Generation of a human somatic LEDGF/p75 KO cell line

To clarify the role of LEDGF/p75 during spreading HIV-1 infection, we generated a human somatic KO in Nalm-6 cells, a human pre-B acute lymphoblastic leukemia cell line [26], [27]. We eliminated the LEDGF/p75 isoform while preserving the LEDGF/p52 splice variant. Deletion of exon 11 to 14 in the PSIP1 gene fuses exon 10 to exon 15 resulting in a frame shift that yields a truncated LEDGF/p75 in which the C-terminus, including the IBD (aa 326–530) is replaced by a 9 aa tail (Figure S1A, referred to as LEDGF^KO). Targeting plasmids were designed carrying the genomic flanking regions of LEDGF/p75 exon 11 and 14, interspersed with a floxed selection cassette (Figure 1A). Following transfection of wild-type Nalm-6 cells (Nalm^+/+) with the first targeting plasmid and subsequent selection, three heterozygous clones (cl) (denoted as Nalm^+/c; cl 31, cl 97 and cl 147, respectively) were obtained (Figure 1B). We continued with Nalm^+/c cl 31. Transfection of Nalm^+/c cl 31 with the second targeting plasmid resulted in the selection of a homozygous KO clone carrying both resistance cassettes (Nalm^c/c 31 cl 73). Selection cassettes were removed by Cre-mediated excision, resulting in seven LEDGF/p75 KO clones, referred to as Nalm^−/− cl 1-7.

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Figure 1. Generation and validation of human LEDGF/p75 KO cell line.

(A) Scheme for PSIP1 gene targeting by homologous recombination. The 2.3 and 2.0 kb arm indicated on the targeting plasmids enable homologous recombination and harbor a puromycin (Puro^r) or hygromycin B resistance (Hyg^r) cassette (c) flanked by loxP sites (arrows). Exon 11 of p52 and p75 are indicated separately as well as the IBD coding region. BamHI restriction sites are indicated (scissors). DT-A denotes the gene encoding for Diphteria toxin A. (B) Schematic overview of KO and intermediates: Nalm-6 wild-type (Nalm^+/+) contains 2 PSIP1 genes; Nalm^+/c clones (cl) 31, 97, 147, contain a puromycin resistance cassette in one allele; Nalm^c/c 31 cl 73, contains both a puromycin and a hygromycin B resistance cassette; after CRE-mediated excision cl 1-7 are generated and termed Nalm^−/−. (C) Genomic PCR on DNA from different clones. Primer binding sites are indicated in panel A (see also Table S2). Indicated bands confirm amplification of a 1.6 kb fragment by primers D and E in full length PSIP1 as shown in Nalm^+/c and Nalm^+/+, but not in Nalm^c/c and Nalm^−/− cl 1. (D) Southern blot on genomic DNA after BamHI digestion. Probe and restriction sites are indicated in panel A. Intact PSIP1 generates a 16.2 kb fragment. After insertion of a resistance cassette a 7.5 kb fragment is generated, after CRE-mediated excision a 14.6 kb fragment is formed indicating KO of a 1.6 kb fragment containing exon 11–14. (E) Western blot analysis for LEDGF protein of whole cell extracts. Marker heights (right), LEDGF/p75 (arrow) and LEDGF^KO (arrowhead) are indicated. (F) Nalm-6 cells were transduced with HIV-fLuc. Luciferase expression is shown as percentage relative light units (RLU) per µg protein as compared to Nalm^+/c cl 31. (G) In parallel, the number of integrated proviral copies was evaluated for HIV-fLuc. Following transduction, cells were grown for at least 10 days to eliminate non-integrated viral DNA and analyzed by quantitative PCR. (H) HIV-1 integration site distribution analysis. Left panel shows relative number of experimentally derived HIV-1 integration events in genes according to the RefSeq annotation, versus computationally generated matched random control (MRC). The right panel shows integration events occurring ±2 kb around CpG islands as compared with MRC. Average ± standard deviations are shown from experiments performed at least in triplicate.

https://doi.org/10.1371/journal.ppat.1002558.g001

Correct homologous recombination of the genomic region was verified via genomic PCR (Figure 1C), Southern blot analysis (Figure 1D) and sequencing of the genomic and mRNA region (Figure S1A). The absence of wild-type LEDGF/p75 in the KO cells was corroborated by RT-PCR (Figure S1B and S1C), qRT-PCR (Figure S1D) and Western blot analysis (Figure 1E, arrow). A band of 52 kDa appears in the Nalm^+/c and Nalm^−/− cell lines; it corresponds to the truncated form, LEDGF^KO (Figure 1E, arrowhead), and is absent in wild-type cells. Throughout the manuscript Nalm^−/− cl 1 and cl 2 monoclonal cell lines are used. Wild-type Nalm-6 cells, referred to as Nalm^+/+, were used as controls, next to Nalm^+/c cl 31, referred to as Nalm^+/c, the closest clonal ancestor of the Nalm^−/− cells.

Single round lentiviral transduction of LEDGF/p75 KO cells is hampered at the integration step

We first evaluated whether the LEDGF/p75 KO cells (Nalm^−/−) support transduction by a single round HIV-based viral vector. We challenged the abovementioned engineered cell lines with a VSV-G pseudotyped HIV reporter virus encoding firefly luciferase under control of the viral long terminal repeat promoter (HIV-fLuc). Transduction efficiency (RLU/µg protein) was 6.7-fold lower in Nalm^−/− cells (cl 1 and cl 2) compared to control Nalm^+/+ and Nalm^+/c cells (Figure 1F) (15±3.7% residual reporter activity; n = 10). Quantitative PCR revealed 2.4-fold lower integrated copies comparing Nalm^−/− with Nalm^+/c (Figure 1G), whereas late RT products (Figure S1E) and 2-LTR circles remained unaffected (Figure S1F). Together these data indicate a block between reverse transcription and integration.

