Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair
© Smith et al. 2008
Received: 16 November 2007
Accepted: 22 January 2008
Published: 22 January 2008
Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.
Post-integration repair (PIR) is an essential step in the retroviral lifecycle, and yet it remains incompletely understood. PIR occurs after the retroviral integrase has removed two nucleotides from the 3'-ends of viral DNA and then joined the newly exposed hydroxyl groups to staggered phosphates in complementary strands of the host chromosomal DNA, through non-blunt cleavage of host DNA in concert with the ligation reaction [1, 2]. This initial integrase-mediated linkage between viral and host DNA produces an intermediate, in which the proviral DNA is flanked by short, single-stranded gaps in the host-cell DNA. PIR completes integration through four distinct steps: trimming the 2-bp flaps from the 5'-ends of the proviral DNA, filling in the single-stranded gaps that arose from the original staggered cleavage of host DNA, ligation of the trimmed 5' viral DNA ends to the filled-in host DNA strands, and reconstitution of appropriate chromatin structure at the integration site.
It has been proposed that that the virus exploits host-cell double-strand DNA break (DSB) repair pathways to complete the integration process, and initial evidence suggests that it involves the NHEJ (non-homologous end joining) pathway, as well as the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3 related) kinases [3–9]. Nevertheless, several key issues remain. First, the earliest known sensor of DSBs, the Nijmegen breakage syndrome-1 protein (NBS1), has not been examined in the context of retroviral PIR. NBS1 is the crucial initiating component of the MRN complex, which comprises three proteins: MRE11 (meiotic recombination 11 homologue), a combined exo- and endo-nuclease ; RAD50, which binds DNA duplexes and may function as an anchor to hold the DNA ends together at a DSB ; and NBS1 itself. NBS1 associates with DSBs immediately after the DNA damage occurs  and recruits MRE11 and RAD50 [13, 14]. In addition, NBS1 recruits the ATM kinase to DSB sites , and NBS1  and ATM  are then both required to recruit the ATR kinase . Activation of the ATM and ATR kinases allows them to phosphorylate several DNA repair and checkpoint proteins, including NBS1 itself [17–21]. Nijmegen breakage syndrome (NBS), which is caused by a hypomorphic mutation in the NBS1 gene, and ataxia telangiectasia (A-T), which is caused by mutations in the ATM gene, highlight the significance of NBS1 in DSB repair [22, 23]. NBS and A-T cells exhibit similar DNA repair deficiencies, including hypersensitivity to γ-irradiation, which causes DSBs, and defective cell-cycle checkpoints that fail to arrest cell proliferation when unrepaired DSBs are present [21, 24]. Because of the central role of NBS1 in DSB repair, we now hypothesize that this protein might initiate cellular responses leading to retroviral PIR as well.
The second key issue in understanding retroviral PIR concerns conflicting data in the literature about the roles of the ATM and ATR kinases. Although many publications demonstrated the participation of other NHEJ proteins in PIR [3, 5, 6, 8, 25, 26], the precise roles for ATM and ATR remain less clear. For example, we reported only a minor function for the ATM protein, which became apparent mainly in the absence of other NHEJ components . In contrast, some laboratories reported that ATM is required for efficient PIR even in the presence of NHEJ [8, 27], whereas others reported efficient transduction even in the absence of ATM [28, 29]. One explanation is that these discrepancies arose from the use of different immortalized cell lines in these studies. Therefore, in the current study we addressed the role of ATM in PIR in primary human cells.
Third, although a great deal is known about DSB repair, details of PIR have yet to be delineated. Retroviruses hijack numerous DSB repair proteins [3, 5, 6, 8, 25, 26, 30], but the geometry of retroviral integration differs considerably from DSB repair, which is limited to linking two blunt ends together. We now hypothesize that the two repair processes may crucially diverge. Initial supportive evidence comes from our recent finding that phosphorylation of the histone H2AX on its Ser 139 residue is crucial to DSB repair, but not for efficient PIR . Importantly, differences between the two repair processes might allow strategies to inhibit PIR while still allowing NHEJ.
