The hepatitis E virus (HEV) poses a major global health challenge; however, no specific antiviral treatments are available, and the understanding of viral life cycle steps including cellular entry is very limited. While it is known that HEV infection depends on endolysosomal trafficking of virions to lysosomal compartments to initiate host cell entry, the underlying molecular determinants governing this process remain poorly understood.

Using a mini-arrayed CRISPR/Cas9 screen, we identified the lysosomal cholesterol transporter Niemann–Pick disease type C1 (NPC1) as a novel host dependency factor required for HEV infection. The importance of NPC1 for enveloped and non-enveloped HEV infection was confirmed through siRNA-mediated knockdown and CRIPSR/Cas9 mediated knockout. Moreover, pharmacological inhibition of NPC1 with itraconazole or U18666A efficiently lowered HEV infection of different strains and genotypes in vitro. Furthermore, NPC1 inhibition in primary human hepatocytes significantly decreased HEV infectivity and led to reduced viral RNA levels in feces in HEV-infected gerbils. Time-of-addition and subgenomic replicon assays revealed a role for NPC1 during viral entry without impacting replication. Ultimately, mechanistic studies using fluorescence microscopy indicate that NPC1 disruption leads to cholesterol accumulation and lysosomal enlargement, trapping viral particles and RNA within endo-/lysosomal compartments.

Collectively, our findings identify NPC1 as a critical host factor required for HEV entry and suggest that repurposing NPC1-targeting drugs could offer a promising new antiviral strategy for HEV treatment.