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. 1993 Jul;67(7):3739–3747. doi: 10.1128/jvi.67.7.3739-3747.1993

The full-length Tat protein is required for TAR-independent, posttranscriptional trans activation of human immunodeficiency virus type 1 env gene expression.

Y S Kim 1, A T Panganiban 1
PMCID: PMC237737  PMID: 8510203

Abstract

Tat is a protein that dramatically increases the expression of all genes expressed from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat through interaction with a cis-acting target sequence referred to as TAR (for trans-acting responsive region). The tat gene is divided into two coding exons which, when translated, result in the synthesis of an 86-amino-acid protein. However, the 72-amino-acid segment encoded by the first coding exon of tat is sufficient to encode a fully active Tat protein in known assays. We examined expression of the env gene from an LTR that lacks TAR (designated dTAR-env). Surprisingly, only the full-length Tat peptide trans activated expression of the env gene from dTAR-env. Comparison of RNA and protein expression of the env gene in the presence of Tat indicated that the mechanism of trans activation is posttranscriptional rather than transcriptional. To test whether the TAR-independent Tat function is specific to the HIV-1 env gene, we analyzed expression of heterologous genes from the long terminal repeat lacking TAR. These heterologous genes were not trans activated by Tat in the absence of a TAR element, which suggests that the second-exon peptide of Tat has a sequence-specific role in TAR-independent trans activation of the HIV-1 env gene. Analysis of a mutant in the 5' end of the env gene was used to identify a cis-acting sequence required for Tat responsiveness.

