Fig 1.
Inhibition of de novo HBV infection by MnCl2 in HepG2-NTCP cells.
(A) HepG2-NTCP cells were treated with or without MnCl2 (40 μM) for 10 h, followed by an additional 12 h incubation with HBV inoculum (see Materials and Methods). Left, secreted HBeAg was determined at the indicated time points (n = 3). The dotted line indicates the cutoff value. Right, representative images of immunofluorescence labeling of intracellular HBc (red) at 7 days post-inoculation (dpi). Cell nuclei were stained with DAPI (blue). (B) Dose-response curve showing the cytotoxicity of MnCl2 in HepG2-NTCP cells determined by the CCK8 assay performed in triplicate. The CC50 value was calculated with GraphPad Prism software. (C) Time-of-addition assay of the effect of Mn2+ (40 μM) on de novo HBV infection of HepG2-NTCP cells. Upper, the experimental scheme. Middle, supernatant HBeAg levels at the indicated time points were plotted. Comparisons were made between pre-infection and post-infection treatment. Lower, immunofluorescence staining of HBc as described in (A). (D and E) HepG2 cells were transfected with a plasmid pHBV1.3 carrying a 1.3-mer overlength HBV genome. After 6 h, the cells were treated with different concentrations of MnCl2 for further 3 days. Capsid-associated HBV DNA species were assessed by Southern blotting, with two replicates at each concentration, representing three independent experiments. RC, relaxed circular DNA; DSL, double-stranded linear DNA; SS, single-stranded linear DNA (D). Viral protein expression and secretion were assessed by immunoblotting (two replicates at each concentration) and ELISA (n = 3), respectively (E). Error bars indicate mean ± SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.
Fig 2.
Hyperactivation of mTORC1 in HepG2-NTCP cells treated with MnCl2.
(A) Transcriptomic analysis performed on HepG2-NTCP cells mock-treated or treated with 10 μM MnCl2 for 2 h. KEGG pathway enrichment analysis was used to analyze the DEGs. The size of dots indicates the proportion of genes enriched in the corresponding pathway. (B) Histogram showing DEGs ranked in the top 60 according to adjusted P values. Genes involved in regulating mTORC1 activation or regulated by mTORC1 activity are highlighted. Asterisks indicate genes encoding ribosomal components essential for protein synthesis. (C) Time course of mTORC1 activation and AKT phosphorylation in HepG2-NTCP cells following exposure to Mn2+. Cells maintained in DMEM medium containing 10% FBS were stimulated with MnCl2 (10 μM) for various times, followed by immunoblotting with the indicated antibodies. The blots were analyzed by densitometry, with the intensity of the phospho-protein signal normalized to the corresponding total protein band. Cells starved in EBSS medium (amino acid deprivation) for 4 h were used as control for mTORC1 inactivation. (D) HepG2-NTCP cells maintained in DMEM medium containing 10% FBS or 0.1% FBS were stimulated with or without MnCl2 (10 μM) for 2 h, followed by immunoblotting with the indicated antibodies. Cells starved in EBSS medium (for 4 h) were used as control. Note that in the setting of EBSS starvation, Mn2+ had a potential effect on the forms of total 4E-BP1. Each blot is a representative of at least three independent experiments. (E) HepG2-NTCP cells maintained in DMEM medium containing 10% FBS were stimulated with MnCl2 at the indicated concentration for 2 h with or without 1 μM Torin 1, followed by immunoblotting with the indicated antibodies. Results represent two independent experiments.
Fig 3.
Suppression of mTORC1 promotes de novo HBV infection in HepG2-NTCP cells.
(A) HepG2-NTCP cells were maintained in DMEM medium containing indicated concentrations of FBS for 10 h, followed by a further 12 h incubation with HBV inoculum (still with FBS at the indicated concentrations). Supernatant HBeAg was determined by ELISA at the indicated time points (n = 3). (B) HepG2-NTCP cells cultured in DMEM medium supplemented with 10% FBS were treated with either Torin1 (1 μM) or PP242 (1 μM) for 10 h, followed by infection with HBV in the presence of the inhibitors. The cells were then maintained in fresh medium (without inhibitors) for subsequent HBeAg analysis (n = 3). Control, cells were mock-treated with DMSO. (C) Huh7 cells transiently transfected with a NTCP-EGFP-mCherry–expressing plasmid were treated with or without 1 μM Torin1 for 4 h, and then incubated with HBV inoculum for the indicated times. The subcellular distribution of the NTCP fusion protein was monitored by confocal microscopy. Red-only puncta are within acidic endolysosomal compartments because of the quenching of GFP fluorescence (lower, arrows). Upper left, the principle of NTCP-EGFP-mCherry to monitor the lysosomal engulfment. Upper right, the number of red dots in each cell was calculated in 8–10 fields of view over three independent experiments. (D) Huh7 cells transiently expressing NTCP-EGFP-mCherry were transfected with siRNA against HRS or TSG101 for 48 h. Cells were then stimulated with HBV inoculum for an additional 1 h before confocal microscopy examination. (E and F) HepG2-NTCP cells were transfected with siRNA against HRS (E) or TSG101 (F). After 48 h, cells were infected with HBV for an additional 12 h. Left, secreted HBeAg was determined by ELISA at the indicated time points (n = 3). Cells transfected with siNC (negative control siRNA) or mock transfected were used as controls. Right, the knockdown efficiency of individual siRNA was assessed by immunoblotting. (G) HepG2-NTCP cells were treated with Torin1 (1 μM) or serum starvation for 4 h. Left, representative images for the subcellular colocalization of endogenous RAB5 (green) and HRS (red) assessed by confocal immunofluorescence microscopy. Right, cells were analyzed by immunoblotting with the indicated antibodies. All the data shown are representative results from at least three independent experiments. Error bars indicate mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001; ns, not significant.
