Archives of Virology

, Volume 139, Issue 1–2, pp 133–154

Spatial association of HIV-1tat protein and the nucleolar transport protein B23 in stably transfected Jurkat T-cells

  • W. A. Marasco
  • A. M. Szilvay
  • K. H. Kalland
  • D. G. Helland
  • H. M. Reyes
  • R. J. Walter
Original Papers

Summary

The human immunodeficiency virus (HIV-1) encodes a transactivator protein, the product of the tat gene (tat), which is essential for virus replication. In this study, immunogold electron microscopy was used in a stably transfected Jurkat T-cell line that constitutively expresses HIV-1tat protein to determine the subcellular and intranuclear distribution oftat protein. Two nucleocytoplasmic shuttle proteins C23/nucleolin and B23 and a third nucleolar antigen that was detected by monoclonal antibody MAb 1277 were also examined. In addition, spatial association of C23 and B23 withtat protein at several subcellular locations was examined in dual-labeling experiments. The results showed thattat protein was found in both the cytoplasm and nucleus but was especially prominent within the dense fibrillar and granular components of the nucleolus. There was little labeling oftat protein in the fibrillar centers where MAb 1277 antigen was localized at a comparatively high level. The subcellular and intranucleolar distribution oftat protein was virtually identical to the pattern seen with C23 and B23. Although the intranuclear distributions of C23, B23 andtat protein were very similar, C23 andtat protein were seldom spatially associated. In contrast, B23 andtat protein were frequently spatially associated in the nucleolus and in several other subcellular locations including the cytoplasm, nucleoplasm, at the nuclear envelope and plasma membrane. While a physical association was not directly demonstrated in this study, the spatial association between B23 andtat protein strongly suggest that such an association may exist.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Adam SA, Marr RS, Gerace L (1975) Nuclear envelope permeability. Nature 254: 109–114Google Scholar
  2. 2.
    Aldovini A, Debouck C, Feinberg MB, Rosenberg M, Arya SK, Wong-Staal F (1988) Synthesis of the complete trans-activation gene product of human T-lymphotropic virus type III inEscherichia coli: demonstration of immunogenicity in vivo and expression in vitro. Proc Natl Acad Sci USA 88: 6672–6676Google Scholar
  3. 3.
    Biggiogera M, Fakan S, Kaufmann SH, Black A, Shaper JH, Busch H (1989) Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus. J Histochem Cytochem 37: 1371–1374Google Scholar
  4. 4.
    Borer RA, Lehner CF, Eppenberger HM, Nigg EA (1989) Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56: 379–390.Google Scholar
  5. 5.
    Bouche G, Caizergues-Ferrer M, Bugler B, Amalric F (1984) Interrelations between the maturation of a 100 kDa nucleolar protein and pre rRNA synthesis in CHO cells. Nucleic Acids Res 12: 3025–3035Google Scholar
  6. 6.
    Bugler B, Bourbon H, Lapeyre B, Wallace MO, Chang JH, Olson MOJ (1987) RNA binding fragments from nucleolin contain the ribonucleoprotein consensus sequence. J Biol Chem 262: 10922–10925Google Scholar
  7. 7.
    Calnan BJ, Tidor B, Biancalana S, Hudson D, Frankel AD (1991) Arginine-mediated RNA recognition: the arginine fork. Science 252: 1167–1171Google Scholar
  8. 8.
    Caputo A, Sodroski JG, Haseltine WA (1990) Constitutive expression of HIV-1tat protein in human Jurkat T cell using a BK virus vector. J AIDS 3: 372–379Google Scholar
  9. 9.
    Chemicon International, Inc., Temecula, CA (1991) Mouse anti-human subcellular particles. Monoclonal antibodies. Technical BulletinGoogle Scholar
  10. 10.
    Clawson GA, Woo CH, Button J, Smuckler EA (1984) Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope. Biochemistry 23: 3501–3507Google Scholar
  11. 11.
    Clawson GA, Button J, Smuckler EA (1985) Photoaffinity labeling of a nuclear matrix nucleoside triphosphatase and its modulation in the acute-phase response. Exp Cell Res 159: 171–175Google Scholar
  12. 12.
    Dang CV, Lee WMF (1989) Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70 and HIV tat proteins. J Biol Chem 264: 18019–18023Google Scholar
  13. 13.
    Dayton AI, Sodroski JG, Rosen CA, W Chun Goh, Haseltine WA (1986) The transactivator gene of the human T-cell lymphotropic virus type III is required for replication. Cell 4: 941–947Google Scholar
