Human immunodeficiency virus-1 (HIV-1) has infected more than 60 million people and caused nearly 30 million deaths worldwide1, ultimately the consequence of cytolytic infection of CD4+ T cells. In humans and in macaque models, most of these cells contain viral DNA and are rapidly eliminated at the peak of viraemia2,3,4, yet the mechanism by which HIV-1 induces helper T-cell death has not been defined. Here we show that virus-induced cell killing is triggered by viral integration. Infection by wild-type HIV-1, but not an integrase-deficient mutant, induced the death of activated primary CD4 lymphocytes. Similarly, raltegravir, a pharmacologic integrase inhibitor, abolished HIV-1-induced cell killing both in cell culture and in CD4+ T cells from acutely infected subjects. The mechanism of killing during viral integration involved the activation of DNA-dependent protein kinase (DNA-PK), a central integrator of the DNA damage response, which caused phosphorylation of p53 and histone H2AX. Pharmacological inhibition of DNA-PK abolished cell death during HIV-1 infection in vitro, suggesting that processes which reduce DNA-PK activation in CD4 cells could facilitate the formation of latently infected cells that give rise to reservoirs in vivo. We propose that activation of DNA-PK during viral integration has a central role in CD4+ T-cell depletion, raising the possibility that integrase inhibitors and interventions directed towards DNA-PK may improve T-cell survival and immune function in infected individuals.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Joint. United Nations Programme on HIV/AIDS. Global Report fact sheet: The global AIDS epidemic http://www.unaids.org/documents/20101123_FS_Global_em_en.pdf (2010)
Brenchley, J. M. et al. CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J. Exp. Med. 200, 749–759 (2004)
Mattapallil, J. J. et al. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 434, 1093–1097 (2005)
Nishimura, Y. et al. Resting naive CD4+ T cells are massively infected and eliminated by X4-tropic simian-human immunodeficiency viruses in macaques. Proc. Natl Acad. Sci. USA 102, 8000–8005 (2005)
Aiken, C. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A. J. Virol. 71, 5871–5877 (1997)
Butler, S. L., Hansen, M. S. & Bushman, F. D. A quantitative assay for HIV DNA integration in vivo. Nature Med. 7, 631–634 (2001)
Leavitt, A. D., Robles, G., Alesandro, N. & Varmus, H. E. Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. J. Virol. 70, 721–728 (1996)
Li, L. et al. Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection. EMBO J. 20, 3272–3281 (2001)
Chen, H., Boyle, T. J., Malim, M. H., Cullen, B. R. & Lyerly, H. K. Derivation of a biologically contained replication system for human immunodeficiency virus type 1. Proc. Natl Acad. Sci. USA 89, 7678–7682 (1992)
Doitsh, G. et al. Abortive HIV infection mediates CD4 T cell depletion and inflammation in human lymphoid tissue. Cell 143, 789–801 (2010)
Daniel, R., Katz, R. A. & Skalka, A. M. A role for DNA-PK in retroviral DNA integration. Science 284, 644–647 (1999)
Baekelandt, V. et al. DNA-dependent protein kinase is not required for efficient lentivirus integration. J. Virol. 74, 11278–11285 (2000)
Daniel, R. et al. Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces the efficiency of stable transduction by retroviruses. Mol. Cell. Biol. 21, 1164–1172 (2001)
Ariumi, Y., Turelli, P., Masutani, M. & Trono, D. DNA damage sensors ATM, ATR, DNA-PKcs, and PARP-1 are dispensable for human immunodeficiency virus type 1 integration. J. Virol. 79, 2973–2978 (2005)
Callén, E. et al. Essential role for DNA-PKcs in DNA double-strand break repair and apoptosis in ATM-deficient lymphocytes. Mol. Cell 34, 285–297 (2009)
Engelman, A., Mizuuchi, K. & Craigie, R. HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell 67, 1211–1221 (1991)
Morozov, V. E., Falzon, M., Anderson, C. W. & Kuff, E. L. DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps. J. Biol. Chem. 269, 16684–16688 (1994)
Lau, A., Kanaar, R., Jackson, S. P. & O'Connor, M. J. Suppression of retroviral infection by the RAD52 DNA repair protein. EMBO J. 23, 3421–3429 (2004)
Hill, R. & Lee, P. W. The DNA-dependent protein kinase (DNA-PK): More than just a case of making ends meet? Cell Cycle 9, 3460–3469 (2010)
