Egration eventsdetected on d5 occurred whether or not drugs that prevent
Egration eventsdetected on d5 occurred whether or not drugs that prevent successive infection rounds (SAQ, T-20 or AZT) were added at the time of RAL removal on d3. Thus, they fully reflected de novo integration arising from pre-accumulated unintegrated viral DNA originating from infection at d0 and still present at d3. A last condition was performed adding RAL at d0 and AZT at d1 (AZT was maintained until d7), allowing reverse transcription to occur but preventing a weak replication from unintegrated viral DNA as highlighted by Trinite and colleagues [23]. In this condition, when RAL was removed at d3, the amount of DNAi at d5 was similar to the one quantified in the condition without AZT, excluding a major role of the replication from unintegrated viral DNA in the detection of new integration events after RAL removal (Figure 2D). In contrast, the further increase in DNAi on d7 (compared to d5) in the absence of SAQ, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26795252 T-20 or AZT reflected subsequent rounds of infection (Figure 2D). Newly integration events are thus compatible with synthesis of new viral progeny highlighting that integration from pre-accumulated unintegrated viral DNA is biologically relevant.Newly integration events after RAL removal result from a DNAL intermediate generated from 2-LTRcTo confirm that the de novo integration events originate strictly from accumulated unintegrated DNA forms, weThierry et al. Retrovirology (2015) 12:Page 4 ofFigure 2 Reversibility of RAL action. Quantification of total viral DNA at various time post-infection (A-B). (A) MT4 cells were infected on d0 with pNL4-3 WT or D116N (Additional file 1: Figure S2C) +/-RAL (500 nM), +/-SAQ (1 M). On d3, RAL and/or SAQ were removed (RAL or SAQ) or maintained (+); Efavirenz (500 nM) was added (+) or not (-) to the experiment. Black triangle, curve 8: D116N infection; black diamond, curve 9: D116N + SAQ infection. (B) RAL blockade from d0-3 (black diamonds) compared to blockade from d1-3 (open circles). On d3 RAL was removed from all cultures, and the RAL d1-3 culture was diluted 1/10, 1/100 or 1/1000 with uninfected cells or left undiluted. (C) Quantification of DNAi on d3 and d7 for the experiments shown in panels A-B. (D) Quantification of DNAi with RAL added and removed on d0 and d3, respectively. SAQ (1 M), or T-20 (1 M) was added on d3, when RAL was removed. AZT (25 M) was added at d3 when RAL was removed or at d1 and maintained until d7. Results are the mean from three representative independent experiments ?standard deviation (error bars).then assessed RAL reversal using single cycle eGFP reporter viruses that prevent successive rounds of infection [24]. Cells were infected at low multiplicity of infection (m.o.i.) (Figure 3, 40 ng of p24Gag per 106 cells) or at a higher m.o.i. (200 ng of p24Gag per 106 cells, Additional file 1: Figure S5). Cell fluorescence intensities had bimodal distributions (Figure 3A, right). High mean fluorescence signal (HMFS) originates from the strong Stattic manufacturer transcriptional activity of DNAi, whereas the low mean fluorescence signal (LMFS) originates from the weaker transcriptional activity of unintegrated DNA as already described [24]. In the absence PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29072704 of RAL (WT), 3.08 of the total cell population was GFP+. 59.4 of the GFP+ cells displayed LMFS and 40.6 displayed HMFS (Figure 3A, panel 1). In contrast, HMFS was negligible when integration was prevented: LMFS was99.3 with RAL treatment or when a D116N mutant reporter virus was used (Figure 3A, panels 2?). Upon RAL removal,.
