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. 1996 Mar 5;93(5):2089–2094. doi: 10.1073/pnas.93.5.2089

Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II.

G Mavankal 1, S H Ignatius Ou 1, H Oliver 1, D Sigman 1, R B Gaynor 1
PMCID: PMC39914  PMID: 8700889

Abstract

The Tat-responsive region (TAR) element is a critical RNA regulatory element in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactivator protein Tat. Recently, we demonstrated by gel-retardation analysis that RNA polymerase II binds to TAR RNA and that Tat prevents this binding even when Tat does not bind to TAR RNA. These results suggested that direct interactions between Tat and RNA polymerase II may prevent RNA polymerase II pausing and lead to Tat-mediated increases in transcriptional elongation. To test this possibility, we performed protein interaction studies with RNA polymerase II and both the HIV-1 and the closely related HIV-2 Tat protein. These studies indicated that both the HIV-1 and HIV-2 Tat proteins could specifically interact with RNA polymerase II. Mutagenesis of both HIV-1 and HIV-2 Tat demonstrated that the basic domains of both the HIV-1 and HIV-2 Tat proteins were required for this interaction. Furthermore, "far Western" analysis suggested that the largest subunit of RNA polymerase II was the site for interaction with Tat. The interactions between Tat and RNA polymerase II were of similar magnitude to those detected between RNA polymerase II and the cellular transcription factor RAP30, which stably associates with RNA polymerase II during transcriptional elongation. These studies are consistent with the model that RNA polymerase II is a cellular target for Tat resulting in Tat-mediated increases in transcriptional elongation from the HIV long terminal repeat.

