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<?xml version="1.0" ?> <tei> <teiHeader> <fileDesc xml:id="0"/> </teiHeader> <text xml:lang="en"> <p>Mammalian cells encounter a variety of DNA-damaging agents and<lb/> possess a number of different response pathways to maintain genomic<lb/> integrity <ref type="biblio" >1</ref> . One of the less well defined DNA damage responses results<lb/> from retroviral infections such as those induced by HIV-1. Integration<lb/> of the double-stranded cDNA reverse transcriptase product into the<lb/> host genome is an essential step in the retroviral life cycle. This event is<lb/> catalysed in part by the viral integrase protein that cleaves the host DNA<lb/> and promotes a strand transfer reaction resulting in short staggered DNA<lb/> breaks at the site of attachment <ref type="biblio">2</ref> . The gapped DNA intermediates then<lb/> have to be detected and efficiently repaired by host cell proteins if there<lb/> is to be a completed integration process and a productive infection. In<lb/> addition, it has been suggested that the unintegrated linear viral cDNA<lb/> may itself invoke a DNA damage response that, if left unchecked, will<lb/> result in the death of the host cell. This raises the possibility that the<lb/> inhibition of host DNA damage response factors could provide a new<lb/> therapeutic approach for the treatment of HIV-1 infections, especially<lb/> because some of these factors are not essential for cell survival.<lb/></p> <p>A number of groups have provided evidence that the Ku-dependent<lb/> non-homologous end-joining (NHEJ) pathway that is normally associ-<lb/>ated with the repair of DNA double-strand breaks (DSBs) is required<lb/> to support efficient retroviral infection <ref type="biblio">3–6</ref> and to prevent viral-induced<lb/> cell death <ref type="biblio">3,4</ref> . However, there is some controversy as to whether cell<lb/> death following retroviral infection is dependent on integrase activity <ref type="biblio">3,4</ref> .<lb/> Additional questions have also been raised about the involvement of<lb/> the ATM and ataxia-telangiectasia-and Rad3-related (ATR) proteins<lb/> in retroviral-induced DNA damage responses <ref type="biblio">7,8</ref> . Both ATM and ATR<lb/> are phosphatidylinositol-3-OH-kinase (PI(3)K)-like serine/threonine<lb/> kinases (PIKKs) that regulate cellular responses to DNA damage by con-<lb/>trolling cell-cycle arrest and DNA repair pathways <ref type="biblio" >9,10</ref> . Although many of<lb/> the protein substrates of ATM and ATR overlap <ref type="biblio">9</ref> , activation of the ATM<lb/> or ATR kinases largely depends on the type of DNA damage lesion and<lb/> the stage of the cell cycle in which it is encountered. ATM predomi-<lb/>nantly detects DNA DSBs, such as those caused by ionizing radiation,<lb/> and responds very rapidly to these lesions at all stages of the cell cycle,<lb/> whereas ATR is activated by agents such as ultraviolet radiation and<lb/> replication-fork collapse during S phase <ref type="biblio">10</ref> .<lb/></p> <p>Here, by using both genetic and pharmacological approaches, we dem-<lb/>onstrate that ATM activity has an important role in retroviral replication.<lb/> By screening a small molecule compound library developed for the PIKK<lb/> family, we have identified KU-55933, a novel, specific and potent inhibi-<lb/>tor of ATM <ref type="biblio" >11</ref> . This compound, which is not effective in targeting ATR,<lb/> DNA-dependent protein kinase (DNA-PK) or PI(3)K, suppresses HIV-1<lb/> replication and provides important proof of concept that the inhibition<lb/> of ATM, a non-essential cellular target, may represent a new approach<lb/> to the treatment of HIV-1 infections.<lb/></p> <head>RESULTS<lb/></head> <head> ATM facilitates efficient HIV-1 infection<lb/></head> <p>To investigate whether the absence of ATM affects HIV-1 infection,<lb/> both ATM knockout mouse embryonic stem (ES) cells and human<lb/> fibroblast cells from ataxia telangiectasia (AT) patients were tested for<lb/> their ability to support HIV-1 integrase-proficient (HIV-1 IN + ) vector<lb/> transduction <ref type="figure">(Fig. 1a)</ref> using both a colony formation assay based on<lb/> antibiotic selection and a luciferase reporter gene assay <ref type="biblio">12</ref> . For both the<lb/> ATM knockout mouse ES cells and the human AT fibroblast cells the<lb/> results demonstrated a clear reduction in efficiency of HIV-1 vector<lb/> transduction compared with the control cells expressing functional<lb/> ATM. To confirm these results, we performed identical assays using<lb/> previously described AT fibroblast cells <ref type="biblio" >13</ref> that had been complemented<lb/> with an ATM expression vector <ref type="figure">(Fig. 1b)</ref>. Consistent with the results<lb/> in <ref type="figure">Fig. 1a</ref>, ATM expression resulted in higher transduction rates com-<lb/>pared with the AT cells that contained vector alone. Because these<lb/> findings were different from those described in a previous study <ref type="biblio" >7</ref> , we<lb/> wished to extend these experiments from single-step transduction<lb/> assays to an investigation of wild-type HIV-1 replication. Replication-<lb/>competent wild-type HIV-1 virus was used to infect lymphoblast cells<lb/> derived from AT patients (GM01526) or control cells that contained<lb/> functional ATM (GM14680). HIV-1 replication was assessed by meas-<lb/>uring the levels of viral p24 antigen that were present in cell-free super-<lb/>natants after 7 days <ref type="figure">(Fig. 1c)</ref>. The reduced levels of viral replication<lb/> in ATM-deficient lymphoblasts, together with the results from the<lb/> transduction assays, support the idea that ATM has an important role<lb/> in HIV-1 infections. This role can probably be extended to retroviruses<lb/> in general because a similar dependency on ATM was seen for viral<lb/> vector transduction assays that used murine embryonic stem cell virus<lb/> (see <ref type="figure">Supplementary Information, Fig. S1</ref>).