1887

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Volume 92, Issue 7

DNA-dependent protein kinase interacts functionally with the RNA polymerase II complex recruited at the human immunodeficiency virus (HIV) long terminal repeat and plays an important role in HIV gene expression Free

Abstract

DNA-dependent protein kinase (DNA-PK), a nuclear protein kinase that specifically requires association with DNA for its kinase activity, plays important roles in the regulation of different DNA transactions, including transcription, replication and DNA repair, as well as in the maintenance of telomeres. Due to its large size, DNA-PK is also known to facilitate the activities of other factors by providing the docking platform at their site of action. In this study, by running several chromatin immunoprecipitation assays, we demonstrate the parallel distribution of DNA-PK with RNA polymerase II (RNAP II) along the human immunodeficiency virus (HIV) provirus before and after activation with tumour necrosis factor alpha. The association between DNA-PK and RNAP II is also long-lasting, at least for up to 4 h (the duration analysed in this study). Knockdown of endogenous DNA-PK using specific small hairpin RNAs expressed from lentiviral vectors resulted in significant reduction in HIV gene expression and replication, demonstrating the importance of DNA-PK for HIV gene expression. Sequence analysis of the HIV-1 Tat protein revealed three potential target sites for phosphorylation by DNA-PK and, by using kinase assays, we confirmed that Tat is an effective substrate of DNA-PK. Through peptide mapping, we found that two of these three potential phosphorylation sites are recognized and phosphorylated by DNA-PK. Mutational studies on the DNA-PK target sites of Tat further demonstrated the functional significance of the Tat–DNA-PK interaction. Thus, overall our results clearly demonstrate the functional interaction between DNA-PK and RNAP II during HIV transcription.

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© 2011 SGM
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2011-07-01
2024-04-20
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References

  1. Ammosova T., Berro R., Jerebtsova M., Jackson A., Charles S., Klase Z., Southerland W., Gordeuk V. R., Kashanchi F., Nekhai S. 2006; Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription. Retrovirology 3:78 [View Article][PubMed]
     [Google Scholar]
  2. An J., Yang T., Huang Y., Liu F., Sun J., Wang Y., Xu Q., Wu D., Zhou P. 2011; Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair. BMC Biochem 12:2 [View Article][PubMed]
     [Google Scholar]
  3. Anderson C. W., Carter T. H. 1996; The DNA-activated protein kinase – DNA-PK. Curr Top Microbiol Immunol 217:91–111[PubMed]
     [Google Scholar]
  4. Anderson C. W., Lees-Miller S. P. 1992; The nuclear serine/threonine protein kinase DNA-PK. Crit Rev Eukaryot Gene Expr 2:283–314[PubMed]
     [Google Scholar]
  5. Arias J. A., Peterson S. R., Dynan W. S. 1991; Promoter-dependent phosphorylation of RNA polymerase II by a template-bound kinase. Association with transcriptional initiation. J Biol Chem 266:8055–8061[PubMed]
     [Google Scholar]
  6. Ariumi Y., Turelli P., Masutani M., Trono D. 2005; DNA damage sensors ATM, ATR, DNA-PKcs, and PARP-1 are dispensable for human immunodeficiency virus type 1 integration. J Virol 79:2973–2978 [View Article][PubMed]
     [Google Scholar]
  7. Baekelandt V., Claeys A., Cherepanov P., De Clercq E., De Strooper B., Nuttin B., Debyser Z. 2000; DNA-dependent protein kinase is not required for efficient lentivirus integration. J Virol 74:11278–11285 [View Article][PubMed]
     [Google Scholar]
