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. 1997 Jan;71(1):75–83. doi: 10.1128/jvi.71.1.75-83.1997

The human T-cell lymphotropic virus type 1 Tof protein contains a bipartite nuclear localization signal that is able to functionally replace the amino-terminal domain of Rex.

D M D'Agostino 1, V Ciminale 1, L Zotti 1, A Rosato 1, L Chieco-Bianchi 1
PMCID: PMC191026  PMID: 8985325

Abstract

The X region of human T-cell lymphotropic virus type 1 (HTLV-1) encodes two nucleolar/nuclear proteins, the posttranscriptional regulator of mRNA expression Rex and a protein of unknown function named Tof. To gain insight into the possible biological role of Tof, we investigated the mechanism governing its intracellular trafficking and identified its nucleolar/nuclear localization signal (NLS). Mutational analysis of Tof revealed that its NLS was located between amino acids 71 and 98 and contained two arginine-rich domains that functioned in an interdependent manner. Studies of Tof-Rex hybrid proteins showed that the Tof NLS could functionally replace the NLS of Rex at the level of nuclear targeting. As the NLS of Rex is known to mediate its interaction with its RNA target, the Rex-responsive element (RXRE), we tested whether the NLS of Tof could replace that of Rex in mediating activation of a RXRE-containing mRNA. Results showed that the NLS of Tof was indeed able to mediate activation of RXRE-containing mRNAs, suggesting that Tof itself may function as a regulator of RNA expression and utilization. A comparison of their compartmentalization in response to actinomycin D treatment indicated that Tof did not share Rex's shuttling pathway. Expression of Tof from its natural multiply spliced mRNA required the presence of Rex, suggesting that Tof may regulate viral or cellular mRNA expression during the later stages of viral replication.

