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Journal of Virology, March 2005, p. 2973-2978, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.2973-2978.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

DNA Damage Sensors ATM, ATR, DNA-PKcs, and PARP-1 Are Dispensable for Human Immunodeficiency Virus Type 1 Integration

Yasuo Ariumi,¹ Priscilla Turelli,¹ Mitsuko Masutani,² and Didier Trono^1*

Department of Microbiology and Molecular Medicine and "Frontiers in Genetics" Research Program, Faculty of Medicine, University of Geneva, Geneva, Switzerland,¹ Biochemistry Division, National Cancer Center Research Institute, Tokyo, Japan²

Received 13 August 2004/ Accepted 8 October 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Integration of a DNA copy of the viral RNA genome is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses. While the virally encoded integrase is key to this process, cellular factors yet to be characterized are suspected to participate in its completion. DNA damage sensors such as ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related), DNA-PK (DNA-dependent protein kinase), and PARP-1 [poly(ADP-ribose) polymerase 1] play central roles in responses to various forms of DNA injury and as such could facilitate HIV integration. To test this hypothesis, we examined the susceptibility to infection with wild-type HIV-1 and to transduction with a vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1-derived lentiviral vector of human cells stably expressing small interfering RNAs against ATM, ATR, and PARP-1. We found that integration normally occurred in these knockdown cells. Similarly, the VSV-G-pseudotyped HIV-1-based vector could effectively transduce ATM and PARP-1 knockout mouse cells as well as human cells deficient for DNA-PK. Finally, treatment of target cells with the ATM and ATR inhibitors caffeine and wortmannin was without effect in these infectivity assays. We conclude that the DNA repair enzymes ATM, ATR, DNA-PKcs, and PARP-1 are not essential for HIV-1 integration.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Viral DNA integration is an essential step in the retroviral life cycle. Following human immunodeficiency virus type 1 (HIV-1) entry, reverse transcriptase synthesizes a double-stranded DNA copy of the viral genomic RNA. A so-called preintegration complex is formed that contains, in addition to the virus genetic material, the viral proteins integrase (IN), nucleocapsid (NC), virion protein R (VPR), and matrix (MA), as well as host factors such as HMGA1, BAF, INI1, Ku, and LEDGF/p75 (25). The initial DNA cutting and joining steps of the integration process are mediated by the viral integrase, but the final repair of residual DNA gaps must be performed by host factors. In vitro experiments indicate that cellular DNA polymerase and DNA ligase can mediate this process (27). However, which cellular factors are involved in vivo remains undefined. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) was identified as the product of the severe combined immunodeficiency (SCID) gene by genetic complementation (13). DNA-PK is a multimeric nuclear kinase, comprising a 465-kDa catalytic subunit (DNA-PKcs) and a DNA-binding heterodimer of Ku70 and Ku80 (Ku86). DNA-PK is implicated in the regulation of the nonhomologous DNA end-joining (NHEJ) pathway and in V(D)J recombination (13). DNA-PKcs belongs to the phosphatidylinositol 3-kinase-related family, which also includes the ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases, and are involved in cell cycle regulation and DNA repair as well (23). PARP-1 is another nuclear enzyme participating in this type of event, as it catalyzes the transfer of an ADP-ribosyl moiety from its substrate NAD⁺ to various nuclear proteins, including PARP-1 itself, histone H1, DNA topoisomerase, and DNA-dependent protein kinase, in response to DNA double-stranded breaks (1, 6). These DNA damage sensors play central roles in responses to various forms of DNA injury and, as such, could facilitate HIV integration. As well, these molecules could prevent apoptotic events that might be triggered by the exposure of free DNA ends created during reverse transcription and integration, be it at the extremities of the linear viral DNA or within the target cell chromosome. To probe a possible involvement of these cellular proteins in these crucial aspects of HIV-1 replication, we used single-round assays to measure the infectivity of HIV-1 and HIV-1-derived lentiviral vectors in cells rendered defective for DNA damage sensors by RNA interference or gene knockout or by pharmacological inhibition.

