The guanosine nucleotide analog bemnifosbuvir inhibits hepatitis E virus infection in cell and organoid models

Figures

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  Fig. 1. AT-527 inhibited HEV replication in Huh7-based cell models. A The effects of AT-527 treatment for 24, 48 or 72 hours on HEV replication-related luciferase units (RLU) in Huh7 cells harboring the GT3 replicon of the p6 clone. The untreated group serves as control (CTR) (set as 1) (n = 9). B The 50% inhibitory concentration (IC50; n = 9) and 50% cytotoxic concentration (CC50; n = 5–6) were calculated using GraphPad Prism software. C, D The effects of ribavirin were tested under the identical experimental conditions (n = 5), and IC50 and CC50 values were calculated accordingly. E Huh7 cells harboring the infectious HEV (p6) clone were treated with indicated concentrations of AT-527 (n = 6–8) or ribavirin (n = 4) for 48 hours. The effects on viral RNA were quantified by qRT-PCR, and F the IC50 values were calculated accordingly. G Western blot analysis and quantification of HEV capsid protein level in infected Huh7 cells treated with AT-527 for 48 hours. The uninfected group (mock) serves as the negative control, whereas the infected but untreated group was set as 1 (n = 4). H Immunofluorescence analysis of viral ORF2-encoded capsid protein (red) in Huh7 cells treated with 10 μM…

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  Fig. 2. AT-527 inhibited HEV replication in human liver-derived organoids. A Schematic representation of the experimental procedure. B The effects of AT-527 treatment for 24, 48 or 72 hours on viral replication-related luciferase units (RLU) in organoids harboring GT3 subgenomic replicon. The untreated group serves as control (CTR) (set as 1) (n = 4). C Immunofluorescence analysis of viral ORF2-encoded capsid protein after treatment with 10 μM AT-527 for 48 hours in organoids. Uninfected organoids incubated with the anti-HEV capsid protein antibody serve as mock. HEV infected but untreated organoids serve as the positive control. DAPI (blue) and Epcam (green) were applied to visualize nuclei and cytomembrane. D Organoids harboring the infectious HEV clone were treated with indicated concentrations of AT-527 for 48 hours (1 and 10 μM; n = 4). The effects on viral RNA were quantified by qRT-PCR. E The cytotoxicity was assessed by AlamarBlue assay in organoids treated with AT-527 for 24, 48 and 72 hours (1 and 10 μM; n = 6). F HEV RNA in organoids harboring infectious HEV was quantified by qRT-PCR after 48 hours of treatment with 10 μM AT-527 by adding 1 μg/mL exogenous guanosine (n = 4).…

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  Supplementary Figure S1. Illustrated chemical structures. The structures of AT-527, AT-511 (the free base form of AT-527), and guanosine.

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  Supplementary Figure S2. The effects of AT-527 on replication of GT1 HEV in Huh7 cells. Viral replication-related luciferase activity of a Huh7 cell-based genotype 1 HEV replicon (Sar55 clone) was assessed after treatment with AT-527 for 24, 48, or 72 hours, and the untreated group serves as control (CTR) (set as 1) (n = 8). Data are presented as means ± SEM; ∗P < 0.05, ∗∗∗P < 0.001. Mann-Whitney test.

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  Supplementary Figure S3. Assessing the anti-HEV effects of AT-527 by a re-infection assay. Naïve Huh7 cells were inoculated with HEV particles harvested from HEV-infected Huh7 cells at 48 hours post-treatment with different concentrations of AT-527 (untreated cells served as the control). After 48 hours of inoculation, Huh7 cells were subjected to immunofluorescence staining for the HEV ORF2-encoded capsid protein (A) or lysed for qRT-PCR analysis of HEV RNA (B) (n = 4).

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  Supplementary Figure S4. The anti-HEV effects of AT-511, the free base form of AT-527. The Huh7 infectious cell model was treated with indicated concentrations of AT-511 for 48 hours (n = 4). Viral RNA levels were quantified by qRT-PCR.

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  Supplementary Figure S5. The effects of long-term treatment with AT-527. A The effects of 7 days of treatment with AT-527 on HEV replication in Huh7 cells harboring GT3 replicon quantified by luciferase activity. B The effects of 7 days treatment with AT-527 on HEV replication in Huh7 cells harboring GT1 replicon measured by luciferase units, and the untreated (CTR) group serves as control (set as 1) (n = 4).

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  Supplementary Figure S6. The effects of AT-527 on replication of GT1 HEV in human liver-derived organoids. Viral replication-related luciferase activity of GT1 HEV replicon in liver organoids (Sar55 clone) was measured after treatment with AT-527 for 24, 48, or 72 hours (n = 4). Data are presented as means ± SEM; ∗P < 0.05. Mann-Whitney test.

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  Supplementary Figure S7. AT-527 inhibited HEV variant in cell and organoid models. A The infectious Huh7-p6G1634R cell model was treated with indicated concentrations of AT-527(n = 4) or ribavirin (n = 4) for 48 hours. The effects on viral RNA were quantified by qRT-PCR. B Immunofluorescence analysis of HEV ORF2-encoded capsid protein in Huh7-p6G1634R cells treated with 10 μM AT-527 for 48 hours. C Naïve Huh7 cells were inoculated with HEV particles harvested from HEV-infected Huh7 cells 48 hours post-treatment with 10 μM AT-527 (untreated set as control group). After inoculation for 48 hours, immunofluorescence analysis of HEV ORF2 protein was performed. Huh7 cells incubated with the anti-HEV capsid protein antibody serves as mock. HEV-infected Huh7 cells untreated and incubated with the anti-HEV capsid protein antibody serves as the positive control, and the nuclei were stained using DAPI (blue). (Scale bar, 100 μm. 40 × oil immersion objective). D Human liver organoids harboring infectious GT3 HEV were treated with indicated concentrations of AT-527 for 48 hours. The effects on viral RNA were quantified by qRT-PCR (n = 4). E Immunofluorescence analysis of viral ORF2-encoded capsid protein after…

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  Supplementary Figure S8. Combination of AT-527 with ribavirin or IFN-α. A The antiviral effects of AT-527 in combination with ribavirin, treated for two days in Huh7 cells harboring the GT3 replicon, showing synergy distribution of the pairwise combination (n = 4). B The antiviral effects of AT-527 in combination with IFN-α, showing synergy distribution of the pairwise combination (n = 4). Data were normalized to the untreated control (set as 1) and presented as means ± SEM.