  14. 14.
    Desai K, Loewenstein PM, Green M (1991) Isolation of a cellular protein that binds to the human immunodeficiency virus TAT protein and can potentiate transactivation of the viral promotor. Proc Natl Acad Sci USA 88: 8875–8879Google Scholar
  15. 15.
    Fankhauser C, Izaurralde E, Adachi Y, Wingfield P, Laemmli UK (1991) Specific complex of human immunodeficiency virus type I rev and nucleolar B23 proteins: Dissociation by the rev response element. Mol Cell Biol 11: 2567–2575Google Scholar
  16. 16.
    Feng S, Holland EC (1988) HIV-1 tat trans-activation requires the loop sequence within tar. Nature 334: 165–167Google Scholar
  17. 17.
    Fisher AG, Feinberg MB, Josephs SF, Harper ME, Marselle LM, Reyes G, Gonda MA, Aldovini A, Debouk C, Gallo RC, Wong-Staal F (1986) Thetrans-activator gene of HTLV-III is essential for virus replication. Nature 320: 2367–2371Google Scholar
  18. 18.
    Frankel AD, Bredt DS, Pabo CO (1988) Tat protein from human immunodeficiency virus forms a metal-linked dimer. Science 240: 70–73Google Scholar
  19. 19.
    Garcia JA, Harrich D, Soultanakis E, Wu F, Mitsuyasu R, Gaynor RB (1989) Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation. EMBO J 8: 775–778Google Scholar
  20. 20.
    Goh WC, Rosen CA, Sodroski JG, Ho DD, Haseltine WA (1986) Identification of a protein encoded by the transactivator gene,tat III, of a human T-cell lymphotrophic retrovirus type III. J Virol 59: 181–184Google Scholar
  21. 21.
    Hainfield JF (1987) A small gold-conjugated antibody label: Improved resolution for electron microscopy. Science 236: 450–453Google Scholar
  22. 22.
    Haseltine WA (1991) Regulation of HIV-1 replication bytat andrev. In: Haseltine WA, Wong-Staal F (eds) Genetic structure and regulation of HIV. Raven Press, New York, pp 1–42Google Scholar
  23. 23.
    Hauber J, Malim MH, Cullen BR (1989) Mutational analysis of the conserved basic domain of human immunodeficiency virus tat protein. J Virol 63: 1181–1187Google Scholar
  24. 24.
    Herrera AH, Olson MOJ (1986) Association of protein C23 with rapidly labeled nucleolar RNA. Biochemistry 25: 6258–6264Google Scholar
  25. 25.
    Horisberger M (1984) Electron-opaque markers; a review. In: Polak J, Varndell J (eds) Immunolabeling for electron microscopy. Elsevier, Amsterdam New York, pp 17–26Google Scholar
  26. 26.
    Jordan G (1987) At the heart of the nucleolus. Nature 329: 489–490Google Scholar
  27. 27.
    Kalderon D, Richardson WD, Markham AF, Smith AE (1984) Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 311: 33–38Google Scholar
  28. 28.
    Miyazaki Y, Tatamatsu T, Nosaka T, Fujita S, Hatanaka M (1992) Intranuclear topological distribution of HIV-1 trans-activators. FEBS 305: 1–5Google Scholar
  29. 29.
    Muesing MA, Smith DH, Capon DJ (1987) Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell 48: 691–701Google Scholar
  30. 30.
    Müller WEG, Wenger R, Reuter P, Renneisen K, Schröder HC (1989) Association of Tat protein and viral mRNA with nuclear matrix from HIV-1-infected H9 cells. Biochim Biophys Acta 1008: 208–212Google Scholar
  31. 31.
    Müller WEG, Okamoto T, Reuter P, Ugarkovic D, Schröder HC (1990) Functional characterization of tat protein from human immunodeficiency virus. Evidence that tat links viral RNAs to nuclear matrix. J Biol Chem 265: 3803–3808Google Scholar
  32. 32.
    Nelbock P, Dillon PJ, Perkins A, Rosen CA (1990) A cDNA for a protein that interacts with the human immunodeficiency virus tat transactivator. Science 248: 1650–1653Google Scholar
  33. 33.
    Newmeyer DD, Forbes DJ (1988) Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell 52: 641–653Google Scholar
  34. 34.
    Nigg EA, Baeuerle PA, Luhrmann R (1991) Nuclear-import-export: in search of signals and mechanisms. Cell 66: 15–22Google Scholar
  35. 35.
    Olson MOJ, Rivers ZM, Thompson BA, Kao WY, Case ST (1983) Interaction of nucleolar phosphoprotein C23 with cloned segments of rat ribosomal deoxyribonucleic acid. Biochemistry 22: 3345–3351Google Scholar
  36. 36.
    Paine PL, Moore LC, Horowitz SB (1990) Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol 111: 807–816Google Scholar
  37. 37.
    Pearson L, Garcia J, Wu F, Modesti N, Nelson J, Gaynor R (1990) A transdominant tat mutant that inhibits tat-induced gene expression from the human immunodeficiency virus long terminal repeat. Proc Natl Acad Sci USA 87: 5079–5083Google Scholar