Therefore, we now sought to examine the presence, interactions, and function of several DSB repair proteins in retroviral PIR, namely, the initial DSB sensor NBS1 and the ATM and ATR kinases. Our comparisons of PIR with DSB repair continue to reveal fundamental similarities and differences.
Primary human fibroblasts and lymphoid cell lines
All human cells were purchased from the Coriell Cell Repository (Camden, New Jersey): primary NBS fibroblast cells (deficient in the wild type NBS1 protein – GM07166) and matched control cells (GM04506); A-T primary fibroblasts (deficient in the ATM protein – GM02052) and matched controls (GM01661); EBV transformed NBS B-Lymphocytes (GM15818) and matched control EBV transformed cells (GM15817). All cells were maintained in RPMI-1640 medium in the presence of 10% fetal bovine serum (FBS), 5 × 10-6M 2-mercaptoethanol, non-essential amino acids, and 1% Pen/Strep.
All VSV G-pseudotyped HIV-1-based vectors were prepared as described previously [3, 32], and carried either a lacZ or EGFP reporter gene. A multiply attenuated vector (lacking the accessory proteins vpr, nef, vpu and vif) carrying the lacZ reporter is denoted as MAV .
Primary fibroblasts were plated at a density of 2 × 104 cells/well in 24-well plates, 105 cells/60-mm dish, or 3 × 105/100-mm dish. B-Lymphocytes were plated at a density of 3 × 105/ml in 24-well plates. Cells were infected the next day for 6 hours or overnight in the presence of 5 or 10 μg of DEAE-dextran per ml. Cultures were then assayed for reporter gene expression at multiple time points from two to seven days post-infection (dpi). Cells infected with lacZ-encoding viruses were stained overnight to detect β-galactosidase activity directly in dishes (Stratagene protocol) and blue cells were counted the following day. EGFP reporter gene expression was detected by flow cytometry. As a control to rule out non-specific effects of NBS1 deficiency on transient expression of lacZ, NBS1-deficient and control primary fibroblasts were plated at a density of 2 × 104 cells/well in a 24-well plate. The following day, cells were transfected with the lacZ plasmid, which encodes the lacZ reporter under control of the CMV promoter  using a ProFection Mammalian Calcium Chloride Transfection system (Promega). Cells were stained three days later for β-galactosidase activity (Stratagene protocol). To evaluate the effect of re-introduction of wild-type NBS1 on HIV-1 transduction, NBS1-deficient cells were plated at a density of 3 × 104 cells/well in a 24-well plates. The following day, cells were transfected with either an NBS1 expression plasmid or an empty vector, using the ProFection Mammalian Calcium Chloride Transfection system (Promega). One day post-transfection cells were infected with the HIV-1-based vector at a multiplicity of infection (m.o.i.) of 0.005. Cells were stained eight days later using a β-galactosidase assay as described above.
To detect and quantify fully integrated proviral DNA, a two-step nested PCR technique was conducted. Primary NBS1-deficient fibroblasts and control cells were infected with HIV-1-based vector (lacZ reporter) at m.o.i 1, m.o.i. 0.01, or mock infected. Three days post-infection genomic DNA was extracted (Promega kit A1120). First round of Alu-PCR employed a primer targeting the cellular Alu sequence 5'-GCC TCC CAA AGT GCT GGG ATT ACA G-3' as well as the M661 primer targeting the HIV-1 LTR/gag region, 5'-CCT GCG TCG AGA GAG CTC CTC TGG-3'. This initial amplification step used 150 ng of genomic DNA as template. Samples were subjected to 30 PCR cycles of 95C – 30 s, 60C – 45 s, and 72C – 5 min, and after the final round, samples were kept at 72°C for 10 min. Products of the first round were diluted 1/1,000 and used in the 30-cycle second round (nested) with viral LTR primers: 5'-GGA TTG TGC TAC AAG CTA GTA CC-3'; and 5'-TGA GGG ATC TCT AGT TAC CAG AGT-3'. Second-round PCR was cycled as follows: 95°C for 5 min; 30 cycles of 95°C for 40 s, 55°C for 45 s, 72°C for 60 s, and the last round was followed by 72°C for 10 min. PCR products from the second round were resolved by electophoresis on an agarose gel and subjected to Southern blotting with an HIV-1- LTR probe.
