Figure 4.

VP30 reduces GP- and Tg-induced UPRE-dependent signalling. (a) HuH7 cells were transfected with plasmids encoding the indicated MARV proteins and the UPRE-specific luciferase reporter plasmids as described in the legend to Figure 1. To express viral proteins, 0.5 µg of each plasmid was used in the transfection. In the iVLP setting, which involved the use of a combination of plasmids encoding all MARV proteins, the plasmid amounts used in transfection were as described by Wenigenrath et al. [25]. The experiment was repeated 4 times. (b) Equal amounts of lysates of transfected HuH7 cells were subjected to Western blot analysis using monoclonal antibodies. NP, GP, VP30, and tubulin were detected simultaneously; VP40 was stained afterwards on the same blot. The asterisk indicates remaining VP30 staining; irrelevant lines have been removed. (c) VP30-dependent reduction of Tg-induced UPR. Tg (5 nM) was used to induce UPRE-dependent reporter gene expression in VP30-, VP35-, and GP-expressing cells that had been transfected as described in the legend to Figure 1. The experiment was repeated 4 times. (d) Western blot analysis of cell lysates obtained from c. VP35 was stained with a polyclonal antibody against VP35; GP, VP30, and tubulin were detected afterwards on the same blot using monoclonal antibodies. Each circle represents a sample from an individual experiment, data are shown as the means ± SD. (e) To analyse UPRE-dependent luciferase activity, HAP1 cells (wt, shown in yellow) or HAP1 IRE1 KO cells (shown in pink) were transfected, treated and harvested as described for HuH7 cells. To restore IRE1 signalling in KO cells, the KO cells were transfected with a plasmid encoding IRE1 (100 ng); The cells were treated either with vehicle (DMSO) or with 5 nM Tg for 16 h. The experiments were performed three times. Each circle represents a sample from an individual experiment, data are shown as the means ± SD.