Table 1.
Demographic data, ancestry, and sequence alterations in ADAR1 mutation–positive donors.
Fig 1.
ADAR1 deficiency restricts HIV-1 replication in activated human CD4+ T cells.
(a-b) PBMCs from healthy donors (n = 4) and ADAR1-mutated patients (n = 3) were challenged with equivalent infectious units of HIV-1 GFP virus and analyzed on day 5 after infection. The reverse transcriptase inhibitor (RTI) cocktail (10μM AZT, 20μM DDI, and 10μM 3TC) was used as specificity control. (a) Representative dot plots of flow cytometry analysis from PBMCS of an AGS patient and a healthy control. The viable cells (gate, left graphs) were stained and selected for CD3+CD4+ expression (b). The percentages of infected (GFP+) CD4+ T cells (c) are shown in the upper right quadrant. (d) Bar graph representing the mean±s.e.m. of GFP+ infected cells (n = 3), ***P<0.001. (e) Bars represent luciferase activity in relative luciferase units (RLU) of cells infected with a VSV-G virus. The activity was measured in non-stimulated (NS), IL-2-stimulated or RTI-treated cells. Bars represent mean±s.e.m (n = 3). (f) Bar graphs represent mean±s.d (n = 3) of HIV-1 p24 protein amounts released into the cell culture supernatant on day 5 after infection, *P<0.05. Relative quantification of viral DNA pol (g) and viral pol mRNA (h) expressed as fold versus NS control cells. (i) Relative quantification of ISG15, IFIT27, RSAD2, IFIT44L, and OAS1 from HIV-1-infected PBMCs from AGS patients (n = 3) and healthy donors (n = 3) presented as fold over NS control expression.
Fig 2.
Silencing ADAR1 restricts HIV-1 replication in Jurkat T cells.
(a) Jurkat T cells were stable transduced with shRNA control, GFP control or ADAR1 shRNAs. Bar graph shows normalized ADAR1 mRNA levels. Panel shows western blot of the two best shRNA targeting ADAR1. (b) Cells were transduced at MOI 10 or 20. Panels show representative western blot of ADAR1 protein reduction in ADAR1 shRNA transduced cells. Bar graphs show densitometry analysis of bands compared to Control shRNA. Actin was used as loading control. (c) Control shRNA (n = 4) and ADAR1 shRNA (n = 4) transduced Jurkat T cells were challenged with equivalent infectious units of HIV-1 GFP virus (MOI 0.1 or 1) and analyzed by FACS on day 5 after infection. Panels show representative dot plots of FACS analysis from transduced cells. The viable cells (gate, left graphs) were analyzed for GFP fluorescence. Histograms show GFP signal of Control shRNA (black line) and ADAR1 shRNA cells (red line) after HIV1-GFP infection at different MOIs. (d) Bar graph representing the mean±s.d. of GFP+ infected cells (n = 4), **P<0.01. (e) Bar graph representing the mean±s.d. of GFP fluorescence of infected cells (n = 4), **P<0.01. (f) Bar graphs represent mean±s.d. (n = 3) of HIV-1 p24 protein amounts released into the cell culture supernatant on day 5 after infection, *P<0.05. (g) Histograms show GFP signal of control shRNA (black line) and ADAR1 shRNA cells (red line) after VSV-G-GFP infection at different MOIs. (h) Bar graph representing the mean±s.d. of GFP+ infected cells (n = 3) **P<0.01, *P<0.05.
Fig 3.
Relative quantification (mean±s.e.m) of viral DNA pol (a) and viral pol mRNA (b) expressed as fold versus NT-shRNA control cells (n = 3). (c) Gag (n = 2) and (d) GFP (n = 3) mRNA expressed as fold versus NT-shRNA control cells (e) Bar graphs show the ratio of Gag protein versus Gag mRNA levels on infected cells (n = 3), *P<0.05. (f) Time course ratio expression of Gag protein versus Gap mRNA from day 1 to day 5 after infection (n = 2 =). (g) Relative quantification of ISG15, IFIT27, RSAD2, IFIT44L, and OAS1 of control shRNA and ADAR1 shRNA cells. (h) Western blot of eIF2α and the phosphorylated form of eIF2α. Actin was used as loading control.