Hepatitis E virus replication and interferon responses in human placental cells

Abstract

Hepatitis E virus (HEV) is a member of the genus Orthohepevirus in the family Hepeviridae and the causative agent of hepatitis E in humans. HEV is a major health problem in developing countries, causing mortality rates up to 25% in pregnant women. However, these cases are mainly reported for HEV genotype (gt)1, while gt3 infections are usually associated with subclinical courses of disease. The pathogenic mechanisms of adverse maternal and fetal outcome during pregnancy in HEV-infected pregnant women remain elusive. In this study, we observed that HEV is capable of completing the full viral life cycle in placental-derived cells (JEG-3). Following transfection of JEG-3 cells, HEV replication of both HEV gts could be observed. Furthermore, determination of extracellular and intracellular viral capsid levels, infectivity, and biophysical properties revealed production of HEV infectious particles with similar characteristics as in liver-derived cells. Viral entry was analyzed by infection of target cells and detection of either viral RNA or staining for viral capsid protein by immunofluorescence. HEV gt1 and gt3 were efficiently inhibited by ribavirin in placental as well as in human hepatoma cells. In contrast, interferon-α sensitivity was lower in the placental cells compared to liver cells for gt1 but not gt3 HEV. Simultaneous determination of interferon-stimulated gene expression levels demonstrated an efficient HEV-dependent restriction in JEG-3. Conclusion: We showed differential tissue-specific host responses to HEV genotypes, adding to our understanding of the mechanisms contributing to fatal outcomes of HEV infections during pregnancy. Using this cell-culture system, new therapeutic options for HEV during pregnancy can be identified and evaluated. (Hepatology Communications 2018;2:173-187).

Figure 1

Replication of subgenomic HEV genotype 1 and 3 in different placental‐derived cell lines. Different cell lines JEG‐3, BeWo, JAR, and HepG2 were transfected by electroporation with HEV RNA of gt1 strain Sar55/S17 and gt3 strain Kernow‐C1 p6 reporter constructs and were left untreated (black circles) or treated with 100 µM RBV (gray triangles) 4 hours p.t. Gaussia luciferase activity was assessed at 4, 24, 48, and 72 hours p.t. and normalized to 4‐hour values. Depicted are the mean values of three independent experiments ± SEM. Abbreviation: RLU, relative light unit.

Figure 2

Replication of full‐length HEV and detection of intracellular and extracellular capsid protein. JEG‐3 and HepG2 cells were transfected by electroporation with full‐length HEV RNA gt3 Kernow‐C1 p6 and were left untreated (black bars) or treated with 100 µM RBV (gray bars) 4 hours p.t. (A) Viral replication in each cell line was measured by qRT‐PCR 4, 24, 48, and 72 hours p.t. in cell lysates. (B) Immunofluorescence detection of HEV antigen in paraformaldehyde‐fixed JEG‐3 cells 48 hours p.t. with ORF2‐encoded antigen (green) and with DAPI (blue). Abbreviation: DAPI, 4’,6‐diamidino‐2‐phenylindole.

Figure 3

Determination of HEV infectious particle production, infectivity, and biophysical properties of HEVcc. JEG‐3 and HepG2 cells were transfected by electroporation with full‐length authentic HEV RNA gt3 Kernow‐C1 p6. (A) Cells were left untreated (black bars) or treated with 100 µM RBV (gray bars) 4 hours p.t. A commercially available HEV antigen ELISA detecting the ORF2‐encoded capsid protein was used to determine extracellular and intracellular capsid protein amounts over time. All values were normalized to 4‐hour values. Depicted are the mean values of three independent experiments ± SEM. (B) Intracellular (black bars) and extracellular (white bars) virus produced in JEG‐3 and HepG2 cells was harvested and used to infect HepG2/C3A cells. Infected cells were stained for ORF2‐specific antigen (green) and DAPI (blue) 7 days postinfection. Quantitative infectivity was determined manually by counting infectious foci. (C) Iodixanol density gradients of extracellular and intacellular HEVcc produced in JEG‐3 and HepG2 cells either treated with the detergent NP‐40 for 1 hour (black) or left untreated (white). Fractions were analyzed for buoyant density with a refractometer for HEV antigen (circles) and HEV RNA (boxes). All antigen values below the cut‐off value log₁₀ OD 0.202 are reported as negative by the ELISA. RNA levels are depicted as percentage of total RNA level. Abbreviations: DAPI, 4’,6‐diamidino‐2‐phenylindole; OD, optical density.

