Hepadnavirus tropism determined by diversified NTCP functionality in Feline species

Hepatitis B virus (HBV) exhibits a narrow host range, largely determined by sodium taurocholate cotransporting polypeptides (NTCP), a hepatic bile acid transporter acting as the HBV receptor. NTCP among animals appears to have evolved following trade-off mutations to lose its viral receptor activity while preserving its transporter function. Here, through profiling of the NTCP homologs from 23 mammalian species, we uncover unexpected functional heterogeneity of feline NTCPs. NTCPs derived from cat, tiger, and leopard supported viral preS1 binding and HBV infection, whereas lion-NTCP was deficient for preS1 binding and, notably, exhibited markedly reduced bile acid transport. Cat-NTCP was further demonstrated to have viral receptor function by productive infection in primary cat hepatocytes. Experimental mutagenesis identified double substitution at positions 191 and 268 as key determinants: introducing lion-type substitutions into cat-NTCP (G191R and T268A) abolished preS1 binding, whereas reciprocal substitutions in lion-NTCP restored receptor function. Molecular dynamics simulations suggested that lion-NTCP adopts conformation in which a transmembrane helix bending enlarges the whole bile acid tunnel and disrupts preS1 compatibility. Such differences also determined host susceptibility to another feline-infected domestic cat HBV. These findings reveal diversified NTCP functionality with evolutionary plasticity that acquires resistance to hepadnavirus infection at the expense of physiological transporter function.