Figure 3
From: Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration
2-LTR circles account for de novo integration and the resumption of viral replication after RAL removal. (A) Flow cytometry analysis of envelope-pseudotyped viruses NLENG1-ES-IRES expressing eGFP (Additional file 1: Figure S2A-B). 1: WT without RAL. 2: D116N without RAL. 3–5: WT + 500 nM RAL (added at d0). RAL was maintained (3) or removed on d2 (4) or d3 (5). 6: NT: non-transduced cells. We monitored eGFP expression on d4 (4) or d5 (1–3, 5–6). Left, percentage of GFP+ cells. Right, gating on the GFP+ cells to discriminate DNAi expression (HMFS: high mean fluorescence signal) from unintegrated viral DNA expression (LMFS: low mean fluorescence signal). Data are representative of five independent experiments. (B) Quantification of DNAi, 2-LTRc, 1-LTRc and DNAL corresponding to experiments 1, 3 and 5. RAL was removed (ΔRAL) or maintained (RAL) at d3 post-infection. Percentage of 1-LTRc (dark grey column), 2-LTRc (black column), percentage of DNAi (white column) and percentage of DNAL (light grey column) are calculated. Raw data in copy number are reported. Results are the mean from five representative independent experiments ± standard deviation (error bars).