Old) (Figure 4A, lower panel). This phenomenon represents an experimental case
Old) (Figure 4A, lower panel). This phenomenon represents an experimental case in which integration inhibition does not systematically lead to strong 2-LTRc accumulation. Indeed, in the TL3.4 cells, 2-LTRc accumulation was observed only with RAL treatment or D116N (Figure 4A, lower panel). This also suggests that LEDGF/p75 is not an essential factor for 2-LTRc formation. It is important to note that the inhibition of the remaining integration between conditions of TL3.4 infected with WT, and TL3.4 infected with D116N or in the presence of RAL (Figure 4A, upper panel, columns 3 and 4), cannot explain the difference of 2-LTRc accumulation in these conditions of infection (Figure 4A, lower panel, columns 3 and 4). Indeed, the observed 7-fold decrease in integration is expected PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28945807 to display 85 of maximal 2-LTRc accumulation based on the correlation shown in Figure 1D. Moreover, as described previously in other cell lines, RAL reversal produced a fall in the percentage of 2-LTR circles in both cell lines (Additional file 1: Figure S8). These results indicate that when integration is inhibited without blocking IN catalytic competence (i.e. in the absence of LEDGF), the inhibition is not necessarily associated with 2-LTRc accumulation since IN is still competent to GW0742 cost cleave 2-LTRc (see model in Figure 4B). Accordingly, inhibiting the catalytic activity of IN (RAL treatment or D116N infection) leads to accumulation of 2-LTRc due to the inability of IN to cleave these circular DNA forms. Our data imply that IN plays a key role in controlling the balance between the amounts of DNAL PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27484364 and 2-LTRc through direct effects on 2-LTRc – > DNAL conversion. In this context, RAL removal leads to 2LTRC cleavage, which in turn produces new DNAL that can integrate and support resumption of viral replication. Taken together with the above-mentioned observation of new DNAL forms after RAL removal, our data indicate that IN catalytic activity is directly involved in the 2-LTRc – > DNAL conversion.Thierry et al. Retrovirology (2015) 12:Page 7 ofFigure 4 2-LTRc are related to the IN catalytic activity. Cells expressing (TC3) or depleted (TL3.4) of LEDGF/p75 were infected with NLENG1-ES-IRES-WT +/- 500 nM RAL or NLENG1-ES-IRES-D116N. (A) Percentage of integration efficiency (upper panel) and 2-LTRc (lower panel) at d3 post-infection for both cell lines. Results are representative of four independent experiments; p value is reported in the histogram. (B) Model explaining the modulation of 2-LTRc amount in different contexts: LEDGF+ (TC3) or LEDGF- (TL3.4) with IN+ (WT) or IN- (D116N or WT + RAL). The numbers above the bars in panel A are related to the corresponding model in panel B.Integrase is found at the palindromic junction of 2-LTRcThe IN-dependent conversion of 2-LTR circles into DNAL implies that IN should be physically present at the palindromic junction. It has been demonstrated by Bukrinsky and collaborators that HIV-1 IN was not detected in association with 2-LTRc [27]. Importantly, this result was obtained under condition where no 2-LTRc accumulation was observed (i.e. WT infection in the absence of any INSTI compound). We then performed ChIP (chromatin immunoprecipitation) experiments to assess the presence of IN on 2-LTRc within cells under 2-LTRc accumulation conditions at 24 and 72 hours post-infection. According to Bukrinsky’s study [27], IN was not found at the palindromic junction during WT infection (Figure 5), probably due to the small amount of 2-LT.