Fig. 7: nHEV and eHEV particles require lysosomal cathepsin activity for cell entry. | Nature Communications

Fig. 7: nHEV and eHEV particles require lysosomal cathepsin activity for cell entry.

From: Integrin beta 1 facilitates non-enveloped hepatitis E virus cell entry through the recycling endosome

Fig. 7

(A) S10-3 LAMP-1-GFP cells were inoculated with eHEV (MOI = 20 GE/cell) or nHEV (MOI = 30 GE/cell) for 6 h or 2 h on ice and incubated at 37 °C for 10 h or 7 h, respectively. Genomes (magenta) were detected by RNA-FISH using the ORF1 probe. Scale bar = 5 μm. (B) and (C) S10-3 cells were treated with cathepsin inhibitor E64 or DMSO and infected with (B) nHEV (MOI = 30 GE/cell, n = 7) or (C) eHEV (MOI = 20 GE/cell, n = 6). E64 was added with virus for 24 h (“during”), or 24 h post-infection and throughout the course of infection (“after”). Infectivity was assessed 5 days post-infection. (D) and (E) S10-3 cells were treated with E64 or DMSO and infected with (D) nHEV (MOI = 30 GE/cell, n = 9) or (E) eHEV (MOI = 20 GE/cell, n = 7). 8 h later, the inoculum was replaced with fresh media containing drugs. 24 h post-inoculation, HEV capsid were detected by staining and genomes as in (A) and quantified using CellProfiler. (F) Maximum projections of E64-treated S10-3 LAMP-1-GFP cells inoculated with nHEV (MOI = 30 GE/cell). 24 h later, capsids (magenta) and genomes (yellow) were detected as in (C). Representative of n = 6 microscope fields. (G) S10-3 LAMP-1-GFP cells were treated with E64 or DMSO 30 min prior to inoculation with nHEV (MOI = 30 GE/cell). After 8 h at 37 °C, inoculum was replaced with fresh media containing drugs. Genomes were detected and analysed as in (A). (H) Proposed working model on HEV cell entry. The interaction of nHEV with ITGB1 triggers internalisation through Rab11+ recycling endosomes, while eHEV is routed into Rab5a+ early endosomes. Both particles traffic through Rab7+ late endosomes and reach Lamp1+ lysosomes. The capsid and envelope are degraded by lysosomal cathepsins, allowing the release of viral genomes into the cytosol through an unknown penetration mechanism. This figure was created in BioRender. Dao Thi, V. (2025). https://BioRender.com/f6k0uqw. All replicates are from three independent experiments. Statistical analysis was performed by unpaired two-tailed Student’s t test (A, D, E) or one-way ANOVA (B, C, G). ****: p < 0.0001; ns, non-significant.

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