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

Abstract

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.