The vesicular stomatitis virus glycoprotein (VSVG) expression vector pHIT/G , the HIV-1 proviral construct pNL4-3 , pNL-ADA , and the HIV-1 proviral indicator constructs pNL-Luc-E(−) and pNL-Luc-E(−)R(−)  have been described previously. To introduce D64A mutation into IN (adenine of nucleotide 4420 to cytosine) to create pNL-IN-D64A, site-directed mutagenesis (QuikChange; Stratagene) was performed using pNL4-3 as a template. To create pNL-ADA-IN-D64A and pNL-Luc-IN-D64A-E(−) that contained D64A mutants, the SpeI-PflMI fragment (nucleotides 1507–5297) of pNL-IN-D64A was replaced with those of pNL-ADA and pNL-Luc-E(−), respectively. To create the Vpr-deficient construct pNL-ADA-R(−), pNL-ADA-IN-D64A-R(−), and pNL-Luc-IN-D64A-E(−)R(−), the PflMI-SalI fragment (nucleotides 5297–5785) of pNL-Luc-E(−)R(−) was replaced with those of pNL-ADA, pNL-ADA-IN-D64A, and pNL-Luc-IN-D64A-E(−), respectively.
The neomycin-resistant marker expressing vector pNL-Neo-E(−)R(−) was created by inserting a PCR-amplified neomycin-resistant gene into the NotI-XhoI site of pNL-Luc-E(−)R(−). To create a neomycin-resistant marker expressing D64A, the mutant pNL-Neo-IN-D64A-E(−)R(−) was created by the SpeI-PflMI fragment (nucleotides 1507–5297) of pNL-IN-D64A and replaced with that of pNL-Neo-E(−)R(−). To create pIRES2-EGFP-I-SceI, a pIRES2-EGFP (Clontech)-based plasmid with an I-SceI recognition site, a synthetic double-stranded oligonucleotide (I-SceI-sense and I-SceI-antisense oligonucleotides; see Additional file 1: Table S2) was inserted into the EcoRI and BamHI sites of pIRES2-EGFP.
To make the adenoviral vector Ad-I-PpoI, I-PpoI cDNA was amplified from pBabe-HA-ER-I-PpoI using the Adeno-PpoI-DraI-F and Adeno-PpoI-DraI-R primers (Additional file 1: Table S2) and cloned into the SwaI site of the pAxCALNLwtit2 cosmid vector (NIPPON GENE). To generate the EGFP-expressing lentiviral vector (pLenti6-EGFP), EGFP cDNA from pENTR1a-EGFP was cloned into pLenti6/V5-DEST (Invitrogen) using LR Clonase (Invitrogen). The IN D64V mutation of the gag/pol-expressing plasmid pLP1 (Invitrogen; pLP1-IN-D64V) was introduced using pLP1 as a template with site-directed mutagenesis (QuikChange; Stratagene).
THP-1, HT1080, HEK293, and HEK293T cell lines were obtained from the RIKEN Cell Bank. TIG-3 (primary human fibroblast cells) and MT-4 cells were obtained from the Health Science Research Resources Bank (Osaka, Japan). HT1080, HEK293, HEK293T, MAGIC5, and TIG-3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MT-4 cell was maintained in RPMI 1640 supplemented with 10% FBS. To obtain macrophage-like cells, THP-1 cells, maintained in Iscove’s modified Dulbecco’s medium supplemented with 10% FBS, were treated for 2 d with 5.0 × 10–8 M PMA. As described previously , PMA-treated THP-1 cells were positive for Mac-1, a specific marker of macrophages. Peripheral blood was derived from healthy donors who worked within the institute and gave informed consent. Experimental procedures were approved by the internal review board. PBMCs and MDMs were prepared and cultured as previously described . MDMs were prepared from healthy volunteers who gave informed consents. The experimental protocol was approved by the internal review board.
HIV-1 and lentiviral vector preparation
The preparation and titration of replication-competent and VSVG-pseudotyped viruses are described elsewhere [36, 37, 62, 63]. The lentiviral vectors were prepared using pLenti6-EGFP and the ViraPower Lentiviral Packaging Mix (Invitrogen) according to the manufacturer’s protocol. Viral supernatants were centrifuged at 120 × g for 5 min, filtered through a 0.2-μm filter, and stored at −80°C. To exchange the medium for DMEM supplemented with 0.1% FBS, the viruses were ultracentrifuged at 86,000 × g for 1 h.