<lb/></p> <head>HIV-1 infection activates the ATM-dependent DNA damage<lb/> response pathway<lb/></head> <p>There are a number of studies that describe both the activation of ATM<lb/> in response to ionizing radiation-induced DSBs <ref type="biblio">9,10,14,15</ref> and the subsequent<lb/> phosphorylation of cellular targets. We investigated the activation of ATM<lb/></p> <figure>b<lb/> 0<lb/> 1<lb/> 2<lb/> 3<lb/> 4<lb/> AT<lb/> ATM<lb/> complemented<lb/> a<lb/> 0<lb/> 20<lb/> 40<lb/> 60<lb/> 80<lb/> 100<lb/> 120<lb/> ATM +/+<lb/> 1BR<lb/> AT5<lb/> Human fibroblast<lb/> c<lb/> GM14680<lb/> (WT ATM)<lb/> GM01526<lb/> (AT)<lb/> 0<lb/> 3.5<lb/> 3.0<lb/> 2.5<lb/> 2.0<lb/> 1.5<lb/> 1.0<lb/> 0.5<lb/> Transduction efficiency (%)<lb/> Mouse ES<lb/> ATM −/−<lb/> Relative transduction efficiency<lb/> HIV p24 (pg ml<lb/> −1 ) (×10<lb/> 4 )<lb/> Figure 1 ATM function is required for efficient HIV-1 infection. (a) Retroviral<lb/> transduction assays using HIV-1 IN + viral vectors in 7–10-day colony formation<lb/> (hatched), or 3-day luciferase assays (solid), were performed for matched<lb/> ATM −/− knockout mouse ES cells, and also with AT5 human AT fibroblasts<lb/> or 1BR fibroblasts containing wild-type ATM. Data are shown as the mean<lb/> percentage of viral transduction (transduction efficiency) compared with ATM-<lb/>expressing control cells (± s.d. from at least two independent experiments). For<lb/> all data in Fig. 1, wild-type or complemented control cells are shown in blue<lb/> and ATM-deficient cells are shown in orange. (b) Colony formation (hatched)<lb/> and luciferase (solid) HIV-1 IN + vector transduction assays were performed<lb/> using matched AT22IJE-T human AT fibroblast cells (AT22IJE-T pEBS7) and<lb/> AT cells complemented with the ATM cDNA (AT22IJE-T pEBS7-YZ5). Data<lb/> are shown as relative viral transduction efficiency compared with the AT cells<lb/> (± s.d. from two independent experiments). (c) HIV-1 replication assays for<lb/> GM14680 wild-type (WT) and GM01526 AT human lymphoblast cells infected<lb/> with HIV-1 RF wild-type virus strain. Data are shown for the mean amount of<lb/> HIV-1 p24 antigen present in cell-free supernatants 7 days after infection (±<lb/> s.d. from three replicate infections.<lb/> a<lb/> b<lb/></figure> <figure>Time after addition (h)<lb/> Virus<lb/> IR<lb/> HIV-1 IN +<lb/> HIV-1 IN +<lb/> HIV-1 IN D64V<lb/> HIV-1 IN D64V<lb/> HIV-1 IN +<lb/> HIV-1 IN D64V<lb/> p53<lb/> β-actin<lb/> Ser 15 p53<lb/> P<lb/> β-actin<lb/> ATR<lb/> DNA-PK<lb/> ATM<lb/> Ser 1981 ATM<lb/> P<lb/> p53<lb/> Ser 15 p53<lb/> P<lb/> NBS1<lb/> Ser 345 NBS1<lb/> P<lb/> GM14680<lb/> (WT ATM)<lb/> GM01526<lb/> (AT)<lb/> CHK2<lb/> Thr 68 CHK2<lb/> P<lb/> U T<lb/> H IV -1 IN +<lb/> H IV -1 IN D 6 4 V<lb/> IR<lb/> U V<lb/> U T<lb/> H IV -1 IN +<lb/> H IV -1 IN D 6 4 V<lb/> IR<lb/> U V<lb/> 0<lb/> 1<lb/> 2<lb/> 4<lb/> 6<lb/> 8<lb/> 2 Gy<lb/> Figure 2 Functional HIV-1 integrase is required to elicit an ATM-dependent<lb/> DNA damage response. (a) DNA damage responses of GM14680 wild-type<lb/> and GM01526 AT cells transduced with viral vectors. Results of whole-cell<lb/> lysate immunoblots from untreated cells (UT) or those infected with equal<lb/> amounts of integrase-proficient (HIV-1 IN + ) or D64V integrase mutant<lb/> (HIV-1 IN D64V ) vectors at 6 h post-infection. Also shown are immunoblots of<lb/> cells collected 1 h after exposure to 2 Gy ionizing radiation (IR) or 20 J m −2<lb/> ultraviolet radiation (UV). Immunoblots were sequentially probed with<lb/> nonphospho-and phospho-specific antibodies against ATM, CHK2, p53 and<lb/> NBS1. Immunoblots were also probed with nonphospho-specific antibodies<lb/> against ATR, DNA-PK and β-actin (loading controls). (b) Time course of p53<lb/> Ser 15 phosphorylation after transduction with HIV-1 IN + and HIV-1 IN D64V<lb/> mutant viral vectors. Results of whole-cell lysate immunoblots from U2OS<lb/> cells infected with equal amounts of viral vectors at increasing time points<lb/> after infection. Also shown are immunoblots of U2OS lysates from cells<lb/> collected 15 min after exposure to 2 Gy ionizing radiation. Immunoblots<lb/> were probed with antibodies specific to p53 P-Ser 15 before re-probing for<lb/> total p53 or β-actin (loading controls).<lb/></figure> <p>after HIV-1 infection by monitoring a number of these well characterized<lb/> ATM-dependent phosphorylation events. We infected both GM14680<lb/> and GM01526 AT cells with either functional HIV-1 IN + luciferase vec-<lb/>tors or those containing the inactivating integrase mutation D64V (ref.<lb/> 16) (HIV-1 IN D64V ). We then monitored the status of ATM phosphoser-<lb/>ine 1981 (P-Ser 1981) <ref type="biblio">17</ref> and CHK2 phosphothreonine 68 (P-Thr 68) <ref type="biblio">18</ref> ,<lb/> both specific markers for ATM activation, as well as p53 P-Ser 15 <ref type="biblio">(refs 19,<lb/> 20)</ref> and NBS1 P-Ser 343 <ref type="biblio">(ref. 21)</ref>, which can be phosphorylated by both<lb/> ATM or ATR depending on the type of DNA damage involved.<lb/></p> <p>To illustrate the presence or absence of functional ATM and ATR<lb/> activity in GM14680 and GM01526 AT cells, both were exposed to either<lb/> ionizing radiation, which is predominantly signalled through ATM, or<lb/> ultraviolet radiation, which is signalled through ATR <ref type="biblio">10</ref> . GM14680 cells<lb/> showed phosphorylation of all tested substrates in response to either<lb/> ionizing radiation or ultraviolet radiation, whereas GM01526 AT cells<lb/> only demonstrated phosphorylation of p53 Ser 15 and NBS1 Ser 343 in<lb/> response to ultraviolet radiation, confirming the loss of ATM function<lb/> but intact ATR activity in these cells <ref type="figure">(Fig. 2a)</ref>.<lb/></p> <p>Analysis of cells at 6 h post-infection with HIV-1 IN + vectors showed<lb/> an increase in phosphorylation for all the DNA damage signalling tar-<lb/>gets tested in GM14680 cells but not in GM01526 AT cells <ref type="figure">(Fig. 2a)</ref>,<lb/> indicating that DNA damage signalling in response to HIV-1 infec-<lb/>tion proceeds predominantly through activation of ATM kinase. In<lb/> addition, infection with HIV-1 IN D64V mutant vectors demonstrated<lb/> a reduction in phosphorylation for most targets when compared with<lb/> HIV-1 IN + vectors, suggesting that integrase-induced host DNA damage<lb/> but not unintegrated viral cDNA predominantly elicits this response.<lb/> Interestingly, an exception to this seems to be ATM auto-phosphoryla-<lb/>tion on Ser 1981, which is also enhanced during infection with HIV-1<lb/> IN D64V mutant vectors. The fact that this ATM phosphorylation event<lb/> is not translated into the phosphorylation of downstream ATM targets<lb/> such as CHK2, p53 or NBS1 is discussed later.