Since LEDGF/p75 determines lentiviral integration site selection, we analyzed the distribution of HIV-1 integration sites in the absence of LEDGF/p75. A total of 2535 HIV-1 integration sites were obtained in Nalm-6 cells of which 799 in Nalm^−/− (Table 1). Random control sites were generated computationally and matched to experimental sites with respect to the distance to the nearest MseI cleavage site (matched random control, MRC) [2]. LEDGF/p75 KO significantly reduced the preference of HIV-1 to integrate in RefSeq genes (P<0.0001 for comparison of Nalm^−/− cl 1 or 2 with Nalm^+/+ or Nalm^+/c) and instead, a preference for CpG islands (P<0.05 for comparison of Nalm^−/− cl 1 or 2 with Nalm^+/+ or Nalm^+/c and P<0.0001 for pooled comparison) emerged (Figure 1H and Table 1). Similar results were obtained using the Ensembl and UniGene annotation (Figure S1G and S1H). HIV-1 integration events in RefSeq genes remained nevertheless significantly favored over MRC in the KO cells (P<0.0001). The target DNA consensus proved to be LEDGF/p75 independent (compare Figure S1I with S1J). The consensus sequence for the different cell lines was similar to that determined previously [28]–[30].

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Table 1. Integration frequency near mapped genomic features in the human genome.

https://doi.org/10.1371/journal.ppat.1002558.t001

In LEDGF/p75 KO cells residual replication is observed with laboratory strains but not with clinical isolates of HIV-1

In human LEDGF/p75 KD cells HIV-1 replication is hampered, but not completely blocked which can be attributed to the remaining minute amounts of LEDGF/p75 [5], [6], [20]. Although single round viral vector transduction was severely reduced in LEDGF KO MEFs [11], [17], [21], spreading HIV-1 infection in the absence of LEDGF/p75 could not be tested. To test HIV-1 replication, we introduced the CD4 receptor into the Nalm-6 cells that express CXCR4 [31], a co-receptor for HIV-1 replication. All selected transgenic cell lines (Nalm^+/+, Nalm^+/c and Nalm^−/− cl 1 and cl 2) showed similar growth rates (Figure S6A and S6C) and CD4 and CXCR4 expression levels (Figure S6D and S6E). We then challenged the respective cell lines with the laboratory strain HIV_NL4.3 (Figure 2A). Both Nalm^+/+ (Figure S2A) and Nalm^+/c cells supported viral replication to the same extent (Figure 2A). Peak viral replication was consistently observed between day 7 and 9 post infection depending on the multiplicity of infection (MOI; compare MOI 0.5 and 0.1 in Figure 2A). In Nalm^−/− cells infected with HIV_NL4.3, low-level p24 production was observed, eventually leading to a breakthrough albeit after a lag-period of 14 to 18 days compared to control cells (Figure 2A, n = 6, a representative experiment is shown). Comparable data showing this delay were obtained with another laboratory strain, HIV_IIIb (data not shown).

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Figure 2. Residual HIV-1 replication in LEDGF/p75 KO cells is only observed after challenge with laboratory strains.

Control Nalm^+/c and KO Nalm^−/− cl 1 and cl 2 cell lines were challenged with the laboratory strain HIV_NL4.3 (A) and clinical isolates #1, 93TH053 (B) and #2, 96USSN20 [32] (C). Cells were infected at different MOI as indicated. Replication was monitored by measuring the p24 content of the supernatant. Experiments were repeated at least three times; representative experiments are shown.

https://doi.org/10.1371/journal.ppat.1002558.g002

Next, we challenged the different cell lines with two clinical isolates of HIV-1 (93TH053, denoted as #1 and 96USSN20 [32], denoted as #2). Viral breakthrough was observed 17 to 20 days post infection in the control cell line (Figure 2B and 2C). In the first two weeks after infection of the KO cell lines only a discrete increase in p24 was observed; at 35 days after infection p24 levels were below detection limit (Figure 2B and 2C).

Residual virus production in KO cells is caused by spreading infection which is hampered at the integration step

We next evaluated whether the rise in p24 titers observed in Nalm^−/− cells after challenge with laboratory HIV-1 strains could be explained by virus release from cells infected in the first round, rather than ongoing replication cycles. Therefore we challenged Nalm^+/c and Nalm^−/− cells with HIV_NL4.3 and resuspended the cells at 8 hrs post infection (Figure S2B) in fresh medium containing either zidovudine (AZT), ritonavir (RIT) or no inhibitor. AZT, a reverse transcriptase inhibitor, blocks infection of new cells but allows monitoring of virus release from already infected cells whereas RIT, a protease inhibitor, blocks processing of GAG-precursor processing in the virus released from infected cells. In Nalm^−/− cells as well as in control Nalm^+/c cells the p24 production clearly decreased in the presence RIT or AZT. The decrease in p24 in Nalm^+/c without inhibitor at day 6 was due to the cytopathic effect of the virus. This indicates that the p24 increase observed in Nalm^−/− cells results from spreading infection and not solely from virus release from cells infected in the first round.

The observed delay in multiple round HIV-1 replication in the absence of LEDGF/p75 was further analyzed by quantification of the different HIV-1 DNA species at different time points after infection. Late RT products at 10 hrs post infection and 2-LTR circles at 24 hrs post infection were comparable in Nalm^+/c and Nalm^−/− cells (Figure S3A and S3B). Addition of the IN strand transfer inhibitor (INSTI) raltegravir (RAL) in Nalm^+/c and Nalm^−/− cell lines resulted in a comparable increase in 2-LTR circles at 24 hrs post infection. The number of integrated proviral copies (Alu-qPCR, Figure S3C) was severely reduced in the presence of RAL. In Nalm^−/− a reduction in the number of integrants was detected after 24 and 48 hrs compared to Nalm^+/c cell lines.

We next characterized the virus harvested from Nalm^−/− at day 18 after infection with the laboratory strain HIV_NL4.3 (referred to as HIV^−/−). Challenging Nalm^+/c cells with this virus demonstrated that HIV^−/− is replication competent (Figure S2C, right panel, HIV^−/− on Nalm^+/c). In addition, we evaluated whether HIV^−/− virus was phenotypically adapted to the absence of LEDGF/p75. HIV^−/− replication remained impaired in Nalm^−/− compared to Nalm^+/c cells (Figure S2C, right panel). The proviral IN sequence of HIV^−/− was unaltered compared with the consensus sequence of HIV_NL4.3 (data not shown). Control HIV harvested from Nalm^+/c cells (denoted as HIV^+/c) demonstrated a phenotype that was comparable to that of HIV_NL4.3 (Figure S2C, left panel). Serial passaging (N = 10) of HIV-1 on LEDGF/p75 KO cells did not result in phenotypic adaptation or changes in the proviral IN sequence (data not shown).

