Fig 2: PIKfyve kinase activity maintains endolysosomal organisation and function required during HEV entry. (A–B) HepG2/C3A cells were treated with DMSO or 100 nM apilimod for 5 hours. Cells were then stained with LysoTracker (A) or immunostained for either LAMP1 or CD63 (B). Scale bars=10 µm. (C) Left: representative RNA fluorescence in situ hybridisation (RNA-FISH) images of HepG2/C3A cells treated with either DMSO or apilimod (100 nM) and inoculated with neHEVCC (Kernow-C1/p6) for 5 hours. HEV RNA was visualised using an ORF1-specific probe, and cells were counterstained with LAMP1 (green) and DAPI (blue). Scale bars=10 µm (full size images) and 5 µm (insets). Right: quantification of HEV RNA-positive LAMP1 vesicles per cell (53 to 58 cells per condition from three independent experiments). Effect size was calculated as Cohen’s d (d). (D) HepG2/C3A lysates were immunoblotted for cathepsin B (CTSB), cathepsin L (CTSL) and GAPDH. (E) Quantification of CTSL (left) and CTSB (right) maturation expressed as the ratio of pro-CTS to mature CTS (mean±SD; n=3). Statistical significance against DMSO was determined using a one-way ANOVA with Dunnett’s post hoc test (****p<0.0001, ***p<0.001). (F) Left: HepG2/C3A cells were incubated with the green B cathepsin substrate for 24 hours in the presence of DMSO, apilimod or K11777 (500 nM each). Nuclei were stained with Hoechst. Scale bars=100 µm. Right: Quantification of RNA spots per cell (3772 to 6.963 cells per condition from three independent experiments). Effect size was calculated as Cohen’s d (d). ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; HEV, hepatitis E virus.

Fig 3: PIKfyve kinase activity is required during HEV entry. (A) Left: effect of apilimod (16 nM), YM201636 (80 nM) and APY0201 (16 nM) on replication of an HEV subgenomic reporter replicon (Kernow-C1/p6-Gaussia luciferase). Normalised relative light units (RLU; mean±SD; n=3) were measured 72 hours post-electroporation, relative to DMSO control. Right: normalised cell viability (%) following treatment with apilimod, YM201636 and APY0201, relative to DMSO (mean±SD; n=3). (B) Schematic of the time-of-drug-addition assay. HepG2/C3A cells were inoculated with non-enveloped HEVCC (Kernow-C1/p6) at 0 hours and treated with DMSO, apilimod, K11777, YM201636, APY0201 (all 100 nM) or RBV (25 µM) at different time points: 1 hour before infection (−1 hour, h.b.i.), at infection (0 hours), or 2, 4, 8, 12, 16, 24, 48 and 72 hours post-infection (h.p.i.), followed by fixation at 96 h.p.i. At 8 h.p.i., the inoculum was removed and replaced with fresh drug-containing medium. RBV served as a positive control for replication inhibition and K11777 served as a positive control for entry inhibition. (C) Inhibitory effects of different compounds on HEV infection when added either before infection (h.b.i.) or after infection (h.p.i.) of HepG2/C3A cells. Statistical significance against DMSO was determined using a one-way ANOVA with Dunnett’s post hoc test (****p<0.0001, ***p<0.001, **p<0.01 and *p<0.05). ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; HEV, hepatitis E virus; RBV, ribavirin.

Fig 4: Inhibition of PIKfyve kinase activity restricts HEV infection. (A–C) Left: Immunofluorescence images of HepG2/C3A cells at 3 days post-infection (d.p.i.) with non-enveloped HEVCC (Kernow-C1/p6) after treatment with apilimod (A), YM201636 (B) and APY0201 (C) at the indicated concentrations or DMSO as vehicle control. HEV ORF2 is shown in green and nuclei (DAPI) in blue. Scale bars=100 µm. Right: dose-dependent effects of apilimod (A), YM201636 (B) and APY0201 (C) in HepG2/C3A cells at 3 d.p.i. with non-enveloped HEVCC (Kernow-C1/p6). Coloured lines represent compound effects; black dotted lines indicate cell viability (mean±SD; n=3). (D) Full well images of HepG2/C3A cells infected with enveloped HEVCC (Kernow-C1/p6) on treatment with apilimod, YM201636, APY0201, the cathepsin inhibitor K11777 or DMSO. Images were stitched and processed in FIJI. ORF2-positive cells are indicated in black. (E) Quantification of HEV infectivity, relative to DMSO (mean±SD; n=3). Statistical significance was determined using a one-way ANOVA with Dunnett’s post hoc test. (F) Immunofluorescence images of HepG2/C3A cells at 3 d.p.i. with non-enveloped HEVCC (83-2-27) treatment with apilimod, YM201636, and APY0201, the cathepsin inhibitor K11777 (100 nM) or DMSO. HEV ORF2 is shown in green and nuclei (DAPI) in blue. Scale bars=100 µm. (G) Quantification of HEV infectivity, relative to DMSO (mean±SD; n=3). Statistical significance was determined using a one-way ANOVA with Dunnett’s post hoc test. (****p<0.0001, ***p<0.001 and *p<0.05, non-significant values are not shown). ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; HEV, hepatitis E virus.