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Selected References

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  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arya S. K., Guo C., Josephs S. F., Wong-Staal F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science. 1985 Jul 5;229(4708):69–73. doi: 10.1126/science.2990040. [DOI] [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. Braddock M., Thorburn A. M., Chambers A., Elliott G. D., Anderson G. J., Kingsman A. J., Kingsman S. M. A nuclear translational block imposed by the HIV-1 U3 region is relieved by the Tat-TAR interaction. Cell. 1990 Sep 21;62(6):1123–1133. doi: 10.1016/0092-8674(90)90389-v. [DOI] [PubMed] [Google Scholar]
  5. Braddock M., Thorburn A. M., Kingsman A. J., Kingsman S. M. Blocking of Tat-dependent HIV-1 RNA modification by an inhibitor of RNA polymerase II processivity. Nature. 1991 Apr 4;350(6317):439–441. doi: 10.1038/350439a0. [DOI] [PubMed] [Google Scholar]
  6. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  7. Cullen B. R. Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism. Cell. 1986 Sep 26;46(7):973–982. doi: 10.1016/0092-8674(86)90696-3. [DOI] [PubMed] [Google Scholar]
  8. Dayton A. I., Sodroski J. G., Rosen C. A., Goh W. C., Haseltine W. A. The trans-activator gene of the human T cell lymphotropic virus type III is required for replication. Cell. 1986 Mar 28;44(6):941–947. doi: 10.1016/0092-8674(86)90017-6. [DOI] [PubMed] [Google Scholar]
  9. Desai K., Loewenstein P. M., Green M. Isolation of a cellular protein that binds to the human immunodeficiency virus Tat protein and can potentiate transactivation of the viral promoter. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8875–8879. doi: 10.1073/pnas.88.20.8875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dingwall C., Ernberg I., Gait M. J., Green S. M., Heaphy S., Karn J., Lowe A. D., Singh M., Skinner M. A., Valerio R. Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6925–6929. doi: 10.1073/pnas.86.18.6925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ensoli B., Barillari G., Salahuddin S. Z., Gallo R. C., Wong-Staal F. Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients. Nature. 1990 May 3;345(6270):84–86. doi: 10.1038/345084a0. [DOI] [PubMed] [Google Scholar]
  12. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  13. Feinberg M. B., Jarrett R. F., Aldovini A., Gallo R. C., Wong-Staal F. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell. 1986 Sep 12;46(6):807–817. doi: 10.1016/0092-8674(86)90062-0. [DOI] [PubMed] [Google Scholar]
  14. Feng S., Holland E. C. HIV-1 tat trans-activation requires the loop sequence within tar. Nature. 1988 Jul 14;334(6178):165–167. doi: 10.1038/334165a0. [DOI] [PubMed] [Google Scholar]
  15. Fisher A. G., Feinberg M. B., Josephs S. F., Harper M. E., Marselle L. M., Reyes G., Gonda M. A., Aldovini A., Debouk C., Gallo R. C. The trans-activator gene of HTLV-III is essential for virus replication. 1986 Mar 27-Apr 2Nature. 320(6060):367–371. doi: 10.1038/320367a0. [DOI] [PubMed] [Google Scholar]
  16. Garcia J. A., Harrich D., Soultanakis E., Wu F., Mitsuyasu R., Gaynor R. B. Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation. EMBO J. 1989 Mar;8(3):765–778. doi: 10.1002/j.1460-2075.1989.tb03437.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  18. Gorman C. M., Merlino G. T., Willingham M. C., Pastan I., Howard B. H. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. doi: 10.1073/pnas.79.22.6777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  20. Harrich D., Garcia J., Mitsuyasu R., Gaynor R. TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes. EMBO J. 1990 Dec;9(13):4417–4423. doi: 10.1002/j.1460-2075.1990.tb07892.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hauber J., Cullen B. R. Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type I long terminal repeat. J Virol. 1988 Mar;62(3):673–679. doi: 10.1128/jvi.62.3.673-679.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hauber J., Perkins A., Heimer E. P., Cullen B. R. Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6364–6368. doi: 10.1073/pnas.84.18.6364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hopkins N., Besmer P., DeLeo A. B., Law L. W. High-frequency cotransfer of the transformed phenotype and a tumor-specific transplantation antigen by DNA from the 3-methylcholanthrene-induced Meth A sarcoma of BALB/c mice. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7555–7559. doi: 10.1073/pnas.78.12.7555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jakobovits A., Smith D. H., Jakobovits E. B., Capon D. J. A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator. Mol Cell Biol. 1988 Jun;8(6):2555–2561. doi: 10.1128/mcb.8.6.2555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim Y. S., Risser R. TAR-independent transactivation of the murine cytomegalovirus major immediate-early promoter by the Tat protein. J Virol. 1993 Jan;67(1):239–248. doi: 10.1128/jvi.67.1.239-248.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kimpton J., Emerman M. Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. J Virol. 1992 Apr;66(4):2232–2239. doi: 10.1128/jvi.66.4.2232-2239.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laspia M. F., Rice A. P., Mathews M. B. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell. 1989 Oct 20;59(2):283–292. doi: 10.1016/0092-8674(89)90290-0. [DOI] [PubMed] [Google Scholar]
  28. Laspia M. F., Rice A. P., Mathews M. B. Synergy between HIV-1 Tat and adenovirus E1A is principally due to stabilization of transcriptional elongation. Genes Dev. 1990 Dec;4(12B):2397–2408. doi: 10.1101/gad.4.12b.2397. [DOI] [PubMed] [Google Scholar]