Quantitative data are displayed as means ± standard deviations. Comparisons between two groups were performed using the two tailed Student t-test.
The NBS1 protein is required for association of ATM, but not ATR, with viral DNA
Normal and NBS1-deficient primary human fibroblasts were infected with the pseudotyped HIV-1-based vector (lacZ reporter) at an m.o.i. of 0.1 and harvested at the indicated time points (Figure 1A). ChIP analysis was used to identify accumulation of NBS1, ATM, and ATR at sites of proviral DNA integration. Nuclear DNA and its associated proteins were crosslinked, immunoprecipitated with the indicated antibodies (anti-NBS1, ATM, or ATR), and associated viral DNA was amplified by PCR. Figure 1A shows an agarose gel of the amplified PCR products. In normal primary fibroblasts, the presence of viral DNA in NBS1, ATM, and ATR immunoprecipitates was first detected 12 hrs post-infection. In NBS1-deficient cells, however, we did not observe any association of viral DNA at any timepoint with NBS1, as expected, nor with ATM (Figure 1A). Surprisingly, NBS1 deficiency and the failure to recruit ATM did not block the association of viral DNA with ATR (Figure 1A, third row), even though NBS1 and ATM are each required for recruitment of ATR to DSB sites [15, 16]. As a negative control, no viral DNA was detected in any sample immunoprecipitated with the irrelevant anti-PI-3K kinase antibody (Figure 1A, bottom row).
To verify that the failure of ATM association with viral DNA in NBS1-deficient cells arises specifically from the mutation in the NBS1 gene, rather than from some other difference between these and control cells, we performed NBS1 reconstitution studies. Normal and NBS1-deficient fibroblasts were transfected with either an expression plasmid for wild-type NBS1 or an empty vector . Transfected cells were then infected with the HIV-1-based vector. The right half of Figure 1B shows ATM association with viral DNA in NBS1-deficient cells that were transfected with the NBS1 expression plasmid, but not in NBS1-deficient cells transfected with the empty vector, thereby confirming the essential role of NBS1. The NBS1 expression plasmid brought the amount of viral DNA associated with ATM to roughly the same level as in normal control cells (Figure 1B). Interestingly, overexpression of NBS1 in normal cells enhanced the association of viral DNA with ATM (Figure 1B, left half), suggesting that the NBS1 protein could be a limiting factor for ATM-mediated PIR even in normal cells. Taken together, our results demonstrate that NBS1 is required for association of ATM, but not ATR, with vector DNA.
The NBS1 protein is required for efficient stable transduction of human fibroblasts by HIV-1-based vectors
To test whether the transduction deficiency of NBS1-deficient cells can be observed using another reporter gene, control and NBS1-deficient primary human fibroblasts were infected with an HIV-1-based vector carrying the EGFP reporter . At an m.o.i. of 0.1, 13.45% of control cells expressed the reporter gene whereas EGFP expression was detected in only about one third as many NBS1-deficient fibroblasts (4.79%, Figure 2D). Based on the results of these different assays, we conclude that NBS1 deficiency substantially decreases stable retroviral transduction of primary human fibroblasts. We note that a drop of transduction efficiency of NBS1-deficient cells was noted previously (about two fold), but the data were not further analyzed .
