Viral entry defines the hepatitis E virus species barrier in murine hepatocytes

Figures

• 

  Figure 1.. Primary murine hepatocytes are refractory to HEV-3 infection. (A) Heat maps of brain and liver tissue specific marker gene expression in primary murine hepatocytes. Transcripts per million (TPM) values from in total 4 individual donor mice of the CD-1 and C57BL6JR strain. (B) Representative immunofluorescence images stained for HEV-3 ORF2-encoded capsid protein in primary human hepatocytes (PHH, 1 donor) and primary mouse hepatocytes (PMH, 3 donors strain CD-1, 1 donor strain C57BL6JR) at 3 days post HEV-3 inoculation. Primary cells were either (pre-)treated with baricitinib [2 µM], NITD008 [0.5 µM] or with DMSO as solvent control. ORF2-encoded capsid protein = green; DAPI = blue; scale bar = 200 µm. (C) Progeny virus was harvested from lysates of primary cells at 3 days post HEV-3 inoculation and quantified via focus forming unit (FFU) assay on HepG2/C3A cells. Dashed line represents the limit of detection (LOD) with non-detectable samples that were set to LOD. Data presented derives from 1 donor each. (D) Depth of reads mapping to the HEV-3 Kernow-C1/p6 reference genome (GenBank accession no. JQ679013) in total RNA extracted from HEV-3 inoculated PHH (data was published…

• 

  Figure 2.. Murine liver cell lines support HEV-3 replication. (A) Heat maps of brain and liver tissue specific marker gene expression in human hepatoma HepG2 cells as well as murine hepatic cell lines MLT, Hep56.1D and AML12. Transcripts per million (TPM) values from three individual passages of cells are shown. (B, C, D) Replication fitness of HEV-3 (Kernow-C1/p6 and 83-2-27) and HEV-1 (Sar-55/S17) subgenomic replicons in human and murine liver cell lines with and without Ribavirin (RBV, 50 µM) treatment. Replication was measured at indicated time points in correlation to Gaussia luciferase activity. The presented data are relative light units (RLU) normalized to 4 h post electroporation. Mean values ± SD from n = 4 experiments are displayed.

• 

  Figure 3.. Infectious HEV-3 particles can be harvested from murine hepatic cells. (A) Representative immunofluorescence images (n = 3) of ORF2 capsid protein expression at 3 days post electroporation of in vitro transcribed full-length HEV-3 RNA. ORF2-encoded capsid protein = green; DAPI = blue; scale bar = 200 µm. (B) Viral titers of infectious HEV-3 particles harvested from human and murine cell lines at indicated time points after electroporation of in vitro transcribed full-length viral RNA. Naked virions were harvested from cell lysates whereas enveloped virions were collected from supernatant. Infectivity of harvested viruses were quantified via focus forming unit (FFU) assay on HepG2/C3A cells. Mean values ± SD from n = 3 experiments are shown. Non-detectable values for viral titres in independent experiments were set to the limit of detection (LOD).

• 

  Figure 4.. HEV-3 exhibits increased sensitivity against the murine interferon-stimulated response. (A) Dose-dependent antiviral activity of human and murine interferon-α (IFN-α) against HEV-3 replication. HEV replication was normalized to cells treated with the solvent control (ctr.). Normalized replication levels [%] were used for linear regression to determine half-maximal inhibitory concentration (IC50). Mean values ± SD are shown for n = 3 experiments. (B) Comparison of host transcriptional responses between untreated and IFN-α treated [100 IU/mL] HEV-transfected human and murine cells. N.s., not significant with p-value >0.05; p < 0.05 = *; p < 0.0001 = ****; FC, fold change (C) Comparison of the mean log2 fold change of n = 47 (human) and 38 (murine) selected IFN-stimulated genes (ISGs) of HEV-3 or tRNA transfected cells upon IFN-α treatment [100 IU/mL] compared to untreated. Statistical significance was calculated using two-tailed, paired t-test, p < 0.05 = *, p < 0.001 = ***. Presented RNA sequencing data derive from n = 3 experiments.

• 

  Figure 5.. Absence of a strong innate immune response to HEC-3 infection in murine hepatic cells. (A) Impact of JAK inhibitors ruxolitinib and baricitinib [2 µM] as well as species-specific IFN-α treatment [100 IU/ml] on HEV-3 replication (upper), naked HEV-3 (middle) and enveloped HEV-3 (lower) particle production in HepG2, MLT and Hep56.1D cells at 72 h post transfection of sub genomic or infectious full-length RNA. The presented replication data are relative light units (RLU) normalized to the solvent control treated cells. Infectivity of harvested viruses were quantified via focus forming unit (FFU) assay on HepG2/C3A cells. Non-detectable values for viral titres were set to the limit of detection (LOD). Mean and individual values + SD from n = 3-4 experiments are shown. Statistical significance was calculated using an ordinary one-way ANOVA with Dunnett multiple comparison correction. Data of the virus production were log transformed prior to statistical analyses. N.s., not significant with p-value >0.05; * = p-value <0.05; ** = p-value <0.01; *** = p-value <0.001; **** = p-value <0.0001. (B, C) ISRE-promoter activity in HEV-3 transfected human and murine hepatic cell lines. Cells…

• 

  Figure 6.. Interspecies heterokaryon formation promotes HEV-3 replication. (A) Fusion of GFP-expressing HepG2 cells (green) transfected with a HEV-3 subgenomic reporter replicon to either H2B-mCherry (red) expressing naïve HepG2 or murine hepatic cells (MLT or Hep56.1D) using polyethylene glycol (PEG). Representative immunofluorescence images of n = 4 at 24 h post fusion. Scale bar = 200 µm. (B) HEV replication was quantified by luciferase readout at 24 h post fusion. HEV-transfected HepG2 cells without co-cultivation were used as a control (ctr.) and for normalization (norm.). Mean and individual values + SD from n = 4 experiments are shown. Statistical significance was calculated on log transformed data using an ordinary two-way ANOVA with Dunnett multiple comparison correction. N.s., not significant with p-value >0.05; * = p-value <0.05; ** = p-value <0.01.

• 

  Figure 7.. Entry of infectious HEV-3 into murine liver cells is blocked after viral attachment. (A) Representative immunofluorescence images. HEV capsid protein was stained in human HepG2 and murine MLT, Hep56.1D and AML12 cells at 5 days post HEV-3 inoculation (n = 3). Cells were either (pre-)treated with baricitinib [2 µM], NITD008 [0.5 µM] or with DMSO as solvent control. ORF2 capsid protein = green; DAPI = blue; scale bar = 1000 µm. (B) Binding of HEV virus-like particles (VLPs) of the species Paslahepevirus balayani (HEV-A) to the human liver cell line HepG2 and murine hepatic cell lines (AML12 and Hepa1-6) was measured by immunofluorescence assay. The relative fluorescent intensity was quantified by ImageJ and normalized to HepG2 cells incubated with wildtype (WT) VLPs. Representative images and mean values as well as individual values + SD from n = 2 experiments with 4 technical replicates are shown. VLPs = red; nuclei = blue; scale bar = 50 µm. (C) Binding of infectious HEV-3 particles to murine hepatic cell lines after 2 h incubation at 4 °C. HEV capsid protein was detected by immunofluorescence staining and HEV RNA by RNAscope. Representative images of n = 3 independent…