Quantitative PCR of provirus DNA
For the quantification of early RT, late RT, 2-LTR circle, and integrated DNA, qPCR was performed as described elsewhere [64, 65]. Briefly, cells were harvested at 48 hpi, and genomic DNA was prepared by QuickGene (FujiFilm). For the quantification of early RT, late RT, and 2-LTR circle products, the primers and probe sets M667/AA55/R-U5, M667/M661/R-U5, and MH535/2-LTR-AS/NL4-3-U3 were used, respectively. TaqMan Universal PCR Master Mix with UNG (Applied Biosystems) and ABI7000 (Applied Biosystems) were used according to the manufacturer’s instructions. For Alu-PCR (quantification of integrated DNA), the primer and probe sets first-Alu-F/first-Alu-R/first-gag-R and second-tag-R/2-LTR-S/probe-2 were used for the first and second rounds of qPCR, respectively. The amplification conditions for the first round of PCR, using AmpliTaq Gold 360 Master Mix (Applied Biosystems), were as follows: 95°C for 10 min, followed by 12 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 10 min. The second round of qPCR was conducted using TaqMan Universal PCR Master Mix according to the manufacturer’s instructions. To generate a standard curve for Alu-PCR, HEK293T cells (approximately one million cells) were infected with VSVG-pseudotyped NL-Luc-E(−)R(−) virus (200 ng p24), then harvested at 30 d post-infection (dpi), and genomic DNA was prepared. For the quantification of β-globin DNA copy numbers, the primer set globin-F/globin-R was used with SYBR Premix ExTaq (TaKaRa). Sequence information for primers and probes is listed in Additional file 1: Table S2.
Cleavage of I-SceI and I-PpoI sites
Ad-I-SceI and Ad-LacZ were prepared as described previously . PMA-treated THP-1 cells were infected with Ad-I-SceI or Ad-LacZ at 1 h post–HIV-1 infection for 1 h at a multiplicity of infection (MOI) of 100. In Figure 1E–1H, HT1080 cells were transfected with plasmid DNA that encoded a chmeric protein of estrogen receptor-I-PpoI (ER-I-PpoI), and then 4-hydroxytamoxifen (4-OHT) was added to activate the endonuclease and induce DSB. The pAxCALNLwtit2 cosmid vector harboring I-PpoI cDNA was digested with BspT104I and transduced into HEK293 cells to produce Ad-I-PpoI. The adenoviral vector encoding Cre recombinase, AxCANCre (TaKaRa), was co-infected with Ad-I-PpoI at an MOI of 30 to remove the floxed stuffer between the CAG promoter and I-PpoI cDNA.
Quantification of the I-SceI site specific viral integration
PMA-treated THP-1 cells were co-infected with WT virus and Ad-I-SceI or Ad-LacZ, and then extracted genomic DNA was subjected to I-SceI-qPCR analysis. I-SceI site targeting integration rate of HIV-1 DNA was estimated by PCR amplification using primer sets pIRES2eGFP+543F/pNL4-3+9207R and pIRES2eGFP+574F/pNL4-3+98R+9173R for the first and second rounds of qPCR, respectively. The amplification conditions for the first round of PCR, using ExTaq polymerase (TaKaRa), were as follows: 30 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 30 s. The second round of qPCR was conducted using SYBR Premix ExTaq polymerase (TaKaRa) according to the manufacturer’s instructions. For the second round PCR template, 1/25 the volume of the first PCR amplicon was used. To prepare a standard sample for the I-SceI-qPCR, the 5'-LTR DNA fragment of HIV-1 was amplified using the pNL4-3+9074F-Sce-RI and pNL4-3+9423R-BamHI and cloned into the EcoRI and BamHI sites of pIRES2-EGFP (pIRES2-EGFP-5'-LTR). Then, HT1080 cells were transfected with pIRES2-EGFP-5'-LTR and HT1080/pIRES2-EGFP-5'-LTR cell was obtained. By Southern blot and sequence analyses we confirmed that two copies of the DNA fragment of pIRES2-EGFP-5'-LTR vector were present in HT1080/pIRES2-EGFP-5'-LTR diploid cells. Sequence information for primers and probes is listed in Additional file 1: Table S2.
