Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Sep;13(9):5245–5254. doi: 10.1128/mcb.13.9.5245

Inducible transcriptional activation of the human immunodeficiency virus long terminal repeat by protein kinase inhibitors.

F L Brown 1, E Tahaoglu 1, G J Graham 1, J J Maio 1
PMCID: PMC360213  PMID: 8355680

Abstract

The protein kinase inhibitor 2-aminopurine (2-AP) greatly stimulated expression in human promonocytes-macrophages of plasmid constructs carrying various reporter genes (chloramphenicol acetyltransferase, lacZ, firefly luciferase [luc], and Salmonella typhimurium histidinol dehydrogenase [his]) driven by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. Adenine, adenosine, and caffeine were also effective inducers, but other purine or pyrimidine derivatives were ineffective. Experiments with mutant derivatives of the HIV-1 long terminal repeat revealed no specific eukaryotic promoter elements necessary for 2-AP induction but indicated the need for some minimum combination of such elements. Induction of HIV-1-directed gene expression appeared not to require action of the transcription factor NF-kappa B. The mechanism of induction was investigated by using the luc and his genes linked to the HIV-1 long terminal repeat. 2-AP induced marked, steady rises in mRNA accumulation from both transfected and chromosomally integrated HIV-1 constructs but no increases from an endogenous gene encoding gamma-actin or glucose 6-phosphate dehydrogenase. Thus, induction is selective and not an artifact induced by transfecting DNA into cells. In run-on transcription experiments, the rates of transcription initiation of both transfected and integrated copies of the his gene increased about sixfold in cells treated with 2-AP. Thus, while increased initiation accounted for a portion of 2-AP induction, it could not cause the far greater increase in steady-state mRNA levels. 2-AP induction did not change mRNA decay rates and differed from the phorbol ester (phorbol myristate acetate)-induced activation of the protein kinase C-NF-kappa B pathway in its time course and in its requirement for new protein synthesis. Gel retardation assays showed that unlike phorbol myristate acetate induction, 2-AP induction is enhancer independent. Whereas many previous studies have implicated the activation of various protein kinases in gene induction, we here describe a mechanism of gene activation that appears to involve protein kinase inhibition as a component of the induction response.

Full text

PDF
5245

Images in this article

5250Image
on p.5250
5250Image
on p.5250
5251Image
on p.5251
5251Image
on p.5251
5252Image
on p.5252