HRP-2 KD inhibits residual HIV-1 replication in LEDGF/p75 KO cells

Although residual HIV-1 replication in KO cells was only detectable after infection with laboratory strains, we performed additional experiments to understand this phenotype. Residual viral replication in the absence of LEDGF/p75 can either be explained by cofactor independent replication, or by the presence of a second cofactor that substitutes for LEDGF/p75. Like LEDGF/p75, HRP-2 also harbors a PWWP-domain and an IBD-like domain shown to interact with HIV-1 IN in vitro [12]. In order to determine whether HRP-2 can act as an alternative co-factor for HIV integration, we targeted the HRP-2 mRNA using miRNA-based short hairpins (miR HRP-2). As controls we employed a vector lacking the miRNA expression cassette (denoted as control) (Figure S7B). We generated stable HRP-2 KD cells, termed Nalm^+/+_{miR HRP-2}, Nalm^+/c_{miR HRP-2} and Nalm^−/−_{miR HRP-2} and matched controls Nalm^+/+_control, Nalm^+/c_control and Nalm^−/−_control. HRP-2 KD cells showed 65, 75 and 80% depletion of HRP-2, respectively, as determined by qPCR (Figure 3A). No effect on cellular growth kinetics was observed (data not shown). Upon single round transduction with HIV-fLuc no difference was observed in Nalm^+/c cells with or without HRP-2 KD (Figure 3B, left panel), whereas luciferase activity was reduced 5-fold in the Nalm^−/−_control cell line (20.0±1.5%, n = 3) due to LEDGF/p75 KO. An additional 2.4-fold reduction was observed in Nalm^−/−_{miR HRP-2} when compared to Nalm^−/−_control (8.4±0.6%, n = 3) (Figure 3B, left panel) that correlated with a 2-fold reduction in integrated copies (Figure 3B, right panel).

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Figure 3. Additional HRP-2 KD hampers residual replication in LEDGF/p75 KO cells.

Stable HRP-2 KD (miR HRP-2) and control (control) Nalm^+/+, Nalm^+/c and Nalm^−/− were generated. (A) HRP-2 mRNA levels were determined by qPCR. (B) In the left panel luciferase activity following HIV-fLuc transduction is shown. Cells were grown for an additional 10 days to eliminate all non-integrated viral DNA species before total viral DNA was measured as demonstrated in the right panel. (C,D,E) Stable cell lines were challenged with the laboratory strain HIV_NL4.3 at different MOIs (5-0.05). Replication was monitored by measuring the p24 content of the supernatant. In (F) the number of integrated copies as determined by Alu-qPCR is shown. In (G) we quantified total viral DNA at 5 days post infection, in the presence of Ritonavir (RIT), added 4 hrs post infection. Average ± standard deviations are shown from experiments performed at least in triplicate.

https://doi.org/10.1371/journal.ppat.1002558.g003

We next challenged these cells with the laboratory strain HIV_NL4.3 at different MOI (Figure 3C–E). In the control Nalm^+/+ and Nalm^+/c cell lines, we observed a minor reduction in viral replication upon HRP-2 KD but only at lower MOI (compare Figure 3C and 3D, E). However, HRP-2 KD in LEDGF/p75 KO cells additionally inhibited HIV-1 replication 2- to 3-fold compared to control cells (Figure 3C–E, compare Nalm^−/−_control and Nalm^−/−_{miR HRP-2}, detail panel). We generated a second LEDGF/p75 KO HRP-2 KD cell line to corroborate our results. Single round transduction with HIV-fLuc resulted in an additional 4.7-fold reduction of luciferase reporter activity when compared with LEDGF KO cells (Figure S5F), whereas HIV_NL4.3 replication was affected 10-fold at day 8 post infection when comparing LEDGF/p75 KO and LEDGF/p75 KO HRP-2 KD cells (compare Figure S5D with S5E, condition without compounds). To corroborate that additional KD of HRP-2 results in an increased block of integration in LEDGF/p75 KO cells, we analyzed the number of integrated viral copies at 24 hrs and at 5 days post infection, the latter in the presence of RIT (Figure 3F and 3G, respectively). A 2-fold drop in proviral copies upon HRP-2 KD was observed.

HRP-2 KD further hampers HIV-1 replication in LEDGF/p75 KD cells

To extend our findings in LEDGF/p75 KO cells, we tested whether HRP-2 KD resulted in additional reduction of viral replication in LEDGF/p75 KD HeLaP4 (Figure S4), PM1 (Figure 4A–C) and SupT1 (Figure 4D–F) cell lines. First, wild-type HeLaP4 (wild-type) and LEDGF/p75 KD (miR LEDGF) cells [18] were transduced with miR HRP-2 or miR control vectors, the latter containing a miRNA-hairpin directed against monomeric red fluorescent protein (DsRed) mRNA [33] (Figure S7C). Following zeocin selection, single HRP-2 KD (wild-type/miR HRP-2) and double KD (miR LEDGF/miR HRP-2) cells showed 20–25% of residual HRP-2 mRNA levels compared to the control cell lines (wild-type, wild-type/miR control and miR LEDGF/miR control cells) as determined by qPCR (Figure S4A and S4C). Loss of HRP-2 protein was corroborated by Western blot analysis and immunocytochemistry (data not shown). Of note, LEDGF/p75 levels remained unaffected upon additional HRP-2 KD (data not shown) and growth rates of the respective cell lines were comparable (Figure S6B and S6C). KD of HRP-2 in wild-type HeLaP4 cells did not affect multiple round HIV-1 replication (Figure S4B), confirming previous findings by Llano et al. [6]. LEDGF/p75 KD on the other hand reduced HIV-fLuc transduction 5-fold (luciferase reporter activity = 19.2±3.5% of wild-type) (Figure S4D). Additional KD of HRP-2 in LEDGF/p75-depleted cells diminished HIV-fLuc reporter activity an additional 3-fold, to 6.3±2% of control cells (miR LEDGF/miR control) (Figure S4D). This reduction was accompanied with a 2-fold decrease in the number of integrated copies (Figure S4E). Transfection of the cell lines with the plasmid encoding HIV-fLuc (pHIV-fLuc) did not demonstrate any difference (Figure S4F), ruling out transcriptional effects upon HRP-2 KD. Next, we infected double KD (miR LEDGF/miR HRP-2) cells and control (miR LEDGF/miR control) cells together with wild-type and LEDGF/p75 back-complemented (LEDGF BC) cells with the laboratory strain HIV_NL4.3 (Figure S4G). Viral replication was inhibited in miR LEDGF cells and rescued upon LEDGF/p75 back-complementation (Figure S4G, compare wild-type and LEDGF BC). Additional KD of HRP-2 in LEDGF/p75 depleted cells (miR LEDGF/miR HRP-2) inhibited viral replication more than LEDGF/p75 KD alone (miR LEDGF/miR control). The latter demonstrated a breakthrough around day 30 post infection (Figure S4G, open diamonds), whereas cells with double KD did not demonstrate viral breakthrough (Figure S4G, open squares). Analysis was ended at 48 days post infection. Comparable data were obtained in HeLaP4 cell lines generated with other LV constructs (Figure S7B and S7D) using hygromycin B selection or eGFP sorting (data not shown). The additional block of HIV-1 replication upon HRP-2 KD in LEDGF/p75 depleted cell lines was also measured by quantifying the number of integrated proviral copies. At day 39, 45 and 48 post infection the number of integrated copies was low in double KD (miR LEDGF/miR HRP-2) cells compared to the control LEDGF/p75 KD (miR LEDGF/miR control) cells (Figure S4H) with proviruses numbering 0.032 (±0.012) and 0.038 (±0.012) per RNaseP genomic copy on day 39 and 48 respectively, compared to 1.39 (±0.18) and 0.79 (±0.23) in the control LEDGF/p75 KD cell lines. In addition, we quantified different HIV-1 DNA species at different time points post infection in wild-type, LEDGF/p75 KD (miR LEDGF/miR control) and double KD cells (miR LEDGF/miR HRP-2). We observed no difference in late RT products at 10 hrs post infection (Figure S4I). The number of 2-LTR circles in LEDGF/p75 KD (miR LEDGF/miR control) and both LEDGF/p75 and HRP-2 KD (miR LEDGF/miR HRP-2) cells was elevated compared to wild-type cells (Figure S4J). Together with the data in the LEDGF/p75 KO cells, these data indicate that HRP-2 KD blocks HIV-1 at a step between reverse transcription and integration but only after potent depletion of LEDGF/p75.

