• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. May 1994; 68(5): 3343–3353.
PMCID: PMC236825

Effects of the Tat basic domain on human immunodeficiency virus type 1 transactivation, using chemically synthesized Tat protein and Tat peptides.

Abstract

To study the structure relationship of different Tat domains, the full-length Tat protein Tat1-86, the gene product of the first exon Tat1-72 which retains full activity of the protein, and a panel of shorter peptides mimicking different regions of the primary structure of the Tat protein were chemically synthesized by the solid-phase method, using an efficient protocol. Synthetic Tat1-86 and Tat1-72 transactivated beta-galactosidase activity in HeLa cells containing the lacZ gene under the control of the human immunodeficiency virus type 1 long terminal repeat. Analyses of the activity of Tat1-86 and Tat1-72 with the sulfhydryl of cysteine residues free or protected by the acetamidomethyl group showed that only the Tat fragments with deprotected cysteine residues retain transactivation ability. In contrast, peptide Tat1-48 was inactive, with cysteine residues either free or protected. Similarly, other shorter synthetic peptides covering the different Tat domains were inactive. Interestingly, when peptides Tat1-48 and Tat38-60 were used simultaneously, a significant transactivation was obtained. This result suggests that both peptide domains are implicated in transactivation, probably by acting at two different sites. This permits us to propose a fundamentally new step in the understanding of the molecular mechanism of Tat transactivation.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anfinsen CB. Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223–230. [PubMed]
  • Arya SK, Guo C, Josephs SF, Wong-Staal F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science. 1985 Jul 5;229(4708):69–73. [PubMed]
  • Barry PA, Pratt-Lowe E, Unger RE, Luciw PA. Cellular factors regulate transactivation of human immunodeficiency virus type 1. J Virol. 1991 Mar;65(3):1392–1399. [PMC free article] [PubMed]
  • Braddock M, Chambers A, Wilson W, Esnouf MP, Adams SE, Kingsman AJ, Kingsman SM. HIV-1 TAT "activates" presynthesized RNA in the nucleus. Cell. 1989 Jul 28;58(2):269–279. [PubMed]
  • Charneau P, Alizon M, Clavel F. A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication. J Virol. 1992 May;66(5):2814–2820. [PMC free article] [PubMed]
  • Chavez LG, Jr, Scheraga HA. Intrinsic stabilities of portions of the ribonuclease molecule. Biochemistry. 1980 Mar 4;19(5):1005–1012. [PubMed]
  • Chun R, Glabe CG, Fan H. Chemical synthesis of biologically active tat trans-activating protein of human immunodeficiency virus type 1. J Virol. 1990 Jun;64(6):3074–3077. [PMC free article] [PubMed]
  • Cordingley MG, LaFemina RL, Callahan PL, Condra JH, Sardana VV, Graham DJ, Nguyen TM, LeGrow K, Gotlib L, Schlabach AJ, et al. Sequence-specific interaction of Tat protein and Tat peptides with the transactivation-responsive sequence element of human immunodeficiency virus type 1 in vitro. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8985–8989. [PMC free article] [PubMed]
  • Cullen BR. The HIV-1 Tat protein: an RNA sequence-specific processivity factor? Cell. 1990 Nov 16;63(4):655–657. [PubMed]
  • Desai K, Loewenstein PM, 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. [PMC free article] [PubMed]
  • Dingwall C, Ernberg I, Gait MJ, Green SM, Heaphy S, Karn J, Lowe AD, Singh M, Skinner MA, 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. [PMC free article] [PubMed]
  • Frankel AD, Biancalana S, Hudson D. Activity of synthetic peptides from the Tat protein of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7397–7401. [PMC free article] [PubMed]
  • Frankel AD, Bredt DS, Pabo CO. Tat protein from human immunodeficiency virus forms a metal-linked dimer. Science. 1988 Apr 1;240(4848):70–73. [PubMed]
  • Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell. 1988 Dec 23;55(6):1189–1193. [PubMed]
  • Furie B, Schechter AN, Sachs DH, Anfinsen CB. An immunological approach to the conformational equilibrium of staphylococcal nuclease. J Mol Biol. 1975 Mar 15;92(4):497–506. [PubMed]
  • Gatignol A, Buckler-White A, Berkhout B, Jeang KT. Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science. 1991 Mar 29;251(5001):1597–1600. [PubMed]
  • Gatignol A, Kumar A, Rabson A, Jeang KT. Identification of cellular proteins that bind to the human immunodeficiency virus type 1 trans-activation-responsive TAR element RNA. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7828–7832. [PMC free article] [PubMed]
