Fig 1: Effect of FACT complex on in vitro HIV-1 integration. A concerted integration assay was performed using 200 nM of HIV-1 IN, 10 ng of donor DNA, 50 ng of p5S naked or chromatinized plasmid DNA (PN DNA), and increasing concentrations of FACT complex. The reaction products were loaded onto 1% agarose gels, and a representative set of experiments performed with the wild-type integrase is reported in (a). The positions and structures of the donor substrate and the different half-site (HSI), full-site (FSI) and donor/donor integration (d/d) products are shown. Quantification of the total integration products (FSI, HSI and donor/donor) was performed via gel detection and is reported in (b) as the percentage relative to activity without FACT. Effect of FACT complex or SSRP1 or Spt16 proteins [optimal FACT concentration as determined in (b)] on integration into PN DNA was analyzed and integration efficiency was reported in (c). Effect of FACT nucleosome remodeling activity on integration catalyzed by HIV-1 IN on chromatinized substrates was analyzed by comparing chromatinized p5S vector (histone/DNA ratio = 1) treated with UV or untreated as reported in “Methods” section. The treated or untreated substrates were then used in concerted integration assays without FACT or with increasing concentrations of the complex. Quantification of the total integration products detected on gel (see representative experiment in Additional file 4: Figure S4) (FSI, HSI and donor/donor) was performed via gel detection and is reported in (d) as the percentage relative to activity detected on naked DNA (pre-normalized data obtained in the control experiments using naked DNA and without FACT are also reported as the percentage of integrated substrate). All values are shown as the mean ± standard deviation (error bars) of at least three independent sets of experiments
Fig 2: Effect of FACT knock down on early steps of HIV-1 derived lentiviral vectors in HEK293T cells. SSRP1 encoding gene knock down was conducted essentially as previously described [31] by performing two successive siRNA transfections (see “Methods” section). The amount of SSRP1 protein monitored by western blotting is reported in (a). This method allowed us to achieve more than 80% of apparent SSRP1 extinction. After knock down, FAIRE analyses were performed as indicated in “Methods” section and the results obtained in cells depleted for SSRP1 are reported in (b). The early steps of replication of lentiviral vectors carrying the GFP encoding gene were determined by measuring GFP-positive cells with flux cytometry (c) and quantification of the viral DNA populations at 0–48 h post-transduction using quantitative PCR (data obtained after a 20 nM siRNA treatment are reported in d for the 24 and 48 h time points, see full time course analysis in Additional file 7: Figure S7). Data obtained with the interferin transfector agent alone and control siRNA are also reported in the figure. All values are shown as the mean ± standard deviation (error bars) of four independent sets of experiments. The p-values were calculated by Student’s t-test and are shown as *p < 0.05 and **p < 0.005 to represent the probability of obtaining significant differences compared with untreated conditions
Fig 3: Model for FACT action on HIV-1 integration steps. HIV-1 intasomes are targeted to the PolII-transcribed region of the chromatin thanks to the association of IN with LEDGF/p75 and binding to H3K36me3. In these regions the FACT complex and LEDGF/p75 are both enriched owing to the LEDGF/p75 and SSRP1 interaction. Chromatin remodeling mediated by FACT occurring in the vicinity of the targeted region may generate partially dissociated nucleosomes leading to chromatin structures that are preferential substrates for both RNA transcription and HIV-1 integration. FACT action on chromatin at the vicinity of the integration sites may lead to increased accessibility to nucleosomal DNA as well as histone tails. Integration could then occur efficiently on partially dissociated nucleosome or accessible native nucleosome. Further FACT-mediated regulation of the viral genes transcription can then occur. The expected effect of curaxin treatment on HIV-1 and PFV integration is reported on the basis of the drugs effect on FACT chromatin remodeling activity and on the retroviruses preferences for open or compact chromatin structure
Fig 4: Interaction between HIV-1 IN, LEDGF/p75 and FACT complex. A Schematic representation of the previously reported interactions between HIV-1 IN, LEDGF/p75 and FACT complex is shown in a. IN/FACT, IN/LEDGF, LEDGF/FACT, IN/LEDGF/FACT and IN/IBD/FACT interactions were analyzed by co-immunoprecipitation using recombinant cofactors and polyclonal anti-HIV-1 IN antibodies. IBD/FACT interactions were analyzed by GST-pull down using IBD-GST and FACT recombinant proteins. The interactions were monitored either direct gel staining using colloidal blue or western blot using the corresponding antibodies and quantified by Image J software (see quantification in b and representative experiments in Additional file 3: Figure S3). All values are shown as the mean ± standard deviation (error bars) of at least three independent sets of experiments. The p-values were calculated by Student’s t test and are shown as *p < 0.05 and **p < 0.005 to represent the probability of obtaining significant differences compared with the data obtained with the negative background obtained with the beads alone. Cellular interaction between SSRP1, LEDGF/p75 and IN was checked by immunoprecipitation with an anti-FLAG antibodies in cells lysates obtained from LEDGF/p75-deficient cells (si1340/1428 cells) and transfected with plasmid expressing SSRP1-Myc and HIV-1 IN-Myc, and either FLAG-LEDGF/p75 (lane 1) or an empty plasmid (lane 2) (c). Then, immunoprecipitated proteins were evaluated for the presence of the expressed proteins by immunoblotting with tag-specific antibodies. (**) represents a longer exposure of (*). Detection of light chain Igs were used as loading control. The experiment was performed twice with identical results
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