Inhibition of Hepatitis E Virus Spread by the Natural Compound Silvestrol

Figures

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  Figure 1. Repressive effect of silvestrol on release of infectious viral particles from A549 cells persistently infected with hepatitis E virus (HEV) strain 47842c. (A) qPCR quantification of extracellular viral RNA in the supernatant derived from silvestrol treated cells (2 nM and 50 nM). The values were referred to the DMSO control; (B) Absolute tissue culture infective dose (TCID50) values determined after A549/D3 infection (logarithmic scale); (C) Relative TCID50 values compared to DMSO on the basis of (B). n = 3 for all experiments; * p < 0.05, ** p < 0.01, **** p < 0.0001.

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  Figure 2. Lactate dehydrogenase (LDH) and PrestoBlue assays to determine silvestrol-mediated cytotoxicity and cytostaticity. (A,B) Processed data for LDH-activity in cell-culture supernatants derived from cells treated with 2 or 50 nM silvestrol as compared to respective DMSO controls in (A) A549 cells persistently infected with HEV strain 48932c or (B) in non-infected A549/D3. (C,D) Processed data for redox-metabolic activity in cells treated with 2 or 50 nM silvestrol when compared to respective DMSO controls in (C) A549 cells persistently infected with HEV strain 47832c or (D) in non-infected A549/D3 cells. * p < 0.05, ** p < 0.01, **** p < 0.0001.

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  Figure 3. Effect of silvestrol on the intracellular amount of HEV-specific RNA in A549 cells persistently infected with HEV strain 47842c. qPCR analysis using ORF2-specific primers of total RNA isolated from A549 cells persistently infected with HEV; data were normalized to intracellular RPL27 RNA levels. Cells were treated with 2 nM or 50 nM silvestrol, DMSO treatment served as control. n = 3; * p < 0.05.

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  Figure 4. Effects of silvestrol on intracellular HEV capsid protein levels in A549 cells persistently infected with HEV strain 47832c. (A) Representative Western Blot of cellular lysates derived from cells treated with either 2 nM or 50 nM silvestrol. DMSO treatment served as control. For detection, a capsid protein specific antiserum and a beta-actin-specific antibody (loading control) were used; (B) Relative quantification of Western Blot band intensities normalized to intracellular beta-actin levels referred to the DMSO control. n = 3; * p < 0.05, **** p < 0.0001.

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  Figure 5. Immunofluorescence staining of silvestrol-treated A549 cells persistently infected with HEV strain 47832c. (A) Persistently HEV-infected A549 cells were seeded on cover-slips and treated with respective concentrations of silvestrol for the indicated time points. Nuclei were stained using 4′,6-Diamidin-2-phenylindole (DAPI) (blue) and an anti-HEV capsid protein (core) antiserum (green), scale bar =50 µm (B) Fluorescence intensity quantification of HEV capsid protein immunostaining and referred to the respective DMSO control to gain fold-change; and, (C) Comparison of the proportion of HEV capsid protein expressing cells depicted in Figure 5A referred to the respective DMSO control. n = 4 for all experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 6. Effect of silvestrol on the genome replication of HEV genotype 3 strain p6Kernow Gaussia luciferase reporter constructs in A549 cells. Intracellular luciferase activity measured in RLU/s normalized to total protein amount after silvestrol treatment (2 and 50 nM) compared to the DMSO-treated cells. n = 3 for all experiments; * p < 0.05, ** p < 0.01.

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  Figure 7. Induction of MVP expression upon HEV infection and subsequent cytosolic decrease by 10 nM silvestrol treatment (24 h) as measured by ultradefinition mass spectrometry (UDMSE) and depicted in Table 1. n = 3; ** p < 0.01.

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  Figure 8. MVP is upregulated in HEV-infected A549 cells and is downregulated and translocated to the perinuclear region upon 10 nM silvestrol treatment. (A) Western Blot analysis of cellular lysates derived from persistently HEV-infected A549 cells as compared to A549/D3. An MVP-specific antibody was used for detection, detection of beta-actin served as loading control. (B) Shows the corresponding quantification of (A) normalized to beta-actin. n = 4; ** p < 0.01. (C) Western Blot analysis of cellular lysates derived from persistently HEV-infected A549 cells treated with 10 nM silvestrol for 24 h. Treatment with DMSO served as a control. (D) Shows the corresponding quantification of (C) normalized to beta-actin. n = 4; ** p < 0.01. (E) A549/D3 and persistently HEV-infected A549 cells were seeded on cover-slips and treated with 10 nM silvestrol for 24 h. Nuclei were stained with DAPI (blue), HEV capsid protein-specific antibodies (green) and MVP-specific antibodies (red) were used for detection. (F) Comparison of the proportion of HEV capsid protein expressing cells after treatment with 10 nM silvestrol depicted in (E) to the respective DMSO control; n = 3, scale bar = 40 µm (G) Western…