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Selected References

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  1. Benko D. M., Robinson R., Solomin L., Mellini M., Felber B. K., Pavlakis G. N. Binding of trans-dominant mutant Rev protein of human immunodeficiency virus type 1 to the cis-acting Rev-responsive element does not affect the fate of viral mRNA. New Biol. 1990 Dec;2(12):1111–1122. [PubMed] [Google Scholar]
  2. Berneman Z. N., Gartenhaus R. B., Reitz M. S., Jr, Blattner W. A., Manns A., Hanchard B., Ikehara O., Gallo R. C., Klotman M. E. Expression of alternatively spliced human T-lymphotropic virus type I pX mRNA in infected cell lines and in primary uncultured cells from patients with adult T-cell leukemia/lymphoma and healthy carriers. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3005–3009. doi: 10.1073/pnas.89.7.3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhat N. K., Adachi Y., Samuel K. P., Derse D. HTLV-1 gene expression by defective proviruses in an infected T-cell line. Virology. 1993 Sep;196(1):15–24. doi: 10.1006/viro.1993.1450. [DOI] [PubMed] [Google Scholar]
  4. Bogerd H. P., Huckaby G. L., Ahmed Y. F., Hanly S. M., Greene W. C. The type I human T-cell leukemia virus (HTLV-I) Rex trans-activator binds directly to the HTLV-I Rex and the type 1 human immunodeficiency virus Rev RNA response elements. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5704–5708. doi: 10.1073/pnas.88.13.5704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bogerd H., Greene W. C. Dominant negative mutants of human T-cell leukemia virus type I Rex and human immunodeficiency virus type 1 Rev fail to multimerize in vivo. J Virol. 1993 May;67(5):2496–2502. doi: 10.1128/jvi.67.5.2496-2502.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caputo A., Haseltine W. A. Reexamination of the coding potential of the HTLV-1 pX region. Virology. 1992 Jun;188(2):618–627. doi: 10.1016/0042-6822(92)90516-r. [DOI] [PubMed] [Google Scholar]
  7. Ciminale V., D'Agostino D. M., Zotti L., Franchini G., Felber B. K., Chieco-Bianchi L. Expression and characterization of proteins produced by mRNAs spliced into the X region of the human T-cell leukemia/lymphotropic virus type II. Virology. 1995 Jun 1;209(2):445–456. doi: 10.1006/viro.1995.1277. [DOI] [PubMed] [Google Scholar]
  8. Ciminale V., Pavlakis G. N., Derse D., Cunningham C. P., Felber B. K. Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I. J Virol. 1992 Mar;66(3):1737–1745. doi: 10.1128/jvi.66.3.1737-1745.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cochrane A. W., Jones K. S., Beidas S., Dillon P. J., Skalka A. M., Rosen C. A. Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression. J Virol. 1991 Oct;65(10):5305–5313. doi: 10.1128/jvi.65.10.5305-5313.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cullen B. R., Hauber J., Campbell K., Sodroski J. G., Haseltine W. A., Rosen C. A. Subcellular localization of the human immunodeficiency virus trans-acting art gene product. J Virol. 1988 Jul;62(7):2498–2501. doi: 10.1128/jvi.62.7.2498-2501.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. D'Agostino D. M., Ciminale V., Pavlakis G. N., Chieco-Bianchi L. Intracellular trafficking of the human immunodeficiency virus type 1 Rev protein: involvement of continued rRNA synthesis in nuclear retention. AIDS Res Hum Retroviruses. 1995 Sep;11(9):1063–1071. doi: 10.1089/aid.1995.11.1063. [DOI] [PubMed] [Google Scholar]
  12. Derse D., Mikovits J., Polianova M., Felber B. K., Ruscetti F. Virions released from cells transfected with a molecular clone of human T-cell leukemia virus type I give rise to primary and secondary infections of T cells. J Virol. 1995 Mar;69(3):1907–1912. doi: 10.1128/jvi.69.3.1907-1912.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  14. Franchini G. Molecular mechanisms of human T-cell leukemia/lymphotropic virus type I infection. Blood. 1995 Nov 15;86(10):3619–3639. [PubMed] [Google Scholar]
