(A) Pre-S1 binding and HDV infection on cells expressing wild-type or mutant NTCPs. Corresponding amino acids (one-letter form) at the mutated positions of NTCP are shown for hNTCP, crab-eating monkey NTCP (mkNTCP), and tsNTCP. Huh-7 cells were transfected with plasmids encoding tsNTCP, hNTCP, mkNTCP, or NTCP mutants as indicated. The mutant NTCPs include hNTCP-bearing mutations of mkNTCP residues and mkNTCP-bearing mutations of human residues at indicated positions. The transfected cells were maintained in PMM for 24 hr and then either stained with 200 nM FITC-pre-S1 or infected with 500 mge HDV. HDV delta antigen in infected cells was detected with mAb 4G5 on 7 dpi. Replacing aa 157–165 of mkNTCP with human counterpart rendered mkNTCP an efficient receptor for pre-S1 binding and HDV infection. (B) All NTCP variants expressed comparable levels of NTCP. Huh-7 cells transfected as in panel A were biotinylated 24 hr after the transfection, then lysed and analyzed for cell surface NTCP expression (top), total NTCP expression (middle), and GAPDH (bottom), respectively. For cell surface expression, cell lysates were pulled down with streptavidin T1 Dynabeads and subsequently examined by western blot with mAb 1D4 recognizing a C9 tag at the C-terminus of each NTCP variant. For total NTCP expression, cell lysates were directly subjected to SDS-PAGE, followed by Western blot analysis with 1D4. (C) Effects of NTCP mutations on HBV infection. HepG2 cells were transfected with plasmids encoding hNTCP, mkNTCP, or hNTCP variants bearing the indicated monkey residues, or mkNTCP variants with the indicated human residues. Transfected cells were maintained in PMM for 24 hr, and subsequently infected with HBV at 100 mge. HBeAg and HBV 3.5 kb RNA were assayed on 6 dpi. Similar to panel B, comparable NTCP surface expression levels in the transfected HepG2 cells were confirmed for all the NTCP variants tested (Figure 7—figure supplement 2). NTCP: sodium taurocholate cotransporting polypeptide; HDV: hepatitis D virus; hNTCP: human NTCP; tsNTCP: Tupaia NTCP; PMM: primary hepatocytes maintenance medium; mge: multiplicities of genome equivalents; mAb: monoclonal antibody; HBV: hepatitis B virus; HBeAg: HBV e antigen; dpi: days post-infection.