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. 2020 May 26;8(6):790. doi: 10.3390/microorganisms8060790

Figure 1.

Figure 1

UL35 downmodulates transcription of IFNβ in luciferase reporter assays. (A) HEK293T cells were co-transfected with a reporter plasmid coding for firefly luciferase under the control of the murine IFNβ promoter (IFNβ-Luc) together with a Renilla luciferase normalization control (pRL-TK) and expression plasmids for IRES-GFP (unstimulated) or cGAS-GFP (stimulated), Cherry-STING and either empty vector (ev), HA-tagged M35, HA-tagged UL35 or untagged UL35. Cells were lysed 20 h later and luciferase activity was analyzed. An anti-HA antibody was used for detection of HA-tagged versions of UL35 and M35, and expression of untagged UL35 was verified with a UL35-specific antibody. (B–E) HEK293T cells were transfected with expression plasmids for IFNβ-Luc, pRL-TK and (B) RIG-I N, (C) MAVS, (D) TBK1, (E) IRF3-5D, or the respective ev. Cells were further co-transfected with either ev, HA-tagged UL35 or V5-tagged M35. Luciferase activity was analyzed as described in (A). (F) HEK293T cells were co-transfected with a reporter plasmid coding for firefly luciferase under the control of the human ISG56 promoter (ISG56-Luc), pRL-TK, and either ev, UL35-HA, M35-V5, or M27-V5. 24 h later, 0.1 ng/mL recombinant human IFNβ or vehicle only (unstimulated) was added. Cells were lysed 16 h post stimulation and luciferase activity was assessed. (A–F) Luciferase fold induction was calculated based on the firefly luciferase values normalized to Renilla from stimulated and unstimulated samples. Data was combined from at least three independent experiments and is shown as mean ± SD. Values were normalized to ev control. Significance compared to ev was calculated using the Student’s t-test (unpaired, two tailed), ns = not significant, *** p < 0.001, **** p < 0.0001.