  15. Franchini G., Mulloy J. C., Koralnik I. J., Lo Monico A., Sparkowski J. J., Andresson T., Goldstein D. J., Schlegel R. The human T-cell leukemia/lymphotropic virus type I p12I protein cooperates with the E5 oncoprotein of bovine papillomavirus in cell transformation and binds the 16-kilodalton subunit of the vacuolar H+ ATPase. J Virol. 1993 Dec;67(12):7701–7704. doi: 10.1128/jvi.67.12.7701-7704.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gessain A., Barin F., Vernant J. C., Gout O., Maurs L., Calender A., de Thé G. Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. Lancet. 1985 Aug 24;2(8452):407–410. doi: 10.1016/s0140-6736(85)92734-5. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hidaka M., Inoue J., Yoshida M., Seiki M. Post-transcriptional regulator (rex) of HTLV-1 initiates expression of viral structural proteins but suppresses expression of regulatory proteins. EMBO J. 1988 Feb;7(2):519–523. doi: 10.1002/j.1460-2075.1988.tb02840.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hinuma Y., Nagata K., Hanaoka M., Nakai M., Matsumoto T., Kinoshita K. I., Shirakawa S., Miyoshi I. Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6476–6480. doi: 10.1073/pnas.78.10.6476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hope T. J., Bond B. L., McDonald D., Klein N. P., Parslow T. G. Effector domains of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex are functionally interchangeable and share an essential peptide motif. J Virol. 1991 Nov;65(11):6001–6007. doi: 10.1128/jvi.65.11.6001-6007.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kalland K. H., Szilvay A. M., Brokstad K. A., Saetrevik W., Haukenes G. The human immunodeficiency virus type 1 Rev protein shuttles between the cytoplasm and nuclear compartments. Mol Cell Biol. 1994 Nov;14(11):7436–7444. doi: 10.1128/mcb.14.11.7436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Katahira J., Ishizaki T., Sakai H., Adachi A., Yamamoto K., Shida H. Effects of translation initiation factor eIF-5A on the functioning of human T-cell leukemia virus type I Rex and human immunodeficiency virus Rev inhibited trans dominantly by a Rex mutant deficient in RNA binding. J Virol. 1995 May;69(5):3125–3133. doi: 10.1128/jvi.69.5.3125-3133.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kiyokawa T., Seiki M., Iwashita S., Imagawa K., Shimizu F., Yoshida M. p27x-III and p21x-III, proteins encoded by the pX sequence of human T-cell leukemia virus type I. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8359–8363. doi: 10.1073/pnas.82.24.8359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Koralnik I. J., Fullen J., Franchini G. The p12I, p13II, and p30II proteins encoded by human T-cell leukemia/lymphotropic virus type I open reading frames I and II are localized in three different cellular compartments. J Virol. 1993 Apr;67(4):2360–2366. doi: 10.1128/jvi.67.4.2360-2366.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koralnik I. J., Gessain A., Klotman M. E., Lo Monico A., Berneman Z. N., Franchini G. Protein isoforms encoded by the pX region of human T-cell leukemia/lymphotropic virus type I. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8813–8817. doi: 10.1073/pnas.89.18.8813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kubota S., Hatanaka M., Pomerantz R. J. Nucleo-cytoplasmic redistribution of the HTLV-I Rex protein: alterations by coexpression of the HTLV-I p21x protein. Virology. 1996 Jun 15;220(2):502–507. doi: 10.1006/viro.1996.0339. [DOI] [PubMed] [Google Scholar]
  27. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Maldarelli F., Martin M. A., Strebel K. Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation. J Virol. 1991 Nov;65(11):5732–5743. doi: 10.1128/jvi.65.11.5732-5743.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Melchior F., Gerace L. Mechanisms of nuclear protein import. Curr Opin Cell Biol. 1995 Jun;7(3):310–318. doi: 10.1016/0955-0674(95)80084-0. [DOI] [PubMed] [Google Scholar]
  30. Mermer B., Felber B. K., Campbell M., Pavlakis G. N. Identification of trans-dominant HIV-1 rev protein mutants by direct transfer of bacterially produced proteins into human cells. Nucleic Acids Res. 1990 Apr 25;18(8):2037–2044. doi: 10.1093/nar/18.8.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meyer B. E., Malim M. H. The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm. Genes Dev. 1994 Jul 1;8(13):1538–1547. doi: 10.1101/gad.8.13.1538. [DOI] [PubMed] [Google Scholar]
  32. Nagashima K., Yoshida M., Seiki M. A single species of pX mRNA of human T-cell leukemia virus type I encodes trans-activator p40x and two other phosphoproteins. J Virol. 1986 Nov;60(2):394–399. doi: 10.1128/jvi.60.2.394-399.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nosaka T., Miyazaki Y., Takamatsu T., Sano K., Nakai M., Fujita S., Martin T. E., Hatanaka M. The post-transcriptional regulator Rex of the human T-cell leukemia virus type I is present as nucleolar speckles in infected cells. Exp Cell Res. 1995 Jul;219(1):122–129. doi: 10.1006/excr.1995.1212. [DOI] [PubMed] [Google Scholar]