Quantification of the I-PpoI site specific viral integration
Serum starved HT1080 cells were co-infected with Ad-I-PpoI and lentiviruses, which were generated by pLenti6-EGFP or pLP1-IN-D64V. To estimate I-PpoI site targeting or total integration of the lentiviral vector, I-PpoI-qPCR or EGFP-qPCR was conducted using the TaqMan Universal PCR Master Mix (Applied Biosystems). For I-PpoI-qPCR in the direct or inverted repeat orientation, the primer sets rDNA+11725R/pLenti6+5237F or rDNA+11645F/pLenti6+5237F were used, respectively; pLenti6-LTR was used as the TaqMan probe. For EGFP-qPCR, the primers EGFP-F/EGFP-R and TaqMan EGFP-probe were used. As a standard sample for estimating copy numbers of viral DNA integrated in the I-PpoI site, genomic DNA of HT1080/Lenti6-EGFP-std cells were was used. We have confirmed by Southern blot and sequence analyses that HT1080/Lenti6-EGFP-std cells harbored two copies of Lenti6-EGFP proviral DNA in both orientations in the I-PpoI site. On the other hand, as a standard sample for total provirus DNA, genomic DNA of HT1080/pIRES2-EGFP-5'-LTR cells, which possessed two copies of EGFP, were used. Sequence information for primers and probes is listed in Additional file 1: Table S2.
PCR and sequence analysis
To amplify the host DNA/5'-LTR junction at the I-SceI site, the primer sets pIRES2eGFP+543F/pNL4-3+9207R and pIRES2eGFP+574F/pNL4-3+98R+9173R were used for the first and second rounds of PCR, respectively. To amplify the host DNA/3'-LTR junction at the I-SceI site, the primer sets pIRES2eGFP+1910R/L-M667 and pIRES2eGFP+887R/LambdaT were used for the first and second rounds of PCR, respectively. The amplification conditions for the host DNA/5'-LTR and host DNA/3'-LTR were as follows: 40 cycles for the first round of PCR or 30 cycles for the second round of PCR at 98°C for 10 s, 60°C for 30 s, and 72°C for 30 s and 35 cycles at 98°C for 10 s, 60°C for 30 s, and 72°C for 60 s for the first round of PCR and 30 s for the second round of PCR, respectively. ExTaq polymerase (TaKaRa) was used for the PCR. PCR amplicons were used directly or cloned into pCR2.1-TOPO (Invitrogen) as a template for sequence analysis. To amplify the rDNA/lentiviral vector at the I-PpoI site in the direct repeat orientation, the primer sets rDNA+11784R/pLenti6+5208F and rDNA+11747R/pLenti6+5232F were used for the first and second rounds of PCR, respectively. To amplify the rDNA/lentiviral vector at the I-PpoI site in the inverted repeat orientation, the primer sets rDNA+11589F/pLenti6+5208F and rDNA+11612F/pLenti6+5232F were used for the first and second rounds of PCR, respectively. The amplification conditions for the rDNA/lentiviral vector at the I-PpoI site were as follows: 40 cycles for the first and second rounds of PCR at 98°C for 10 s, 60°C for 30 s, and 72°C for 30 s. ExTaq polymerase (TaKaRa) was used for PCR. PCR amplicons were used directly or cloned into pCR2.1-TOPO (Invitrogen) as a template for sequence analysis. To analyze the IN mutations of NL-ADA and NL-IN-D64A-ADA viruses in Figure 5B and 5C, viral RNAs were isolated from conditioned medium (14 dpi; WT/RAL and D64A/DMSO, 16 dpi; D64A/RAL) and amplified by primer set pNL+4207F/pNL+5120R and Titan One-Tube RT-PCR Kit (Roche diagnostics). The amplicons were cloned into pCR2.1-TOPO and sequenced. The primers are listed in Additional file 1: Table S2.
To estimate the rate of insertion and/or deletion, the LAM-PCR method was performed as described previously [27, 66]. Briefly, 1.0 × 106 HT1080 cells were infected with VSVG-pseudotyped NL-Neo-E(−)R(−) virus (200 ng p24) in the presence of RAL or DMSO, and G418-resistant cells were harvested at 28 dpi and subjected to LAM-PCR. The sequence information for primers is listed in Additional file 1: Table S2.
