Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Immunofluorescence microscopy quantification of NFκB translocation, phosphorylated SP1 residue Ser101, phosphorylated cJun residues Ser63 and Ser73, and phosphorylated RNA polymerase II residues Ser2 and Ser5 in differentiated THP1 cells infected with Vpr WT or control viruses in the presence or absence of ATM, ATR, or DNA-PK inhibition ( n = 50 cells). Cells were infected for 24 hours, treated with vehicle or the indicated inhibitor for 24 hours, and then subjected to immunofluorescence microscopy. Analyses were performed using a one-way ANOVA; ns, not significant; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . (B) Representative immunofluorescence microscopy images of R-loop abundance in primary MDM and HeLa cells infected with indicated viruses 48 hours post-infection ( n = 50 cells). Analyses performed using a student t test; *** p < 0.001. (C–E) Immunofluorescence microscopy quantification of R-loop abundance in HeLa cells infected with indicated viruses 48 hours post-infection. Samples were left untreated (C) or treated with RNaseH (D, left), triptolide (D, right), or with the indicated DDR inhibitors (E), respectively ( n = 50 cells). For RNaseH treatment, cells were infected for 48 hours prior to methanol fixation and addition of recombinant RNaseH to deplete RNA associated with R-loops. For inhibitor treatments, HeLa cells were infected for 24 hours, treated with the indicated inhibitor for 24 hours, and then subjected to immunofluorescence microscopy. Analyses were performed using a one-way ANOVA; ns, not significant; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . (F) Immunofluorescence microscopy quantification of DDR activation (left) and R-loop abundance (right) in HeLa cells infected with indicated viruses and transfected with RNaseH constructs ( n = 50 cells). Cells were infected for 24 hours before transfection of control or RNaseH-expressing plasmids for 24 hours prior to being prepared for immunofluorescence microscopy. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001; * p < 0.05. The data underlying this Figure can be found in . (G) Flow cytometric histograms of HeLa cells infected with control or Vpr WT LTR-mCh viruses for 24 hours prior to transient expression of RNaseH for 24 hours and quantification of LTR-mCh MFI in transfected cells via flow cytometry ( n = 3 experiments). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ).

Article Snippet: For inhibitor experiments, cells were treated 24-hours post-infection with either 10 nM ATM inhibitor (Fisher, #AZD1390), 10 μM ATR inhibitor (Fisher, #NU6027), 3 mM caffeine, 16 μM DNA-PK inhibitor #1 (MedChemExpress, #NU7026), or 16 μM DNA-PK inhibitor #2 (MedChemExpress #NU7441) for 24 hours.

Techniques: Immunofluorescence, Microscopy, Translocation Assay, Residue, Infection, Control, Inhibition, Recombinant, Activation Assay, Transfection, Construct, Expressing, Flow Cytometry