Molecular and Cellular Biochemistry

, Volume 345, Issue 1–2, pp 105–118 | Cite as

Effect of the redox state on HIV-1 tat protein multimerization and cell internalization and trafficking

  • Raffaella Pierleoni
  • Michele Menotta
  • Antonella Antonelli
  • Carla Sfara
  • Giordano Serafini
  • Sabrina Dominici
  • Maria Elena Laguardia
  • Annalisa Salis
  • Gianluca Damonte
  • Lucia Banci
  • Marco Porcu
  • Paolo Monini
  • Barbara Ensoli
  • Mauro MagnaniEmail author


The redox state of the cysteine-rich region of the HIV Tat protein is known to play a crucial role in Tat biological activity. In this article, we show that Tat displays two alternative functional states depending on the presence of either one or three reduced sulphydryl groups in the cysteine-rich region, respectively. Using different approaches, a disulfide pattern has been defined for the Tat protein and a specific DTT-dependent breaking order of disulfide bonds highlighted. The Tat redox state deeply influences macrophage protein uptake. Immunoistochemistry analysis shows that the oxidized protein does not enter cells, whereas partially reduced protein reaches the cytosol and, to a limited extent, the nucleus. Finally electrophoretic analysis shows Tat high-molecular weight multi-aggregation, resulting in the loss of biological activity. This is due to strong electrostatic and metal-binding interactions, whereas Tat dimerization involves metal-binding interactions as well as disulfide bond formation.