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Figure 4. HRP-2 KD additionally hampers HIV-1 replication in LEDGF/p75 KD cell lines.

Stable LEDGF/p75 and/or HRP-2 KD and control PM1 and SupT1 cells were generated. In (A) qPCR results for LEDGF/p75 and (B) HRP-2 mRNA expression levels, normalized to RNaseP for PM1 are shown. Average with standard deviations from experiments in triplicate, are shown. The constructs used, are shown below the graph. (C) Multiple round HIV-1 replication after challenge with the laboratory strain HIV_NL4.3. On the right a detail panel for LEDGF/p75 KD cells is shown. In (D) LEDGF/p75 and (E) HRP-2 expression levels in SupT1 cells, analogous to (A) and (B) are shown. (F) Multiple round HIV-1 replication in the different SupT1 cells, analogous to (C), is shown.

https://doi.org/10.1371/journal.ppat.1002558.g004

Next, we expanded our findings to relevant T-cell lines, PM1 and SupT1. We generated cell lines with stable KD of LEDGF/p75, HRP-2 or both, together with their respective controls (constructs shown in Figure S7B). For PM1 cells KD efficiency was 85–92% for LEDGF/p75 (Figure 4A) and 79–81% for HRP-2 (Figure 4B), for SupT1 cells it amounted to 81–88% for LEDGF/p75 (Figure 4D) and 75–80% for HRP-2 (Figure 4E). In both cell lines HRP-2 KD alone did not affect HIV-1 replication, whereas a clear reduction in HIV-1 replication was observed upon LEDGF/p75 KD (Figure 4C and 4F, left panel, for PM1 and SupT1 respectively). Consistent with our findings in LEDGF/p75 KO cells and LEDGF/p75 depleted HeLaP4 cells, also in PM1 and SupT1 cells, HRP-2 KD in LEDGF/p75 depleted cells further hampered HIV-1 replication (Figure 4C and 4F, detail panels, for PM1 and SupT1 respectively).

LEDGINs block residual HIV-1 replication in Nalm^−/− cells

Recently, we reported a new class of antiretrovirals termed LEDGINs that bind to the LEDGF/p75 binding pocket of HIV-1 IN and block HIV-1 integration and replication in cell culture [25]. We assayed their activity in the LEDGF/p75 KO cells. We challenged Nalm^+/+ and Nalm^+/c cells together with Nalm^−/− cells with the laboratory strain HIV_IIIb in the presence of different concentrations of LEDGIN 7 [25]. LEDGIN 7 blocked HIV-1 replication in all cell lines in a concentration dependent manner (Figure 5A and 5C). Similar data were obtained with the laboratory strain HIV_NL4.3 (Figure S5A). The toxicity profile in Nalm-6 cells corresponded to that elaborated previously in MT4 cells [25]. No significant toxicity was observed in the concentrations used (data not shown). Of note, LEDGINs were also active against HIV harvested from LEDGF/p75 KO cells (HIV^−/−, data not shown). RAL served as a positive control, demonstrating equal inhibition of HIV-1 replication in the different cell lines (Figure 5B and 5D). Dose response curves (Figure 5E and 5F) enabled determination of IC₅₀ values, listed in Table S1.

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Figure 5. LEDGINs block residual HIV-1 replication in LEDGF/p75 KO cells.

Nalm^+/c and Nalm^−/− cell lines were challenged with HIV_IIIb in the presence of varying concentrations of HIV inhibitors. Supernatant was harvested for p24 ELISA. (A,C) LEDGIN 7 was added at different concentrations (0 µM, circles; 0.2 µM, squares; 3.5 µM, triangles or 31 µM, triangles pointing downwards). (B,D) Identical conditions were used as shown in (A,C), but the INSTI raltegravir (RAL) was added at different concentrations (0 µM, circles; 0.002 µM, squares; 0.025 µM, triangles or 0.3 µM, triangles pointing downwards). (A–D) Averages from duplicate experiments ± standard deviations are shown. (E) Dose-response curves in Nalm^+/c and Nalm^−/− cell lines were generated from p24 ELISA values obtained at day 6 for LEDGIN 7 concentrations of 0 µM, 0.06 µM, 0.2 µM, 0.7 µM, 3.5 µM, 8.9 µM, 31 µM. Data are fitted to a sigmoidal dose-response (variable slope) curve, from which IC₅₀ values were calculated (Table S1). (F) In analogy with (E), p24 values obtained at day 6 for RAL were plotted to enable determination of IC₅₀. RAL was used in concentrations of 0 µM, 0.002 µM, 0.007 µM, 0.02 µM, 0.08 µM and 0.3 µM.