  29. Maddon P. J., Dalgleish A. G., McDougal J. S., Clapham P. R., Weiss R. A., Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell. 1986 Nov 7;47(3):333–348. doi: 10.1016/0092-8674(86)90590-8. [DOI] [PubMed] [Google Scholar]
  30. Maniatis T., Goodbourn S., Fischer J. A. Regulation of inducible and tissue-specific gene expression. Science. 1987 Jun 5;236(4806):1237–1245. doi: 10.1126/science.3296191. [DOI] [PubMed] [Google Scholar]
  31. Marciniak R. A., Calnan B. J., Frankel A. D., Sharp P. A. HIV-1 Tat protein trans-activates transcription in vitro. Cell. 1990 Nov 16;63(4):791–802. doi: 10.1016/0092-8674(90)90145-5. [DOI] [PubMed] [Google Scholar]
  32. Marciniak R. A., Garcia-Blanco M. A., Sharp P. A. Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1990 May;87(9):3624–3628. doi: 10.1073/pnas.87.9.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Muesing M. A., Smith D. H., Capon D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987 Feb 27;48(4):691–701. doi: 10.1016/0092-8674(87)90247-9. [DOI] [PubMed] [Google Scholar]
  34. Nelbock P., Dillon P. J., Perkins A., Rosen C. A. A cDNA for a protein that interacts with the human immunodeficiency virus Tat transactivator. Science. 1990 Jun 29;248(4963):1650–1653. doi: 10.1126/science.2194290. [DOI] [PubMed] [Google Scholar]
  35. Peterlin B. M., Luciw P. A., Barr P. J., Walker M. D. Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9734–9738. doi: 10.1073/pnas.83.24.9734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Puri R. K., Aggarwal B. B. Human immunodeficiency virus type 1 tat gene up-regulates interleukin 4 receptors on a human B-lymphoblastoid cell line. Cancer Res. 1992 Jul 1;52(13):3787–3790. [PubMed] [Google Scholar]
  37. Rappaport J., Lee S. J., Khalili K., Wong-Staal F. The acidic amino-terminal region of the HIV-1 Tat protein constitutes an essential activating domain. New Biol. 1989 Oct;1(1):101–110. [PubMed] [Google Scholar]
  38. Rosen C. A., Sodroski J. G., Goh W. C., Dayton A. I., Lippke J., Haseltine W. A. Post-transcriptional regulation accounts for the trans-activation of the human T-lymphotropic virus type III. Nature. 1986 Feb 13;319(6054):555–559. doi: 10.1038/319555a0. [DOI] [PubMed] [Google Scholar]
  39. Rosen C. A., Sodroski J. G., Haseltine W. A. The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell. 1985 Jul;41(3):813–823. doi: 10.1016/s0092-8674(85)80062-3. [DOI] [PubMed] [Google Scholar]
  40. Roy S., Delling U., Chen C. H., Rosen C. A., Sonenberg N. A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation. Genes Dev. 1990 Aug;4(8):1365–1373. doi: 10.1101/gad.4.8.1365. [DOI] [PubMed] [Google Scholar]
  41. Ruben S., Perkins A., Purcell R., Joung K., Sia R., Burghoff R., Haseltine W. A., Rosen C. A. Structural and functional characterization of human immunodeficiency virus tat protein. J Virol. 1989 Jan;63(1):1–8. doi: 10.1128/jvi.63.1.1-8.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sastry K. J., Reddy H. R., Pandita R., Totpal K., Aggarwal B. B. HIV-1 tat gene induces tumor necrosis factor-beta (lymphotoxin) in a human B-lymphoblastoid cell line. J Biol Chem. 1990 Nov 25;265(33):20091–20093. [PubMed] [Google Scholar]
  43. Seigel L. J., Ratner L., Josephs S. F., Derse D., Feinberg M. B., Reyes G. R., O'Brien S. J., Wong-Staal F. Transactivation induced by human T-lymphotropic virus type III (HTLV III) maps to a viral sequence encoding 58 amino acids and lacks tissue specificity. Virology. 1986 Jan 15;148(1):226–231. doi: 10.1016/0042-6822(86)90419-8. [DOI] [PubMed] [Google Scholar]
  44. Selby M. J., Bain E. S., Luciw P. A., Peterlin B. M. Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat. Genes Dev. 1989 Apr;3(4):547–558. doi: 10.1101/gad.3.4.547. [DOI] [PubMed] [Google Scholar]
  45. Selby M. J., Peterlin B. M. Trans-activation by HIV-1 Tat via a heterologous RNA binding protein. Cell. 1990 Aug 24;62(4):769–776. doi: 10.1016/0092-8674(90)90121-t. [DOI] [PubMed] [Google Scholar]
  46. Siomi H., Shida H., Maki M., Hatanaka M. Effects of a highly basic region of human immunodeficiency virus Tat protein on nucleolar localization. J Virol. 1990 Apr;64(4):1803–1807. doi: 10.1128/jvi.64.4.1803-1807.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sodroski J., Patarca R., Rosen C., Wong-Staal F., Haseltine W. Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III. Science. 1985 Jul 5;229(4708):74–77. doi: 10.1126/science.2990041. [DOI] [PubMed] [Google Scholar]
  48. Southgate C., Zapp M. L., Green M. R. Activation of transcription by HIV-1 Tat protein tethered to nascent RNA through another protein. Nature. 1990 Jun 14;345(6276):640–642. doi: 10.1038/345640a0. [DOI] [PubMed] [Google Scholar]
  49. Tada H., Rappaport J., Lashgari M., Amini S., Wong-Staal F., Khalili K. Trans-activation of the JC virus late promoter by the tat protein of type 1 human immunodeficiency virus in glial cells. Proc Natl Acad Sci U S A. 1990 May;87(9):3479–3483. doi: 10.1073/pnas.87.9.3479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Taylor J. P., Pomerantz R., Bagasra O., Chowdhury M., Rappaport J., Khalili K., Amini S. TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation. EMBO J. 1992 Sep;11(9):3395–3403. doi: 10.1002/j.1460-2075.1992.tb05418.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  52. Weeks K. M., Ampe C., Schultz S. C., Steitz T. A., Crothers D. M. Fragments of the HIV-1 Tat protein specifically bind TAR RNA. Science. 1990 Sep 14;249(4974):1281–1285. doi: 10.1126/science.2205002. [DOI] [PubMed] [Google Scholar]
  53. Wright C. M., Felber B. K., Paskalis H., Pavlakis G. N. Expression and characterization of the trans-activator of HTLV-III/LAV virus. Science. 1986 Nov 21;234(4779):988–992. doi: 10.1126/science.3490693. [DOI] [PubMed] [Google Scholar]

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