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. 1995 Aug;63(8):3073–3078. doi: 10.1128/iai.63.8.3073-3078.1995

Purification and characterization of a Shiga toxin A subunit-CD4 fusion protein cytotoxic to human immunodeficiency virus-infected cells.

A Y al-Jaufy 1, S R King 1, M P Jackson 1
PMCID: PMC173419  PMID: 7622233

Abstract

In a previous paper, we reported that a chimeric toxin composed of the enzymatic domain of the Shiga toxin A polypeptide (StxA1) genetically fused to the human CD4 (hCD4) molecule selectively kills cells infected with human immunodeficiency virus type 1 (HIV-1). Although other hCD4-containing chimeras cytotoxic to HIV-infected cells have been developed, there is limited information regarding their receptor binding and internalization. Therefore, the goals of this study were to purify the StxA1-hCD4 fusion protein, identify the receptor(s), and investigate the cytosolic trafficking route used by the chimeric toxin. Sufficient quantities of the StxA1-hCD4 hybrid were isolated for this investigation by using the pET expression and purification system. Cos-1 cells were rendered sensitive to the StxA1-hCD4 chimera by transfection with the env gene, which encodes HIV-1 envelope glycoproteins. The entry and translocation pathway used by the StxA1-hCD4 hybrid toxin was investigated by assessing the protective capacities of chemical reagents which interfere with microfilament movement, acidification of endosomes, and the integrity of the Golgi apparatus. Our findings indicated that the chimera uses HIV-1 glycoprotein gp120, and perhaps gp41, as a receptor which directs its entry through receptor cycling. Uptake is pH independent, and the StxA1-hCD4 hybrid is apparently translocated to the Golgi complex as with other bipartite toxins.

