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. 2012 Nov 13;1:e00049. doi: 10.7554/eLife.00049

Figure 5. NTCP expression confers Huh-7 susceptibility to HDV infection.

(A) NTCP mRNA expression level in the indicated cell lines and primary hepatocytes. The Huh-7 was used to normalize the relative expression levels in other cells. (B) 1 × 105 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 × 105 Huh-7 cells were transfected, as in panel B. Transfected cells were infected with increasing mge of HDV as indicated. HDV delta antigen was detected as in panel B on 8 dpi. (F) HDV infection of cells with increasing levels of hNTCP. About 1 × 105 Huh-7 cells were transfected with a vector pcDNA6 or hNTCP/pcDNA6 at indicated amounts and cells were inoculated with 500 mge of HDV. HDV delta antigen was detected on 8 dpi as in panel B. NTCP: sodium taurocholate cotransporting polypeptide; PMM: primary hepatocytes maintenance medium; HBV: hepatitis B virus; HDV: hepatitis D virus; mAb: monoclonal antibody; HBsAg: HBV S antigen.

DOI: http://dx.doi.org/10.7554/eLife.00049.013

Figure 5.

Figure 5—figure supplement 1. Infection of HDV on HepG2 cells expressing hNTCP.

Figure 5—figure supplement 1.

About 2 × 105 HepG2 cells were transiently transfected with 0.3 µg hNTCP/pcDNA6 or a vector control and maintained in PMM. Cells were infected at 500 mge with HDV 24 hr after transfection. On 8 dpi, HDV delta antigen in infected cells was stained with mAb 4G5 (left) and the HDV viral RNAs were determined by real time RT-PCR. The competing Myr-59 peptide was tested at 200 nM (right).
DOI: http://dx.doi.org/10.7554/eLife.00049.014
Figure 5—figure supplement 2. Infection of HDV on HepG2 cells expressing hNTCP.

Figure 5—figure supplement 2.

HepG2 stable cell line stably expressing hNTCP (HepG2-hNTCP) was established by G418 selection after transient transfection. The expression of hNTCP was confirmed by staining with FITC-pre-S1 peptide.
DOI: http://dx.doi.org/10.7554/eLife.00049.015
Figure 5—figure supplement 3. Infection of HDV on HepG2 cells expressing hNTCP.

Figure 5—figure supplement 3.

Infection of HDV on HepG2-hNTCP stable cells was conducted in the presence or absence of entry inhibitor 200 nM Myr-59. HDV total RNA, HDV genomic RNA, and antigenomic RNA were assayed with real time qPCR on 8 dpi.
DOI: http://dx.doi.org/10.7554/eLife.00049.016