<lb/></p> <p>To monitor activation of the ATM pathway by HIV-1 infection in a time<lb/> course study and in an additional cell type, we infected U2OS cells that have<lb/> functional ATM and p53 with either HIV-1 IN + or IN D64V mutant vectors and<lb/> monitored p53 P-Ser 15 levels at various time points after infection <ref type="figure">(Fig. 2b)</ref>.<lb/> It can be seen that infection with the HIV-1 IN + vector induces the phospho-<lb/>rylation of p53 Ser 15, peaking in these cells at around 4–6 h post-infection, a<lb/> time consistent with integration and cleavage of the host DNA by integrase <ref type="biblio">2</ref> .<lb/> In contrast, no such response was observed for the integrase-defective HIV-1<lb/> vector, in agreement with the data for the GM14680 cells <ref type="figure">(Fig. 2a)</ref>.<lb/></p> <head>HIV-1 infection of ATM-deficient cells results in enhanced host<lb/> cell death<lb/></head> <p>To determine whether there was enhanced cell death associated with<lb/> HIV-1 infection in ATM-deficient cells, we monitored the matched<lb/> ATM +/+ and ATM −/− ES cells infected with IN + or IN D64V mutant HIV-1<lb/> viral vectors. Cell death was assessed by determining the percentage of<lb/> annexin-V-positive cells at time points up to 48 h post-infection. We<lb/> observed a marked increase in the level of host cell death when ATM-<lb/>deficient cells were infected with HIV-1 IN + vectors, but this was not<lb/> the case when integrase-deficient virus was used or when the host cells<lb/> possessed functional ATM <ref type="figure">(Fig. 3)</ref>. Together, the results presented in<lb/> <ref type="figure">Figs 1–3</ref> suggest that the ATM DNA damage response pathway facilitates<lb/> efficient retroviral infection by contributing to the survival of host cells<lb/> in response to integrase-induced DNA damage.<lb/></p> <head>Suppression of HIV-1 transduction by a small-molecule ATM inhibitor<lb/></head> <p>The genetic data described above suggests that ATM could represent a<lb/> valid target for the suppression of HIV-1 infection by small molecule<lb/> kinase inhibitors. KU-55933 (2-morpholin-4-yl-6-thianthren-1-yl-<lb/>pyran-4-one; <ref type="figure">Fig. 4a</ref>) is a novel ATP competitive inhibitor of ATM with<lb/> an in vitro inhibitory concentration that gives half-maximal activity (IC 50 )<lb/> of 13 nM and cellular activity in the low micromolar range <ref type="biblio">11</ref> . Cellular<lb/> activity of KU-55933 has been demonstrated through both radiosensiti-<lb/>zation experiments and the abrogation of ionizing-radiation-dependent<lb/> phosphorylation of a range of known ATM targets including p53, γH2AX<lb/> and NBS1. Notably, this compound shows specificity with respect to the<lb/> inhibition of other PIKK family members. In particular, it is important<lb/> to note that the in vitro IC 50 of KU-55933 for ATR is greater than 100 µM<lb/> and cellular ATR-dependent phosphorylation events are not inhibited by<lb/> the drug in response to ultraviolet-radiation-induced damage <ref type="biblio">11</ref> .<lb/></p> <p>To test the ability of KU-55933 to inhibit HIV-1 infection, we<lb/> exposed Jurkat lymphoblast cells to HIV-1 IN + luciferase vectors with<lb/> increasing concentrations of compound <ref type="figure">(Fig. 4b)</ref>. KU-55933 effectively<lb/> inhibited HIV-1 transduction in these experiments with an IC 50 value<lb/> of approximately 1 µM <ref type="figure">(Fig. 4b; left panel)</ref>. The significant reduction<lb/> in transduction efficiency could not be attributed to cytotoxic effects<lb/> of the compound, as demonstrated by the effect of drug alone on the<lb/> viability of the Jurkat cells <ref type="figure">(Fig. 4b; right panel)</ref>. We confirmed that<lb/></p> <figure>HIV-1 IN +<lb/> HIV-1 IN D64V<lb/> Inactivated<lb/> No virus<lb/> ATM +/+<lb/> 0<lb/> 10<lb/> 20<lb/> 30<lb/> 40<lb/> Hours post-infection<lb/> ATM −/−<lb/> 0<lb/> 10<lb/> 20<lb/> 30<lb/> 40<lb/> a<lb/> b<lb/> 0<lb/> 1 2<lb/> 2 4<lb/> 3 6<lb/> 4 8<lb/> Hours post-infection<lb/> 0<lb/> 1 2<lb/> 2 4<lb/> 3 6<lb/> 4 8<lb/> Annexin-V<lb/> + cells (%)<lb/> Annexin-V<lb/> + cells (%)<lb/> Figure 3 ATM-deficient cells show enhanced cell death when transduced<lb/> with recombinant HIV-1 IN + vectors. (a, b) Cytopathicity studies in matched<lb/> ATM +/+ (a) and ATM −/− (b) mouse ES cells infected with HIV-1 vectors. Cells<lb/> were mock-infected (no virus; pink circles) or infected with equal amounts of<lb/> wild-type integrase (HIV-1 IN + ; blue squares), D64V integrase mutant (HIV-1<lb/> IN D64V ; orange triangles) or heat-inactivated IN + (inactivated; green crosses)<lb/> HIV-1 viral stocks. Cells were collected at increasing time points after<lb/> infection and annexin-V-stained cells were detected by flow cytometry. Data<lb/> are presented as the mean percentage of annexin-V-positive cells within the<lb/> total cell population ± s.d. from two independent experiments.<lb/></figure> <p>KU-55933 was able to inhibit an ATM-dependent response to viral-<lb/>induced DNA damage by infecting lymphoblast cells (GM14680) with<lb/> HIV-1 IN + luciferase vectors in the presence of 10 µM of compound<lb/> and monitoring the phosphorylation of p53 Ser 15 <ref type="figure">(Fig. 4c)</ref>. KU-55933<lb/> prevented this virus-dependent phosphorylation event and because the<lb/> compound does not demonstrate cellular activity against ATR, these<lb/> data provide further evidence that the phosphorylation of p53 Ser 15<lb/> after HIV-1 infection results from the activity of ATM. The use of KU-<lb/>55933 on both GM14680 and GM01526 AT cells <ref type="figure">(Fig. 4d)</ref> shows that<lb/> the ATM inhibitor reduces transduction efficiency of the GM14680<lb/> cells to a level that is close to that of the ATM-deficient GM01526 cells,<lb/> while having little effect on transduction in AT cells; this provides<lb/> further evidence that the anti-retroviral activity of KU-55933 results<lb/> predominantly from the inhibition of ATM.<lb/></p> <head>KU-55933 targets the post-integration DNA repair phase of the<lb/> retroviral life cycle<lb/></head> <p> Having demonstrated that KU-55933 inhibits HIV-1 vector transduc-<lb/> tion, we wanted to confirm that these results were not owing to the<lb/> inhibition of viral proteins such as reverse transcriptase or integrase.