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akusjärvi G., Svensson C., Nygård O. A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay. Mol Cell Biol. 1987 Jan;7(1):549–551. doi: 10.1128/mcb.7.1.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andreassen P. R., Margolis R. L. 2-Aminopurine overrides multiple cell cycle checkpoints in BHK cells. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2272–2276. doi: 10.1073/pnas.89.6.2272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berkhout B., Gatignol A., Rabson A. B., Jeang K. T. TAR-independent activation of the HIV-1 LTR: evidence that tat requires specific regions of the promoter. Cell. 1990 Aug 24;62(4):757–767. doi: 10.1016/0092-8674(90)90120-4. [DOI] [PubMed] [Google Scholar]
  4. Brasier A. R., Tate J. E., Habener J. F. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques. 1989 Nov-Dec;7(10):1116–1122. [PubMed] [Google Scholar]
  5. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fields A. P., Bednarik D. P., Hess A., May W. S. Human immunodeficiency virus induces phosphorylation of its cell surface receptor. Nature. 1988 May 19;333(6170):278–280. doi: 10.1038/333278a0. [DOI] [PubMed] [Google Scholar]
  7. Goldfeld A. E., Maniatis T. Coordinate viral induction of tumor necrosis factor alpha and interferon beta in human B cells and monocytes. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1490–1494. doi: 10.1073/pnas.86.5.1490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Graham G. J., Maio J. J. RNA transcripts of the human immunodeficiency virus transactivation response element can inhibit action of the viral transactivator. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5817–5821. doi: 10.1073/pnas.87.15.5817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gunning P., Ponte P., Okayama H., Engel J., Blau H., Kedes L. Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol Cell Biol. 1983 May;3(5):787–795. doi: 10.1128/mcb.3.5.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guy B., Kieny M. P., Riviere Y., Le Peuch C., Dott K., Girard M., Montagnier L., Lecocq J. P. HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature. 1987 Nov 19;330(6145):266–269. doi: 10.1038/330266a0. [DOI] [PubMed] [Google Scholar]
  11. Harada S., Koyanagi Y., Nakashima H., Kobayashi N., Yamamoto N. Tumor promoter, TPA, enhances replication of HTLV-III/LAV. Virology. 1986 Oct 30;154(2):249–258. doi: 10.1016/0042-6822(86)90451-4. [DOI] [PubMed] [Google Scholar]
  12. Hartman S. C., Mulligan R. C. Two dominant-acting selectable markers for gene transfer studies in mammalian cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8047–8051. doi: 10.1073/pnas.85.21.8047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hauber J., Bouvier M., Malim M. H., Cullen B. R. Phosphorylation of the rev gene product of human immunodeficiency virus type 1. J Virol. 1988 Dec;62(12):4801–4804. doi: 10.1128/jvi.62.12.4801-4804.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Huang J. T., Schneider R. J. Adenovirus inhibition of cellular protein synthesis is prevented by the drug 2-aminopurine. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7115–7119. doi: 10.1073/pnas.87.18.7115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hunter T., Karin M. The regulation of transcription by phosphorylation. Cell. 1992 Aug 7;70(3):375–387. doi: 10.1016/0092-8674(92)90162-6. [DOI] [PubMed] [Google Scholar]
  16. Jakobovits A., Rosenthal A., Capon D. J. Trans-activation of HIV-1 LTR-directed gene expression by tat requires protein kinase C. EMBO J. 1990 Apr;9(4):1165–1170. doi: 10.1002/j.1460-2075.1990.tb08223.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kalvakolanu D. V., Bandyopadhyay S. K., Tiwari R. K., Sen G. C. Enhancement of expression of exogenous genes by 2-aminopurine. Regulation at the post-transcriptional level. J Biol Chem. 1991 Jan 15;266(2):873–879. [PubMed] [Google Scholar]
  18. Kaufman R. J., Davies M. V., Pathak V. K., Hershey J. W. The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs. Mol Cell Biol. 1989 Mar;9(3):946–958. doi: 10.1128/mcb.9.3.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kaufman R. J., Murtha P. Translational control mediated by eucaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells. Mol Cell Biol. 1987 Apr;7(4):1568–1571. doi: 10.1128/mcb.7.4.1568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laurence J., Sikder S. K., Jhaveri S., Salmon J. E. Phorbol ester-mediated induction of HIV-1 from a chronically infected promonocyte clone: blockade by protein kinase inhibitors and relationship to tat-directed trans-activation. Biochem Biophys Res Commun. 1990 Jan 15;166(1):349–357. doi: 10.1016/0006-291x(90)91952-o. [DOI] [PubMed] [Google Scholar]
  21. Legon S., Brayley A., Hunt T., Jackson R. J. The effect of cyclic AMP and related compounds on the control of protein synthesis in reticulocyte lysates. Biochem Biophys Res Commun. 1974 Feb 4;56(3):745–752. doi: 10.1016/0006-291x(74)90668-8. [DOI] [PubMed] [Google Scholar]
  22. Mahadevan L. C., Wills A. J., Hirst E. A., Rathjen P. D., Heath J. K. 2-Aminopurine abolishes EGF- and TPA-stimulated pp33 phosphorylation and c-fos induction without affecting the activation of protein kinase C. Oncogene. 1990 Mar;5(3):327–335. [PubMed] [Google Scholar]
