Niemann-Pick C1 maintains cholesterol homeostasis in late endosomes to drive hepatitis B virus endosomal escape.

Hepatitis B virus (HBV) infection poses a major global health burden, as current nucleos(t)ide analogs and interferon-alpha (IFN-α) cannot eradicate the viral reservoir. Host factors play an indispensable regulatory role in enabling HBV to establish persistent infection. While host factors governing early HBV infection processes-including attachment, endocytic internalization, endosomal trafficking, and covalently closed circular DNA (cccDNA) formation-are largely elucidated, the regulators mediating HBV release from late endosomes remain elusive. This study investigates the role of Niemann-Pick C1 (NPC1), a late endosomal cholesterol transporter, in early HBV infection. Using primary human hepatocytes and HepG2-NTCP cells, we demonstrate that NPC1 knockdown prior to infection significantly reduces HBV susceptibility, as measured by cccDNA, viral RNAs and antigen levels, whereas NPC1 overexpression exerts only a modest enhancing effect. Moreover, NPC1 depletion did not impair viral transcription or replication, but caused HBV retention in late endosomes independent of viral-endosomal membrane fusion, accompanied by late endosomal cholesterol accumulation. Expression of cholesterol transport-defective NPC1 mutant (P691S) failed to restore HBV infection in NPC1-deficient cells, while HβCD-mediated cholesterol efflux rescued viral release from late endosomes. Finally, pharmacological inhibition of NPC1 using either U18666A or itraconazole potently inhibited HBV infection in dose-dependent manner. Collectively, these findings establish that NPC1 is a novel host factor that promotes HBV escape into the cytoplasm by sustaining late endosomal cholesterol homeostasis, providing a potential target for anti-HBV intervention.