<lb/> To achieve this, 293 cells were transduced with HIV-1 IN + viral vec-<lb/>tors in the absence or presence of 10 µM KU-55933 or 1 µM zidovu-<lb/>dine (also known as AZT), a well characterized inhibitor of reverse<lb/> transcriptase. Total DNA was extracted from cells at increasing times<lb/> post-infection and the formation of late reverse transcriptase cDNA<lb/> (late-RT), 2-LTR circle cDNA, as well as integrated proviral DNA,<lb/> were assessed by PCR <ref type="figure">(Fig. 5)</ref>. Comparative analysis of the formation<lb/> of late-RT <ref type="figure">(Fig. 5a)</ref> and 2-LTR circle <ref type="figure">(Fig. 5b)</ref> HIV-1 cDNA shows<lb/> little effect of the presence of 10 µM KU-55933 but is reduced when<lb/> cells are treated with zidovudine. Analysis of integrated proviral<lb/> HIV-1 DNA through Alu-viral LTR PCR <ref type="figure">(Fig. 5c)</ref> also shows that<lb/> KU-55933 has little effect on integration up to 24 h after infection,<lb/> suggesting no direct activity against HIV-1 integrase. However, at<lb/> 48 h after infection in the presence of KU-55933, the amount of<lb/> integrated HIV-1 DNA is markedly reduced and this is consistent<lb/> with the loss of the ATM-inhibited infected cells through cell death<lb/> <ref type="figure">(Fig. 3)</ref>. Using purified integrase and reverse transcriptase proteins<lb/> in standard in vitro assays, KU-55933 was also shown to have no<lb/> inhibitory activity at concentrations up to 50 µM (data not shown).<lb/> Together, these data show that KU-55933 does not inhibit HIV-1<lb/> integrase or reverse transcriptase activities and the effects on HIV-1<lb/> vector transduction are consistent with ATM inhibition, resulting in<lb/> a failure to efficiently repair integration-induced DNA damage, and<lb/> ultimately leading to the death of the host cell.<lb/></p> <head>The ATM inhibitor KU-55933 suppresses HIV-1 replication<lb/></head> <p>The results that demonstrate the ability of KU-55933 to inhibit single-<lb/>round HIV-1 vector transduction encouraged us to assess whether the<lb/> ATM inhibitor would also suppress non-attenuated virus replication.<lb/> We therefore compared the ability of KU-55933 to inhibit HIV-1 rep-<lb/>lication in C8166 human T-lymphocyte cells with that of the existing<lb/> anti-retroviral nucleoside reverse transcriptase inhibitors abacavir<lb/> and lamivudine (also known as 3TC). It can be seen from <ref type="figure" >Fig. 6</ref> that<lb/> KU-55933 was indeed able to suppress HIV-1 replication with an IC 50<lb/> value of 2.3 µM compared with 0.9 µM for abacavir and 0.2 µM for<lb/> lamivudine. None of the compounds demonstrated overt toxicity in<lb/> the C8166 cells, having half-maximal viability (CC 50 ) values greater<lb/> than 30 µM (data not shown).<lb/></p> <figure>a<lb/> O<lb/> O<lb/> O<lb/> N<lb/> S<lb/> S<lb/> KU-55933<lb/> 0<lb/> 0.4<lb/> 0.8<lb/> 1.2<lb/> 1.6<lb/> 2.0<lb/> 2.4<lb/> d<lb/> c<lb/> − + − +<lb/> − +<lb/> − +<lb/> −<lb/> KU-55933:<lb/> −<lb/> 2<lb/> 4<lb/> 6<lb/> 8<lb/> Time after exposure (h)<lb/> β-actin<lb/> HIV-1 IN +<lb/> Ser 15 p53<lb/> P<lb/> 0<lb/> 20<lb/> 40<lb/> 60<lb/> 80<lb/> 100<lb/> 120<lb/> KU-55933 (µM)<lb/> KU-55933 (µM)<lb/> KU-55933 (µM)<lb/> 0<lb/> 20<lb/> 40<lb/> 60<lb/> 80<lb/> 100<lb/> 120<lb/> b<lb/> Transduction efficiency (%)<lb/> Viability (%)<lb/> 0<lb/> 2<lb/> 4<lb/> 6<lb/> 8<lb/> 1 0<lb/> 0<lb/> 2<lb/> 4<lb/> 6<lb/> 8<lb/> 10<lb/> 0<lb/> 2<lb/> 4<lb/> 6<lb/> 8<lb/> 1 0<lb/> 2 Gy IR<lb/> HIV-1 IN +<lb/> 2 Gy IR<lb/> Transduction luciferase activity (c.p.s.) (×10<lb/> 5 )<lb/> Figure 4 Suppression of HIV-1 vector transduction by KU-55933. (a) The<lb/> chemical structure of KU-55933. (b) HIV-1 IN + luciferase vector transduction<lb/> assays for Jurkat cells treated with KU-55933. Cells were infected with<lb/> HIV-1 luciferase vector 1 h before treatment with KU-55933 (left graph). In<lb/> parallel, Jurkat cells were mock infected (no virus) before treatment with KU-<lb/>55933 and viable cells determined by MTS assay (right graph). Data show<lb/> the mean percentage of luciferase activity (transduction efficiency) or viability<lb/> compared with untreated controls (± s.d. from two independent experiments).<lb/> (c) Immunoblots of GM14680 lymphoblast whole-cell lysates after infection<lb/> with HIV-1 IN + luciferase vectors in the presence or absence of 10 µM KU-<lb/>55933. Lysates from ionizing radiation (IR)-treated cells are also shown for<lb/> comparison. Immunoblots were probed with antibodies against p53 P-Ser 15<lb/> and β-actin. (d) HIV-1 IN + luciferase vector transduction assays for GM14680<lb/> normal (blue diamonds) and GM01526 AT (orange squares) cells treated<lb/> with KU-55933. Data show mean luciferase activity (measured in counts per<lb/> second, c.p.s.) ± s.d. from 10 replicates.<lb/></figure> <p>We were also interested to see how well KU-55933 would work in<lb/> combination with approved anti-HIV therapies such as the nucleoside<lb/> reverse transcriptase inhibitors or protease inhibitors. Using HIV-1 rep-<lb/>lication (cytopathicity) assays and standard methods for determining<lb/> drug synergism or antagonism <ref type="biblio" >22</ref> we constructed an isobologram plot for<lb/> KU-55933 and lamivudine <ref type="figure">(Fig. 6b)</ref>. The isobologram plot obtained for<lb/> KU-55933 and lamivudine demonstrates a synergistic inhibitory effect<lb/> on HIV-1 replication when using these two drugs in combination. A<lb/> similar finding was made in combination studies of KU-55933 with<lb/> zidovudine, and at least an additive effect was observed for the protease<lb/> inhibitor saquinavir (data not shown).<lb/></p> <head>KU-55933 inhibits drug-resistant HIV-1 as efficiently as<lb/> wild-type virus<lb/></head> <p>One of the key advantages of using an inhibitor that targets a cellular<lb/> factor required for the HIV-1 life cycle, rather than a viral protein, is that<lb/> it should be just as effective against drug-resistant HIV-1 strains that will<lb/> emerge after conventional anti-retroviral therapies. To test this principle<lb/> directly, we investigated the activity of KU-55933 against a number of<lb/> recombinant HIV-1 viruses containing mutations in their reverse tran-<lb/>scriptase genes known to be associated with drug resistance. <ref type="table">Table 1<lb/></ref> provides the IC 50 values of KU-55933, abacavir and lamivudine against<lb/> wild-type and drug-resistant HIV-1 strains that contain defined muta-<lb/>tions in the reverse transcriptase gene in 6-day replication assays. It can<lb/> be seen that whereas the IC 50 values of abacavir and lamivudine are much<lb/> higher for the drug-resistant virus compared with the wild-type virus,<lb/> KU-55933 values do not show any significant changes and demonstrate<lb/> comparable efficacy against all mutant HIV-1 strains tested. Moreover,<lb/> the shift in the ranking of compound effectiveness against drug-resistant<lb/> strains of HIV-1, in which KU-55933 is above both abacavir and lami-<lb/>vudine, illustrates the potential value of a cellular target, particularly in<lb/> patients for whom existing anti-retroviral therapy has failed.<lb/></p> <head>KU-55933 suppresses replication of clinical isolates of HIV-1<lb/> in primary blood mononuclear cells<lb/></head> <p>In addition to testing KU-55933 against recombinant HIV-1 in a T-cell line,<lb/> we investigated the ability of the ATM inhibitor to suppress the replication<lb/> of clinical isolates of HIV-1 in primary blood mononuclear cells (PBMCs).<lb/> Data were obtained from 7-day replication assays based on viral p24 antigen<lb/> production. The IC 50 values for KU-55933 against the three wild-type clinical<lb/> HIV-1 isolates HIV-1 CC15 , HIV-1 CC1 and HIV-1 105A are 1.7, 6.5 and 2.9 µM,<lb/> respectively. These values are comparable to previous results obtained with<lb/> recombinant HIV-1. The IC 50 values for KU-55933 against the zidovudine-<lb/>and lamivudine-resistant clinical isolates of HIV-1 are 4.1 and 5.5 µM,<lb/> respectively, illustrating that the ATM inhibitor is just as effective against<lb/> these drug-resistant isolates of HIV-1 as it is against wild-type isolates.<lb/></p> <head>DISCUSSION<lb/></head> <p>The data obtained in this study demonstrate that ATM is required for<lb/> efficient HIV-1 infection. To a large extent this has been facilitated by the<lb/> use of a potent and specific small molecule inhibitor of ATM, KU-55933,<lb/> and by using this inhibitor in studies that extend beyond vector-based<lb/> transduction experiments, to include HIV-1 replication assays and clini-<lb/>cal HIV-1 isolate infections of primary cells.<lb/></p> <p>The current confusion surrounding the role of DNA damage response<lb/> proteins in retroviral infections <ref type="biblio">7,8,23,24</ref> probably stems from the use of<lb/> non-specific inhibitors such as caffeine and wortmannin, the ectopic<lb/> overexpression of dominant-negative proteins, over-interpretation of<lb/> short interfering RNA (siRNA) knockdown data and the use of very dif-<lb/>ferent vector-based transduction assay conditions. For example, caffeine<lb/> has been implicated in inhibiting ATM, ATR <ref type="biblio">25</ref> , DNA-PK <ref type="biblio">26</ref> , CHK2 <ref type="biblio">(ref.<lb/> 27)</ref>, PI(3)K <ref type="biblio">28</ref> and mTOR <ref type="biblio">29</ref> , meaning that it cannot be used to conclusively<lb/> differentiate between these kinase activities in vivo <ref type="biblio">26</ref> . Wortmannin is not<lb/> much more specific, with inhibitory activity primarily against PI(3)K but<lb/> with the compound also effectively inhibiting DNA-PK, ATM and ATR<lb/> at micromolar concentrations <ref type="biblio">29</ref> . In published siRNA knockdown experi-<lb/>ments, non-specific control siRNA molecules alone have been shown<lb/> to activate retroviral transduction readouts <ref type="biblio">23</ref> , whereas dominant-nega-<lb/>tive kinase dead ATR constructs <ref type="biblio">30</ref> — used in an attempt to differentiate<lb/> between ATM and ATR contributions to retroviral transduction <ref type="biblio">7</ref> — have<lb/> previously been demonstrated to also have inhibitory effects on ionizing-<lb/>radiation-induced, ATM-driven phosphorylation events <ref type="biblio">31</ref> . With such a<lb/> combination of different methods used, it is perhaps not surprising that<lb/> conflicting interpretations have arisen.<lb/></p> <p>The earliest published study of the involvement of ATM in retro-<lb/>viral transduction came to the conclusion that this kinase only had<lb/> a minor role in retroviral infections <ref type="biblio">7</ref> . In contrast, our findings dem-<lb/>onstrate that ATM has a significant role in HIV-1 replication and the<lb/> data obtained with the ATM kinase inhibitor KU-55933 provide proof<lb/> of concept that this approach may have potential as an anti-retroviral<lb/> therapy. Possible reasons for these different conclusions may hinge<lb/> on Skalka and colleagues' use of wortmannin along with their use of<lb/> different control cell lines. What is less easy to explain is why their<lb/> study reported no difference in transduction efficiencies between the<lb/> ATM-deficient cell line AT22IJE-T and the matched ATM-reconsti-<lb/>tuted AT22IJE-T cells, whereas in this study a clear difference was<lb/> observed <ref type="figure" >(Fig. 1b)</ref>. One possible explanation may come from the fact<lb/> that ATM expression can be lost from the complimented cells and it<lb/> is important to check the phenotype of the reconstituted cells. In the<lb/> experiments presented in this study, both lines were assayed to deter-<lb/>mine their response to ionizing-radiation-induced DNA damage in<lb/> parallel with the HIV-1 transduction experiments, and the results for<lb/> both the radiation survival assays (see <ref type="figure">Supplementary Information,<lb/></ref></p> <figure type="table">Table 1 KU-55933 inhibits drug-resistant HIV-1 as well as wild type<lb/> Drug IC 50 (µM)<lb/> HIV RT mutant<lb/> KU-55933<lb/> Abacavir<lb/> Lamivudine<lb/> WT<lb/> 3.8<lb/> 2.9<lb/> 0.6<lb/> L74V/M184V<lb/> 5.2<lb/> 20<lb/> >100<lb/> K65R/M184V<lb/> 4.9<lb/> 11.3<lb/> >100<lb/> K65R/L74V/M184V<lb/> 2.8<lb/> 11.0<lb/> >100<lb/> Values given represent data from two independent experiments.<lb/></figure> <p><ref type="figure">Fig. S2</ref>) and the HIV-1 vector transduction assays <ref type="figure">(Fig. 1b)</ref> are<lb/> consistent. In addition, although the ATM-reconstituted AT22IJE-T<lb/> line is more resistant to ionizing radiation and more capable of sup-<lb/>porting HIV-1 vector transduction than the ATM-deficient AT22IJE-<lb/>T cells, they are not fully complemented for the AT defect <ref type="biblio">31</ref> .<lb/></p> <p>Our analyses of DNA damage response markers following HIV-1 infec-<lb/>tion also provide a number of important insights into the involvement of<lb/> ATM in this process. First, in response to retroviral infection, phosphor-<lb/>ylation of ATM Ser 1981, CHK2 Thr 68, p53 Ser 15 and NBS1 Ser 343 are<lb/> only observed in cells with functional ATM. Second, the results that we<lb/> obtained from infection with IN D64V mutant HIV-1 demonstrated reduced<lb/> levels of ATM substrate phosphorylation compared with infection with<lb/> integrase-proficient virus, suggesting that ATM signalling is in response<lb/> to the integrase-induced cleavage of host genomic DNA and not as a<lb/> consequence of the presence of double-stranded viral cDNA. Perhaps<lb/> the one exception to this is the autophosphorylation of ATM Ser 1981.<lb/> This phosphorylation event is a very sensitive marker of cellular stress<lb/> that is not absolutely dependent upon the generation of DNA DSBs and<lb/> can probably occur as a consequence of changes in chromatin structure <ref type="biblio">17</ref> .<lb/> The fact that our observations show HIV-1 IN D64V mutant virus failing<lb/> to generate an extended ATM signalling response (with CHK2 and p53<lb/> phosphorylation), suggests that either a critical threshold of DNA damage<lb/> has not been reached or that there are differences in composition between<lb/> the protein complexes (and therefore available substrates) associated with<lb/> linear viral cDNA ends and viral–host DNA integration intermediates.<lb/> Although not strictly a DNA double-strand break, the close proximity of<lb/> integrase-induced DNA breaks (see <ref type="figure">Supplementary Information, Fig. S3</ref>)<lb/> indicate that it may be recognized and dealt with as such. Notably, a recent<lb/> study by Skalka and colleagues described the phosphorylation and foci<lb/> formation of histone H2AX in response to retroviral infection <ref type="biblio">32</ref> . ATM is<lb/> thought to be the predominant kinase that phosphorylates H2AX Ser 139<lb/> in response to DNA DSBs and this process is thought to be ATR-inde-<lb/>pendent in non-replicating cells <ref type="biblio">33</ref> . These data are therefore consistent with<lb/> ATM activation at a time after infection during which we also observe<lb/> other ATM-dependent phosphorylation events <ref type="figure">(Fig. 2)</ref>.<lb/></p> <p>Activation of an ATM signalling cascade after integrase-induced<lb/> DNA damage is also consistent with a number of additional observa-<lb/>tions in this study, which provide an insight into the mechanism of<lb/> action of KU-55933 as an anti-retroviral agent. The infection with an<lb/> integrase-proficient HIV-1 vector induces cell death in ATM-deficient<lb/> cells <ref type="figure">(Fig. 3)</ref>, suggesting that ATM provides the host cell with protec-<lb/>tion against HIV-1 integrase-induced DNA damage. Consequently, in<lb/> the absence of functional ATM the host cell is more likely to die, thus<lb/> preventing the establishment of an infection. KU-55933 reduces the<lb/> efficiency of HIV-1 transduction down to a level that is seen for AT<lb/> cells, and ablates ATM-signalling as indicated by the lack of p53 Ser 15<lb/> phosphorylation <ref type="figure">(Fig. 4)</ref>, indicating that the effect of the compound in<lb/> these experiments is through the inhibition of ATM. KU-55933 does<lb/> not affect HIV-1 reverse transcriptase activity, viral nuclear import or<lb/> the HIV-1 integrase protein's ability to catalyse the joining of viral cDNA<lb/> with the host genomic DNA <ref type="figure">(Fig. 5)</ref>. Also consistent with the inhibition<lb/> of post-integration repair by KU-55933, is the loss of integrated viral<lb/> DNA from the cell population after 48 h, consistent with the induction<lb/> of host cell death. Further evidence that the compound is not inhibiting<lb/> a retroviral target such as reverse transcriptase or protease activity in<lb/> replication assays, is the lack of any antagonism in drug combination<lb/> studies <ref type="figure">(Fig. 6)</ref> and the ability of KU-55933 to work just as effectively<lb/> against drug-resistant HIV-1 <ref type="table">(Table 1)</ref>.<lb/></p> <p>The data presented therefore suggest that small molecule inhibitors of<lb/> ATM have the potential to suppress HIV-1 infections. Although other DNA<lb/> damage response proteins have been implicated in modulating retroviral<lb/> infections <ref type="biblio" >3–8,34–38</ref> many of these may not be suitable targets for anti-retroviral<lb/> therapy. For example, ATR seems to be essential for normal cell function <ref type="biblio">39,40</ref><lb/> and it is likely that if specific ATR kinase inhibitors could be identified,<lb/> they would result in cellular lethality. Other targets may be implicated from<lb/> genetic data but their enzymatic activities may not be involved. For example,<lb/> both DNA-PK and PARP-1 have been shown to positively influence retrovi-<lb/>ral transduction <ref type="biblio">3–6,34,35</ref> , but our potent and specific small molecule inhibitors<lb/> of DNA-PK <ref type="biblio">29</ref> and PARP <ref type="biblio">41</ref> have little or no effect on retroviral transduction<lb/> (data not shown). Ultimately, whichever cellular target is being considered<lb/> for anti-HIV-1 therapy, there will be associated challenges. Although not<lb/> essential for cell survival (AT patients generally live for 20–30 years), the<lb/> benefit-to-risk equation of targeting ATM for HIV-1 infections will have to<lb/> be carefully investigated. In spite of the challenges, perhaps intelligent and<lb/> intermittent use of inhibitors of cellular targets such as ATM will provide a<lb/> much needed additional weapon in the fight against HIV-1 infections.