  8. Bannister A. J., Gottlieb T. M., Kouzarides T., Jackson S. P. 1993; c-Jun is phosphorylated by the DNA-dependent protein kinase in vitro; definition of the minimal kinase recognition motif. Nucleic Acids Res 21:1289–1295 [View Article][PubMed]
     [Google Scholar]
  9. Blunt T., Finnie N. J., Taccioli G. E., Smith G. C., Demengeot J., Gottlieb T. M., Mizuta R., Varghese A. J., Alt F. W. et al. 1995; Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 80:813–823 [View Article][PubMed]
     [Google Scholar]
  10. Brand S. R., Kobayashi R., Mathews M. B. 1997; The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR. J Biol Chem 272:8388–8395 [View Article][PubMed]
     [Google Scholar]
  11. Carter T., Vancurová I., Sun I., Lou W., DeLeon S. 1990; A DNA-activated protein kinase from HeLa cell nuclei. Mol Cell Biol 10:6460–6471[PubMed]
     [Google Scholar]
  12. Chun R. F., Semmes O. J., Neuveut C., Jeang K. T. 1998; Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat. J Virol 72:2615–2629[PubMed]
     [Google Scholar]
  13. Dahmus M. E. 1995; Phosphorylation of the C-terminal domain of RNA polymerase II. Biochim Biophys Acta 1261:171–182[PubMed] [CrossRef]
     [Google Scholar]
  14. Dahmus M. E. 1996; Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J Biol Chem 271:19009–19012[PubMed] [CrossRef]
     [Google Scholar]
  15. Daniel R., Katz R. A., Skalka A. M. 1999; A role for DNA-PK in retroviral DNA integration. Science 284:644–647 [View Article][PubMed]
     [Google Scholar]
  16. de Vries E., van Driel W., Bergsma W. G., Arnberg A. C., van der Vliet P. C. 1989; HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA–multimeric protein complex. J Mol Biol 208:65–78 [View Article][PubMed]
     [Google Scholar]
  17. Dvir A., Stein L. Y., Calore B. L., Dynan W. S. 1993; Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. J Biol Chem 268:10440–10447[PubMed]
     [Google Scholar]
  18. Dynan W. S., Yoo S. 1998; Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. Nucleic Acids Res 26:1551–1559 [View Article][PubMed]
     [Google Scholar]
  19. Endo-Munoz L., Warby T., Harrich D., McMillan N. A. 2005; Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription. Virol J 2:17 [View Article][PubMed]
     [Google Scholar]
  20. Feinberg M. B., Baltimore D., Frankel A. D. 1991; The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation. Proc Natl Acad Sci U S A 88:4045–4049 [View Article][PubMed]
     [Google Scholar]
  21. Felber B. K., Pavlakis G. N. 1988; A quantitative bioassay for HIV-1 based on trans-activation. Science 239:184–187 [View Article][PubMed]
     [Google Scholar]
  22. Gottlieb T. M., Jackson S. P. 1993; The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72:131–142 [View Article][PubMed]
     [Google Scholar]
  23. Hartley K. O., Gell D., Smith G. C., Zhang H., Divecha N., Connelly M. A., Admon A., Lees-Miller S. P., Anderson C. W., Jackson S. P. 1995; DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell 82:849–856 [View Article][PubMed]
     [Google Scholar]
  24. Hill C. S., Treisman R. 1995; Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80:199–211 [View Article][PubMed]
     [Google Scholar]
  25. Holmes A. M. 1996; In vitro phosphorylation of human immunodeficiency virus type 1 Tat protein by protein kinase C: evidence for the phosphorylation of amino acid residue serine-46. Arch Biochem Biophys 335:8–12 [View Article][PubMed]
     [Google Scholar]
  26. Hunter T. 1995; Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80:225–236 [View Article][PubMed]
     [Google Scholar]
  27. Isel C., Karn J. 1999; Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation. J Mol Biol 290:929–941 [View Article][PubMed]
     [Google Scholar]
