Innate immune responses in human hepatocyte-derived cell lines alter genotype 1 hepatitis E virus replication efficiencies

Abstract

Hepatitis E virus (HEV) is a significant health problem in developing countries causing sporadic and epidemic forms of acute viral hepatitis. Hepatitis E is a self-limiting disease; however, chronic HEV infections are being reported in immunocompromised individuals. The disease severity is more during pregnancy with high mortality (20-25%), especially in third trimester. Early cellular responses after HEV infection are not completely understood. We analyzed innate immune responses associated with genotype-I HEV replication in human hepatoma cell lines (Huh7, Huh7.5 and HepG2/C3A) using HEV replicon system. These cells supported HEV replication with different efficiencies due to the cell type specific innate immune responses. HepG2/C3A cells were less supportive to HEV replication as compared to Huh7.5 and S10-3 cells. Reconstitution of the defective RIG-I and TLR3 signaling in Huh7.5 cells enabled them to induce higher level antiviral responses and restrict HEV replication, suggesting the involvement of both RIG-I and TLR3 in sensing HEV RNA and downstream activation of interferon regulatory factor 3 (IRF3) to generate antiviral responses. Inhibition of IRF3 mediated downstream responses in HepG2/C3A cells by pharmacological inhibitor BX795 significantly improved HEV replication efficiency implying the importance of this study in establishing a better cell culture system for future HEV studies.

Figure 1. Differential replication efficiencies of HEV in different human hepatocyte derived cell lines.

S10-3 (a), Huh7.5 (b) and HepG2/C3A (c) cells were transfected with either wild-type HEV Rluc or HEV Rluc GAA (GDD mutant) RNA (2 μg/well). Cells were co-transfected with firefly luciferase plasmid DNA (pGL-3 promoter vector, 100 ng/well) to normalize cell transfection efficiency and the Renilla luciferase signal. Cell associated Renilla luciferase activity was checked to monitor HEV replication from 1-6 days. The data represents mean ± SD of three independent triplicate set of experiments, [** and ***represent p < 0.01 and p < 0.001 respectively, statistical comparisons were done by one-way analysis of variance (ANOVA) between mock transfected cells, and cells transfected with HEV-Rluc RNA].

Figure 2. Interferon-α treatment inhibits HEV replication in Huh7.5 cells.

(a) Huh7.5 cells transfected with HEV Rluc replicon RNA were treated with IFN-α (1,000 U/ml) 4 h post transfection. Cells were assayed for luciferase activity to monitor HEV RNA replication as described in Fig. 1, [* and ***represent p < 0.05 and p < 0.001 respectively, statistical comparisons were done by one-way analysis of variance (ANOVA) between cells transfected with HEV-Rluc RNA, and cells transfected with HEV-Rluc RNA and treated with IFN-α]. (b) Expression of ISGs; ISG56, Mda5, Mx1 and PKR genes in Huh7.5 cells was checked by SYBR-green based qRT-PCR, 24 h post RNA transfection (mRNA levels (fold) were calculated in relation to the levels of respective genes in mock transfected cells). The data represents mean ± SD of two independent triplicate sets of experiments [*represents p < 0.05, comparison between IFN-α treated cells and cells transfected with HEV-Rluc RNA and treated with IFN-α].

Figure 3. RIG-I and TLR3 are the pattern recognition receptors in sensing HEV RNA replication.

(a) RIG-I expression in Huh7.5 cells: Cells either mock transfected or transfected with RIG-I expression plasmid (pUNO-hRIG-I) were analyzed by immunoblotting for RIG-I protein levels at 24, 48, 72 and 96 h respectively (lanes 1–4). Actin was used as a loading control. (b) RIG-I complementation in Huh7.5 cells inhibits HEV RNA replication: Huh7.5 cells were either mock transfected or transfected with empty vector or with RIG-I expression plasmid. After 24 hours, cells were transfected with HEV Rluc RNA and monitored by luciferase assay at 2 and 4 days post RNA transfection respectively. (c) TLR-3 and TLR3-ΔTIR expression in Huh7.5 cells: Cells either mock transfected or transfected with TLR3 expression plasmids (pUNO-hTLR3 and pZERO-TLR3) were analyzed by immunoblotting for TLR3 protein levels at 24 and 48 h respectively. TLR3-ΔTIR is a TIR domain-less form of the TLR3 gene showing lower molecular weight compared to wild-type TLR3 (TLR3-ΔTIR recognizes its ligands but is unable to induce the signaling). Actin was used as a loading control. (d) TLR3 complementation in Huh7.5 cells inhibits HEV RNA replication: Huh7.5 cells were transfected with TLR3 or TLR3-ΔTIR expression plasmids 24 h prior to transfection with HEV Rluc RNA and monitored by luciferase assay at 2 and 4 days post transfection respectively [**represents statistical comparison by one-way analysis of variance (ANOVA) with cells transfected with empty vector or TLR3-ΔTIR expression plasmid and showed p < 0.01 in figures (b,d)]. (e) RIG-I and TLR3 complementation restore antiviral response in Huh7.5 cells: Cells expressing RIG-I and TLR3 were transfected with HEV Rluc RNA and expression of ISGs ISG56, MDA5, Mx1 and PKR were assessed by SYBR-green based qRT-PCR at 24 h post RNA transfection. (f) Antiviral response in HepG2/C3A cells: HepG2/C3A cells transfected with HEV Rluc RNA were analyzed for ISG expression similarly as in (e) with Huh7.5 cells as a control. The data represents mean ± SD of three independent triplicate sets of experiments. Cropped blots are used in the main figure and full length blots are included in Supplementary Figure 1.

Figure 4. BX795 blocks the phosphorylation and activation of IRF3 leading to suppression of interferon stimulated genes (ISGs).

HepG2/C3A cells, untreated or treated with BX795, were transfected with (a) HEV Rluc RNA, (b) HEV Rluc GAA and (c) double stranded RNA analogue (poly I:C) and processed for immunoblotting for the detection of phosphorylated IRF3 (P-IRF3, Ser396). The blot was reprobed for the detection of total IRF3 (T-IRF3) and actin at indicated time points.The relative protein band density for p-IRF3 was normalized with actin and compared with mock treated and BX795 treated cells at different time points and mentioned as relative fold (RF) values. Cropped blots are used in the main figure and full length blots are included in Supplementary Figure 1. (d–f) HepG2/C3A cells left untreated or treated with BX795 were transfected with HEV Rluc RNA and processed for qRT-PCR of type I IFN genes (IFNA and IFNB) and ISGs (PKR and Mx1) at 24 h post transfection. The data represents mean ± SD of three independent experiments [*, ** and ***represent p < 0.05, p < 0.01 and p < 0.001 respectively, statistical comparisons were done by one-way analysis of variance (ANOVA) between HEV Rluc and BX795 + HEV Rluc].

Figure 5. Inhibition of IFN signaling enhances HEV replication efficiency.

HepG2/C3A cells untreated or treated with BX795 were transfected with HEV Rluc RNA and viral RNA replication was monitored by luciferase assay from 1–5 days post transfection. The data represents mean ± SD of three independent triplicate sets of experiments [*, ** and ***represent p < 0.05, p < 0.01 and p < 0.001 respectively, statistical comparisons were done by one-way analysis of variance (ANOVA) between untreated and BX795 treated cells at indicated time points].