<lb/></p> <figure>Untreated<lb/> 10 µM KU-55933<lb/> 1 µM zidovudine<lb/> a<lb/> b<lb/> c<lb/> Untreated<lb/> 10 µM KU-55933<lb/> 1 µM zidovudine<lb/> Untreated<lb/> 10 µM KU-55933<lb/> 1 µM zidovudine<lb/> Untreated<lb/> 10 µM KU-55933<lb/> 1 µM zidovudine<lb/> Hours post-infection<lb/> Hours post-infection<lb/> Hours post-infection<lb/> Late-RT DNA<lb/> 2-LTR DNA<lb/> Integrated DNA<lb/> 0<lb/> 0 6 12 18 24 30 36 42 48 54<lb/> 0.5<lb/> 1.0<lb/> 1.5<lb/> 2.0<lb/> 2.5<lb/> 3.0<lb/> Hours post-infection<lb/> 0 6 12 18 24 30 36 42 48 54<lb/> Hours post-infection<lb/> 0 6 12 18 24 30 36 42 48 54<lb/> Hours post-infection<lb/> 0<lb/> 0.2<lb/> 0.4<lb/> 0.6<lb/> 0.8<lb/> 1.0<lb/> 1.2<lb/> 0<lb/> 0.5<lb/> 1.0<lb/> 1.5<lb/> 2.0<lb/> 2.5<lb/> 0 3 6 9 24 48<lb/> 0 3 6 9 24 48<lb/> 0 3 6 9 24 48<lb/> Late-RT DNA (relative to GAPDH)<lb/> 2-LTR DNA<lb/> (relative to GAPDH)<lb/> Integrated DNA<lb/> (relative to GAPDH)<lb/> Figure 5 Effect of KU-55933 on HIV-1 cDNA intermediate formation and<lb/> integration. (a–c) PCR of DNA extracted from HIV-1 IN + vector-transduced<lb/> 293 cells at increasing times after virus addition. The amounts of HIV-1<lb/> late-RT cDNA (a), 2-LTR cDNA (b) and integrated proviral DNA (c) were<lb/> estimated from cells that were transduced in the absence (untreated; blue<lb/> squares) or presence of 10 µM KU-55933 (pink crosses) or 1 µM zidovudine<lb/> (orange triangles). Left panels show gel images of PCRs and right panels<lb/> show the quantified, normalized data. All PCR quantification results are<lb/> expressed as a normalized ratio of HIV-1 DNA: GAPDH control DNA. Gel<lb/> images of GAPDH control PCRs are not shown.<lb/></figure> <head>METHODS<lb/></head> <p>Cell lines. The murine ES cell line J1 and its ATM −/− homozygous knockout<lb/> derivative <ref type="biblio">42</ref> were a kind gift from F. Alt and were grown in the absence of feeder<lb/> cells on gelatinized cell culture dishes in DMEM with 15% FBS and supplemented<lb/> with 500 units ml −1 LIF (Chemicon International, Temecula, CA). Human SV40-<lb/>transformed skin fibroblast cells 1BR.3.N, p53-positive U2OS cells and 293 cells<lb/> were obtained from The European Collection of Cell Cultures (ECACC; Salisbury,<lb/> UK). SV40-transformed AT skin fibroblast cells AT5BIVA and EBV-transformed<lb/> GM14680 normal and GM01526 AT lymphoblastoid cells were all obtained from<lb/> Coriell Cell Repositories (Coriell Institute for Medical Research, NJ). ATM-com-<lb/>plimented AT22IJE-T pEBS7-YZ5 and vector-only control AT22IJE-T pEBS7<lb/> human fibroblast cells were a kind gift from Y. Shiloh and have been previously<lb/> described <ref type="biblio">13</ref> . Jurkat and C8166 T-lymphoblastoid cells were grown as suspension<lb/> cultures in RPMI 1640 medium supplemented with 10% FBS.<lb/></p> <p>HIV-1 transduction assays. Single-round transduction assays using HIV-1 vectors<lb/> and based on colony formation and luciferase have been previously described <ref type="biblio">12</ref> .<lb/> Luciferase transduction assays done in the presence of drug were performed by<lb/> infecting cells with retroviral stocks for 1 h (multiplicity of infection (MOI) ~0.25)<lb/> before washing and replacing with fresh medium containing dilutions of KU-55933.<lb/> At least four replicate wells were performed for each drug dilution. Cells were incu-<lb/>bated for 72 h and then assayed for luciferase activity as previously described <ref type="biblio">12</ref> .<lb/></p> <p>HIV-1 replication p24 and XTT cytopathicity assays with recombinant HIV-<lb/>1 vectors. Recombinant HIV-1 RF and HIV-1 HXB2 wild-type viruses and deriva-<lb/>tives containing site-directed mutations in the reverse transcriptase gene were<lb/> obtained from the MRC AIDS Directed Reagent Programme (National Institute<lb/> Biological Standards and Control, Potters Bar, UK). C8166 T-cells were infected<lb/> with an HIV-1 strain for 1–2 h before being washed and re-seeded into fresh<lb/> 96-well plates containing dilutions of KU-55933, abacavir or lamivudine. At<lb/> least three replicate wells were performed for each drug dilution. For HIV-1 p24<lb/> antigen assays, cells were incubated for 7 days before cell-free supernatants were<lb/> collected and virus quantified by HIV-1 p24 antigen enzyme-linked immuno-<lb/>sorbent assay (ELISA; Abbott Laboratories, Abbot Park, IL). The concentration<lb/> of drug that gave a 50% reduction in HIV-1 p24 when compared with untreated<lb/> control cells (IC 50 ) was calculated from sigmoidal plots of the amount of HIV-1<lb/> p24 versus drug concentration. For HIV-1 XTT (a tetrazolium salt) cytopathic-<lb/>ity assays, cells were incubated for 5–6 days and viable cells remaining were<lb/> quantified using the XTT assay <ref type="biblio">43</ref> . In these cytopathicity assays, the number of<lb/> viable cells remaining is inversely proportional to the level of HIV-1 infection.<lb/> The concentration of drug that gave a 50% inhibition of HIV-1 cytopathicity<lb/> relative to the untreated control cells (IC 50 ) was calculated from sigmoidal plots<lb/> of cell viability versus drug concentration. For KU-55933 combination stud-<lb/>ies, C8166 cells were infected with wild-type HIV- 1 HXB2 for 1–2 h before being<lb/> washed and seeded into 96-well plates containing increasing concentrations of<lb/> KU-55933. Different concentrations of lamivudine were then added and cells<lb/> were incubated for 7 days. IC 50 values for inhibitors alone or in combination<lb/> were determined using XTT cytopathicity assays and plotted against each other<lb/> to give isobolograms <ref type="biblio">22</ref> . A curve that essentially follows the line between the IC 50<lb/> values of the two drugs would indicate additivity, whereas a convex curve would<lb/> indicate drug antagonism, and a concave curve drug synergy.<lb/></p> <p>HIV-1 replication assays with HIV-1 clinical isolates. Replication assays<lb/> using HIV-1 clinical isolates were performed on 3-day IL-2-stimulated<lb/> human PBMCs isolated from buffy coat residues (Lymphoprep; Axis-Shield,<lb/> Oslo, Norway) of blood from healthy volunteers (National Blood service,<lb/> UK). PBMCs were infected for 1–2 h with either wild-type clinical isolates of<lb/> HIV-1 CC15 , HIV-1 CC1 and HIV-1 105A , lamivudine-resistant HIV-1 105F , or zido-<lb/>vudine-resistant HIV-1 CC18 clinical HIV-1 virus isolates <ref type="biblio">44 , before washing and<lb/> re-seeding into fresh 48-well plates that contained dilutions of KU-55933. At<lb/> le</ref>ast three replicate wells were performed for each drug dilution. Cells were<lb/> incubated for 7 days and the amount of HIV-1 virus in cell-free supernatants<lb/> was quantified by HIV-1 p24 antigen ELISA as previously described. PBMCs<lb/> and clinical HIV-1 strains were isolated and phenotyped by MRC-Technology<lb/> (London, UK) with all of the relevant ethical approvals.