https://doi.org/10.1371/journal.ppat.1002558.g005

LEDGINs also disrupt the interaction of HIV-1 IN with HRP-2

We have shown that residual replication of HIV-1 laboratory strains in LEDGF/p75 KO cells is predominantly mediated by HRP-2 and that LEDGINs block residual HIV-1 replication in KO cells. This can be explained by allosteric inhibition of LEDGINs or by the fact that binding of LEDGINs to the IN-surface also impedes the interaction with HRP-2 or a combination of both. We evaluated whether LEDGINs inhibit the HRP-2-IN interaction in an AlphaScreen assay. Since IN binds HRP-2 via its IBD (aa 470–593) [12] in vitro, we measured the interaction between recombinant HIV-1 IN and the C-terminal part of HRP-2 (aa 448–670). We generated maltose binding protein (MBP) tagged fusions containing either the C-terminal end of LEDGF/p75 (aa 325–530) or HRP-2 (aa 448–670). These recombinant proteins, MBP-LEDGF/p75_325–530 and MBP-HRP-2_448–670, bound to His₆-IN with apparent K_D's of 6.6 nM (±4.6 nM) or 89.8 nM (±18.1 nM), respectively (Figure 6A). In line with previous observations [25], LEDGINs inhibited the IN-LEDGF_325–530 interaction (Figure 6B; IC₅₀ = 2.60±0.99 µM). LEDGINs also inhibited the IN-HRP-2_448–670 interaction, albeit with a 10-fold lower IC₅₀ (Figure 6B; IC₅₀ = 0.23±0.14 µM). This 10-fold increased potency for LEDGIN 7 to block interaction of IN with MBP-HRP-2_448–670 compared to MBP-LEDGF_325–530 correlates well with the 13-fold lower affinity of MBP-HRP-2_448–670 for IN, as shown in Figure 6A.

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Figure 6. LEDGINs block the interaction of HIV-1 IN with the IBD of either LEDGF/p75 or HRP-2.

(A,B) Interactions between His₆-tagged IN and MBP-LEDGF_325–530 or MBP-HRP-2_448–670 were assessed using AlphaScreen technology. (A) 10 nM MBP-LEDGF_325–530 (circles) or MBP-HRP-2_448–670 (squares) was incubated with various concentrations of IN (2-fold dilutions from 500 to 1.9 nM). Anti-MBP donor and Ni²⁺-chelate acceptor beads were added and the AlphaScreen signal was read on an EnVision Multilabel reader. Boxed data show average apparent K_D values ± S.E.M. for the IN-MBP-LEDGF_325–530 or IN-MBP-HRP-2_448–670 interaction as determined from four independent experiments, each performed in duplicate. A representative experiment is shown. (B) MBP-LEDGF_325–530 or MBP-HRP-2_448–670 (10 nM) was incubated with IN (500 nM) and various concentrations of LEDGIN 7 (10, 2, 0.2, 0.02 and 0 µM, the latter plotted as 0.001 µM). Again, beads were added and the plate was read as described. Boxed data show IC₅₀ values of LEDGIN 7 on the interaction of IN with MBP-LEDGF_325–530 or MBP-HRP-2_448–670. Average IC₅₀ values ± standard deviations were derived from three independent experiments each performed in duplicate. A representative experiment is shown. In (C–F) the homology of LEDGF/p75_IBD and HRP-2_IBD and their interaction with IN is shown. (C) Alignment of LEDGF/p75_347–429 and HRP-2_470–552. Amino acids are colored by type and secondary structure is depicted on top (α-helices in red). Residues involved in the interaction with IN catalytic core domain are boxed. Alignments were generated with ClustalW [63] and further visualized in ALINE [64]. (D–F) Cartoon representations of the IN catalytic core domain (CCD) dimer (pale green and pale yellow) with (D) LEDGIN 6 (sticks, colored by atom), (E) LEDGF/p75_IBD (gray) or (F) HRP-2_IBD (cyan). Side chains of the crucial amino acids, mimicked by LEDGINs, i.e. residues I365, D366, L368 of LEDGF/p75, and corresponding HRP-2 residues V488, D489 and P491, are shown in sticks colored by atom. Structures of LEDGF/p75_IBD in complex with IN CCD dimer were taken from PDB ID: 2B4J [65]. HRP-2_IBD homology models were built with MODELLER [66], based upon the same LEDGF/p75_IBD – IN CCD dimer structure. The structure of the LEDGIN 6 – IN CCD dimer complex was taken from PDB ID: 3LPU [25]. All structures were visualized in PyMol (DeLano Scientific LLC, Palo Alto, USA).

https://doi.org/10.1371/journal.ppat.1002558.g006

Next, we evaluated whether LEDGINs remain active in LEDGF/p75 KO HRP-2 KD cells. The residual HIV-1 replication was sensitive to inhibition by LEDGINs (Figure S5E).

Cells and culture

Nalm-6 cells, SupT1 cells, obtained from the ATCC (Manassas, VA) and PM1 cells, a kind gift from Dorothee von Laer (Innsbruck Medical University, Innsbruck, Austria), were maintained in RPMI 1640 – GlutaMAX-I (Invitrogen, Merelbeke, Belgium) supplemented with 8% heat-inactivated fetal calf serum (FCS; Harlan Sera-Lab Ltd.) and 50 µg/ml gentamycin (Gibco, Invitrogen). HEK293T cells, obtained from O. Danos (Genethon, Evry, France), and HeLaP4 cells, a kind gift from Pierre Charneau, Institut Pasteur, Paris, France, were grown in DMEM (Invitrogen) supplemented with 5% FCS, 50 µg/ml gentamycin and 0.5 mg/ml geneticin (Invitrogen). All cells were grown in a humidified atmosphere with 5% CO2 at 37°C.

Growth kinetics

For growth curve analysis, Nalm-6 cells were seeded at 100,000 in 5 ml of corresponding medium and HeLaP4 cells at 200,000 per well in a 6-well plate. Cell growth was followed on consecutive days by cytometry (Coulter Z1, Beckmann Coulter). Experiments were performed in triplicate.

