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

Abstract 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 β1 (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. Importance Hepatitis 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 β1 (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.