We next cloned human and Tupaia NTCPs and validated the binding of the exogenously expressed NTCPs with the WT b peptide and an N-terminal myristoylated pre-S1 peptide with native residues. Both human NTCP (hNTCP) and tsNTCP could be efficiently cross-linked by WT b but not N9K b when expressed in 293T cells as shown by Western blotting with the anti-WT b antibody 2D3 as well as an anti-C9 antibody recognizing the C-terminal C9 tag of the recombinant hNTCP and tsNTCP proteins ( Figure 3A ). WT b but not the control N9K b peptide bound to 293T cells expressing a green fluorescent protein (GFP)-tagged tsNTCP (tsNTCP-EGFP) and co-localized with tsNTCP-EGFP on the cell surface. This binding was readily competed off by the free Myr-47/WT peptide ( Figure 3B ). Moreover, a native pre-S1 peptide specifically recognized the human hepatocellular carcinoma Huh-7 cell line transfected with hNTCP ( Figure 3C ). Consistently, Huh-7 cells transfected with either tsNTCP or hNTCPs had markedly increased HDV binding to the cells. The Myr-47/WT peptide readily competed with binding of the wild-type HDV, whereas a noninfectious mutant HDV virus bearing a single N9K mutation in the pre-S1 domain of its L envelope protein failed to bind either hNTCP-or tsNTCPexpressing Huh-7 cells ( Figure 3D) . Collectively, these data demonstrated a specific interaction between NTCP and the pre-S1 domain of the L protein, which directly mediates the binding of HDV virions to target cells. 