3 days. For AAV8-HBV infection, PTHs were infected with a recombinant AAV8 carrying 1.05× overlength HBV genome. Secreted HBeAg was assessed on indicated days post-infection. The effect of tsNTCP silencing in all viral infections was independently evaluated with a total of four siRNAs against tsNTCP (see 'Materials and methods'). The data shown are the result of a representative siRNA out of the four tested. (B) Differentiated HepaRG cells express high level of NTCP mRNA and knockdown NTCP in these cells inhibited HDV and HBV infections. HDV and HBV infection of siRNA-transfected HepaRG cells was conducted similarly as in panel A. HDV RNA levels in the infected cells were measured on 9 dpi. For HBV infection, secreted HBeAg was collected every 2 days as indicated and analyzed by ELISA. The copy numbers of HBV total RNA and 3.5 kb RNA in the infected cells were measured at the end of the experiment, 10 dpi. (C) Knockdown hNTCP in PHHs hampered HBV infection. Frozen PHHs were thawed and plated 1 day before transfecting with siRNAs against hNTCP or a control siRNA. Similar to panels A and B, 3 days after transfection, PHHs were inoculated with 100 genome equivalent copies of HBV per cell, and the levels of secreted HBeAg were determined at indicated dpi. HBV RNAs were quantified at the end of the experiment, 9 dpi. The knockdown efficiency of siRNA targeting tsNTCP or hNTCP shown in panels A-C was determined by real time RT-PCR on day 4 after transfection. NTCP: sodium taurocholate cotransporting polypeptide; HBV: hepatitis B virus; HDV: hepatitis D virus; PTH: primary Tupaia hepatocytes; tsNTCP: Tupaia NTCP; siRNA: small interfering RNA; dpi: days post-infection; hNTCP: human NTCP. DOI: 10.7554/eLife.00049.012 The Huh-7 was used to normalize the relative expression levels in other cells. (B) 1 × 10 5 Huh-7 cells were transfected with 100 ng hNTCP/pcDNA6 or a vector control in 24-well plate and maintained in PMM, 24 hr after transfection, transfected cells were infected with HDV at 500 genome equivalent copies per cell. On 8 dpi, HDV delta antigen, which typically locates in nuclei, was stained with 4G5 antibody in green, nuclei were stained with DAPI in blue. (C) Huh-7 cells transfected with hNTCP were infected with HDV similarly as in panel B in the presence or absence of HBV entry inhibitors: HBIG (hepatitis B immune globulin), Myr-59, and anti-HBsAg mAb, 17B9. 4G5 was used as an antibody control. HDV RNA copies of infected cells were quantified by real-time RT-PCR on 6 dpi. (D) Huh-7 cells transfected with hNTCP were infected with HDV similarly as in panel B. The HDV viral RNAs in infected cells at indicated time points were quantified by real-time RT-PCR. (E) HDV infection with increasing multiplicities of genome equivalents (mge). With 100 ng hNTCP/pcDNA6, 1 × 10 5 Figure 5 . Continued on next page transcripts in the infected HepG2-hNTCP cells gradually increased during first several days of infection and reached a steady level after day 5 ( Figure 6F ). Together these data show that NTCP contributes substantially to HBV infection.