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Cell culture. Mouse embryonic fibroblasts (MEFs) derived from PARP-1 wild-type (PARP-1^+/+), PARP-1^–/– (19), ATM wild-type (ATM^+/+), and ATM^–/– mice (26). M059J (DNA-PKcs^–/–), M059K (DNA-PKcs^+/+) (12), 293T, and P4.2 (5) cells were maintained under similar conditions in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum at 37°C. The wild-type J1 and PARP-1-deficient embryonic stem (ES) cells were cultured as described previously (17).

RNA interference. Oligonucleotides with the following sense and antisense sequences were used for the cloning of small hairpin RNA (shRNA)-encoding sequences in lentiviral vectors: ATM, 5'-GATCCCCGGATTTGCGTATTACTCAGTTCAAGAGACTGAGTAATACGCAAATCCTTTTTGGAAA-3' (sense),5'-AGCTTTTCCAAAAAGGATTTGCGTATTACTCAGTCTCTTGAACTGAGTAATACGCAAATCCGGG-3' (antisense); ATR, 5'-GATCCCCGGCGTCGTCTCAGCTCGTCTTCAAGAGAGACGAGCTGAGACGACGCCTTTTTGGAAA-3' (sense), 5'-AGCTTTTCCAAAAAGGCGTCGTCTCAGCTCGTCTCTCTTGAAGACGAGCTGAGACGACGCCGGG-3' (antisense); PARP-1 #1, 5'-GATCCCCGCTCTATCGAGTCGAGTACTTCAAGAGAGTACTCGACTCGATAGAGCTTTTTGGAAA-3' (sense), 5'-AGCTTTTCCAAAAAGCTCTATCGAGTCGAGTACTCTCTTGAAGTACTCGACTCGATAGAGCGGG-3' (antisense); PARP-1 #2, 5'-GATCCCCGAGCGGGCGCGCCTCTTGCTTCAAGAGAGCAAGAGGCGCGCCCGCTCTTTTTGGAAA-3' (sense), 5'-AGCTTTTCCAAAAAGAGCGGGCGCGCCTCTTGCTCTCTTGAAGCAAGAGGCGCGCCCGCTCGGG-3' (antisense); PARP-1 #3, 5'-GATCCCCGGACTCGCTCCGGATGGCCTTCAAGAGAGGCCATCCGGAGCGAGTCCTTTTTGGAAA-3' (sense), 5'-AGCTTTTCCAAAAAGGACTCGCTCCGGATGGCCTCTCTTGAAGGCCATCCGGAGCGAGTCCGGG-3' (antisense); PARP-1 #4, 5'-GATCCCCGCGCTTCTGCACCAACTCATTCAAGAGATGAGTTGGTGCAGAAGCGCTTTTTGGAAA-3' (sense), 5'-AGCTTTTCCAAAAAGCGCTTCTGCACCAACTCATCTCTTGAATGAGTTGGTGCAGAAGCGCGGG-3' (antisense); PARP-1 #5, 5'-GATCCCCGTTGCTGATGGGTAGTACCTTCAAGAGAGGTACTACCCATCAGCAACTTTTTGGAAA-3' (sense), 5'-AGCTTTTCCAAAAAGTTGCTGATGGGTAGTACCTCTCTTGAAGGTACTACCCATCAGCAACGGG-3' (antisense). The oligonucleotides above were annealed and subcloned into the BglII-HindIII site of pSUPER (4). To construct pLVsiRNA against ATM, ATR, and PARP-1 #1 to 5, the BamHI-SalI fragments of the corresponding pSUPER plasmid were subcloned into the BamHI-SalI site of pRDI292 (3).

Constructs, virus production, infection, transduction, and titrations. The wild-type X4 HIV-1 proviral DNA clone (R9) and the vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1-based vector system have been described previously (18). We used the second-generation packaging construct pCMV-R8.91 (28), the envelope plasmid pMDG, and the green fluorescent protein (GFP)-expressing vector pWPTS-GFP. HIV-1 and retroviral vector particles were produced by transient transfection of 293T cells with FuGENE6 (Roche). Titrations were performed using the multinucleate activation of galactosidase indicator assay in CD4⁺ long terminal repeat (LTR)-ß-Gal HeLa-derived P4.2 cells (5) or by GFP fluorescence-activated cell sorter (FACS) analysis with a FACStrak apparatus (Becton Dickinson).