To evaluate the production of functional virion from RAL-treated cells, 1 × 105 MT-4 cells were infected with replication-competent NL4-3 or NL-IN-D64A (20 ng p24). After 2 h of the infection, cells were washed with phosphate buffered saline (PBS) twice and suspended in 1.0 mL of medium. To prepare the culture supernatant, three-quarter of the cultures were harvested every 2 d, and the culture was continued by adding 750 μL of the complete medium into each well. From −1 dpi to harvest, MT-4 cells were treated with 10 μM RAL or DMSO. Conditioned medium (100 μL) was added to 1 × 104 MAGIC5 cells , and at 48 hpi, cells were stained by X-gal to estimate the number of transduced cells. To estimate HIV-1 RNA copy numbers, 1 × 105 MDMs were infected with NL-ADA, NL-ADA-R(−), NL-ADA-IN-D64A, or NL-ADA-IN-D64A-R(−) (20 ng p24) for 2 h, then washed with medium four times. Three-quarters of the conditioned medium was harvested and replaced with fresh medium every 2 d. From −1 dpi to harvest, MDMs were treated with 10 μM RAL or DMSO. HIV-1 RNA of conditioned medium was purified and subjected to RT-qPCR using the Lenti-X qRT-PCR Titration Kit (TaKaRa). To evaluate the effect of DNA damaging agents, 2.5 μM etoposide or 1.25 μM bleomycin were added to MDMs from 0–2 dpi. To exclude a possibility that detected HIV RNA merely reflect the RNA from carry-over virion, fusion inhibitor ENF, dissolved in phosphate buffer saline PBS, was added from 0 hpi to harvest as a negative control.
Colony formation assay
To evaluate the effect of DNA damaging agents on the integration rate of D64A mutant virus, serum-starved HT1080 cells (5 × 105 cells) in DMEM with 0.1% FBS were infected with a neomycin-resistant marker expressing VSVG-pseudotyped NL-Neo-IN-D64A-E(−)R(−) virus (100 ng p24) in the presence of 0 (DMSO), 0.625, 1.25, or 2.5 μM etoposide and 0 (water), 0.313, 0.625, or 1.25 μM bleomycin. Cells were selected with G418 from 2 dpi, then stained with Giemsa at 12 dpi. The G418-resistant colony numbers were normalized by plating efficiency, which represented the cytotoxicity of etoposide and bleomycin. The plating efficiencies after treatment of etoposide and bleomycin at 0, 0.625, 1.25, 2.5 μM were 100, 57.2, 26.0 and 19.5%, and 100, 60.4, 68.8 and 60.4%, respectively.
Detection of phosphorylated histone H2AX (γH2AX) and phosphorylated ATM (pATM) was done, according to the reported method using antibodies against γH2AX (phosphorylated at Ser 139, Millipore, cat no. 05–636) and pATM (Ser 1981, Millipore, cat. no. 05–740) . Briefly, the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS. The fixed cells were permeabilized with 0.2% Triton X-100 in PBS. After treatment with PBS supplemented with 10% goat serum for 30 min, the cells were incubated with antibodies. After 1 h of incubation at 37°C, secondary antibodies conjugated with Alexa 546 (Molecular Probes) were added for 1 h at 37°C. Nuclei were stained by Hoechst33258.
To evaluate the infectivity of viruses, 1.0 × 104 cells were infected with VSVG-pseudotyped luciferase viruses (2 ng p24) for 48 h, then subjected to luciferase assay by using One-Glo (Promega) and a Veritas Microplate luminometer (TURNER BIOSYSTEMS).
To analyze the status of cell cycle, HT1080 cells were labeled with 5 μM BrdU for 30 min and fixed in ice-cold 70% ethanol. Anti-BrdU-fluorescein (Roche Diagnostics) was used to stain the BrdU-labeled cells, according to the manufacturer’s instructions. BrdU-labeled cells were analyzed by BD FACSCalibur flow cytometer (BD Biosciences). To analyze the percentages of EGFP-positive cells, Flow cytometry analysis was performed using a BD FACSCalibur.
Metaphase FISH analysis was performed to estimate the proviral DNA copy number and co-localization of proviral DNA and rDNA using lentiviral vector and rDNA specific probes (Chromosome Science Labo Inc.).
Statistical significance was determined by Student’s t-test, and values of P < 0.05 were considered statistically significant.