Identification of molecular determinants governing KIF4 cargo recognition of NTCP and their role in HBV/HDV infection

The surface availability of the sodium taurocholate co-transporting polypeptide (NTCP), which serves as entry receptor for Hepatitis B virus (HBV) and Hepatitis D virus (HDV), is a key determinant of HBV/HDV infectivity. We previously identified the Kinesin motor protein KIF4 as a key regulator of NTCP trafficking to the plasma membrane. However, the molecular mechanism by which NTCP is recognized as a KIF4 transport cargo has remained undefined. Here, we identified a leucine-rich motif, LLLI (aa 298-301), within the C-terminal transmembrane domain of NTCP as an essential determinant for KIF4 transport. Co-immunoprecipitation analyses revealed its importance for NTCP interaction with KIF4. This motif is conserved in another established KIF4 cargo, integrin {beta}1 (ITGB1), and mutation of the corresponding residues similarly disrupted ITGB1-KIF4 interaction, indicating a shared cargo recognition mechanism employed by KIF4. Functionally, disruption of the NTCP leucine-rich motif markedly impaired NTCP surface localization without affecting total protein expression, resulting in severe attenuation of HBV and HDV infection. Structural modeling and computational protein-protein interaction analysis indicated that the mutation does not alter NTCP structure or NTCP/preS1 binding affinity, supporting a trafficking-specific defect. Substitution mutations identified L298 and L299 as the minimal residues required for interaction with KIF4. Our findings define a recognition motif that regulates KIF4-dependent cargo identification and surface trafficking. It also underscores the potential of targeting KIF4-mediated NTCP recognition and transport to inhibit HBV and HDV entry. ImportanceHepatitis B and D viruses require the hepatocyte surface receptor NTCP for entry. We identified a conserved leucine-rich motif (LLLI, aa 298-301) in NTCP that is necessary for its recognition by the kinesin motor KIF4; disrupting this motif selectively impairs NTCP surface trafficking and markedly attenuates HBV/HDV infection without reducing total NTCP or intrinsic receptor function. Importantly, we identified a similar motif in integrin {beta}1 (ITGB1; aa 747-750) that is required for interaction with KIF4, revealing a similar cargo selection mechanism for different proteins. Scientifically, these findings define a motif-derived cargo-selection determinant implied by KIF4, advancing a general principle for kinesin-dependent cargo recognition. More broadly, this work highlights a possible host-directed antiviral approach that targets cargo-selection interfaces and provides a conceptual framework for manipulating kinesin-mediated trafficking of clinically relevant membrane proteins beyond NTCP, with implications for receptor regulation and membrane protein therapeutics.