Plasmids

The HIV-based lentiviral transfer plasmid pCHMWS_CD4_IRES_Bsd encodes the CD4 receptor driven by a human early cytomegalovirus (CMV) promoter followed by an EMCV internal ribosomal entry site (IRES) and a blasticidin resistance cassette (Bsd). The plasmid was generated by PCR amplification of human CD4 from a T-cell cDNA library using CD4-Fwd and CD4-Rev, followed by digestion with BamHI and XbaI, and cloning into pCHMWS_LEDGF_BC_IRES_Bsd [18], digested with BamHI and SpeI.

The lentiviral transfer plasmids for miRNA-based KD were generated based on miRNA-R30 as previously described [50], [51] (Table S2). For HRP-2 KD, miR HRP-2 was adapted from the sequence validated previously [6]. As negative controls a non-functional, scrambled miRNA30-based short-hairpin sequence (miR scrambled) and a functional, short-hairpin sequence targeting the monomeric red fluorescent protein from Discosoma corallimorpharia, DsRed (miR DsRed) were designed [33]. PCR fragments were introduced into the XhoI–BamHI sites from a modified pN3-eGFP plasmid (Clontech, Saint Quentin Yuelines, France) duplicated and cloned into the XhoI-KpnI sites at the 3′ end of the enhanced green fluorescent protein (eGFP) reporter cDNA, driven by a Spleen focus forming virus LTR (SFFV) promoter, resulting in pCSMWS_eGFP_miR_HRP2 and pCSMWS_eGFP_miR_scrambled. To generate pCSMWS_Zeo_miR_HRP2, the zeocin resistance cassette (Zeo) was amplified with primers Zeo-Fwd and Zeo-Rev from pBUD (Invitogen), digested with NheI-Pfl23II and inserted into the XbaI–Pfl23II digested pCSMWS_eGFP_miR_HRP2 plasmid. To generate pCSMWS_Zeo_miR_DsRed, miR_DsRed was cloned into the XhoI/KpnI digested pCSMWS_Zeo_miR_HRP2 plasmid. To generate pCSMWS_Hygro_miR_HRP2, the hygromycin B resistance cassette (Hygro) was amplified using Hygro-Fwd and Hygro-Rev as primers and pBud (Invitrogen) as a template. The resulting products were digested ClaI-XhoI and ligated into pCSMWS_Zeo_miR_HRP2.

For bacterial expression of C-terminal His₆-tagged HIV-1 IN and MBP-tagged-LEDGF_325–530, the plasmids pKBIN6H [10] and pMBP-Δ325 [4] were used, respectively. To construct pMBP-HRP-2_448–670, the sequence corresponding to aa 448 to 670 of HRP-2 was PCR amplified with primers HRP2-Fwd, and HRP2-Rev, using p3xFlagHRP-2 (a kind gift from E. Poeschla) as a template. The resulting products were digested and ligated into pMAL-c2E (New England Biolabs Inc., USA). The integrity of all plasmids was confirmed by DNA sequencing.

Retroviral vector production and transduction

LV production was performed as described earlier [18], [52]. Briefly, vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector particles were produced by PEI transfection in HEK293T cells using the different transfer plasmids. Single round HIV_{NL4.3ΔNefΔEnv}fLuc (HIV-fLuc) virus was prepared by co-transfection of HEK293T cells with pNL4-3.LucR–E– (pHIV-fLuc, National Institutes of Health AIDS Research and Reference Reagent Program) and pMD.G, that codes for VSV-G.

For lentiviral transduction experiments, Nalm-6 cells were typically plated at 150,000 cells per well in a 96-well plate and transduced overnight. After 72 hrs, 50% of cells were reseeded for luciferase expression quantification and/or FACS analysis. The remainder was cultured for quantitative PCR and integration site analysis during at least 10 days to eliminate non-integrated DNA. HeLaP4 cells were plated at 20,000 cells per well in a 96-well plate and transduced overnight. After 72 hrs, 50% of cells were reseeded for luciferase quantification. The remainder was cultured for quantitative PCR or integration site analysis as described for Nalm-6 cells.

PSIP1 KO targeting plasmids

Targeting plasmids for generation of PSIP1 KO were designed and cloned as described previously [27], [53] utilizing the MultiSite Gateway System (Invitrogen) as described [54]. Briefly, a 2.3 and a 2.0 kb fragment for the left and right arms of the targeting plasmids, respectively, were amplified by genomic PCR using primers LEDGF/p75 attB4 and LEDGF/p75 attB1 for the left arm, and primers LEDGF/p75 attB2 and LEDGF/p75 attB3 for the right arm (Table S2). The resulting fragments were cloned into pDONR/P4-P1R and pDONR/P2R-P3 via recombination, resulting in p5′-ENTR-left and p3′-ENTR-right, respectively. The fragments p5′-ENTR-left, p3′-ENTR-right, pDEST DTA-MLS, and pENTR lox-Puro or pENTR lox-Hygro, were then ligated using recombination to generate the final targeting plasmids pTARGET-LEDGF/p75-Hyg and pTARGET-LEDGF/p75-Puro, respectively.

Generation and validation of LEDGF/p75 KO cell lines

Cell lines were generated as previously described [27]. Briefly, targeting plasmid was transfected with Nucleofector I (Amaxa, Inc., Gaithersburg, MD, USA) using 2×10⁶ Nalm-6 cells and 2 µg of DNA. At 24 hrs after transfection, cells were seeded into 96-well plates at 10³ cells per well, in culture medium supplemented with either 0.2 µg/ml puromycin (BD BioSciences, San Jose, CA, USA) or 0.35 mg/ml of hygromycin B (Clontech, Mountain View, CA, USA). After 2–3 weeks, individual drug resistant colonies were propagated and analyzed by genomic PCR using primers A and B or C (Figure 1A), generating a 3272 bp AB-fragment for Nalm^+/c, Nalm^c/c, Nalm^−/− and a 3123 bp AC-fragment for Nalm^c/c, Nalm^−/− (Figure 1C). Genomic PCR of the targeted region was performed with primers D and E generating a 1624 bp DE-fragment in Nalm^+/+, Nalm^+/c and a 288 bp DE-fragment in Nalm^−/− (Figure 1C). Targeting efficiency was calculated as the ratio of the number of cell clones where the LEDGF/p75 allele was disrupted by homologous recombination to the number of drug-resistant cell clones (Figure 1B). Sequencing of the genomic KO region was performed as follows. The PCR fragments obtained after amplification with primers gFB and gRB spanning a 1885 bp region around exon 11–14 in wild-type cells or a 571 bp region in KO cells, followed by nested PCR with primers gFA and gRA resulting in a 1694 bp in wild-type or 380 bp region in KO cells, were cloned into the pCRII-TOPO plasmid (Invitrogen, Merelbeke, Belgium) and sequenced with primers M13-Fwd and M13-Rev (Figure S1A). Total RNA extracted from KO clones (RNeasy 96 kit, Qiagen) was used for cDNA synthesis using oligo-dT primers (High capacity cDNA RT kit, Applied biosystems). Correct recombination was verified at mRNA level for LEDGF/p52, LEDGF/p75 and truncated LEDGF/p75 by PCR on cDNA using primers RNA-A and RNA-B for LEDGF/p52, LEDGF/p75 and LEDGF^KO, RNA-A and RNA-C for LEDGF/p75 and LEDGF^KO, RNA-A and RNA-D for LEDGF/p52, resulting in fragments of 245 bp, 1.606 kb or 1.163 kb and 1.011 kb, respectively (Figure S1C). Additionally, the cDNA of the truncated protein was sequence verified (Figure S1A) as follows: a PCR product generated by primers d243 and RNA-C, followed by nested PCR with primers d244 and LEDGF-R-exon15, was cloned into pCRII-TOPO (Invitrogen) and sequenced with M13-Fwd and M13-Rev. Primers are listed in Table S2.