<lb/></p> <p>Drug cytotoxicity assays. For all cell-based drug inhibition studies, cytotoxicity<lb/> assays were performed in parallel to monitor effects of drug alone on cell growth.<lb/> Cytotoxicity/cell viability assays were performed using at least three replicate wells<lb/> by either XTT <ref type="biblio">43</ref> or MTS (CellTiter96-aqueous solution; Promega, Madison, WI)<lb/> viability assays. The concentration of drug that gave 50% reduction in cell viability<lb/> when compared with untreated control cells (CC 50 ) was calculated from sigmoidal<lb/> plots of cell viability versus drug concentration.<lb/></p> <p>Cytopathicity assays. Mouse ES cells were plated on gelatinized 96-well plates<lb/> and either mock infected (cell culture supernatants without virus) or infected with<lb/> HIV-1 IN + , HIV-1 IN D64V or heat-inactivated HIV-1 IN + luciferase virus vector<lb/> stocks at an MOI~10. Heat-inactivated HIV-1 IN + stocks were incubated at 60 o C<lb/> for 10 min. At increasing times after virus addition, cells were collected and stained<lb/> with annexin-V–EGFP (Clontech, Palo Alto, CA) according to the manufacturers'<lb/> instructions. The percentage of annexin-V-positive cells was determined by flow<lb/> cytometry analysis using BD FACScalibur and CellQuest software.<lb/></p> <p>HIV-1 induced ATM activation. U2OS, GM14680 or GM01526 AT cells<lb/> (1.5 × 10 6 in each case) were transduced with HIV-1 IN + or IN D64V mutant HIV-<lb/>1 luciferase virus stocks (MOI~5) for 1 h before changing to fresh medium. At<lb/> increasing times after virus exposure, cells were washed twice in PBS, collected,<lb/> and split at a ratio of 2:3 for preparation of protein extracts and 1:3 for DNA<lb/> extraction. Whole-cell protein lysates were prepared by resuspending cells in<lb/> either SDS-loading buffer (50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 0.5 mM<lb/></p> <figure>a<lb/> IC 50<lb/> CC 50<lb/> 2.3 µM<lb/> 0.9 µM<lb/> 0.2 µM<lb/> KU-55933<lb/> Abacavir<lb/> Lamivudine<lb/> 0<lb/> 20<lb/> 40<lb/> 60<lb/> 80<lb/> 100<lb/> 120<lb/> Log drug concentration [µM]<lb/> 1<lb/> 10<lb/> 10 −1<lb/> 10 −2<lb/> 10 −3<lb/> 10 −4<lb/> 100<lb/> 0<lb/> 0.4<lb/> 0.8<lb/> 1.2<lb/> 1.6<lb/> 2.0<lb/> KU-55933 ( µM)<lb/> Fixed [KU-55933]<lb/> Fixed [lamivudine]<lb/> IC 50<lb/> b<lb/> HIV-1 replication<lb/> (level of p24 as percentage control)<lb/> > 30 µM<lb/> > 30 µM<lb/> > 30 µM<lb/> Lamivudine (µM)<lb/> 0<lb/> 2<lb/> 4<lb/> 6<lb/> 8<lb/> 1 0<lb/> 1 2<lb/> Figure 6 Inhibition of HIV-1 replication by KU-55933. (a) HIV-1 replication<lb/> assays for C8166 cells infected with HIV-1 HXB2 wild-type virus in the presence<lb/> of KU-55933 (blue diamonds) or the reverse transcriptase inhibitors abacavir<lb/> (pink crosses) and lamivudine (orange circles). The mean percentage of HIV-1<lb/> p24 antigen compared with untreated control cells from at least two replicate<lb/> infections along with compound IC 50 and CC 50 values are shown. (b) Inhibition<lb/> of HIV-1 replication by KU-55933 used in combination with lamivudine.<lb/> C8166 cells were infected with wild-type HIV-1 HXB2 in the presence of<lb/> increasing concentrations of inhibitor alone and in combination with the other<lb/> inhibitor. IC 50 values were determined for each inhibitor combination by XTT<lb/> cytopathicity assays from at least two replicate infections. The data are shown<lb/> as IC 50 isobologram plots, and inhibitor combinations that fall below the<lb/> dotted line drawn between the IC 50 values for the individual inhibitors (pink<lb/> triangles) indicate synergistic effects.<lb/></figure> <p>MgOAc and 1 mM DTT) or high-salt buffer (20 mM HEPES pH 7.2, 450 mM<lb/> NaCl, 10% glycerol, 0.2 mM EDTA, 1 mM DTT, 5 mM NaF, 1 mM Na 3 VO 4 and<lb/> protease inhibitor cocktail) followed by freeze thaw extraction. Proteins were<lb/> separated on 6% or 10% SDS–PAGE gels, blotted on PVDF membranes and<lb/> probed with phosphospecific rabbit polyclonal antibodies against ATM Ser 1981<lb/> (Rockland, Gilbertsville, PA), CHK2 Thr 68, p53 Ser 15 or NBS1 Ser 343 (Cell<lb/> Signaling Technology, Beverly, MA). Immunoblots were also probed with<lb/> antibodies against ATM (5C2; Abcam, Cambridge, UK), CHK2 (NT2; Prosci,<lb/> Anaheim, CA), p53 (DO-1; Santa Cruz Biotech, Santa Cruz, CA), NBS1 (NB100-<lb/>143; Abcam), ATR (N-19; Santa Cruz Biotech), DNA-PK (Ab-2; Labvision,<lb/> Fremont, CA) or β-actin antibody (Sigma, St Louis, MO). As a control for viral<lb/> entry and reverse transcription of IN D64V mutant virus vectors, PCRs for the<lb/> presence of HIV-1 vector DNA were performed on total DNA that had been<lb/> extracted from each sample. The amount of HIV-1 viral cDNA (late-RT and<lb/> 2-LTR) in IN + and IN D64V mutant transduced cells was found to be similar <ref type="biblio" >12</ref> (see<lb/> also <ref type="figure">Supplementary Information, Fig. S4</ref>). For radiation-treated samples, sub-<lb/>confluent cells were exposed to either 2 Gy ionizing radiation using a Faxitron<lb/> Corporation (Wheeling, IL) X-ray cabinet, or 20 J m −2 ultraviolet radiation using<lb/> a Stratagene (La Jolla, CA) Stratalinker.<lb/></p> <p>PCR analysis of HIV-1 DNA. PCR analysis of HIV-1 late-RT, 2-LTR and glyc-<lb/>eraldehyde-3-phosphate dehydrogenase (GAPDH) DNA from HIV-1 vec-<lb/>tor-transduced 293 cells has been described previously <ref type="biblio" >12</ref> . Integrated proviral<lb/> HIV-1 DNA was quantified using a nested HIV-1 LTR-Alu PCR. First-round<lb/> LTR-Alu PCRs were performed using 50 ng genomic DNA with HIV-1 LTR<lb/> primer MH535 and Alu primer SB704 <ref type="biblio">(ref. 45)</ref>. We then amplified and quanti-<lb/>fied 1:1,000 of first-round PCR product using HIV-1 LTR nested primer pairs<lb/> LTR6 and LTR9 <ref type="biblio">(ref. 46)</ref>. At this dilution, unintegrated HIV-1 LTR DNA car-<lb/>ryover was not detectable in PCR control reactions or in HIV-1 IN D64V infected<lb/> cells (see <ref type="figure">Supplementary Information, Fig. S4</ref>), ensuring that all detectable LTR<lb/> amplification corresponds to integrated proviral DNA only. All PCRs were<lb/> limited in cycle number to ensure linearity of amplification.<lb/></p> </text> </tei>