Effects of hepatitis E virus infection on interferon production via ISG15

Figure 1 Hepatitis E virus infection enhances the production of Interferon-α and -β and induces elevation of Interferon-stimulated gene 15 protein in C3A cells. A: The concentrations of IFN-α and β were evaluated in cell culture supernatants at different time points (0, 8, 12, 24, 48, 72 and 120 h) by enzyme-linked immunosorbent assay; B: The expression of ISG15 protein replication was detected at different time points (0, 8, 12, 24, 48, 72 and 120 h) by western blotting. The data are expressed as mean ± standard error of the mean. ^aP < 0.05, ^bP < 0.01. HEV: Hepatitis E virus; IFN: Interferon; ISG: IFN-stimulated gene.

Figure 2 Hepatitis E virus open reading frame 3 protein enhances the production of Interferon-α and β and the expression of Interferon-stimulated gene 15 protein in C3A cells. C3A cells were transfected with 2 μg of either pORF3-EGFP (ORF3) or pEGFP-N1 empty vector (GFP), and the HEV mutant, leading to the loss of ORF3 protein expression (ΔORF3) for 48 h, untreated cells were used as the control (Con). Then, the cells and cell culture supernatants were harvested at different time points (0, 8, 12, 24, 48, 72 and 120 h). A: The concentrations of IFN-α and β were evaluated in cell culture supernatants at different time points (0, 8, 12, 24, 48, 72, 120 h) by enzyme-linked immunosorbent assay; B: The expression of ISG15 protein was detected at different time points (0, 8, 12, 24, 48, 72 and 120 h) by western blotting. The data are expressed as mean ± standard error of the mean. ^aP < 0.05, ^bP < 0.01. GFP: Green fluorescent protein; HEV: Hepatitis E virus; IFN: Interferon; ISG: IFN-stimulated gene; ORF: Open reading frame.

Figure 3 Interferon-stimulated gene 15 silencing enhances the production of Interferon-α/β in C3A cells. Three siRNA inhibiting ISG15 (siISG15) and control-siRNA (si-control) were constructed, and C3A cells were cotransfected with 20 nmol/L ISG15-targeted siRNA (siISG15) or control-siRNA (si-control) along with HEV RNA. A: The mRNA level of ISG15 was estimated by reverse transcription-PCR; B: The protein level of ISG15 was estimated by western blotting. Next, the siRNA2 was transfected into C3A cells that had been pretreated with HEV RNA, and the impact of siISG15 on IFN was investigated; C: The level of IFN-α/β was tested by enzyme-linked immunosorbent assay. The data are expressed as mean ± standard error of the mean. ^aP < 0.05, ^bP < 0.01. HEV: Hepatitis E virus; IFN: Interferon; ISG: IFN-stimulated gene; si: Small interfering.

Figure 4 Over-expression of Interferon-stimulated gene 15 results in the reduction of Interferon-α/β in C3A cells. C3A cells were transfected with 2 mg ISG15-EGFP plasmid (ISG15) along with HEV RNA. A: ISG15 protein expression was detected by western blotting; B: The levels of IFN-α/β were detected by enzyme-linked immunosorbent assay. ^aP < 0.05. HEV: Hepatitis E virus; IFN: Interferon; ISG: IFN-stimulated gene.