  23. Maio J. J., Brown F. L. Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity. J Cell Physiol. 1991 Dec;149(3):548–559. doi: 10.1002/jcp.1041490326. [DOI] [PubMed] [Google Scholar]
  24. Maio J. J., Brown F. L. Regulation of expression driven by human immunodeficiency virus type 1 and human T-cell leukemia virus type I long terminal repeats in pluripotential human embryonic cells. J Virol. 1988 Apr;62(4):1398–1407. doi: 10.1128/jvi.62.4.1398-1407.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marcus P. I., Sekellick M. J. Interferon induction by viruses. XVI. 2-Aminopurine blocks selectively and reversibly an early stage in interferon induction. J Gen Virol. 1988 Jul;69(Pt 7):1637–1645. doi: 10.1099/0022-1317-69-7-1637. [DOI] [PubMed] [Google Scholar]
  26. Nabel G. J., Rice S. A., Knipe D. M., Baltimore D. Alternative mechanisms for activation of human immunodeficiency virus enhancer in T cells. Science. 1988 Mar 11;239(4845):1299–1302. doi: 10.1126/science.2830675. [DOI] [PubMed] [Google Scholar]
  27. Nabel G., Baltimore D. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature. 1987 Apr 16;326(6114):711–713. doi: 10.1038/326711a0. [DOI] [PubMed] [Google Scholar]
  28. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  29. Parker P. J., Kour G., Marais R. M., Mitchell F., Pears C., Schaap D., Stabel S., Webster C. Protein kinase C--a family affair. Mol Cell Endocrinol. 1989 Aug;65(1-2):1–11. doi: 10.1016/0303-7207(89)90159-7. [DOI] [PubMed] [Google Scholar]
  30. Rosenberg Z. F., Fauci A. S. Immunopathogenesis of HIV infection. FASEB J. 1991 Jul;5(10):2382–2390. doi: 10.1096/fasebj.5.10.1676689. [DOI] [PubMed] [Google Scholar]
  31. Schlegel R., Belinsky G. S., Harris M. O. Premature mitosis induced in mammalian cells by the protein kinase inhibitors 2-aminopurine and 6-dimethylaminopurine. Cell Growth Differ. 1990 Apr;1(4):171–178. [PubMed] [Google Scholar]
  32. Schlegel R., Pardee A. B. Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science. 1986 Jun 6;232(4755):1264–1266. doi: 10.1126/science.2422760. [DOI] [PubMed] [Google Scholar]
  33. Schneider R. J., Shenk T. Impact of virus infection on host cell protein synthesis. Annu Rev Biochem. 1987;56:317–332. doi: 10.1146/annurev.bi.56.070187.001533. [DOI] [PubMed] [Google Scholar]
  34. Schultz M. C., Choe S. Y., Reeder R. H. Specific initiation by RNA polymerase I in a whole-cell extract from yeast. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1004–1008. doi: 10.1073/pnas.88.3.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sen R., Baltimore D. Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism. Cell. 1986 Dec 26;47(6):921–928. doi: 10.1016/0092-8674(86)90807-x. [DOI] [PubMed] [Google Scholar]
  36. Shafit-Zagardo B., Brown F. L., Zavodny P. J., Maio J. J. Transcription of the KpnI families of long interspersed DNAs in human cells. Nature. 1983 Jul 21;304(5923):277–280. doi: 10.1038/304277a0. [DOI] [PubMed] [Google Scholar]
  37. Sodroski J., Rosen C., Wong-Staal F., Salahuddin S. Z., Popovic M., Arya S., Gallo R. C., Haseltine W. A. Trans-acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat. Science. 1985 Jan 11;227(4683):171–173. doi: 10.1126/science.2981427. [DOI] [PubMed] [Google Scholar]
  38. Steffy K., Wong-Staal F. Genetic regulation of human immunodeficiency virus. Microbiol Rev. 1991 Jun;55(2):193–205. doi: 10.1128/mr.55.2.193-205.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Steinmann K. E., Belinsky G. S., Lee D., Schlegel R. Chemically induced premature mitosis: differential response in rodent and human cells and the relationship to cyclin B synthesis and p34cdc2/cyclin B complex formation. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6843–6847. doi: 10.1073/pnas.88.15.6843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tiwari R. K., Kusari J., Kumar R., Sen G. C. Gene induction by interferons and double-stranded RNA: selective inhibition by 2-aminopurine. Mol Cell Biol. 1988 Oct;8(10):4289–4294. doi: 10.1128/mcb.8.10.4289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Usheva A., Maldonado E., Goldring A., Lu H., Houbavi C., Reinberg D., Aloni Y. Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein. Cell. 1992 May 29;69(5):871–881. doi: 10.1016/0092-8674(92)90297-p. [DOI] [PubMed] [Google Scholar]
  42. Vaishnav Y. N., Wong-Staal F. The biochemistry of AIDS. Annu Rev Biochem. 1991;60:577–630. doi: 10.1146/annurev.bi.60.070191.003045. [DOI] [PubMed] [Google Scholar]
  43. Zinn K., Keller A., Whittemore L. A., Maniatis T. 2-Aminopurine selectively inhibits the induction of beta-interferon, c-fos, and c-myc gene expression. Science. 1988 Apr 8;240(4849):210–213. doi: 10.1126/science.3281258. [DOI] [PubMed] [Google Scholar]
  44. van Zonneveld A. J., Curriden S. A., Loskutoff D. J. Type 1 plasminogen activator inhibitor gene: functional analysis and glucocorticoid regulation of its promoter. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5525–5529. doi: 10.1073/pnas.85.15.5525. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES