Fig. 5. YFV-17D-mScarlet replication is disrupted by signaling through endogenous or chimeric IFN receptors.

(A) The yellow fever virus (YFV)-17D viral genome was generated with an in-frame insertion of the mScarlet fluorescent protein between the regions encoding the E and NS1 proteins. (B) U2OS cells infected with YFV-17D-mScarlet were imaged in the RFP and transillumination channels. Scale bars, 200 μm. (C) Gating strategy for quantifying the frequency of YFV-17D-mScarlet–infected cells by flow cytometry. Dashed line, uninfected cells; solid line, infected cells (at an MOI of 1) 3 dpi. (D) Naïve (untransduced) U2OS cells were infected with YFV-17D-mScarlet at an MOI of 1 and fixed at the indicated times. The percentage of infected cells at each time was quantified by flow cytometry. (E) Naïve (untransduced) U2OS cells infected with YFV-17D-mScarlet (at an MOI of 1) were treated with IFN-β (1000 IU/ml) at either 1 or 2 dpi. Cells under all conditions were then fixed at 3 dpi and analyzed by flow cytometry to detect mScarlet as a measure of the percentage of cells that were infected. Uninfected and untreated groups were included as controls. (F) U2OS cells expressing chimeric type I or III IFN receptors were treated with EPO (100 ng/ml) at either 1 or 2 dpi and fixed at 3 dpi for analysis by flow cytometry to detect mScarlet-expressing cells. Naïve cells treated with IFN-β (1000 IU/ml) were included as a positive control. The type I chimera consists of EPOR_het-IFNAR2 paired with EPOR_M150E-IFNAR1, whereas the type III chimera consists of EPOR_het-IFNLR1 paired with EPOR_M150E-IL10RB. Infected cells are shown as a proportion of untreated naïve cells, which was set at 1. Data in (D) to (F) are means ± SD. ***P < 0.001, ****P < 0.0001; ns, not significant; uninfect, uninfected; NT, no treatment.