Fig 4.
Endosome-lysosome fusion is not required for early HBV infection.
(A) HepG2-NTCP cells were transfected with siRNAs against VAMP7 for 48 h, followed by HBV infection for an additional 12 h. Left, supernatant HBeAg was determined by ELISA at the indicated time points (n = 3). Right, the knockdown efficiency of individual siRNA was assessed by immunoblotting. (B) HepG2-NTCP cells were transfected with siVAMP7-1 for 48 h. Representative images of cells stained with LysoSensor (left) and LysoTracker (right) probes. The integrated density of five or three low-magnification (40×) fields of view was calculated using ImageJ. The results shown are representative of three independent experiments. (C) HepG2-NTCP cells were treated with Apilimod or YM201636 at the indicated doses for 10 h, followed by HBV infection for an additional 12 h in the presence of the inhibitors. Cells were then maintained in fresh media (without inhibitors) for subsequent analysis. The supernatant HBeAg was determined by ELISA at the indicated time points (n = 3). Right, representative immunofluorescence images of intracellular HBc (red) at 7 dpi. (D and E) HepG2-NTCP cells were treated with 10 μM CQ (D) or 10 mM NH4Cl (E) for 10 h, followed by an additional 12–h incubation with HBV inoculum (in the presence of the chemicals). Supernatant HBeAg and intracellular HBc were determined as depicted in (C). Error bars indicate mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Fig 5.
Mn2+ promotes lysosomal function and acidification.
(A) HepG2-NTCP cells were treated with MnCl2 at indicated doses for 24 h with or without CQ (10 μM). Endogenous LC3B was determined by immunoblotting. (B) Huh7 cells were stimulated with 10 ng/mL EGF for 8 h in the presence of MnCl2 at the indicated doses with or without CQ (10 μM). Left, the endogenous EGFR levels were examined by immunoblotting. Right, EGFR bands were analyzed by densitometry normalized to that of ACTB. (C) HepG2-NTCP cells treated with or without MnCl2 (100 μM) for 12 h were examined by transmission electron microscopy. Arrows indicate the vacuolar degradative compartments (v-DGCs). Arrowheads indicate the nonvacuolar DGCs. Ly, lysosome; AP, autophagosome; Mito, mitochondria. Right, the ratio of v-DGCs to total DGCs per cell section was analyzed over two independent experiments with 9–10 images each. (D) Representative pictures of LysoSensor (upper panels) and LysoTracker (middle panels) staining of HepG2-NTCP cells treated with MnCl2 at indicated doses for 12 h. The integrated density of 4–5 low-magnification (40×) fields of view was calculated using ImageJ (right). (E) HepG2-NTCP cells treated with or without MnCl2 (100 μM) for 12 h were incubated with FITC-Dextran (5 μg/mL) for an additional 4 h, followed by confocal microscopy examination. The integrated density of five low-magnification fields of view (40×) was calculated using ImageJ. All the data shown are representative results from 2–3 independent experiments. Error bars indicate mean ± SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.
Fig 6.
Sustained inhibition of mTORC1 leads to lysosomal dysfunction.
(A and B) Representative images for LysoSensor (left) and LysoTracker (right) staining of HepG2-NTCP cells treated with or without Torin1 (1μM) for 3 h (A) or 12 h (B). The integrated density of five fields of view (40×) was calculated using ImageJ. (C) HepG2-NTCP cells treated with or without Torin1 (1μM) for 3 h (left) or 12 h (right) were incubated with FITC-Dextran (5 μg/mL) for an additional 4 h. Left, representative images of confocal microscopy. Right, the integrated density of five fields of view (40×) was calculated using ImageJ. (D) HepG2-NTCP cells were transfected with siRNAs against TFEB for 48 h, followed by incubation with HBV inoculum for an additional 12 h. Left, secreted HBeAg was determined by ELISA at the indicated time points (n = 3). Cells transfected with siNC or mock transfected were used as controls. Right, the knockdown efficiency of individual siRNA was assessed by immunoblotting of HEK 293T cells stably expressing TFEB-GFP. All the data shown are representative results from three independent experiments. Error bars indicate mean ± SD. ***, P < 0.001; ns, not significant.