  34. Nosaka T., Siomi H., Adachi Y., Ishibashi M., Kubota S., Maki M., Hatanaka M. Nucleolar targeting signal of human T-cell leukemia virus type I rex-encoded protein is essential for cytoplasmic accumulation of unspliced viral mRNA. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9798–9802. doi: 10.1073/pnas.86.24.9798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Orita S., Saiga A., Takagi S., Tanaka T., Okumura K., Aono Y., Hinuma Y., Igarashi H. A novel alternatively spliced viral mRNA transcribed in cells infected with human T cell leukemia virus type 1 is mainly responsible for expressing p21X protein. FEBS Lett. 1991 Dec 16;295(1-3):127–134. doi: 10.1016/0014-5793(91)81402-t. [DOI] [PubMed] [Google Scholar]
  36. Osame M., Usuku K., Izumo S., Ijichi N., Amitani H., Igata A., Matsumoto M., Tara M. HTLV-I associated myelopathy, a new clinical entity. Lancet. 1986 May 3;1(8488):1031–1032. doi: 10.1016/s0140-6736(86)91298-5. [DOI] [PubMed] [Google Scholar]
  37. Perry R. P., Kelley D. E. Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species. J Cell Physiol. 1970 Oct;76(2):127–139. doi: 10.1002/jcp.1040760202. [DOI] [PubMed] [Google Scholar]
  38. Poiesz B. J., Ruscetti F. W., Gazdar A. F., Bunn P. A., Minna J. D., Gallo R. C. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7415–7419. doi: 10.1073/pnas.77.12.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Prasher D. C., Eckenrode V. K., Ward W. W., Prendergast F. G., Cormier M. J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992 Feb 15;111(2):229–233. doi: 10.1016/0378-1119(92)90691-h. [DOI] [PubMed] [Google Scholar]
  40. Richard N., Iacampo S., Cochrane A. HIV-1 Rev is capable of shuttling between the nucleus and cytoplasm. Virology. 1994 Oct;204(1):123–131. doi: 10.1006/viro.1994.1516. [DOI] [PubMed] [Google Scholar]
  41. Rizzuto R., Brini M., Pizzo P., Murgia M., Pozzan T. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol. 1995 Jun 1;5(6):635–642. doi: 10.1016/s0960-9822(95)00128-x. [DOI] [PubMed] [Google Scholar]
  42. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  43. Roithmann S., Pique C., Le Cesne A., Delamarre L., Pham D., Tursz T., Dokhélar M. C. The open reading frame I (ORF I)/ORF II part of the human T-cell leukemia virus type I X region is dispensable for p40tax, p27rex, or envelope expression. J Virol. 1994 May;68(5):3448–3451. doi: 10.1128/jvi.68.5.3448-3451.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Schwartz S., Felber B. K., Benko D. M., Fenyö E. M., Pavlakis G. N. Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J Virol. 1990 Jun;64(6):2519–2529. doi: 10.1128/jvi.64.6.2519-2529.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schwartz S., Felber B. K., Pavlakis G. N. Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein. J Virol. 1992 Jan;66(1):150–159. doi: 10.1128/jvi.66.1.150-159.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Seiki M., Hattori S., Yoshida M. Human adult T-cell leukemia virus: molecular cloning of the provirus DNA and the unique terminal structure. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6899–6902. doi: 10.1073/pnas.79.22.6899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Silver P. A. How proteins enter the nucleus. Cell. 1991 Feb 8;64(3):489–497. doi: 10.1016/0092-8674(91)90233-o. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Siomi H., Shida H., Nam S. H., Nosaka T., Maki M., Hatanaka M. Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing. Cell. 1988 Oct 21;55(2):197–209. doi: 10.1016/0092-8674(88)90043-8. [DOI] [PubMed] [Google Scholar]
  51. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  52. Solomin L., Felber B. K., Pavlakis G. N. Different sites of interaction for Rev, Tev, and Rex proteins within the Rev-responsive element of human immunodeficiency virus type 1. J Virol. 1990 Dec;64(12):6010–6017. doi: 10.1128/jvi.64.12.6010-6017.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stauber R., Gaitanaris G. A., Pavlakis G. N. Analysis of trafficking of Rev and transdominant Rev proteins in living cells using green fluorescent protein fusions: transdominant Rev blocks the export of Rev from the nucleus to the cytoplasm. Virology. 1995 Nov 10;213(2):439–449. doi: 10.1006/viro.1995.0016. [DOI] [PubMed] [Google Scholar]
  54. Weichselbraun I., Berger J., Dobrovnik M., Bogerd H., Grassmann R., Greene W. C., Hauber J., Böhnlein E. Dominant-negative mutants are clustered in a domain of the human T-cell leukemia virus type I Rex protein: implications for trans dominance. J Virol. 1992 Jul;66(7):4540–4545. doi: 10.1128/jvi.66.7.4540-4545.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Zazopoulos E., Sodroski J. G., Haseltine W. A. p21rex protein of HTLV-1. J Acquir Immune Defic Syndr. 1990;3(12):1135–1139. [PubMed] [Google Scholar]

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