Previous reports demonstrated that retroviral infectivity decreased in cells lacking DSB repair enzymes such as ATM and DNA-PKcs 141617. To confirm whether the enzymes affect HIV-1 infectivity, mutant cell lines and complemented cell lines were transduced with an HIV-based vector encoding a GFP reporter gene. As shown in Figure 1A, the transduction efficiency was impaired in the mutant cells lacking ATM compared to the complemented cells, indicating that ATM is involved in HIV-1 transduction. We also found that DNA-PKcs-deficient M059J cells showed a significantly lower level of transduction efficiency compared to DNA-PKcs-positive M059K cells (Figure 1B), indicating that DNA-PKcs is also required for stable transduction of HIV-1.

The influences of NBS1 and Mre11 on retroviral infectivity were controversial in previous reports 2122. In our cell lines, NBS1 and Mre11 deficiencies did not influence transduction efficiency (Figure 1C and 1D), suggesting that the MRN complex might not affect HIV-1 transduction.

We also investigated whether defects in these DSB repair enzymes affected MLV infectivity by using an MLV-based vector encoding a GFP reporter gene. As for the HIV-based vector, the infectivity of the MLV-based vector significantly decreased in DNA-PKcs-deficient cells, indicating the conserved role of DNA-PKcs in retroviral infection (Additional file S1B). Mre11-deficient cells also showed impaired MLV infectivity compared to the complemented cells (Additional file S1D). However, infectivity of MLV vector remained intact in the mutant cells lacking NBS1, which is the other component of the MRN complex (Additional file S1C). This might be due to the different extents of deficiencies of Mre11 and NBS1. In contrast to the HIV-based vector, ATM-deficient cells showed similar transduction efficiency of the MLV-based vector compared to the complemented cells (Additional file S1A). These results suggest that DSB repair enzymes are differentially required for the stable transduction of HIV-1 and MLV.

Since one of the potential targets of DNA repair enzymes is the junction between provirus and the host DNA 181923, we postulated that abnormal junctions would be generated in cells deficient in DNA repair enzymes. We therefore analyzed the sequences of the host-virus junctions. After amplification of integration sites by Alu PCR, we used inverse PCR to amplify the sequences around the integration sites with primers specific to LTRs and Alu repeat elements 24. With this method, we could identify integration sites efficiently, with few non-specific amplification products.

We analyzed 216 3' junctions between HIV-1 provirus and the host DNA in a control cell line, MRC5SV, and found one abnormal junction with a single nucleotide insertion, and seven junctions with deletions in viral DNA ends (Figure 2). In mutant cells lacking DSB repair enzymes, there were more abnormal junctions with inserted nucleotides between provirus and the host DNA. There were two different groups of abnormal nucleotides. One was a GT dinucleotides (or a G mononucleotide) adjacent to the provirus that is normally removed by integrase in 3'-processing. They did not originate from the host DNA. The other type of abnormal junction contained inserted nucleotides of unknown origin. The number of abnormal junctions with insertions was 1 of 216 (0.5%) events in the control cells, but 8 of 161 (5.0%) events in ATM-deficient cells (Figure 2 and Table 1). In ATM-complemented cells, 1 of 151 (0.7%) junctions had abnormal insertions, which was a significantly lower frequency than that of ATM-deficient cells. Although GFP reporter assays showed that defect of the MRN complex did not affect HIV-1 infectivity, the junctions in the MRN complex deficient cells also had abnormal insertions: 11 of 147 (7.5%) junctions in Mre11-deficient cells and 6 of 145 (4.1%) junctions in NBS1-deficient cells. It is of note that some of the abnormal junctions in Mre11-deficient cells also included 2, 4, 11, or 15 nucleotides of the primer binding site (PBS) sequences (Figure 2). In contrast, abnormal junctions with insertions were less frequent in Mre11-complemented cells (2 of 144: 1.4%) and NBS1- complemented cells (1 of 168: 0.6%). These results indicate that both Mre11 and NBS1 are indeed associated with HIV-1 replication. In contrast, in DNA-PKcs-deficient cells, only 3 of 153 (2.0%) junctions had abnormal insertions (Additional file S2), which is not a statistically significant difference compared to control MRC5SV cells.

Abnormal junctions with insertions were also found in 10 of 136 (7.4%) junctions in cells deficient in Artemis (Figure 2 and Table 1), which is a target of phosphorylation by ATM and DNA-PKcs 2526. Since Artemis-complemented cells could not be established, we could not conclude that these abnormalities observed in Artemis deficient cells were due to the deficiency of Artemis. However, the frequency was much higher than that of control MRC5SV cells (P = 0.0003), indicating the potential effects of Artemis on HIV-1 replication.

