Figure 3. Vpr inhibition of innate immune activation is dependent on DCAF1 but independent of cell cycle arrest.

(A) Immunoblot detecting p24 (capsid) and Vpr in pelleted VSV-G pseudotyped VLP lacking genome used in (B). Size markers in kDa are indicated on the right. (B) Fold induction of IFIT1-Luc after activation of STING by cGAMP (5 μg/ml) and infection with VLP bearing WT or mutant Vpr, or lacking Vpr (1 RT U/ml) in IFIT1-Luc reporter THP-1 cells. Cells were infected at the same time as cGAMP treatment. (C) Flow cytometry plots showing cell cycle phases of THP-1 cells transduced with an empty vector, WT Vpr, or mutant Vpr, encoding vector (MOI 1) or left untransduced as a control and stained with propidium iodide to label DNA. Percentage cells in each cell cycle stage are shown. (D) Fold induction of IFIT1-Luc after activation of STING by cGAMP (5 μg/ml) in cells expressing Vpr from a lentiviral vector, or expressing empty vector, or in untransduced IFIT1-Luc reporter THP-1 cells expressing a control, or a DCAF1 targeting shRNA. Mean +/- SEM n = 3 independent experiments. (E) Immunoblot detecting DCAF1, or actin as a loading control, from extracted THP-1 cells expressing a non-targeting, or DCAF1-targeting, shRNA. Size markers are shown in kDa on the right. (F) Fold induction of IFIT1-Luc after activation of STING by cGAMP (5 μg/ml) in cells expressing WT, or mutant, Vpr from a lentiviral vector (MOI 1), or empty vector (MOI 1) or in untransduced IFIT1-Luc reporter THP-1 cells. (G) Fold induction of MxA mRNA after activation of STING by cGAMP (5 μg/ml) in cells expressing WT, or mutant, Vpr from a lentiviral vector (MOI 1), or after transduction by empty vector (MOI 1) or in untransduced THP-1 cells. Data are mean ± SD (n = 3). Two-way ANOVA test: * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to no Vpr or empty vector controls. Data are representative of three (B–D, F) or two (A, E, G) independent experiments.

Figure 3—figure supplement 1. Vpr inhibition of innate immune activation is dependent on DCAF1 but independent of cell cycle arrest.

(A) Percentage of THP-1 cells in Figure 3C transduced by the vector encoding Vpr and GFP, or empty vector encoding GFP alone, at the indicated MOI and treated with cGAMP (5 μg/ml) or left untreated. (B) NMR structure of full-length Vpr showing position of Vpr mutants (PDB: 1M8L). White region (c-terminus) of Vpr shown in (B) is unresolved in the crystal structure (C). (C) Crystal structure of Vpr (orange) with its target protein UNG2 (blue) and cofactors DCAF1(pink) and DDB1 (green) showing position of Vpr mutations (PDB: 5JK7). (D) Percentage of THP-1 cells in Figure 3F transduced by the vector encoding WT, or mutant, Vpr and GFP (MOI 1), or empty vector encoding GFP alone (MOI 1), and treated with cGAMP (5 μg/ml), or left untreated as a control. (E) Fold induction of IFIT1-Luc after HT-DNA (5 μg/ml) transfection in cells expressing WT, or mutant, Vpr from a lentiviral vector (MOI 1), or empty vector (MOI 1), or in untransduced IFIT1-Luc reporter THP-1 cells. (F) Percentage of THP-1 cells in Figure 3E transduced with HIV-1 vector encoding WT, or mutant, Vpr and GFP (MOI 1), or empty vector encoding GFP alone (MOI 1), and transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (G) Percentage of THP-1 cells in G2/M phase of cell cycle after transduction with an empty vector (MOI), or vector encoding WT Vpr, or mutant Vpr, (MOI 1) or left untransduced as a control. Mean+/-SEM n = 2. Unless stated data are expressed as means ± SD (n = 3). 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 (A), (D) or two (E–G) independent experiments.