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. 2020 Dec 10;9:e60821. doi: 10.7554/eLife.60821

Figure 5. Vpr inhibits IRF3 nuclear translocation.

(A) Immunoblot detecting Phospho-STING (Ser366), total STING, phospho-TBK1 (Ser172), total TBK1, phospho-IRF3 (Ser386), total IRF3, or actin as a loading control, from extracted THP-1 cells expressing Vpr from a lentiviral vector (MOI 1), expressing empty vector, or THP-1 left untransduced as a control and transfected with HT-DNA (5 μg/ml) or left untransfected as a control. Size markers are shown in kDa. (B) Mean fold induction of IFIT1-Luc in cells from A and B (C) Flow cytometry plot (forward scatter vs pIRF3-S396 fluorescence) of THP-1 cells infected with Vpr bearing virus-like particles (VLP) lacking genome (1 RT U/ml), or Vpr free VLP, stimulated with cGAMP (5 μg/ml) or HT-DNA transfection (5 μg/ml). Lower panel shows the flow cytometry data as a bar graph, plotting pIRF3-S396-positive cells. (D) Single-cell immunofluorescence measurement of IRF3 nuclear translocation in PMA differentiated THP-1 cells treated with cGAMP, or left untreated, and infected with HIV-1 GFP bearing Vpr, lacking Vpr or left untransduced. Cells were fixed and stained 3 hrs after infection/transfection. Red line shows the translocation coefficient threshold. Blue lines represent mean translocation coefficient. (E) Percentage of cells in D with IRF3 translocation coefficient greater than 0.5 (above red line). (F) Single-cell immunofluorescence measurement of IRF3 nuclear translocation in PMA-differentiated THP-1 cells stimulated with cGAMP (5 μg/ml), or left unstimulated, and infected with HIV-1 GFP lacking Vpr or bearing WT Vpr or Vpr mutants as shown (1 RT U/ml) or left uninfected. (G) Single cell immunofluorescence measurement of IRF3 nuclear translocation in PMA differentiated THP-1 cells transfected with HT-DNA (5 μg/ml), or left untransfected, and infected with HIV-1 GFP lacking Vpr, or bearing WT or mutant Vpr (1 RT U/ml) or left uninfected. Data in B is expressed as means ± SEM (n = 2). Data is analyzed using two-way ANOVA: * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to data from infection with HIV-1 lacking Vpr. Data are representative of three (C–G) or two (A, B) independent experiments.

Figure 5.

Figure 5—figure supplement 1. Vpr inhibits IRF3 nuclear translocation.

Figure 5—figure supplement 1.

(A) Percentage of THP-1 cells in Figure 5A transduced by HIV-1 GFP vector bearing Vpr, or HIV-1 GFP lacking Vpr, transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (B) Immunoblot detecting Phosho-STING (Ser366), total STING, phospho-TBK1 (Ser172), total TBK1, phospho-IRF3 (Ser386) or total IRF3 from extracted THP-1 cells expressing Vpr, empty vector or left untransduced as a control, and transfected with HT-DNA (5 μg/ml), or left untransfected as a control. Size markers are shown (kDa). (C) Fold induction of IFIT1-Luc in cells from gel in Figure 5A, expressing Vpr, or empty vector, and transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (D) Percentage of THP-1 cells from Figure 5B transduced by HIV-1 GFP bearing Vpr, or lacking Vpr, transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (E) Fold induction of IFIT1-Luc in cells from second experiment (gel presented in Figure 5B) expressing Vpr, or empty vector, and transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (F) Single cell measurement of IRF3 nuclear translocation in PMA-differentiated THP-1 cells stimulated with LPS, or left unstimulated, and infected with HIV-1 GFP lacking Vpr or bearing Vpr (1 RT U/ml), or left uninfected (top panel). Percentage of cells with IRF3 translocation coefficient greater than 0.5 plotted as a percentage (bottom panel). (G) Percentage of cells with IRF3 translocation coefficient greater than 0.5 plotted as a percentage from Figure 5F. (H) Percentage of cells with IRF3 translocation coefficient greater than 0.5 plotted as a percentage from Figure 5G. (I) Single-cell measurement of IRF3 nuclear translocation in PMA differentiated THP-1 cells transfected with poly I:C, or left untransfected, and infected with HIV-1 GFP lacking Vpr or bearing WT or mutant Vpr (1 RT U/ml), or left uninfected. (J) Percentage of cells with IRF3 translocation coefficient greater than 0.5 plotted as a percentage from Figure 5I. Data is analyzed using two-way ANOVA test: * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to empty vector. Data are representative of three (F–K) or two (A–E) independent experiments.
Figure 5—figure supplement 2. Nuclear translocation of IRF3 after cGAMP stimulation in the presence or absence of Vpr.

Figure 5—figure supplement 2.