Figure 4

Infection of JEG‐3 cells with HEV gt3 and gt1. (A) JEG‐3 cells were infected with HEVcc originating from the gt3 p6 strain and were left untreated (black circles) or incubated with 50 µM RBV (gray triangles). Samples were taken every 2‐3 days, and viral RNA was measured by qRT‐PCR. Depicted are the mean values of three independent experiments ± SEM. (B) Immunofluorescence staining of JEG‐3 or S10‐3 cells infected with gt1 Sar55 primary stool suspension or HEVcc gt3 p6‐derived particles. Cells were stained for HEV capsid ORF2 (red) or with DAPI (blue) 7 days postinfection. White arrows indicate HEV‐positive cells. Abbreviation: DAPI, 4’,6‐diamidino‐2‐phenylindole; p.i., postinfection.

Figure 5

Antiviral activity of sofosbuvir in placental‐derived JEG‐3 cells. (A) JEG‐3 and (B) HepG2 cells were transfected by electroporation with HEV RNA of gt1 strain Sar55/S17 and gt3 strain Kernow‐C1 p6 reporter constructs. Cells were treated with either 100 µM RBV (gray inverted triangles), 10 µM SOF (gray triangles), or with a combination of 10 µM SOF and 100 µM RBV (dark gray boxes) 4 hours p.t. Gaussia luciferase activity was determined at 4, 24, 48, and 72 hours p.t. and normalized to 4‐hour values. Bar plots of data points representing the luciferase activity 72 hours p.t. for each cell line and gt illustrate the maximum replication inhibition. Depicted are the mean values of at least three independent experiments ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; repeated measures one‐way analysis of variance of log‐transformed values followed by Dunnett's multiple comparison test). Abbreviations: ns, not significant; RLU, relative light unit.

Figure 6

Antiviral properties of IFN‐α2a and analysis of STAT1 phosphorylation after treatment. (A) JEG‐3 or (B) HepG2 cells were transfected by electroporation with HEV RNA of gt1 strain Sar55/S17 (light gray triangles and bars) and gt3 strain Kernow‐C1 p6 (dark gray circles and bars) reporter constructs and treated with serially diluted IFN‐α2a 4 hours p.t. Gaussia luciferase counts were assessed 72 hours p.t. and normalized to untreated control values. Side by side plotting of the data points representing the highest IFN concentration (250 ng/mL) illustrates the maximum replication inhibition (A and B, right panels). Depicted are the mean values of four independent experiments ± SEM (*P < 0.05; unpaired t test with Welch's correction). (C) Western blot‐based analysis of STAT1 phosphorylation in JEG‐3 and HepG2 cells after transfection with HEV RNA of gt1 strain Sar55/S17, gt3 strain Kernow‐C1 p6 reporter constructs, and tRNA. Cells were either treated with 1,000 IU IFN‐α2a 12 hours p.t. for 30 minutes or left untreated and were lysed directly afterward. Depicted are the 90‐kDa bands for pSTAT1 as well as 37‐kDa bands for GAPDH (one out of two independent experiments shown). Abbreviations: GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; ns, not significant; pSTAT1, phosphorylated STAT1; RLU, relative light unit.

Figure 7

HEV restriction in expression of selected ISGs under treatment with IFN‐α2a. (A) JEG‐3 and (B) HepG2 cells were transfected by electroporation with HEV RNA of gt1 strain Sar55/S17 (light gray bars), gt3 strain Kernow‐C1 p6 (dark gray bars) reporter constructs, or tRNA as control (hatched bars), following treatment with 1,000 IU/mL (ca. 2.6 ng/mL) IFN‐α2a for 24 hours. Respective cell lines were lysed and expression of depicted ISGs was assessed by qRT‐PCR 4 hours and 24 hours posttreatment. Values were normalized to untreated control values. Depicted are the mean values of four independent experiments ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001; two‐way analysis of variance of log‐transformed values followed by Tukey's multiple comparisons test).