  • Gaynor R, Soultanakis E, Kuwabara M, Garcia J, Sigman DS. Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4858–4862. [PMC free article] [PubMed]
  • Green M, Loewenstein PM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell. 1988 Dec 23;55(6):1179–1188. [PubMed]
  • Hauber J, Malim MH, Cullen BR. Mutational analysis of the conserved basic domain of human immunodeficiency virus tat protein. J Virol. 1989 Mar;63(3):1181–1187. [PMC free article] [PubMed]
  • Hurrell JG, Smith JA, Leach SJ. Immunological measurements of conformational motility in regions of the myoglobin molecule. Biochemistry. 1977 Jan 25;16(2):175–185. [PubMed]
  • Jeyapaul J, Reddy MR, Khan SA. Activity of synthetic tat peptides in human immunodeficiency virus type 1 long terminal repeat-promoted transcription in a cell-free system. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7030–7034. [PMC free article] [PubMed]
  • Jeyapaul J, Seshamma T, Khan SA. Synthetic HIV-1 Tat can dissociate HeLa nuclear protein-TAR RNA complexes in vitro: a novel Tat-nuclear protein interaction. Oncogene. 1991 Sep;6(9):1507–1513. [PubMed]
  • Kamine J, Chinnadurai G. Synergistic activation of the human immunodeficiency virus type 1 promoter by the viral Tat protein and cellular transcription factor Sp1. J Virol. 1992 Jun;66(6):3932–3936. [PMC free article] [PubMed]
  • Kato H, Sumimoto H, Pognonec P, Chen CH, Rosen CA, Roeder RG. HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors. Genes Dev. 1992 Apr;6(4):655–666. [PubMed]
  • Krippl B, Ferguson B, Jones N, Rosenberg M, Westphal H. Mapping of functional domains in adenovirus E1A proteins. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7480–7484. [PMC free article] [PubMed]
  • Mann DA, Frankel AD. Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J. 1991 Jul;10(7):1733–1739. [PMC free article] [PubMed]
  • Marciniak RA, Garcia-Blanco MA, Sharp PA. 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. [PMC free article] [PubMed]
  • Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. [PMC free article] [PubMed]
  • Nagy JA, Meinwald YC, Scheraga HA. Immunochemical determination of conformational equilibria for fragments of the A alpha chain of fibrinogen. Biochemistry. 1982 Apr 13;21(8):1794–1806. [PubMed]
  • Rappaport J, Lee SJ, 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]
  • Rice AP, Carlotti F. Mutational analysis of the conserved cysteine-rich region of the human immunodeficiency virus type 1 Tat protein. J Virol. 1990 Apr;64(4):1864–1868. [PMC free article] [PubMed]
  • Rice AP, Chan F. Tat protein of human immunodeficiency virus type 1 is a monomer when expressed in mammalian cells. Virology. 1991 Nov;185(1):451–454. [PubMed]
  • Rocancourt D, Bonnerot C, Jouin H, Emerman M, Nicolas JF. Activation of a beta-galactosidase recombinant provirus: application to titration of human immunodeficiency virus (HIV) and HIV-infected cells. J Virol. 1990 Jun;64(6):2660–2668. [PMC free article] [PubMed]
  • Rosen CA, Sodroski JG, Haseltine WA. 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. [PubMed]
  • Rosen CA, Terwilliger E, Dayton A, Sodroski JG, Haseltine WA. Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2071–2075. [PMC free article] [PubMed]
  • Ruben S, Perkins A, Purcell R, Joung K, Sia R, Burghoff R, Haseltine WA, Rosen CA. Structural and functional characterization of human immunodeficiency virus tat protein. J Virol. 1989 Jan;63(1):1–8. [PMC free article] [PubMed]
  • Sachs DH, Schechter AN, Eastlake A, Anfinsen CB. Antibodies to a distinct antigenic determinant of staphylococcal nuclease. J Immunol. 1972 Dec;109(6):1300–1310. [PubMed]
  • Seigel LJ, Ratner L, Josephs SF, Derse D, Feinberg MB, Reyes GR, O'Brien SJ, 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. [PubMed]
  • Sheline CT, Milocco LH, Jones KA. 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Sodroski J, Rosen C, Wong-Staal F, Salahuddin SZ, Popovic M, Arya S, Gallo RC, Haseltine WA. Trans-acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat. Science. 1985 Jan 11;227(4683):171–173. [PubMed]
  • Subramanian T, Kuppuswamy M, Venkatesh L, Srinivasan A, Chinnadurai G. Functional substitution of the basic domain of the HIV-1 trans-activator, Tat, with the basic domain of the functionally heterologous Rev. Virology. 1990 May;176(1):178–183. [PubMed]
  • Weeks KM, Ampe C, Schultz SC, Steitz TA, Crothers DM. Fragments of the HIV-1 Tat protein specifically bind TAR RNA. Science. 1990 Sep 14;249(4974):1281–1285. [PubMed]
  • Wright CM, Felber BK, Paskalis H, Pavlakis GN. Expression and characterization of the trans-activator of HTLV-III/LAV virus. Science. 1986 Nov 21;234(4779):988–992. [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...