Western blotting. Cells were lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 4 mM EDTA, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Supernatants from these lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting analysis using anti-ATM (NB100-104; GeneTex), anti-ATR (RB-ATR10-UP50; GeneTex), anti-Chk2 (NT; ProSci), anti--tubulin (B-5-1-2; Sigma), or anti-PARP-1 (C2-10; Alexis) antibodies as previously described (1).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Neither ATM nor ATR is essential for HIV-1 integration. To analyze the effect of ATM and ATR on HIV-1 integration efficiency, we first used lentiviral vector-mediated RNA interference to knock down these proteins stably in CD4⁺ LTR-ß-Gal HeLa-derived P4.2 cells (5). shRNA-encoding sequences were cloned downstream from a polymerase III promoter within the context of an HIV-1-derived self-inactivating lentiviral vector, a puromycin marker allowing for the selection of transduced cells (3). Western blotting of lysates demonstrated very effective downregulation of both kinases, either alone or in combination, in cells transduced with lentivectors expressing the corresponding shRNAs (Fig. 1a). Importantly, ATM/ATR knockdown cells exhibited normal growth rates (not illustrated). We thus exposed these and control P4.2 cells to a VSV-G-pseudotyped GFP-expressing HIV-1-derived lentiviral vector and analyzed transduction efficiency by FACS (Fig. 1b). The various P4.2 derivatives were equally susceptible to lentivector-mediated transduction. Moreover, wild-type HIV-1 exhibited similar levels of infectivity in control and knockdown cells (Fig. 1c). As well, the transduction efficiency of the HIV-derived lentiviral vector was comparable in ATM knockout and ATM wild-type MEFs (Fig. 1d) (26). Finally, P4.2 cells were treated with 4 mM caffeine or 4 µM wortmannin, conditions previously shown to inhibit the ATM and ATR kinases (20, 21), before exposure to wild-type HIV-1 or a GFP-expressing lentiviral vector. Neither drug affected HIV-1 infection (Fig. 2a) or transduction (Fig. 2b) efficiency. Of note, under none of the above conditions did we observe significant retrovirus-induced cell death (not illustrated). We conclude that ATM and ATR are not required for efficient HIV-1 integration.

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FIG. 1. Both ATM and ATR are dispensable for HIV-1 integration. (a) Inhibition of ATM and/or ATR expression by shRNA-producing lentiviral vectors. Western blotting of cellular lysates with anti-ATM, anti-ATR, and anti-Chk2 antibodies in ATM- (ATMi), ATR- (ATRi), and double-knockdown (DKD) P4.2 cells as well as in P4.2 cells transduced with a control (C) lentiviral vector results are shown. (b) Transduction efficiency of VSV-G-pseudotyped GFP-expressing HIV-1-derived lentiviral vector in control, ATM, ATR, and DKD cells. Two days after transduction at an MOI of 0.1, transduction efficiency was determined by FACS. For this and other figures, experiments were done in duplicate and columns represent the mean percentage of transduced cells, with mean fluorescence intensity (MFI) indicated below. (c) Single-round wild-type HIV-1 infectivity in the same cells, using an MOI of 0.005. The LTR-LacZ-containing cells produce ß-galactosidase upon Tat production from integrated proviruses, which is revealed by 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining (performed 2 days after infection). (d) Transduction susceptibility of ATM wild-type (+/+) or ATM-knockout (–/–) MEFs, determined by FACS 2 days after exposure to GFP-expressing HIV-derived vector at the indicated MOI.

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FIG. 2. Effect of caffeine and wortmannin on HIV-1 infection. (a) Single-round HIV-1 infectivity in P4.2 cells treated with 4 mM caffeine (dissolved in water) or 4 µM wortmannin (dissolved in dimethyl sulfoxide). Drugs were added 1 h prior to infection, and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining was performed 2 days later. (b) Transduction efficiency of VSV-G-pseudotyped GFP-expressing HIV-1 vector under similar conditions.