Some of the abnormal junctions also exhibited micro-homologies in the host sequences, in which 1-4 nucleotides were identical to a part of the GT dinucleotides and/or the PBS sequence following the inserted part (Figure 2). This observation suggests that at least some proviruses with such abnormal junctions might be integrated by a recombination mechanism using these micro-homologies.

To investigate whether the abnormalities were common to both ends of provirus, we also analyzed the sequences of 5' junctions. The junctions between the HIV-1 5' LTR and the host DNA also exhibited similar abnormalities (Figure 3A). Abnormal nucleotides were observed in 10 of 164 (6.1%) junctions in ATM-deficient cells and 13 of 134 (9.7%) junctions in Mre11-deficient cells, compared to 2 of 178 (1.1%) junctions in MRC5SV cells (Figure 3B). In 5' junctions, the remaining nucleotides were AC dinucleotides, which are complementary to the GT dinucleotides detected in 3' junctions. In Mre11 deficient cells, 3' polypurine tract (PPT) sequences were also identified. Thus, defects in DSB repair enzymes enhanced the abnormal joining of both ends of the HIV-1 DNA.

To determine whether these abnormalities are specific to HIV-1, we also analyzed sequences of the 3' junctions of the MLV provirus. Junctions with abnormal nucleotides increased from 5 of 228 (2.2%) events in Mre11-complemented cells to 20 of 256 (7.8%) events in Mre11-deficient cells (Figure 4). The abnormal junctions also included TT dinucleotides, which are usually removed by MLV integrase in 3'-processing. Taken together, these results show that defects in DSB repair enzymes increase abnormal host-virus junctions in both HIV-1 and MLV.

To study whether both 5'- and 3'-junctions of the same provirus were abnormal, we analyzed both 5' and 3' junctional sequences of the same provirus. Since the method used in Figure 2, 3 and 4 could detect only one end of provirus, we next adopted a traditional inverse PCR method. We identified three HIV-1 proviruses with abnormal junctions in Mre11-deficient cells (Figure 5). All three proviruses had the abnormal nucleotides at the 3' junctions. A single G was inserted in case 1, while both GT dinucletotides and part of a PBS were inserted in cases 2 and 3. These 3' junctions also showed micro-homologies in the host sequences, confirming the abnormalities shown in Figure 2. However, the 5' junctions were intact in these proviruses, indicating that these 5' junctions were processed by integrase as per normal. We also found that the host sequence adjacent to the provirus contained short repeats in case 1 and 2. Although all of the other proviruses had 5-bp short repeats as reported previously (data not shown), case 1 and 2 contained 3-bp and 2-bp short repeats, respectively. Case 3 lacked short repeats. These results suggest that the integration of these proviruses was catalyzed by integrase, but in abnormal ways.

Retrovirus-specific base preferences in the immediate vicinity of integration sites have been reported 272829. Our findings of abnormal host-virus junctions prompted us to investigate whether deficiencies in DSB repair enzymes also influence these preference patterns. We analyzed the nucleotide frequencies for the 8 nucleotides downstream and the 4 nucleotides upstream of the 3' ends of HIV-1 proviruses without insertions and/or deletions (Figure 6B). As shown in Figures 6 and 7, we calculated P values at each position by χ² analysis comparing the base compositions in each cell line and the average base compositions in the human genome (A:29%, T:29%, G:21%, C:21%). At the positions with P < 0.01, the bases with high frequencies or low frequencies were focused and colored in Figure 6 and 7. Compared to the control MRC5SV cells and ATM-complemented cells, which showed a preference pattern similar to that in the previous report 28, ATM-deficient cells showed a partially altered pattern. In the position -2, the different patterns were found in ATM-deficient cells compared to control MRC5SV cells (P < 0.0001) or ATM-complemented cells (P < 10^-14). Especially, ATM-deficient cells showed higher frequency of G compared to the control MRC5SV cells and the complemented cells at the position -2. Similarly, integration sites for the 5' end of the provirus in ATM-deficient cells showed a changed preference pattern in position 7 compared to the control MRC5SV cells (P < 0.001), in which ATM-deficient cells showed a higher frequency of G (Figure 7B). Since the 5 bp sequence (positions 1 to 5) is duplicated next to the 3' and 5' ends of the provirus as short repeats, position 7 for the 5' end of the provirus corresponds to position -2 for the 3' end of the provirus. This indicates that the analyses at both ends of the provirus showed the same change, suggesting the influence of deficiency in ATM in the position. In contrast, NBS1- and Mre11-deficient cells showed no clear change in base preference (data not shown). Thus, deficiency in ATM partially influences the local base preference pattern surrounding HIV-1 integration sites.