Generation of stable CD4 expressing Nalm-6 cell lines

Stable CD4 expressing Nalm-6 cell lines were generated by transducing wild-type Nalm^+/+, Nalm^+/c cl 31 and Nalm^−/− cl 1 and cl 2, with the lentiviral vector pCHMWS_CD4_IRES_Bsd and subsequent selection with blasticidin (3 µg/ml; Invitrogen, Merelbeke, Belgium). CD4 expression was verified by flow cytometry using R-Phytoerythrin-conjugated mouse anti-human CD4 monoclonal antibody (BD pharmigen) according to the manufacturer's protocol.

Generation of LEDGF/p75 and HRP-2 KD cell lines

Stable monoclonal LEDGF/p75 KD cells were generated previously [18]. Additional HRP-2 KD was obtained by transduction of HeLaP4 wild-type cells and LEDGF/p75 KD cells with pCSMWS_Zeo_miR_HRP2, pCSMWS_Hygro_miR_HRP2 or pCSMWS_eGFP_miR_HRP2. Transduced cells were selected with zeocin (200 µg/ml) or hygromycin B (200 µg/ml) or by FACS sorting of eGFP positive cells respectively. Control cell lines were generated likewise by transduction with vectors encoding pCSMWS_Zeo_miR_DsRed, pCSMWS_eGFP_IRES_HygroR and pCSMWS_eGFP_miR_scrambled, respectively. Stable PM1 and SupT1 LEDGF/p75 KD cell lines were generated with LV, encoding a miRNA cassette targeting LEDGF/p75 under control of an SFFV promoter (unpublished data). Additional HRP-2 KD and control PM1, SupT1 and Nalm-6 cell lines were generated by transducing the cells with vectors made with pCSMWS_Hygro_miR_HRP2 and pCSMWS_eGFP_IRES_HygroR, respectively.

Integration site amplification and bioinformatic analysis

Integration sites were amplified by linker-Mediated PCR as described previously [17], [18]. For integration sites to be authentic, sequences needed a best unique hit when aligned to the human genome (hg18 draft) using BLAT. The alignment began within 3 bp of the viral long terminal repeat end, and had >98% sequence identity. Reanalysis of previously obtained integration sites [2], [11], [17] was performed in parallel. Statistical methods are described previously [55]. Integration site counts were compared using a two-tailed Fisher's exact test. Analysis was carried out using Prism 5.0 (GraphPad Software).

Virus strains

The origin of HIV_NL4.3 [56] and HIV_IIIb has been described [57]. Clinical isolate #1 was obtained through the AIDS research and reference reagent program, Division of AIDS, NIAID, NIH: HIV-1 93TH053 from the UNAIDS network for HIV isolation. Clinical isolate #2 was obtained through the AIDS research and reference reagent program, Division of AIDS, NIAID, NIH: HIV-1 96USSN20 from Drs Ellenberger, P. Sullivan and R.B. Lal [32]. The p24 antigen titer was determined for each virus stock. The MOI was determined using flow cytometry analysis of intracellular p24 antigen 24 hrs after infection in control Nalm^+/c cells. Cells were stained with pycoerythrin-anti-p24 (KC57-RD1; Beckman Coulter) using the Fix&Perm (Invivogen) cell fixation and cell permeabilization kit following the manufacturer's protocol.

HIV-1 infection experiments

HIV-1 infection of Nalm-6 cells was typically performed with 1*10⁶ cells in 5 ml of medium with the indicated virus and MOI. After 6–12 hrs of infection, cells were washed twice with PBS and resuspended in the initial volume of culture medium. Infection of HeLaP4 cells was performed as described previously [18]. HIV-1 replication was monitored by quantifying p24 antigen in the supernatant daily via ELISA (Alliance HIV-1 p24 ELISA kit; Perkin Elmer). Cells were split 1/6 every 5–6 days if experiments exceeded 10 days. PM1 and SupT1 cells were infected at 0.01 pg p24/cell.

PCR amplification and DNA sequencing of the coding regions of IN

Proviral DNA extraction of infected cells was performed using the QIAamp blood kit (Qiagen) according to the manufacturer's protocol. PCR amplification and sequencing of IN encoding sequences were done as described previously [58].

Antiviral agents

Zidovudine (AZT) and ritonavir (RIT) were purchased and raltegravir (MK518) was kindly provided by Tibotec (Mechelen, Belgium). LEDGIN 7 was synthesized as described [25].

Luciferase activity assay

Cells harvested from a 96-well plate were lysed with 50 µl lysis buffer (50 mmol/l Tris pH 7.5, 200 mmol/l NaCl, 0.2% NP40, 10% glycerol). The lysate was assayed according to the manufacturer's protocol (ONE-Glow; Promega, Madison, WI). Luciferase activity was normalized for total protein (BCA; Pierce, Rockford, IL). All conditions were run at least in triplicate in each experiment.