Nilsson, A., Sirzen, F., Lewensohn, R., Wang, N. & Skog, S. Cell cycle-dependent regulation of the DNA-dependent protein kinase. Cell Prolif. 32, 239–248 (1999)
Chen, B. P. et al. Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA double strand breaks. J. Biol. Chem. 280, 14709–14715 (2005)
Nagasawa, M. et al. Nuclear translocation of the catalytic component of DNA-dependent protein kinase upon growth stimulation in normal human T lymphocytes. Cell Struct. Funct. 22, 585–594 (1997)
Perelson, A. S., Neumann, A. U., Markowitz, M., Leonard, J. M. & Ho, D. D. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 271, 1582–1586 (1996)
Igarashi, T. et al. Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): Implications for HIV-1 infections of humans. Proc. Natl Acad. Sci. USA 98, 658–663 (2001)
Shan, L. et al. Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation. Immunity 36, 491–501 (2012)
Ho, D. D. et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373, 123–126 (1995)
Wei, X. et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 373, 117–122 (1995)
Martínez, E. et al. Changes in cardiovascular biomarkers in HIV-infected patients switching from ritonavir-boosted protease inhibitors to raltegravir. AIDS 26, 2315–2326 (2012)
Collman, R. et al. An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. J. Virol. 66, 7517–7521 (1992)
Adachi, A. et al. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59, 284–291 (1986)
Bolton, D. L. & Lenardo, M. J. Vpr cytopathicity independent of G2/M cell cycle arrest in human immunodeficiency virus type 1-infected CD4+ T cells. J. Virol. 81, 8878–8890 (2007)
Yamamoto, T. et al. Selective transmission of R5 HIV-1 over X4 HIV-1 at the dendritic cell-T cell infectious synapse is determined by the T cell activation state. PLoS Pathog. 5, e1000279 (2009)
O'Doherty, U., Swiggard, W. J. & Malim, M. H. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J. Virol. 74, 10074–10080 (2000)
Willmore, E. et al. A novel DNA-dependent protein kinase inhibitor, NU7026, potentiates the cytotoxicity of topoisomerase II poisons used in the treatment of leukemia. Blood 103, 4659–4665 (2004)
Leahy, J. J. et al. Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg. Med. Chem. Lett. 14, 6083–6087 (2004)
Komarov, P. G. et al. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 285, 1733–1737 (1999)
Suzuki, Y. et al. Quantitative analysis of human immunodeficiency virus type 1 DNA dynamics by real-time PCR: integration efficiency in stimulated and unstimulated peripheral blood mononuclear cells. Virus Genes 27, 177–188 (2003)
Li, Y. et al. Complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation. J. Virol. 66, 6587–6600 (1992)
O'Doherty, U., Swiggard, W. J., Jeyakumar, D., McGain, D. & Malim, M. H. A sensitive, quantitative assay for human immunodeficiency virus type 1 integration. J. Virol. 76, 10942–10950 (2002)
We thank D. Ambrozak, R. Nguyen, and S. Perfetto for help with cell sorting, U. Olshevsky, J. Casazza, D. Bolton, A. Pegu and M. Louder for discussions and technical help, and A. Tislerics and B. Hartman for manuscript preparation. This research was supported by the Intramural Research Program of the Vaccine Research Center, NIAID, National Institutes of Health. The findings and conclusions in this report are those of the authors and do not necessarily reflect the views of the funding agency.
The authors declare no competing financial interests.
This file contains Supplementary Figures 1-4 and Supplementary References. These figures show that HIV-1 induced cell death depends on provirus integration and is observed in p24- CD4+ lymphocytes from healthy donors infected in vitro and patient samples ex vivo. Blocking death by DNA-PK inhibitors does not result from diminished viral replication or general resistance to cell death. (PDF 621 kb)
About this article
Cite this article
Cooper, A., García, M., Petrovas, C. et al. HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration. Nature 498, 376–379 (2013). https://doi.org/10.1038/nature12274
Gene Therapy (2021)
Integrase-derived peptides together with CD24-targeted lentiviral particles inhibit the growth of CD24 expressing cancer cells
Nature Microbiology (2020)
Nature Communications (2020)
Cell Death Discovery (2020)