Synthetic rewiring and boosting type I interferon responses for visualization and counteracting viral infections

Figures

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  Figure 1.. mIFN-β and doxycycline-controlled expression in Conv cells. (A) Depiction of the intracellular circuit in Conv cells for rewiring and switching-off of expression in presence of doxycycline (Dox). The transcriptional activator tTA is integrated downstream of the Mx2 promoter (Mx-tTA cells, see Supplementary Figure S1 for details) and expressed only upon stimulation of IFNAR with mIFN. tTA binds to its cognate PTet promoter and activates luciferase expression. Doxycycline blocks binding of tTA and thereby switches off expression. Coloured boxes indicate induced genes upon IFN stimulation, white boxes indicate the situation in the repressed state (+Dox). (B) Relative Mx2 and tTA mRNA levels were determined in Mx-tTA embryonic stem cells and in in vitro-differentiated primary cells derived thereof. Upon stimulation of cells with 500 U/ml mIFN-β for 24h, mRNA was isolated. Mx2, tTA and actin levels were quantified by RT-qPCR. The fold induction of induced vs. non-induced cells is depicted. The presented data were derived from a single experiment done in triplicates. Mean values (C) mRNA levels of Mx2 and tTA were determined upon stimulating MxtTA cells with 50 U/ml mIFN-β for the…

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  Figure 2.. Conv cells respond to NDV but not to mCMV infection. (A) The IFN levels were determined in the supernatants of Conv cells upon infection with NDV (80 HA units/ml) and mCMV (MOI 6), respectively, on day 2 upon infection. The presented data are based on six (NDV) or three (mCMV) samples from two and one independent experiments, respectively. (B) Conv cells were infected with NDV (80 HA units/ml) and mCMV (MOI 6) for one hour. Virus was removed and luciferase expression of Conv cells was determined 24 h afterwards. The presented data are based on six (NDV) or three (mCMV) samples of two independent experiments. (C) mCMV-mediated blocking of the IFN response pathway was determined by infecting Conv cells with mCMV (MOI 6) for 1 h and subsequently stimulating with 50 U/ml mIFN-β for 24 h. Luciferase expression was analysed 24 h after infection. The experiment was performed twice in triplicates. (D) Conv cells were infected with mCMV (MOI 6) for 1 h. The relative expression of endogenous Mx2 and synthetic tTA was determined by RT-qPCR in absence of virus as well as at the indicated time points after infection with mCMV. The fold induction was calculated in relation to non-infected…

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  Figure 3.. Implementation of a positive feedback module for increased and sustained IFNAR-driven transgene expression. (A) Schematic depiction upon transient activation of Conv cells as well as the ConvAmp cells, e.g. in presence of antagonistic viral proteins. Coloured boxes indicate constantly induced genes, white boxes indicate the situation upon viral counteraction. ConvAmp cells were generated upon integrating the PTet-tTA positive feedback module into Conv cells. As actuators, PTet-luc and PTet-EGFP are depicted. (B) Luciferase expression of ConvAmp cells was determined 24 h after stimulation with 500 U/ml mIFN-β in the presence and absence of doxycycline. Data are based on one out of two independent experiments performed in triplicates. (C) The kinetics of relative Mx2 and tTA mRNA levels were determined by RT-qPCR 8 h or for 72 h after stimulating Conv and ConvAmp cells with mIFN-β (500 U/ml). The experiment was performed twice in triplicates for the untreated and 72 h timepoints and twice in duplicates and triplicates for the 8 h timepoint. (D) The time course of luciferase expression was determined in ConvAmp cells upon a transient stimulation with 500 U/ml mIFN-β for 24 h (black…

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  Figure 4.. Sensitivity of ConvAmp cells toward mCMV infection. (A) The sensitivity of ConvAmp cells in response to mCMV infection was determined. ConvAmp cells were infected with an tRFP-tagged mCMV using indicated MOIs. Frequencies of infected cells were determined by flow cytometry (left, 20 h after infection). Luciferase responses were evaluated 24 h after infection (right). Shown are the mean of duplicates. (B) ConvAmp-eGFP cells were seeded one day prior mCMV infection. Cells were infected with mCMV (MOI 1) for 1 h and subjected to time-lapse microscopy. Time points indicate image acquisition post-infection. Representative pictures of one out of two experiments are shown. Luciferase expression of mCMV infected ConvAmp cells is provided in Supplementary Figure S10.

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  Figure 5.. Conversion of the trigger mIFN-β to hIFN-β protects human cells from infection. (A) Schematic representation of Conv-hIFN and ConvAmp-hIFN cells and induced protection against viral infection in presence of antagonistic viral proteins. Coloured boxes indicate constantly induced genes, white boxes indicate the situation upon viral counteraction. (B) Conv-hIFN and Conv-Amp-hIFN cells were cultured in the absence or presence of doxycycline and stimulated for 6h with 500 U/ml mIFN-β. The supernatant was harvested 24h after treatment. Human A549 cells were pre-incubated for 16 h with the different supernatants and then infected with eGFP tagged VSV for one hour (MOI 1). Viral eGFP expression was analysed 24h post infection by flow cytometry. The experiment was performed once in triplicates. (C) ConvAmp-hIFN cells were co-cultured with BFP-tagged A549 cells in a 1:5 ratio in the presence or absence of doxycycline. Cells were stimulated for 6 h with 500 U/ml mIFN-β. 16 h later these cells were infected with VSV (MOI 1) and analysed by time-lapse microscopy. Representative pictures are shown from one experiment performed in triplicates. (D) ConvAmp-hIFN cells were co-cultured with…