The transduction deficiency of the NBS1-deficient cells can be rescued by expression of normal NBS1
The NBS1 protein is required for efficient transduction of human lymphoid cells by HIV-1-based vectors
The transduction deficiency of NBS cells does not result from a defect in vpr-mediated cell-cycle arrest
The NBS1 protein is required for efficient joining of viral DNA to host cell DNA, but does not affect other steps in the HIV-1 life cycle
Retroviral infection triggers apoptosis of NBS1-deficient cells in an integrase-dependent manner
The decreased amount of viral DNA that is joined to host cell DNA in NBS1-deficient cells presumably results from a failure of PIR. As a theoretical alternative, the NBS1 protein might be required for the initial integrase-mediated joining reaction. To distinguish between these possibilities, we took advantage of the fact that failure of PIR after integrase-mediated joining of viral and host DNA in other contexts triggers apoptosis through activation of cell-cycle checkpoint proteins by the unrepaired intermediate, leading to a loss of infected cells from the population [3, 5, 6, 8, 25, 26]. Thus, normal and NBS1-deficient fibroblasts were infected at a high m.o.i. (4.0) with an integration-competent HIV-1-based vector or a vector carrying the enzymatically inactive D64V mutation in the integrase protein. Cells were further analyzed by Western blotting for the presence of the 85-kDa PARP fragment, an apoptotic marker generated by caspase-mediated cleavage of the PARP protein . As shown in the Figure 6B, only one infection condition stimulated PARP cleavage, namely, infection of NBS1-deficient cells with the integrase-competent HIV-1-based vector. Neither cell type underwent apoptosis after infection with the integrase-deficient virus, and neither viral construct induced PARP cleavage in the normal cells. Thus, HIV-1 infection induces apoptosis of NBS1-deficient cells in an integrase-dependent manner. This finding is consistent with the failure of PIR rather than a defect in the initial integrase-mediated joining.
The ATM kinase is required for efficient HIV-1 transduction of primary human cells
In this study, we demonstrated that NBS1, an early sensor of DSBs, associates with viral DNA, is required for the association of ATM – but not ATR – with viral DNA, mediates efficient integration of viral DNA, promotes stable retroviral transduction, and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host linkages. These data support a key role for the NBS1 protein in PIR. We and others proposed that retroviral PIR employs the NHEJ pathway, including the ATM and ATR kinases [3–9]. Our current results extend that work, by demonstrating the dependence of PIR on NBS1, an interaction between NBS1 and ATM, and a dependence on ATM for PIR in primary, non-transformed cells. All of these features are shared with cellular DSB repair.
Nevertheless, the integration intermediate structurally differs from a DSB (see the Introduction), and so we now revised our model to include the concept that the two repair processes may diverge in key aspects. Initial evidence that PIR uses somewhat different cellular machinery than DSB repair came from our recent study where we observed that phosphorylation of the histone H2AX isoform, which is mediated by both the ATM and ATR kinases and is required for DSB repair, appears to be dispensable for PIR, although it can be detected at the integration sites . Importantly, our current results establish the surprising finding that recruitment of ATR, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. In this context, we note that some HIV-1 transduction occurs even in the absence of normal NBS1 or ATM (see Results section). It is possible that this residual transduction is mediated by ATR.
One possible explanation for the difference in ATR recruitment in PIR vs. DSB repair could be that the single-stranded DNA gaps, which flank the integration site, are sufficient to recruit the ATR protein. In contrast, MRN-dependent processing of DSBs, which may generate single-stranded DNA through the nuclease activity of MRE11, appears necessary for accumulation of ATR at the DSB sites . Additional differences between these DNA repair processes may exist, and might guide the development of therapeutic strategies to selectively inhibit PIR without blocking DSB repair.
As we and others have suggested, cellular co-factors constitute an attractive target for anti-HIV-1 therapy, since development of resistance against inhibitors of these proteins is unlikely [6, 8, 9, 27, 42]. NBS1 and its interactive partners, being such co-factors, are thus potential targets for anti-HIV-1 therapeutics, particularly at steps where PIR differs from DSB repair.
We thank Dr. David Horn (TJU-Infectious Diseases) for reading the manuscript and helpful comments. This work has been supported by NIH grants CA98090 and CA125272 (R.D.) and MH70279 (K.J.W.) and a W.W. Smith Foundation AIDS Research Award (R.D.).
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