DNA-PK is dispensable for HIV-1 integration. To test the effect of DNA-PKcs on HIV-1 integration efficiency, we compared the lentiviral vector susceptibility of a pair of human malignant glioma cell lines, the DNA-PKcs-deficient M059J and its DNA-PKcs-proficient counterpart, M059K (12). At two different multiplicities of infection (MOI), transduction efficiency of a GFP-expressing HIV-derived vector was twice as high in DNA-PKcs-deficient cells compared to control cells, without significant cell death upon infection (Fig. 3 and data not shown). Therefore, the catalytic subunit of DNA-PK is not essential for HIV-1 integration or for protecting cells from retrovirus-induced apoptosis.

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FIG. 3. Transduction efficiency of GFP-expressing VSV-G-pseudotyped HIV-1-derived vector in human DNA-PKcs wild-type (+/+) M059K or DNA-PKcs-deficient (–/–) M059J cells.

PARP-1 is not crucial for HIV-1 integration. To test a possible effect of PARP-1 on HIV-1 integration, we measured the infectivity of the VSV-G-pseudotyped GFP-expressing HIV-1-derived lentiviral vector in PARP-1 knockout and control MEFs (19). At low MOI (0.7), transduction efficiency was strongly decreased in the PARP-1 knockout cells (Fig. 4a). However, at the MOI of 70, the GFP vector transduced up to 50% of these targets (Fig. 4a). Therefore, we used as targets the PARP-1 knockout ES cells that were used to generate the mice from which the MEFs were subsequently derived (226-47 and 210-58), with PARP-1 wild-type ES cells (J1) as a control (17). The HIV-derived vector transduced both types of ES cells equally well, even at low MOI (0.6) (Fig. 4b). To confirm this finding, we established HeLa-derived cells stably knocked downed for PARP-1 by lentivector-mediated RNA interference (Fig. 5a). We then used these cells as targets for HIV-1 infection or transduction with the GFP-expressing HIV-based vector. Consistent with our results in the mouse PARP-1 knockout ES cells, normal levels of HIV-1 susceptibility were recorded in the PARP-1 knockdown human cells (Fig. 5b and c). Taken together, these data indicate that PARP-1 is not a key player in the early steps of HIV-1 infection.

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FIG. 4. (a) Transduction efficiency of HIV-derived GFP-expressing lentiviral vector in PARP-1 wild-type MEF (+/+) and PARP-1 knockout MEF (–/–) cells, using the indicated MOIs. (b) Similar experiment in PARP-1 wild-type (+/+) and PARP-1 knockout (–/–) ES cells.

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FIG. 5. (a) Inhibition of PARP-1 expression by small interfering RNAs. Results shown are from Western blotting of lysates from P4.2 cells transduced with control (C) or anti-PARP-1 shRNA-expressing lentiviral vectors, using antibodies specific for PARP-1 or -tubulin. (b and c) The same cells were then used to score their susceptibility to infection with wild-type HIV-1 (b) and transduction with a GFP-expressing VSV-G-pseudotyped HIV-1-derived vector (c).

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is strongly suspected that host cellular factors participate in HIV-1 integration (25), and it has been proposed that DNA damage sensors might influence this process. Following the initial cutting and joining reaction mediated by the viral integrase, the host cell DNA harbors a partially repaired double-stranded break, the ends of which are held together by single-strand links to the viral DNA. It has been postulated that this integration intermediate elicits a DNA damage response that repairs the gaps and prevents double-stranded break-triggered apoptosis. Consistent with this model, Daniel et al. reported that HIV-1 and avian sarcoma virus integration was impaired in cells transiently expressing a dominant-negative form of ATR, as well as in the presence of the ATM and ATR inhibitors wortmannin and caffeine (8, 9). Since they did not observe a difference in the retrovirus sensitivity of ATM-deficient versus ATM-proficient cells, they concluded that ATR was the enzyme playing an essential role in the early steps of retroviral infection. However, we found here that neither the knockdown of ATM and ATR by RNA interference nor the treatment of target cells with caffeine or wortmannin at concentrations similar to the ones previously used by Daniel et al. had any effect on HIV-1 transduction efficiency or retrovirus-induced cell death. While it could be that RNA interference left low amounts of functional kinases, our drug-based results are difficult to reconcile with Daniel et al.'s observations. Differences in the experimental approaches may account for this discrepancy, but our data strongly suggest that neither ATM nor ATR is crucial for HIV-1 integration or for preventing retrovirus-induced cell death.