Fig 7.
Tomatidine hyperactivates mTORC1 and promotes lysosomal acidification.
(A) Chemical structure of tomatidine (Td). (B) mTORC1 activity (left) and AKT phosphorylation (right) were evaluated by immunoblotting of HepG2-NTCP cells stimulated with tomatidine (1 μM) for the indicated times. (C) Endogenous levels of LC3B were determined by immunoblotting of HepG2-NTCP cells treated with indicated concentrations of tomatidine for 6 h with or without CQ (10 μM). The blots were analyzed by densitometry over three independent experiments, with the intensity of the protein signal normalized to that of ACTB. (D) HepG2-NTCP cells treated with or without tomatidine (10 μM) for 12 h were incubated with FITC-Dextran (5 μg/mL) for an additional 4 h, followed by confocal microscopy examination. The integrated density of four fields of view (40×) was calculated using ImageJ (right). Representative results from three independent experiments are shown. (E) Representative LysoSensor staining of HepG2-NTCP cells treated with indicated concentrations of tomatidine for 12 h. The integrated density of five fields of view (40×) was calculated using ImageJ (right). (F) HepG2-NTCP cells were treated with or without tomatidine (10 μM) for 12 h and subjected to immunofluorescence confocal microscopy. Representative images of the subcellular distribution of LAMP1 (red) and V-ATPase B2 (ATP6V1B2) (green). The DAPI-stained nuclei are in blue. (G) Cells depicted in (F) were fractionated into cytosolic and crude membrane fractions, and subjected to immunoblotting with the indicated antibodies. Lysosomal LAMP1 was used as a membrane fraction loading control and GAPDH as a cytosolic loading control. All the data shown are representative results from three independent experiments. Error bars indicate the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Fig 8.
Tomatidine inhibits de novo HBV infection in HepG2-NTCP cells and in ProliHH organoids.
(A) Dose response curve showing the cytotoxicity of tomatidine in HepG2-NTCP cells determined by the CCK8 assay performed in triplicate. (B) HepG2-NTCP cells were treated with indicated concentrations of tomatidine (Td) for 10 h, followed by HBV infection for an additional 12 h. Left, the supernatant HBeAg was determined by ELISA at the indicated time points (n = 3). Right, representative immunofluorescence images of intracellular HBc (red) at 7 dpi. (C) Time-of-addition assay of the effect of tomatidine (10 μM) on de novo HBV infection of HepG2-NTCP cells. Upper left, the experimental scheme. The cells were then subjected to analysis as described (B). Comparisons were made between pre-infection and post-infection treatment. (D) HepG2-NTCP cells were treated with the indicated concentrations of tomatidine for 10 h, followed by infection with a recombinant HBV rescued by HBc complementation. Upper panel, the infection scheme of the recombinant HBV complemented with HBc protein in trans (created using PowerPoint). Lower panel, the secreted HBeAg was determined by ELISA at dpi 5 (n = 3). (E) The ProliHH organoids were mock-treated, treated with CQ (10 μM) or NH4Cl (5 mM) for 10 h, followed by HBV infection for an additional 12 h. The secreted HBeAg and HBV DNA in the supernatants were determined at the indicated time points by ELISA (left) and real-time PCR (right), respectively (n = 3). (F) The ProliHH organoids were either mock-treated or treated with tomatidine (10 μM) for 10 h, followed by HBV inoculation for an additional 12 h. Left, secreted HBeAg was quantified by ELISA (n = 3). Right, representative images of intracellular HBc immunofluorescence staining (red) at 15 dpi. Organoids not infected with HBV served as negative controls for the labeling. Data shown are representative results from 2–3 independent experiments. Error bars indicate the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Fig 9.
Schematic diagram depicting the early stages of HBV infection and its regulation.
After HBV-triggered internalization, the NTCP receptor was sorted to late endosomal compartments by the ESCRT machinery in concert with the invading virion. The virus may gain cytosolic access directly from the late endosomes. Lysosomal hyperfunction is detrimental to early HBV infection. mTORC1 regulates de novo HBV infection by controlling endosomal transport. Sustained mTORC1 inactivation facilitates viral infection by depleting lysosomes. EE, early endosome. EV, endocytic vesicle; LE, late edosome. The question mark represents a molecular event that remains to be determined. The graphic was created in BioRender (https://BioRender.com/d35t466).