ZO-2 determines cell membrane localization of receptor NTCP and supports hepatitis B virus infection

Hepatitis B virus (HBV) infects hepatocytes by using sodium taurocholate cotransporting polypeptide (NTCP) as a receptor. NTCP can render non-permissive hepatocyte cell lines such as HepG2 permissive to HBV infection. However, only a few reports investigate the NTCP function in HBV infection beyond virus binding. Here, we performed immunopurification followed by liquid chromatography-tandem mass spectrometry to identify ZO-2 (aka TJP2) as a novel NTCP-binding protein. Knockdown or knockout of ZO-2 in NTCP-transduced HepG2 cells decreased the amount of NTCP at the cell surface, leading to reductions in cellular attachment and infection by HBV. Incubation of cells with HBV surface molecules preS1 resulted in the dissociation of NTCP from ZO-2 and in the formation of NTCP-preS1-actin complexes that were internalized into the cell. Latrunculin A, an inhibitor of actin polymerization, suppressed the preS1 internalization into hepatocytes and HBV infection. In conclusion, ZO-2, together with actin, regulates the function of NTCP as a receptor of HBV, providing a new model for HBV-cell interactions and virus internalization.

Although a number of candidates have been reported to bind to the hepatitis B virus (HBV) envelope, accumulating evidence indicates that NTCP is accepted as a functional receptor for HBV infection. Thus, NTCP is an attractive target for antiviral therapies. Here, we showed that ZO-2 interacts with NTCP. The silencing of ZO-2 decreased HBV infection, whereas ZO-1 and ZO-3 knockdown had no effect on HBV infection. Moreover, knockout of ZO-2 induced the downregulation of NTCP from the cell surface. This aberrant NTCP localization causes the reduction of the half-life of NTCP in ZO-2 knockout cells. PreS1 treatment or HBV infection disrupted the NTCP/ZO-2 complex through the dissociation of the actin-binding domain of ZO-2, leading to internalization of a newly formed preS1/NTCP/actin complex into the cell. The actin polymerization inhibitor latrunculin A suppressed HBV infection. These results suggest that ZO-2 regulates cell surface localization of NTCP.