  38. 38.
    Peters R (1986) Fluorescence microphotolysis to measure nucleocytoplasmic transport and intracellular mobility. Biochim Biophys Acta 864: 305–359Google Scholar
  39. 39.
    Rappaport J, Lee SJ, Khalili K, Wong-Staal F (1989) The acidic amino-terminal region of the HIV-1 tat protein constitutes an essential activating domain. New Biol 1: 101–110Google Scholar
  40. 40.
    Ratner L, Haseltine WA, Patarca R, Livak KJ, Starcich B, Josephs SF, Doran ER, Antoni Rafalski J, Whitehorn EA, Baumeister K, Ivanoff L, Petteway SR, Pearson ML, Lautenberger JA, Papas TS, Ghrayeb J, Chang NT, Gallo RC, Wong-Staal F (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313: 277–284Google Scholar
  41. 41.
    Rosen CA, Pavlakis GN (1990) Tat and Rev: positive regulators of HIV gene expression. AIDS 4: 499–509Google Scholar
  42. 42.
    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: 1–8Google Scholar
  43. 43.
    Rungger-Brandles E, Jamrich M, Bautz EK (1981) Localization of RNA polymerase B and histones in the nucleus of primary spermatocytes of Drosphila hydei, studied by immunofluorscence microscopy. Chromosoma 82: 399–407Google Scholar
  44. 44.
    Sadaie RM, Betner T, Wong-Staal F (1988) Site directed mutagenesis of two trans-regulatory genes (tat-III, trs) of HIV-1. Science 239: 910–913Google Scholar
  45. 45.
    Sanchez-Pescador R, Power MD, Barr PJ, Steimer KS, Stempien MM, Brown-Shimer SL, Gee WW, Renard A, Randolph A, Levy JA, Dina D, Luciw PA (1984) Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science 227: 484–492Google Scholar
  46. 46.
    Sato T (1968) A modified method for lead staining. J Electron Microsc (Tokyo) 17: 158–159Google Scholar
  47. 47.
    Schröder HC, Trölltsch D, Friese U, Bachmann M, Müller WEG (1987a) Mature mRNA is selectively released from the nuclear matrix by an ATP/dATP-dependent mechanism sensitive to topoisomerase inhibitors. J Biol Chem 262: 8917–8925Google Scholar
  48. 48.
    Schröder HC, Bachmann M, Diehl-Seifert B, Müller WEG (1987b) Transport of mRNA from nucleus to cytoplasm. Prog Nucleic Acids Res Mol Biol 34: 89–142Google Scholar
  49. 49.
    Selby MJ, Bain ES, Luciw PA, Peterlin BM (1989) Structure, sequence, and position of the stem-loop structure in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat. Genes Dev 3: 547–558Google Scholar
  50. 50.
    Siomi H, Shida H, Hyun Nam S, Nosaka T, Maki M, Hatanaka M (1988) Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing. Cell 55: 197–209Google Scholar
  51. 51.
    Siomi H, Shida H, Maki M, Hatanaka M (1990) Effects of a highly basic region of human immunodeficiency virus tat protein on nucleolar localization. J Virol 64: 1803–1807Google Scholar
  52. 52.
    Sodroski J, Rosen C, Wong-Staal F, Salahuddin SZ, Popovic M, Arya S, Gallo RC, Haseltine WA (1985)Trans-acting transcriptional regulation of the human T-cell leukemia virus type III long terminal repeat. Science 227: 171–173Google Scholar
  53. 53.
    Thiry M, Thiry-Blaise L (1989) In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA. Eur J Cell Biol 50: 235–243Google Scholar
  54. 54.
    Timms BG (1986) Post embedding immunogold labeling for electron microscopy using LR white resin. Am J Anat 175: 267–275Google Scholar
  55. 55.
    Wain-Hobson S, Sonigo P, Danos O, Cole S, Alizon M (1985) Nucleotide sequence of the AIDS virus, LAV. Cell 40: 9–17Google Scholar
  56. 56.
    Weeks KM, Ampe C, Schultz SC, Steitz TA, Crothers DM (1990) Fragments of the HIV-1 tat protein specifically bind TAR RNA. Science 249: 1281–1285Google Scholar
  57. 57.
    Zeitlin S, Parent A, Silverstein S, Efstratiadis A (1987) Pre-mRNA splicing and the nuclear matrix. Mol Cell Biol 7: 111–120Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • W. A. Marasco
    • 1
  • A. M. Szilvay
    • 2
    • 3
  • K. H. Kalland
    • 2
  • D. G. Helland
    • 2
    • 3
  • H. M. Reyes
    • 4
  • R. J. Walter
    • 4
  1. 1.Division of Human Retrovirology, Dana-Farber Cancer InstituteHarvard Medical SchoolBostonUSA
  2. 2.National Center for Research in VirologyUniversity of BergenBergenNorway
  3. 3.Center of BiotechnologyUniversity of BergenBergenNorway
  4. 4.Department of SurgeryHektoen Institute for Medical ResearchChicagoUSA

Personalised recommendations