HIV Tat Redox state Cell internalization 


  1. 1.
    Beral V, Peterman TA, Berkelman RL, Jaffe HW (1990) Kaposi’s sarcoma among persons with AIDS: a sexually transmitted infection? Lancet 335:123–128CrossRefPubMedGoogle Scholar
  2. 2.
    Levine AM (1992) Acquired immunodeficiency syndrome-related lymphoma. Blood 80:8–20PubMedGoogle Scholar
  3. 3.
    Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-Staal F (1990) Tat protein of HIV-1 stimulates growth of cells derived from Kaposi’s sarcoma lesions of AIDS patients. Nature 345:84–86CrossRefPubMedGoogle Scholar
  4. 4.
    Chang HC, Samaniego F, Nair BC, Buonaguro L, Ensoli B (1997) HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. AIDS 11:1421–1431CrossRefPubMedGoogle Scholar
  5. 5.
    Barillari G, Buonaguro L, Fiorelli V, Hoffman J, Michaels F, Gallo RC, Ensoli B (1992) Effects of cytokines from activated immune cells on vascular cell growth and HIV-1 gene expression. Implications for AIDS-Kaposi’s sarcoma pathogenesis. J Immunol 149:3727–3734PubMedGoogle Scholar
  6. 6.
    Ensoli B, Buonaguro L, Barillari G, Fiorelli V, Gendelman R, Morgan RA, Wingfield P, Gallo RC (1993) Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation. J Virol 67:277–287PubMedGoogle Scholar
  7. 7.
    Cafaro A, Caputo A, Fracasso C, Maggiorella MT, Goletti D, Baroncelli S, Pace M, Sernicola L, Koanga-Mogtomo ML, Betti M, Borsetti A, Belli R, Akerblom L, Corrias F, Butto S, Heeney J, Verani P, Titti F, Ensoli B (1999) Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine. Nat Med 5:643–650CrossRefPubMedGoogle Scholar
  8. 8.
    Re MC, Furlini G, Vignoli M, Ramazzotti E, Roderigo G, De Rosa V, Zauli G, Lolli S, Capitani S, La Placa M (1995) Effect of antibody to HIV-1 Tat protein on viral replication in vitro and progression of HIV-1 disease in vivo. J Acquir Immune Defic Syndr Hum Retrovirol 10:408–416CrossRefPubMedGoogle Scholar
  9. 9.
    Tikhonov I, Ruckwardt TJ, Hatfield GS, Pauza CD (2003) Tat-neutralizing antibodies in vaccinated macaques. J Virol 77:3157–3166CrossRefPubMedGoogle Scholar
  10. 10.
    Rezza G, Fiorelli V, Dorrucci M, Ciccozzi M, Tripiciano A, Scoglio A, Collacchi B, Ruiz-Alvarez M, Giannetto C, Caputo A, Tomasoni L, Castelli F, Sciandra M, Sinicco A, Ensoli F, Butto S, Ensoli B (2005) The presence of anti-Tat antibodies is predictive of long-term nonprogression to AIDS or severe immunodeficiency: findings in a cohort of HIV-1 seroconverters. J Infect Dis 191:1321–1324CrossRefPubMedGoogle Scholar
  11. 11.
    Arya SK, Guo C, Josephs SF, Wong-Staal F (1985) Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 229:69–73CrossRefPubMedGoogle Scholar
  12. 12.
    Chang HK, Gallo RC, Ensoli B (1995) Regulation of cellular gene expression and function by the human immunodeficiency virus type 1 Tat protein. J Biomed Sci 2:189–202CrossRefPubMedGoogle Scholar
  13. 13.
    Ensoli B, Fiorelli V, Ensoli F, Cafaro A, Titti F, Butto S, Monini P, Magnani M, Caputo A, Garaci E (2006) Candidate HIV-1 Tat vaccine development: from basic science to clinical trials. AIDS 20:2245–2261CrossRefPubMedGoogle Scholar
  14. 14.
    Fisher AG, Feinberg MB, Josephs SF, Harper ME, Marselle LM, Reyes G, Gonda MA, Aldovini A, Debouk C, Gallo RC (1986) The trans-activator gene of HTLV-III is essential for virus replication. Nature 320:367–371 andCrossRefPubMedGoogle Scholar
  15. 15.
    Ensoli B, Fiorelli V, Ensoli F, Lazzarin A, Visintini R, Narciso P, Di Carlo A, Monini P, Magnani M, Garaci E (2008) The therapeutic phase I trial of the recombinant native HIV-1 Tat protein. AIDS 22:2207–2209CrossRefPubMedGoogle Scholar
  16. 16.
    Longo O, Tripiciano A, Fiorelli V, Bellino S, Scoglio A, Collacchi B, Alvarez MJ, Francavilla V, Arancio A, Paniccia G, Lazzarin A, Tambussi G, Din CT, Visintini R, Narciso P, Antinori A, D’Offizi G, Giulianelli M, Carta M, Di Carlo A, Palamara G, Giuliani M, Laguardia ME, Monini P, Magnani M, Ensoli F, Ensoli B (2009) Phase I therapeutic trial of the HIV-1 Tat protein and long term follow-up. Vaccine 27:3306–3312CrossRefPubMedGoogle Scholar
  17. 17.
    Frankel AD, Bredt DS, Pabo CO (1988) Tat protein from human immunodeficiency virus forms a metal-linked dimer. Science 240:70–73CrossRefPubMedGoogle Scholar
  18. 18.
    Huang HW, Wang KT (1996) Structural characterization of the metal binding site in the cysteine-rich region of HIV-1 Tat protein. Biochem Biophys Res Commun 227:615–621CrossRefPubMedGoogle Scholar
  19. 19.
    Goldstein G, Tribbick G, Manson K (2001) Two B cell epitopes of HIV-1 Tat protein have limited antigenic polymorphism in geographically diverse HIV-1 strains. Vaccine 19:1738–1746CrossRefPubMedGoogle Scholar
  20. 20.
    Barillari G, Gendelman R, Gallo RC, Ensoli B (1993) The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence. Proc Natl Acad Sci USA 90:7941–7945CrossRefPubMedGoogle Scholar
  21. 21.
    Jeang K-T (1996) HIV-1 Tat: structure and function. In: HIV sequence compendium 1996, Part III. Theoretical Biol and Biophys Group, Los Alamos Nat Lab, pp 3–18Google Scholar
  22. 22.
    Mishra M, Vetrivel S, Siddappa NB, Ranga U, Seth P (2008) Clade-specific differences in neurotoxicity of human immunodeficiency virus-1 B and C Tat of human neurons: significance of dicysteine C30C31 motif. Ann Neurol 63:366–376CrossRefPubMedGoogle Scholar
  23. 23.
    Koken SE, Greijer AE, van Verhoef K, Wamel J, Bukrinskaya AG, Berkhout B (1994) Intracellular analysis of in vitro modified HIV Tat protein. J Biol Chem 269:8366–8375PubMedGoogle Scholar