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Selected References

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  1. Aullo P., Alcami J., Popoff M. R., Klatzmann D. R., Murphy J. R., Boquet P. A recombinant diphtheria toxin related human CD4 fusion protein specifically kills HIV infected cells which express gp120 but selects fusion toxin resistant cells which carry HIV. EMBO J. 1992 Feb;11(2):575–583. doi: 10.1002/j.1460-2075.1992.tb05089.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chaudhary V. K., Mizukami T., Fuerst T. R., FitzGerald D. J., Moss B., Pastan I., Berger E. A. Selective killing of HIV-infected cells by recombinant human CD4-Pseudomonas exotoxin hybrid protein. Nature. 1988 Sep 22;335(6188):369–372. doi: 10.1038/335369a0. [DOI] [PubMed] [Google Scholar]
  3. Dalgleish A. G., Beverley P. C., Clapham P. R., Crawford D. H., Greaves M. F., Weiss R. A. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature. 1984 Dec 20;312(5996):763–767. doi: 10.1038/312763a0. [DOI] [PubMed] [Google Scholar]
  4. Eiden L. E., Lifson J. D. HIV interactions with CD4: a continuum of conformations and consequences. Immunol Today. 1992 Jun;13(6):201–206. doi: 10.1016/0167-5699(92)90154-Y. [DOI] [PubMed] [Google Scholar]
  5. Endo Y., Tsurugi K., Yutsudo T., Takeda Y., Ogasawara T., Igarashi K. Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins. Eur J Biochem. 1988 Jan 15;171(1-2):45–50. doi: 10.1111/j.1432-1033.1988.tb13756.x. [DOI] [PubMed] [Google Scholar]
  6. Feener E. P., Shen W. C., Ryser H. J. Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes. J Biol Chem. 1990 Nov 5;265(31):18780–18785. [PubMed] [Google Scholar]
  7. Folks T. M., Powell D., Lightfoote M., Koenig S., Fauci A. S., Benn S., Rabson A., Daugherty D., Gendelman H. E., Hoggan M. D. Biological and biochemical characterization of a cloned Leu-3- cell surviving infection with the acquired immune deficiency syndrome retrovirus. J Exp Med. 1986 Jul 1;164(1):280–290. doi: 10.1084/jem.164.1.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gentry M. K., Dalrymple J. M. Quantitative microtiter cytotoxicity assay for Shigella toxin. J Clin Microbiol. 1980 Sep;12(3):361–366. doi: 10.1128/jcm.12.3.361-366.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  10. Haddad J. E., al-Jaufy A. Y., Jackson M. P. Minimum domain of the Shiga toxin A subunit required for enzymatic activity. J Bacteriol. 1993 Aug;175(16):4970–4978. doi: 10.1128/jb.175.16.4970-4978.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kimpton J., Emerman M. Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. J Virol. 1992 Apr;66(4):2232–2239. doi: 10.1128/jvi.66.4.2232-2239.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Klatzmann D., Barré-Sinoussi F., Nugeyre M. T., Danquet C., Vilmer E., Griscelli C., Brun-Veziret F., Rouzioux C., Gluckman J. C., Chermann J. C. Selective tropism of lymphadenopathy associated virus (LAV) for helper-inducer T lymphocytes. Science. 1984 Jul 6;225(4657):59–63. doi: 10.1126/science.6328660. [DOI] [PubMed] [Google Scholar]
  13. Lipsky N. G., Pagano R. E. A vital stain for the Golgi apparatus. Science. 1985 May 10;228(4700):745–747. doi: 10.1126/science.2581316. [DOI] [PubMed] [Google Scholar]
  14. Maddon P. J., Dalgleish A. G., McDougal J. S., Clapham P. R., Weiss R. A., Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell. 1986 Nov 7;47(3):333–348. doi: 10.1016/0092-8674(86)90590-8. [DOI] [PubMed] [Google Scholar]
  15. McCune J. M., Rabin L. B., Feinberg M. B., Lieberman M., Kosek J. C., Reyes G. R., Weissman I. L. Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell. 1988 Apr 8;53(1):55–67. doi: 10.1016/0092-8674(88)90487-4. [DOI] [PubMed] [Google Scholar]
  16. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  17. Pelham H. R. The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci. 1990 Dec;15(12):483–486. doi: 10.1016/0968-0004(90)90303-s. [DOI] [PubMed] [Google Scholar]
  18. Perez L. G., O'Donnell M. A., Stephens E. B. The transmembrane glycoprotein of human immunodeficiency virus type 1 induces syncytium formation in the absence of the receptor binding glycoprotein. J Virol. 1992 Jul;66(7):4134–4143. doi: 10.1128/jvi.66.7.4134-4143.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sandvig K., Olsnes S., Brown J. E., Petersen O. W., van Deurs B. Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1. J Cell Biol. 1989 Apr;108(4):1331–1343. doi: 10.1083/jcb.108.4.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sandvig K., Ryd M., Garred O., Schweda E., Holm P. K., van Deurs B. Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP. J Cell Biol. 1994 Jul;126(1):53–64. doi: 10.1083/jcb.126.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schneider J., Kent S. B. Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease. Cell. 1988 Jul 29;54(3):363–368. doi: 10.1016/0092-8674(88)90199-7. [DOI] [PubMed] [Google Scholar]
  22. Stein B. S., Gowda S. D., Lifson J. D., Penhallow R. C., Bensch K. G., Engleman E. G. pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane. Cell. 1987 Jun 5;49(5):659–668. doi: 10.1016/0092-8674(87)90542-3. [DOI] [PubMed] [Google Scholar]
  23. Strockbine N. A., Jackson M. P., Sung L. M., Holmes R. K., O'Brien A. D. Cloning and sequencing of the genes for Shiga toxin from Shigella dysenteriae type 1. J Bacteriol. 1988 Mar;170(3):1116–1122. doi: 10.1128/jb.170.3.1116-1122.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  25. Till M. A., Ghetie V., Gregory T., Patzer E. J., Porter J. P., Uhr J. W., Capon D. J., Vitetta E. S. HIV-infected cells are killed by rCD4-ricin A chain. Science. 1988 Nov 25;242(4882):1166–1168. doi: 10.1126/science.2847316. [DOI] [PubMed] [Google Scholar]
  26. Wagner M., Rajasekaran A. K., Hanzel D. K., Mayor S., Rodriguez-Boulan E. Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells. J Cell Sci. 1994 Apr;107(Pt 4):933–943. doi: 10.1242/jcs.107.4.933. [DOI] [PubMed] [Google Scholar]
  27. al-Jaufy A. Y., Haddad J. E., King S. R., McPhee R. A., Jackson M. P. Cytotoxicity of a shiga toxin A subunit-CD4 fusion protein to human immunodeficiency virus-infected cells. Infect Immun. 1994 Mar;62(3):956–960. doi: 10.1128/iai.62.3.956-960.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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