Transfection of plasmid DNA in HeLaP4 cells

HeLaP4 cells were transfected with pHIV-fLuc using Lipofectamine 2000 (Invitrogen, Merelbeke, Belgium) according to the manufacturer's protocol with minor modifications. Briefly, 70,000 cells were seeded in a 96 well plate and transfected after one day with a mixture of 333 ng DNA and 0.66 µl Lipofetamine 2000 for 4 hrs and washed afterwards twice with PBS. 48 hrs post transfection cells were harvested for luciferase activity quantification.

Quantitative PCR

Quantification of LEDGF/p75 mRNA levels was performed as described previously [18]. Similar settings were used to determine HRP-2 mRNA levels. HRP-2 primer/probe set: HRP2 s4, HRP2 as4 and HRP2 probe. In all cases, RNaseP was used as endogenous house-keeping control (TaqMan RNaseP Control Reagent; Applied Biosystems). All samples were run in triplicate for 3 minutes at 95°C followed by 50 cycles of 10 seconds at 95°C and 30 seconds at 55°C. Data were analyzed with iQ5 Optical System Software (BioRad, Nazareth, Belgium). To quantify the different HIV-1 DNA species qPCR for total viral DNA, 2-LTR circles and integrated copies was performed as described [59], [60], with minor modifications. Nalm-6 cells were seeded one day prior to infection at 2*10⁵ cells per ml. After 4 hrs of incubation with HIV, medium was replaced by RPMI containing 10% FCS. Quantification of proviral copies as shown in Figure 3G was performed accordingly, only RIT (at 50 times IC₅₀) was added to the culture medium after the washing step to ensure only a single replication cycle could take place and genomic DNA was isolated after 5 days to dilute all non-integrated forms. Non-infected cells were incubated in parallel. To quantify the number of integrated copies as shown in Figure S4H, cells were cultured for 10 days in medium containing RIT and AZT both at 25 times IC₅₀ following day 39, 45 or 48, before harvesting genomic DNA. Quantitative Alu-PCR for quantification of proviral copies was done in two steps [60]. The first phase amplifies from Alu sequences to U3 sequences absent in self-inactivating (U3-deleted) HIV-1 vectors using 400 nM AluSINIIfwd, 400 nM qAluRout_SB704. Amplification conditions were 95°C for 30 sec, 60°C for 40 sec, 72°C for 1 min 30 sec, ×13 cycles. The second phase amplifies a nested product using 300 nM sense primer Q-Alu-F-in, 300 nM antisense Q-Alu-R-in and 200 nM Alu-probe. PCR conditions were 95°C for 10 sec, 55°C for 30 sec, ×50 cycles.

Southern blot analysis

Southern blot analysis was performed as described previously in [61], [62]. Briefly, genomic DNA was digested with BamHI, separated by electrophoresis on a 0.7% agarose gel and blotted on positively charged nylon membranes (Biodyne B; Pall Corp., Pensacola, FL, USA). The probe covered a 1024 bp genomic region around exon 10 of PSIP1 and was amplified by PCR using LPROBE-Fwd and LPROBE-Rev, and labeled with α-³²P-dCTP (Megaprime DNA labeling system, GE Healthcare, USA). Signals were detected using autoradiography.

Western blotting

Western blotting was performed as described previously [5]. Briefly, cellular extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. LEDGF was detected using a purified IgG1 mouse anti-human LEDGF monoclonal antibody (mAb, C26) (BD Biosciences Pharmingen, San Diego, CA). Equal loading was verified with β-tubulin (T-4026; Sigma-Aldrich, St Louis, MO). Visualisation was performed by chemiluminescence (ECL+; Amersham Biosciences, Uppsala, Sweden).

Production and purification of recombinant proteins

Recombinant HIV-1 IN containing a C-terminal His₆ tag was purified as described previously [10]. LEDGF_325–530 and HRP-2_448–670 fragments were expressed in E. coli as maltose binding protein (MBP) fusions. The purification of pMBP-LEDGF_325–530 from BL21(DE3) bacterial cells was done as described previously [4]. For purification, cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.2, 500 mM NaCl, 5 mM dithiothreitol, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 U/ml DNase). After complete lysis by ultrasonication, the supernatant was cleared by centrifugation and recombinant proteins were bound to amylose resin (New England Biolabs Inc, United Kingdom). The resin was washed with 20 bed volumes wash buffer (50 mM Tris-HCl, pH 7.2, 500 mM NaCl, 5 mM dithiothreitol), and the MBP-tagged proteins were eluted in 1 ml fractions wash buffer supplemented with 10 mM maltose. The fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for protein content, pooled, and concentrated by dialysis (overnight, 4°C) against storage buffer (50 mM Tris-HCl, pH 7.2, 500 mM NaCl, 50% (vol/vol) glycerol). All protein concentrations were measured using the Bradford assay (Bio-Rad).

AlphaScreen

AlphaScreen measurements were performed in a total volume of 25 µL in 384-well Optiwell microtiter plates (PerkinElmer). All components were diluted to their desired concentrations in assay buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.1% Tween-20 and 0.1% BSA). Anti-MBP coated donor beads were generated by dialyzing biotin-labelled anti-MBP (Vector Laboratories) to the assay buffer and incubating 10 nM of this antibody with the desired amount of Streptavidin donor beads (PerkinElmer) for 1 h at room temperature. For the K_D determinations, HIV-1 IN-His₆ was titrated against a background of 10 nM MBP-LEDGF_325–530 or MBP-HRP-2_448–670. This amount provided minimal binding curve perturbation while maintaining a good signal-to-noise ratio. When performing IC₅₀ determinations, LEDGIN 7 was titrated against a background of 500 nM IN-His₆ and 10 nM MBP-LEDGF_325–530 or MBP-HRP-2_448–670. After addition of the proteins and/or compounds, the plate was incubated for 1 h at 4°C and 20 µg/mL anti-MBP donor and Ni²⁺-chelate acceptor beads (PerkinElmer) were admixed, bringing the final volume to 25 µL. After 1 h of incubation at RT, protected from light, the plate was read on an EnVision Multilabel Reader in AlphaScreen mode (PerkinElmer). Results were analyzed in Prism 5.0 (GraphPad software) after non-linear regression with the appropriate equations: one-site specific binding, taking ligand depletion into account for the K_D measurements and sigmoidal dose-response with variable slope for the IC₅₀ determination.

Accession numbers

The Genbank (http://www.ncbi.nlm.nih.gov/genbank) accession numbers for the proteins discussed in this paper are LEDGF/p52 (NM_021144.3), LEDGF/p75 (NM_033222.3) and HRP-2 (NM_032631.2).