Previous reports suggested that some DSB repair enzymes were involved in the formation of 2-LTR circles and 1-LTR circles 2021. To investigate whether the formation of abnormal host-virus junctions links to circularization of viral cDNA, we quantified total viral cDNA, 2-LTR circles and 1-LTR circles in Mre11-deficient cells and the complemented cells. Quantitative analyses of these viral cDNAs showed that the amount of all three types of viral cDNA was similar in the deficient cells and the complemented cells (Figure 8). This suggested that deficiency in the MRN complex did not influence the formation of viral circular DNAs at least in these cell lines.

293T cells and MRC5SV cells, an SV40-transformed human fibroblast line, were cultured in Dulbecco's modified Eagle's medium (DMEM) and were supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 50 μg/ml streptomycin. Adenovirus-transformed Artemis-deficient cells originated from RS-SCID patients and were cultured in DMEM 48. ATM-deficient and ATM-complemented cells were established by transfecting empty vector and ATM expression vector, respectively, into an A-T cell line, AT5BIVA, as described previously 49, and cultured in DMEM containing 200 μg/ml hygromycin B (Calbiochem, San Diego, CA). NBS1-deficient and NBS1-complemented cells were established by transfecting empty vector and NBS1 expression vector, respectively, into an NBS cell line, GM7166VA7, as described previously 50, and cultured in DMEM containing 500 μg/ml G418 (Nacalai tesque, Kyoto, Japan). Mre11-deficient cells were established by transforming an ATLD2 cell line, D6809 (a generous gift from Dr. P. Concannon), by SV40, and the cells were cultured in DMEM. To obtain Mre11-complemented cells, Mre11-deficient cells were transfected with the Mre11 expression vector pCMV-Tag-Mre11, which was created by cloning Mre11 cDNA between the EcoRI and ApaI sites of pCMV-Tag 2B (Clontech, Mountain View, CA), and the cells were cultured in DMEM containing 500 μg/ml G418. For all experiments, we used antibiotic-free medium before 24 h of experiments.

An HIV-based vector encoding a green fluorescent protein (GFP) reporter was produced as follows. 293T cells were transfected by TransFectin (Bio-Rad, Hercules, CA) with the pCSII-EF-MCS-IRES-hrGFP transfer vector 51, the pCMV-Δ8/9 packaging vector, and pcDNA-VSVG envelope coding vector (generous gift from Dr H Miyoshi, RIKEN, Tsukuba, Japan). Two days after transfection, the supernatant was harvested, passed through a 0.45-μm-pore-size filter, and then subjected to centrifugation at 4°C and 75,000 × g for 2 h to concentrate the virus. The virus-containing pellet was dissolved in DMEM.

To produce an MLV-based vector encoding a GFP reporter, the transfer vector pDON-AI-2-IRES-hrGFP was created by excising IRES-hrGFP from pCSII-EF- MCS-IRES-hrGFP via BamHI/HpaI digestion and inserting the DNA into the corresponding site of pDON-AI-2 (Takara Bio, Ohtsu, Japan). GP293 cells, containing a plasmid expressing MLV gag and pol genes, were transfected with pDON-AI-2-IRES-hrGFP and pcDNA-VSVG. 2 days after transfection, supernatant was harvested, and virus was concentrated.

The titer of these vectors was determined using 293T cells, and scoring of transduction was performed by flow cytometry.

An HIV-based vector encoding a neomycin resistance gene was produced by transfecting the pCMV-Δ8/9 packaging vector, pcDNA-VSVG envelope coding vector, and CSII-CMV-IRES Neo^r, which was constructed by inserting IRES and a neomycin resistance gene into CSII-CMV-MCS (a generous gift from Dr H Miyoshi, RIKEN, Tsukuba, Japan).

The mutant cell lines and the complemented cell lines were transduced with various dilutions of the HIV GFP vector or the MLV GFP vector in the presence of 8 μg/ml of polybrene (Sigma, St Louis, MO) for 12 h before changing the medium. The infected cells were harvested two days post-infection and analyzed by flow cytometry to determine the percentage of GFP-expressing cells in each sample.

