Fig 2: The hantaviral structural proteins affect the transcriptional activity of the promoters of TRAIL/TRAIL-Rs. Firefly luciferase reporter plasmids (400 ng) containing TRAIL, DR4, DR5, DcR1, or DcR2 promoter and hantaviral structural plasmids (400 ng) including pSicoR (vector), pSicoR-S, or pSicoR-M were co-transfected into HEK293T cells to detect the promoter activity. Luciferase activity represented the effect of hantaviral structural proteins on the promoters of TRAIL/TRAIL-Rs. The data were presented with the mean ± SD from three independent experiments of relative luciferase activity (*p < 0.05; **p < 0.01).

Fig 3: shRNA-mediated knockdown and rescue of TRAIL influence HTNV replication and apoptosis in HUVECs. HUVECTRAIL-KD cells were infected with HTNV76-118 (MOI = 1) for 2 h, treated with/without rTRAIL (40 ng/mL), and then followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors and HTNV. The mRNA results shown were the average of three replicates. Values represent the mean ± SD (*p < 0.05; **p < 0.01; ***, p 0.001). (A) HTNV S mRNA, (B) TRAIL mRNA, (C) DR4 mRNA, (D) DR5 mRNA, (E) DcR1 mRNA, (F) DcR2 mRNA, (G) caspase-8 mRNA, and (H) caspase-3 mRNA. (I) HTNV NP and TRAIL-related apoptosis proteins in HTNV-infected HUVECTRAIL-KD cells at 2, 3, and 4 dpi. (J) HTNV NP and TRAIL-related apoptosis proteins of HUVECTRAIL-KD cells with rTRAIL rescue. Proteins were quantified using carestream software and compared with the mock group of 2 dpi; the data shown represents one of three similar independent experiments. The numbers represent the relative density of the bands relative to the corresponding control.

Fig 4: Exogenous rTRAIL inhibits HTNV infection by enhancing caspase-8-dependent apoptosis. HUVECs were infected with HTNV76-118 (MOI = 1) for 2 h, treated with/without rTRAIL (40 ng/mL), and then followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors and HTNV. (A) HTNV S, TRAIL DR4, DR5, caspase-8, and caspase-3 mRNA expression. (B) HTNV NP and TRAIL-related apoptosis proteins. (C) DcR1/DcR2 mRNA and protein. The mRNA results shown are the average of three replicates and values represented the mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001). The protein result represents one of three similar experiments. Numbers represent the relative density of the bands relative to the internal control. (D) Immunofluorescence images and (E) statistical analysis of TUNEL assay on HTNV-infected HUVECs with rTRAIL treatment. Cells were stained for virus infection [NP (sred)], for apoptosis [TUNEL (green)], and for nucleus [Hoechst (blue)]. Images data showed one of three independent experiments with similar results.