We finally investigated the molecular determinants of NTCP for HBV and HDV infections. Crab-eating monkey (Macaca fascicularis) NTCP (mkNTCP) shares high protein sequence identity with hNTCP (96.3%) (Figure 7-figure supplement 1) . However, mkNTCP neither supports HDV infection nor pre-S1 peptide (Myr-59) binding ( Figure 7A) , consistent with the known narrow species specificity of the We then made a series of human NTCP variants to cover all the different amino acids between hNTCP and mkNTCP. In each variant, two or a few residues were mutated to their mkNTCP counterparts (Figure 7-figure supplement 1) . Whereas most mutations did not significantly interfere with Myr-59 binding or HDV infection, alteration of five residues of hNTCP between aa 157-165 and its monkey counterpart (from KGIVISLVL to GRIILSLVP, distinct residues are underlined) completely abolished Myr-59 binding and the ability to support HDV infection. Remarkably, replacing the motif of aa 167-156 in mkNTCP with the corresponding human residues converted mkNTCP to an efficient receptor for HDV infection ( Figure 7A) . All the NTCP variants tested were examined for NTCP expression, and comparable levels of cell surface expression were confirmed ( Figure 7B ). Similar to HDV, HBV infection was also abolished on HepG2 cells expressing hNTCP carrying monkey-like mutations, GRIILSLVP, while mkNTCP-bearing human residues KGIVISLVL within the motif of aa 157-165 largely restored HBV infection ( Figure 7C) . These data show that residues between 157 and 165 of NTCP are crucial for binding to the receptor-binding region of the pre-S1 domain of the L protein of HBV, and critically contribute to NTCP-mediated HBV and HDV infections.