  24. 24.
    Fanales-Belasio E, Cafaro A, Cara A, Negri DR, Fiorelli V, Butto S, Moretti S, Maggiorella MT, Baroncelli S, Michelini Z, Tripiciano A, Sernicola L, Scoglio A, Borsetti A, Ridolfi B, Bona R, ten Haaft P, Macchia I, Leone P, Pavone-Cossut MR, Nappi F, Vardas E, Magnani M, Laguardia E, Caputo A, Titti F, Ensoli B (2002) HIV-1 Tat-based vaccines: from basic science to clinical trials. DNA Cell Biol 21:599–610CrossRefPubMedGoogle Scholar
  25. 25.
    Habeeb AFSA (1972) Reaction of protein sulfhydryl groups with Ellman’s reagent. Methods Enzymol 25:457–464CrossRefGoogle Scholar
  26. 26.
    Leichert LI, Jakob U (2004) Protein thiol modifications visualized in vivo. PLoS Biol 2:e333CrossRefPubMedGoogle Scholar
  27. 27.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  28. 28.
    Ziegler HK, Unanue ER (1982) Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Proc Natl Acad Sci USA 79:175–178CrossRefPubMedGoogle Scholar
  29. 29.
    Siddappa NB, Venkatramanan M, Venkatesh P, Janki MV, Jayasuryan N, Desai A, Ravi V, Ranga U (2006) Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein. Retrovirology 3:53CrossRefPubMedGoogle Scholar
  30. 30.
    Fanales-Belasio E, Moretti S, Nappi F, Barillari G, Micheletti F, Cafaro A, Ensoli B (2002) Native HIV-1 Tat protein targets monocyte-derived dendritic cells and enhances their maturation, function, and antigen-specific T cell responses. J Immunol 168:197–206PubMedGoogle Scholar
  31. 31.
    Jeang KT, Xiao H, Rich EA (1999) Multifaceted activities of the HIV-1 transactivator of transcription, Tat. J Biol Chem 274:28837–28840CrossRefPubMedGoogle Scholar
  32. 32.
    Fink AL (2005) Natively unfolded proteins. Curr Opin Struct Biol 15:35–41CrossRefPubMedGoogle Scholar
  33. 33.
    Linding R, Russell RB, Neduva V, Gibson TJ (2003) GlobPlot: exploring protein sequences for globularity and disorder. Nucleic Acids Res 31:3701–3708CrossRefPubMedGoogle Scholar
  34. 34.
    Uversky VN, Gillespie JR, Fink AL (2000) Why are “natively unfolded” proteins unstructured under physiologic conditions? Proteins 41:415–427CrossRefPubMedGoogle Scholar
  35. 35.
    Uversky VN (2002) Natively unfolded proteins: a point where biology waits for physics. Protein Sci 11:739–756CrossRefPubMedGoogle Scholar
  36. 36.
    Vendel AC, Lumb KJ (2003) Molecular recognition of the human coactivator CBP by the HIV-1 transcriptional activator Tat. Biochemistry 42:910–916CrossRefPubMedGoogle Scholar
  37. 37.
    Shojania S, O’Neil JD (2006) HIV-1 Tat is a natively unfolded protein: the solution conformation and dynamics of reduced HIV-1 Tat-(1–72) by NMR spectroscopy. J Biol Chem 281:8347–8356CrossRefPubMedGoogle Scholar
  38. 38.
    Rice AP, Chan F (1991) Tat protein of human immunodeficiency virus type 1 is a monomer when expressed in mammalian cells. Virology 185:451–454CrossRefPubMedGoogle Scholar
  39. 39.
    Ruben S, Perkins A, Purcell R, Joung K, Sia R, Burghoff R, Haseltine WA, Rosen CA (1989) Structural and functional characterization of human immunodeficiency virus tat protein. J Virol 63:1–8PubMedGoogle Scholar
  40. 40.
    Sadaie MR, Mukhopadhyaya R, Benaissa ZN, Pavlakis GN, Wong-Staal F (1990) Conservative mutations in the putative metal-binding region of human immunodeficiency virus tat disrupt virus replication. AIDS Res Hum Retroviruses 6:1257–1263PubMedGoogle Scholar
  41. 41.
    Tahirov TH, Babayeva ND, Varzavand K, Cooper JJ, Sedore SC, Price DH (2010) Crystal structure of HIV-1 Tat complexed with human P-TEFb. Nature 465:747–751CrossRefPubMedGoogle Scholar
  42. 42.
    Falnes PO, Sandvig K (2000) Penetration of protein toxins into cells. Curr Opin Cell Biol 12:407–413CrossRefPubMedGoogle Scholar
  43. 43.
    Liou LY, Herrmann CH, Rice AP (2004) HIV-1 infection and regulation of Tat function in macrophages. Int J Biochem Cell Biol 36:1767–1775CrossRefPubMedGoogle Scholar
  44. 44.
    Fanales-Belasio E, Moretti S, Fiorelli V, Tripiciano A, Pavone Cossut MR, Scoglio A, Collacchi B, Nappi F, Macchia I, Bellino S, Francavilla V, Caputo A, Barillari G, Magnani M, Laguardia ME, Cafaro A, Titti F, Monini P, Ensoli F, Ensoli B (2009) HIV-1 Tat addresses dendritic cells to induce a predominant Th1-type adaptive immune response that appears prevalent in the asymptomatic stage of infection. J Immunol 182:2888–2897CrossRefPubMedGoogle Scholar
  45. 45.
    Kittiworakarn J, Lecoq A, Moine G, Thai R, Lajeunesse E, Drevet P, Vidaud C, Menez A, Leonetti M (2006) HIV-1 Tat raises an adjuvant-free humoral immune response controlled by its core region and its ability to form cysteine-mediated oligomers. J Biol Chem 281:3105–3115CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Raffaella Pierleoni
    • 1
  • Michele Menotta
    • 1
  • Antonella Antonelli
    • 1
  • Carla Sfara
    • 1
  • Giordano Serafini
    • 1
  • Sabrina Dominici
    • 1
  • Maria Elena Laguardia
    • 2
  • Annalisa Salis
    • 3
  • Gianluca Damonte
    • 3
  • Lucia Banci
    • 4
  • Marco Porcu
    • 4
  • Paolo Monini
    • 5
  • Barbara Ensoli
    • 5
  • Mauro Magnani
    • 1
    Email author
  1. 1.Department of Molecular SciencesUniversity of Urbino “Carlo Bo”UrbinoItaly
  2. 2.Diatheva srlFanoItaly
  3. 3.Department of Experimental Medicine and Center of Excellence for Biomedical ResearchUniversity of GenovaGenovaItaly
  4. 4.Centro Risonanze MagneticheUniversity of FlorenceSesto Fiorentino FlorenceItaly
  5. 5.Centro Nazionale AIDSIstituto Superiore di SanitàRomeItaly

Personalised recommendations