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Selected References

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

  1. Acker J., Wintzerith M., Vigneron M., Kédinger C. Primary structure of the second largest subunit of human RNA polymerase II (or B). J Mol Biol. 1992 Aug 20;226(4):1295–1299. doi: 10.1016/0022-2836(92)91071-v. [DOI] [PubMed] [Google Scholar]
  2. Berkhout B., Silverman R. H., Jeang K. T. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell. 1989 Oct 20;59(2):273–282. doi: 10.1016/0092-8674(89)90289-4. [DOI] [PubMed] [Google Scholar]
  3. Dignam J. D., Martin P. L., Shastry B. S., Roeder R. G. Eukaryotic gene transcription with purified components. Methods Enzymol. 1983;101:582–598. doi: 10.1016/0076-6879(83)01039-3. [DOI] [PubMed] [Google Scholar]
  4. Dingwall C., Ernberg I., Gait M. J., Green S. M., Heaphy S., Karn J., Lowe A. D., Singh M., Skinner M. A. HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure. EMBO J. 1990 Dec;9(12):4145–4153. doi: 10.1002/j.1460-2075.1990.tb07637.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Elangovan B., Subramanian T., Chinnadurai G. Functional comparison of the basic domains of the Tat proteins of human immunodeficiency virus types 1 and 2 in trans activation. J Virol. 1992 Apr;66(4):2031–2036. doi: 10.1128/jvi.66.4.2031-2036.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Emerman M., Guyader M., Montagnier L., Baltimore D., Muesing M. A. The specificity of the human immunodeficiency virus type 2 transactivator is different from that of human immunodeficiency virus type 1. EMBO J. 1987 Dec 1;6(12):3755–3760. doi: 10.1002/j.1460-2075.1987.tb02710.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feaver W. J., Svejstrup J. Q., Henry N. L., Kornberg R. D. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK. Cell. 1994 Dec 16;79(6):1103–1109. doi: 10.1016/0092-8674(94)90040-x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Gaynor R. Cellular transcription factors involved in the regulation of HIV-1 gene expression. AIDS. 1992 Apr;6(4):347–363. doi: 10.1097/00002030-199204000-00001. [DOI] [PubMed] [Google Scholar]
  10. Greenblatt J., Nodwell J. R., Mason S. W. Transcriptional antitermination. Nature. 1993 Jul 29;364(6436):401–406. doi: 10.1038/364401a0. [DOI] [PubMed] [Google Scholar]
  11. Harrich D., Hsu C., Race E., Gaynor R. B. Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants. J Virol. 1994 Sep;68(9):5899–5910. doi: 10.1128/jvi.68.9.5899-5910.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herrmann C. H., Rice A. P. Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor. J Virol. 1995 Mar;69(3):1612–1620. doi: 10.1128/jvi.69.3.1612-1620.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hodo H. G., 3rd, Blatti S. P. Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II. Biochemistry. 1977 May 31;16(11):2334–2343. doi: 10.1021/bi00630a005. [DOI] [PubMed] [Google Scholar]
  14. Horikoshi M., Fujita H., Wang J., Takada R., Roeder R. G. Nucleotide and amino acid sequence of RAP30. Nucleic Acids Res. 1991 Oct 11;19(19):5436–5436. doi: 10.1093/nar/19.19.5436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jeang K. T., Chun R., Lin N. H., Gatignol A., Glabe C. G., Fan H. In vitro and in vivo binding of human immunodeficiency virus type 1 Tat protein and Sp1 transcription factor. J Virol. 1993 Oct;67(10):6224–6233. doi: 10.1128/jvi.67.10.6224-6233.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kashanchi F., Piras G., Radonovich M. F., Duvall J. F., Fattaey A., Chiang C. M., Roeder R. G., Brady J. N. Direct interaction of human TFIID with the HIV-1 transactivator tat. Nature. 1994 Jan 20;367(6460):295–299. doi: 10.1038/367295a0. [DOI] [PubMed] [Google Scholar]
  17. Kato H., Sumimoto H., Pognonec P., Chen C. H., Rosen C. A., Roeder R. G. HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors. Genes Dev. 1992 Apr;6(4):655–666. doi: 10.1101/gad.6.4.655. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Ratnasabapathy R., Sheldon M., Johal L., Hernandez N. The HIV-1 long terminal repeat contains an unusual element that induces the synthesis of short RNAs from various mRNA and snRNA promoters. Genes Dev. 1990 Dec;4(12A):2061–2074. doi: 10.1101/gad.4.12a.2061. [DOI] [PubMed] [Google Scholar]
  22. Reinberg D., Roeder R. G. Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of initiation factors IIB and IIE. J Biol Chem. 1987 Mar 5;262(7):3310–3321. [PubMed] [Google Scholar]
  23. Rhim H., Chan F., Echetebu C. O., Rice A. P. Rapid purification of monomer HIV-2 Tat protein expressed in Escherichia coli. Protein Expr Purif. 1993 Feb;4(1):24–31. doi: 10.1006/prep.1993.1004. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Sheline C. T., Milocco L. H., Jones K. A. Two distinct nuclear transcription factors recognize loop and bulge residues of the HIV-1 TAR RNA hairpin. Genes Dev. 1991 Dec;5(12B):2508–2520. doi: 10.1101/gad.5.12b.2508. [DOI] [PubMed] [Google Scholar]
  26. Sopta M., Carthew R. W., Greenblatt J. Isolation of three proteins that bind to mammalian RNA polymerase II. J Biol Chem. 1985 Aug 25;260(18):10353–10360. [PubMed] [Google Scholar]
  27. Tan S., Conaway R. C., Conaway J. W. Dissection of transcription factor TFIIF functional domains required for initiation and elongation. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6042–6046. doi: 10.1073/pnas.92.13.6042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thompson N. E., Steinberg T. H., Aronson D. B., Burgess R. R. Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. J Biol Chem. 1989 Jul 5;264(19):11511–11520. [PubMed] [Google Scholar]
  29. Wintzerith M., Acker J., Vicaire S., Vigneron M., Kedinger C. Complete sequence of the human RNA polymerase II largest subunit. Nucleic Acids Res. 1992 Feb 25;20(4):910–910. doi: 10.1093/nar/20.4.910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wu F., Garcia J., Sigman D., Gaynor R. tat regulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185. Genes Dev. 1991 Nov;5(11):2128–2140. doi: 10.1101/gad.5.11.2128. [DOI] [PubMed] [Google Scholar]
  31. Yin M. J., Paulssen E. J., Seeler J. S., Gaynor R. B. Protein domains involved in both in vivo and in vitro interactions between human T-cell leukemia virus type I tax and CREB. J Virol. 1995 Jun;69(6):3420–3432. doi: 10.1128/jvi.69.6.3420-3432.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zawel L., Reinberg D. Common themes in assembly and function of eukaryotic transcription complexes. Annu Rev Biochem. 1995;64:533–561. doi: 10.1146/annurev.bi.64.070195.002533. [DOI] [PubMed] [Google Scholar]

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