Full-length MAVS, a mitochondrial antiviral-signaling protein, inhibits hepatitis E virus replication, requiring JAK-STAT signaling

Figures

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  Fig. 1. FL-MAVS inhibits HEV replication. (A) Quantitative RT-PCR analysis of FL-MAVS RNA (n = 7) and immunoblot analysis of the FL-MAVS protein level in Huh7.5-p6 cells transduced with FL-MAVS or empty vector for 48 h. MAVS was detected using anti-FLAG antibody. (B) Quantitative RT-PCR analysis of HEV RNA (n = 7) in Huh7.5-p6 cells transduced with FL-MAVS or empty vector for 48 h. (C) Analysis of HEV-related luciferase in Huh7.5-p6-Luc cells transduced with FL-MAVS or empty vector for the indicated time periods (n = 7). (D) Quantitative RT-PCR analysis of FL-MAVS RNA (n = 4) and HEV RNA (n = 4) in PLC-p6 cells transduced with FL-MAVS or empty vector for 48 h. (E) Analysis of HEV-related luciferase in a genotype 1 HEV cell model transduced with FL-MAVS or empty vector (n = 4) for 48 h. Data are the mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

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  Fig. 2. Overexpression of FL-MAVS induces IFN secretion. (A) Quantitative RT-PCR analysis of ISGs RNA in Huh7.5-p6 cells transduced with FL-MAVS or empty vector (n = 3) for 48 h. (B) Quantitative RT-PCR analysis of IFN RNA in Huh7.5-p6 cells transduced with FL-MAVS or empty vector (n = 4-5) for 48 h. Huh7.5-p6 cells were transduced with FL-MAVS or empty vector for 72 h. After that, the cells were washed five times with PBS, and the cell culture medium was refreshed. After another 72 h, the supernatant was collected as a conditioned medium. (C and D) Analysis of firefly luciferase activity in Huh7-ISRE-Luc cells (n = 12) (C) or in an HCV replicon model (n = 12) (D) treated with Huh7.5-p6-derived conditioned medium for 72 h. (E and F) The Huh7.5-p6 cell model was transduced with FL-MAVS or empty vector or treated with 1000 ng/ml 136R (n = 9-11) (E), 1000 ng/ml B18R (n = 13-14) (F), or 0.1% DMSO control for 48 h. HEV RNA was measured by quantitative RT-PCR. Data in the FL-MAVS group are presented relative to the empty vector group (set as 1). Data in the combination group of FL-MAVS with 136R or B18R are presented relative to the 136R- or B18R-alone-treated group (set as 1). The data are…

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  Fig. 3. The antiviral effect of FL-MAVS against HEV is largely dependent on JAK-STAT activation. (A and B) An Huh7.5-p6 infectious model (n = 4-6) (A) or Huh7.5-p6-Luc replicon model (n = 4-8) (B) was transduced with FL-MAVS or empty vector or treated with 10 μM JAK inhibitor, 1000 IU IFNα, or 0.1% DMSO vehicle control. HEV RNA (48 h treatment) and luciferase expression (72 h treatment) was measured by quantitative RT-PCR. Data in the FL-MAVS group are presented relative to the empty vector group (set as 1). Data in the FL-MAVS group are presented relative to the empty vector group (set as 1). Data in the combination group of FL-MAVS with JAK inhibitor or IFNα are presented relative to JAK inhibitor- or IFNα-alone-treated group (set as 1). The data are the mean ± SEM (*, P < 0.05; **, P <0.01; ***, P < 0.001). (C) Immunoblot analysis of the indicated protein levels in Huh7-p6 cells transfected with empty vector (1 μg) or FL-MAVS vector (1 μg) or treated with JAK inhibitor for 48 h. MAVS was detected using an anti-MAVS antibody. (D) Quantitative RT-PCR analysis of ISG RNA in Huh7.5-p6 cells transduced with FL-MAVS or empty vector (n = 2) or treated with the indicated compounds for 48 h.…