For cloning of retroviral integration sites by the Alu-PCR-based method, cells transduced with the HIV-based vector for 2 days were collected and the genomic DNA was obtained by standard phenol-chloroform methods with proteinase K treatment. 3' junctional sequences of HIV were amplified by 1st long PCR using a primer (HIV3-1) specific to the U5 region in the HIV LTR and a primer (Alu-1) specific to the Alu repeat sequence. The amplification products were blunted using T4 DNA Polymerase (TOYOBO, Osaka, Japan), phosphorylated using T4 Polynucleotide Kinase (TOYOBO), and circularized and/or concatemerized using T4 DNA Ligase (TOYOBO). The ligation products were amplified by 2nd long PCR using a primer (HIV3-2) specific to the U5 region in the HIV LTR and a primer (HIV3-3) spanning the junctions generated by ligation. Similarly, 5' junctional sequences of HIV were amplified by 1st PCR using a primer (HIV5-1) specific to the U3 region in the HIV LTR and a primer (Alu-2) specific to the Alu repeat sequence, and 2nd PCR using a primer (HIV5-2) specific to the U3 region in the HIV LTR and a primer (HIV5-3) spanning the junctions generated by ligation. 3' junctional sequences of MLV were amplified by 1st PCR using a primer (MLV3-1) specific to the U5 region in the MLV LTR and a primer (Alu-1) specific to Alu repeat sequence, and 2nd PCR using a primer (MLV3-2) specific to the U5 region in the MLV LTR and a primer (MLV3-3) spanning the junctions generated by ligation. The 2nd PCR products were cloned into the pGEM-T Easy Vector (Invitrogen, Carlsbad, CA), which allows for isolation of individual clones.

For cloning of integration sites including 5' and 3' ends of the same provirus, Mre11-deficient cells were transduced by the HIV-based vector encoding a neomycin resistance gene and cultured in DMEM containing 500 μg/ml G418 for a month. After DNA extraction, the genomic DNA was digested with EcoRI, circularized using T4 DNA Ligase, and digested with NotI. Then, both of the junctional sequences of HIV provirus were amplified by 1st long PCR using a primer (HIV-U5) specific to the U5 region in the HIV LTR and a primer (HIV5-1) that was previously described and 2nd long PCR using another primer (HIV3-1) that was previously described and a primer (HIV-U3) specific to the U3 region in the HIV LTR. The 2nd PCR products were cloned into the pGEM-T Easy Vector.

The sequences of the primers used in these assays are described in Additional file S1.

Sequencing was performed using the Big Dye Terminator (version 3.1) cycle sequencing kit and an ABI3130 autosequencer (both from Applied Biosystems, Foster City, CA). The BLAT program http://genome.ucsc.edu, hosted at the University of California, Santa Cruz, was used to search each integration clone against the March 2006 freeze of the human genome. Low-quality sequences and sequences with < 20 base pairs (bp) were discarded.

HIV-1 cDNA was quantified by fluorescent-monitored quantitative PCR (Taqman) with an ABI Prism 7700 sequence detection system (Applied Biosystems) essentially as described 24. Cells were infected with the HIV-based vector and the total DNA was extracted with DNAzol (Invitrogen) after 12 h or 24 h for analysis of total cDNA or 2-LTR and 1-LTR circles, respectively. Sequences of primers and probes are as follows; total cDNA forward, late RT F: 5'-TGTGTGCCCGTCTGTTGTGT-3'; total cDNA reverse, late RT R: 5'-GAGTCCTGCGTCGAGAGAGC-3'; total cDNA probe, LRT-P: 5'-(FAM)-CAGTGGCGCCCGAACAGGGA-(TAMRA)-3'; 2-LTR circle forward, 2-LTR F: 5'-AACTAGGGAACCCACTGCTTAAG-3'; 2-LTR reverse, 2-LTR-R: 5'-TCCACAGATCAAGGATATCTTGTC-3'; 2-LTR probe, MH603: 5'-(FAM)-ACACTACTTGAAGCACTCAAGGCAAGCTTT-(TAMRA)-3'; 1-LTR circle forward, 1-LTR F: 5'-CACACCTCAGGTACCTTTAAGA-3'; 1-LTR reverse, 1-LTR-R: 5'-GCGCTTCAGCAAGCCGAGTCCT-3'; 1-LTR probe, MH603: 5'-(FAM)-ACACTACTTGAAGCACTCAAGGCAAGCTTT-(TAMRA)-3'. Under our PCR conditions with 1-LTR-F and 1-LTR-R primers, 1-LTR circle products (~660 bp) were preferentially amplified compared with 2-LTR circle products (~1170 bp), as described previously 52. This was verified by checking the specific amplicon generated by standard PCR with the same conditions. For standard curves, we constructed control plasmids by PCR amplification from the total DNA extracts using the same primers as fluorescent-monitored quantitative PCR and cloning the products into the pGEM-T Easy Vector.