  28. Jeanson L., Subra F., Vaganay S., Hervy M., Marangoni E., Bourhis J., Mouscadet J. F. 2002; Effect of Ku80 depletion on the preintegrative steps of HIV-1 replication in human cells. Virology 300:100–108 [View Article][PubMed]
     [Google Scholar]
  29. Kaczmarski W., Khan S. A. 1993; Lupus autoantigen Ku protein binds HIV-1 TAR RNA in vitro . Biochem Biophys Res Commun 196:935–942 [View Article][PubMed]
     [Google Scholar]
  30. Kao S. Y., Calman A. F., Luciw P. A., Peterlin B. M. 1987; Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature 330:489–493 [View Article][PubMed]
     [Google Scholar]
  31. Karn J. 1999; Tackling Tat. J Mol Biol 293:235–254 [View Article][PubMed]
     [Google Scholar]
  32. Kilzer J. M., Stracker T., Beitzel B., Meek K., Weitzman M., Bushman F. D. 2003; Roles of host cell factors in circularization of retroviral DNA. Virology 314:460–467 [View Article][PubMed]
     [Google Scholar]
  33. Kim Y. K., Bourgeois C. F., Isel C., Churcher M. J., Karn J. 2002; Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation. Mol Cell Biol 22:4622–4637 [View Article][PubMed]
     [Google Scholar]
  34. Kim Y. K., Bourgeois C. F., Pearson R., Tyagi M., West M. J., Wong J., Wu S. Y., Chiang C. M., Karn J. 2006; Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency. EMBO J 25:3596–3604 [View Article][PubMed]
     [Google Scholar]
  35. Lees-Miller S. P., Chen Y. R., Anderson C. W. 1990; Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol Cell Biol 10:6472–6481[PubMed]
     [Google Scholar]
  36. Lees-Miller S. P., Godbout R., Chan D. W., Weinfeld M., Day R. S. III, Barron G. M., Allalunis-Turner J. 1995; Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line. Science 267:1183–1185 [View Article][PubMed]
     [Google Scholar]
  37. Li L., Olvera J. M., Yoder K. E., Mitchell R. S., Butler S. L., Lieber M., Martin S. L., Bushman F. D. 2001; Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection. EMBO J 20:3272–3281 [View Article][PubMed]
     [Google Scholar]
  38. Liu H., Herrmann C. H., Chiang K., Sung T. L., Moon S. H., Donehower L. A., Rice A. P. 2010; 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair. Biochem Biophys Res Commun 397:245–250 [View Article][PubMed]
     [Google Scholar]
  39. Majello B., Napolitano G. 2001; Control of RNA polymerase II activity by dedicated CTD kinases and phosphatases. Front Biosci 6:D1358–D1368 [View Article][PubMed]
     [Google Scholar]
  40. Maldonado E., Shiekhattar R., Sheldon M., Cho H., Drapkin R., Rickert P., Lees E., Anderson C. W., Linn S., Reinberg D. 1996; A human RNA polymerase II complex associated with SRB and DNA-repair proteins. Nature 381:86–89 [View Article][PubMed]
     [Google Scholar]
  41. Meek K., Gupta S., Ramsden D. A., Lees-Miller S. P. 2004; The DNA-dependent protein kinase: the director at the end. Immunol Rev 200:132–141 [View Article][PubMed]
     [Google Scholar]
  42. Nunnari G., Argyris E., Fang J., Mehlman K. E., Pomerantz R. J., Daniel R. 2005; Inhibition of HIV-1 replication by caffeine and caffeine-related methylxanthines. Virology 335:177–184 [View Article][PubMed]
     [Google Scholar]
  43. Nussenzweig A., Chen C., da Costa Soares V., Sanchez M., Sokol K., Nussenzweig M. C., Li G. C. 1996; Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382:551–555 [View Article][PubMed]
     [Google Scholar]
  44. O'Brien T., Hardin S., Greenleaf A., Lis J. T. 1994; Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation. Nature 370:75–77 [View Article][PubMed]
     [Google Scholar]
  45. Ochem A. E., Rechreche H., Skopac D., Falaschi A. 2008; Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation. Arch Biochem Biophys 470:1–7 [View Article][PubMed]
     [Google Scholar]
