Luciferase reporter assays

HuH7 cells (2 × 10⁵ cells) and HAP cells (6 × 10⁵ cells) were seeded in 6-well plates and transfected with plasmids on the next day using TransIT-LT1 reagent (Mirus Bio LCC) according to the manufacturer's instructions. The following plasmids were transfected: 1 µg (6-well format) of p5xUPRE-GL3, which encodes firefly luciferase controlled by a UPRE promoter [8,30], and 0.1 µg of a plasmid encoding Renilla luciferase under the control of the SV40 early enhancer/promoter (pGL4.73, Promega) for normalization purposes. To stimulate UPRE-dependent reporter gene expression, cells were either infected with MARV (MOI = 1), treated with thapsigargin (Tg, Sigma-Aldrich, T9033) or treated with tunicamycin (Tu, Sigma-Aldrich, T7765). To analyse UPRE activation by MARV proteins, the cells were additionally transfected with pCAGGS-based plasmids encoding viral proteins. If combinations of two viral proteins were to be expressed, 0.5 µg (Figure 4(a)) or 1 µg (Figure 6(e)) of each plasmid was used. Single viral proteins were expressed by transfecting 1 µg of the appropriate plasmid (Figure 1 and Figure 4(c)). Transfection within the setting of an infectious virus-like particle assay (iVLP) was performed as described by Wenigenrath et al. [25]. The negative control samples were mock-infected and/or treated with vehicle (DMSO). Stimulation with Tg or Tu was performed 16 or 24 h before the cells were lysed. MARV infection of cells was performed at 24 h post-transfection (p.t.). The cells were lysed at 48 h p.t. or p.i. in passive lysis buffer (Promega). Luciferase assays were performed using the Beetle-Juice and Renilla-Juice BIG KITs (PJK). Renilla luciferase signals were used to normalize for transfection efficiency.

MARV GP activates the unfolded protein response element. (a) HuH7 cells were transfected with plasmids encoding firefly luciferase under the control of an UPRE promoter, with pGL4.73, which encodes Renilla luciferase, and with plasmids encoding NP, VP35 or GP. HuH7 cells transfected with the empty vector were treated with vehicle (DMSO) or with Tg. The cells were lysed at 48 h post transfection (p.t.), and equal amounts of the cell lysates were subjected to Western blotting using monoclonal antibodies against GP, NP and tubulin and polyclonal anti-VP35 serum. The experiment was performed five times; the results of one representative experiment are shown. (b) Equal amounts of cell lysates were subjected to SDS-PAGE, and the gels were subsequently incubated with anti-HA antibodies to detect HA-tagged viral proteins. (c) Cell lysates were analysed using luciferase assays. Firefly luciferase activity was normalized to Renilla activity, and the fold activation in comparison to the DMSO control (set to 1) was calculated. The experiment was performed five times. Statistical analysis was performed for wildtype proteins. (d) HuH7 cells were treated and transfected as described in (a) except that the amount of GP_dMLD-expressing plasmid used for transfection was reduced (25 or 100 ng). The total amount of transfected plasmid was kept constant by the addition of empty vector. The experiment was performed four times. (e) Cell lysates were subjected to Western blotting using monoclonal antibodies to detect MARV GP and tubulin. Protein amount was quantified in each of the four independent experiments shown in d. Each circle represents a sample from an individual experiment, data are shown as the means ± SD.

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.

IRE1-dependent signalling during MARV infection. (a, b) HuH7 cells were infected with MARV at a MOI of 1. Cells were lysed at 24 h (a) and 48 h p.i. (b) and subjected to Western blot analysis to detect endogenous IRE1 (total and phosphorylated) and XBP1s proteins as explained in the legend to Figure 3. Total and phosphorylated IRE1 was quantified in each sample, compared to each other and set in relation to Tg-treated samples (set to 1). XBP1s levels were quantified and presented as relative values to DMSO-treated cells (set to 1). The experiments were performed three times. Each circle represents a sample from an individual experiment, data are shown as the means ± SD. (c) Scheme of XBP1-specific mRNAs and RT-PCR results. If there is no IRE1 activity, XBP1u mRNA is not spliced by IRE1; the PstI restriction site is available and the PCR product can be digested. Under conditions of IRE1 activation, XBP1u is spliced; PstI restriction site is lost and the PCR product cannot be digested by the enzyme. Intermediate phenotype: XBP1u is partially spliced; As published by others [39] we detect that XBP1u and XBP1s form a hybrid (XBP1 h, confirmed by sequencing) that is visible in the agarose gel and resistant to digestion. (d) XBP1-specific RT-PCR of RNA derived from HuH7 cells infected with MARV at a MOI of 1 for the indicated times. XBP1 splicing was induced using 5 nM Tg.