Daniel et al. also observed that retrovirus infection induced apoptosis in DNA-PK-deficient scid cells as well as in Ku86- or XRCC4-defective cells. They further reported that an integrase-inactive viral mutant virus failed to trigger this process in scid cells (7, 8, 10). Based on this result, they proposed that the chromosomal breaks induced by retroviral integration could be lethal unless repaired by components of the NHEJ pathway. In the present study, we found that transduction by an HIV-1-derived lentiviral vector was twice as effective in DNA-PKcs-deficient M059J cells as in control M059K cells. This indicates that DNA-PKcs, if anything, may negatively interfere with HIV-1 integration, reminiscent of its recently proposed inhibition of adeno-associated virus integration (24). Baekelandt et al., using DNA-PKcs-deficient scid MEFs as well as Ku-deficient xrs-5 and xrs-6 cells as targets, also demonstrated that DNA-PK is not required for efficient lentivirus integration (2). As well, Bushman and collaborators observed that cells with marked defects in NHEJ, such as Nalm-6 LIG4^–/– cells, exhibited high levels of retrovirus-induced cytotoxicity but that the latter phenomenon was independent from integration. Instead, they found that the formation of 2-LTR viral circles was defective in these cells, suggesting that the proapoptotic signal originated from the accumulation of free viral DNA ends, not from residual gaps in the chromosomal DNA (14, 16). Noteworthy, the 2-LTR circles defect was not apparent in DNA-PKcs-deficient cells, perhaps because this protein is only required for NHEJ of DNA ends with hairpins or long single-stranded overhangs and is not a feature of the final product of viral reverse transcription (16).

Controversy also exists about a possible effect of PARP-1 on HIV-1 integration. Ha et al. (11) found that transduction by an HIV-1 pseudovirus was almost abolished in PARP-1 knockout MEFs. Based on this result, they suggested that PARP-1 is required for efficient HIV-1 integration. Using the same type of target cells, we confirmed their initial observation. However, we investigated further the issue because we knew that our PARP-1 knockout MEFs show extensive hyperploidy compared with PARP-1 wild-type MEF cells (19). We therefore suspected that PARP-1-independent genetic changes might account for their HIV susceptibility phenotype. Confirming this hypothesis, we did not observe any difference in the HIV transduction sensitivity of the parental PARP-1 knockout ES cells. Moreover, HIV-1 infectivity was not affected by the knockdown of PARP-1 in human cells. Consistent with our results, Siva and Bushman showed that PARP-1 is not strictly required for infection of murine cells by retroviruses (22). Moreover, Kameoka et al. demonstrated that HeLa or human lymphoid T cells expressing PARP-1-specific small interfering RNAs fully supported HIV-1 integration, although viral replication was impaired in these targets, owing to a defect in LTR-mediated transcription (15).

While we did not assess a possible role of DNA damage sensors in HIV integration in cells such as primary human T lymphocytes or macrophages, we note that previous claims about the involvement of these proteins in this crucial step of the retroviral life cycle were based on experimental systems comparable to ours. Furthermore, the extensive experience accumulated with HIV-derived vectors in a broad range of targets has so far not disclosed cell-specific requirements for HIV integration cofactors (25).

In sum, our data indicate that the DNA damage sensors ATM, ATR, DNA-PK, and PARP-1 neither are required for efficient HIV-1 integration nor play crucial roles in protecting the cell against retrovirus-induced cell death. Whether these molecules modulate retroviral integration site selection and what other cellular proteins serve as bona fide integrase cofactors remain open questions.

 
We thank B. Mangeat, S. Vianin, and E. Buhlmann for help with the experiments and H. Suzuki, T. Ochiya, O. Niwa, R. Iggo, A. Telenti, and J. Turner for the gift of reagents.

This work was supported by the Swiss National Science Foundation.

 
* Corresponding author. Mailing address: Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, 1211, Geneva 4, Switzerland. Phone: 41-22-379 5720. Fax: 41-22-379 5721. E-mail: Didier.trono{at}medecine.unige.ch.

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Journal of Virology, March 2005, p. 2973-2978, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.2973-2978.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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