  46. Orphanides G., Lagrange T., Reinberg D. 1996; The general transcription factors of RNA polymerase II. Genes Dev 10:2657–2683 [View Article][PubMed]
     [Google Scholar]
  47. Pearson R., Kim Y. K., Hokello J., Lassen K., Friedman J., Tyagi M., Karn J. 2008; Epigenetic silencing of human immunodeficiency virus (HIV) transcription by formation of restrictive chromatin structures at the viral long terminal repeat drives the progressive entry of HIV into latency. J Virol 82:12291–12303 [View Article][PubMed]
     [Google Scholar]
  48. Price D. H. 2000; P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol Cell Biol 20:2629–2634 [View Article][PubMed]
     [Google Scholar]
  49. Reeves W. H., Sthoeger Z. M. 1989; Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. J Biol Chem 264:5047–5052[PubMed]
     [Google Scholar]
  50. Saunders A., Core L. J., Lis J. T. 2006; Breaking barriers to transcription elongation. Nat Rev Mol Cell Biol 7:557–567 [View Article][PubMed]
     [Google Scholar]
  51. Smith G. C., Jackson S. P. 1999; The DNA-dependent protein kinase. Genes Dev 13:916–934 [View Article][PubMed]
     [Google Scholar]
  52. Taccioli G. E., Gottlieb T. M., Blunt T., Priestley A., Demengeot J., Mizuta R., Lehmann A. R., Alt F. W., Jackson S. P., Jeggo P. A. 1994; Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 265:1442–1445 [View Article][PubMed]
     [Google Scholar]
  53. Trigon S., Serizawa H., Conaway J. W., Conaway R. C., Jackson S. P., Morange M. 1998; Characterization of the residues phosphorylated in vitro by different C-terminal domain kinases. J Biol Chem 273:6769–6775 [View Article][PubMed]
     [Google Scholar]
  54. Tuteja N., Tuteja R., Ochem A., Taneja P., Huang N. W., Simoncsits A., Susic S., Rahman K., Marusic L. et al. 1994; Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen. EMBO J 13:4991–5001[PubMed]
     [Google Scholar]
  55. Tyagi M., Karn J. 2007; CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency. EMBO J 26:4985–4995 [View Article][PubMed]
     [Google Scholar]
  56. Tyagi M., Rusnati M., Presta M., Giacca M. 2001; Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. J Biol Chem 276:3254–3261 [View Article][PubMed]
     [Google Scholar]
  57. Tyagi M., Pearson R. J., Karn J. 2010; Establishment of HIV latency in primary CD4+ cells is due to epigenetic transcriptional silencing and P-TEFb restriction. J Virol 84:6425–6437 [View Article][PubMed]
     [Google Scholar]
  58. Yamamoto S., Watanabe Y., van der Spek P. J., Watanabe T., Fujimoto H., Hanaoka F., Ohkuma Y. 2001; Studies of nematode TFIIE function reveal a link between Ser-5 phosphorylation of RNA polymerase II and the transition from transcription initiation to elongation. Mol Cell Biol 21:1–15 [View Article][PubMed]
     [Google Scholar]
  59. Yaneva M., Wen J., Ayala A., Cook R. 1989; cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen. J Biol Chem 264:13407–13411[PubMed]
     [Google Scholar]
  60. Yoo S., Dynan W. S. 1998; Characterization of the RNA binding properties of Ku protein. Biochemistry 37:1336–1343 [View Article][PubMed]
     [Google Scholar]
  61. Zhang J., Corden J. L. 1991; Identification of phosphorylation sites in the repetitive carboxyl-terminal domain of the mouse RNA polymerase II largest subunit. J Biol Chem 266:2290–2296[PubMed]
     [Google Scholar]
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DNA-dependent protein kinase interacts functionally with the RNA polymerase II complex recruited at the human immunodeficiency virus (HIV) long terminal repeat and plays an important role in HIV gene expression
J. Gen. Virol. 92, 1710 (2011); https://doi.org/10.1099/vir.0.029587-0
/content/journal/jgv/10.1099/vir.0.029587-0
/content/journal/jgv/10.1099/vir.0.029587-0
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