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

Figure 1. HIV-1 replication in cGAMP-stimulated MDMs requires Vpr.

(A) Replication of WT Yu2 HIV-1 or Yu2 HIV-1ΔVpr in MDMs stimulated with 1 μg/ml, 2 μg/ml or 4 μg/ml cGAMP or left unstimulated, infection measured by counting Gag-positive cells stained with anti-p24. Mean+/-SEM n = 3 1 and 2 μg/ml cGAMP; n = 2 4 μg/ml cGAMP. *** = two-way ANOVA p value < 0.001, *=p < 0.05. (B) Fold induction of IFIT1-Luc after activation of STING by cGAMP (5 μg/ml) and infection with HIV-1 virus-like particles (VLP) lacking genome and bearing Vpr (+Vpr) or lacking Vpr (-Vpr) (1 RT U/ml) in IFIT1-Luc reporter THP-1 cells. cGAMP and virus were added to cells at the same time. (C) Fold induction of CXCL10 after infection of THP-1 cells with HIV-GFP -Vpr or HIV-GFP +Vpr at the indicated MOI. (D) Percentage of THP-1 cells infected by HIV-GFP -Vpr or HIV-GFP +Vpr in (C). (E) Fold induction of CXCL10 after infection of THP-1 cells with HIV-GFP -Vpr, HIV-GFP +Vpr, or HIV-1 particles lacking Vpr and genome, at indicated doses measured by reverse transcriptase SG-PERT assay. (F) Percentage of THP-1 cells infected by HIV-GFP viruses in (E). (G) Fold induction of CXCL10 after infection of unmodified control, cGAS-/-or MAVS-/- THP-1 knock out cells with HIV-GFP lacking Vpr (0.3 RT U/ml). (H) Percentage infection of control, cGAS-/-or MAVS-/- THP-1 knock out cells infected with HIV-GFP at indicated doses of RT (SG-PERT). (B–H) Data are expressed as means ± SD (n = 3) with two-way ANOVA * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to virus without genome (B), HIV GFP+Vpr (C, E) and control (G).

Figure 1.

Figure 1—figure supplement 1. HIV-1 replication in cGAMP-stimulated MDMs requires Vpr and Vpr suppresses HIV-1 innate immune sensing by cGAS.

Figure 1—figure supplement 1.

(A) Replication of wild-type (WT) NL4-3 HIV-1, or NL4-3 HIV-1ΔVpr, in activated primary human CD4+ T cells stimulated with 1, 2, or 4 μg/ml cGAMP or left unstimulated as a control. Two representative examples of three are shown with virus replication measured by percentage T-cell p24 positivity, measured by flow, (top panels) or supernatant RT activity (lower panels). This experiment was also performed twice in Jurkat cells with virus replication measured by percentage T-cell p24 positivity, measured by flow, giving similar results as shown.’ Replication of WT NL4-3 HIV-1 or NL4-3 HIV-1ΔVpr in activated CD4+ T cells stimulated with 1 μg/ml, 2 μg/ml, or 4 μg/ml cGAMP or left unstimulated, measured by flow cytometry staining infected cells with anti-p24 antibody. (B) HIV-GFP titre in control, cGAS-/-or MAVS-/- THP-1 cells used in Figure 1G. (C) Immunoblot detecting cGAS, MAVS, or actin as a loading control, from extracted cGAS-/-or MAVS-/- knock out THP-1 cells or their CRISPR/Cas control cells. Size marker positions are shown on the right (kDa).