Purification and characterization of a Shiga toxin A subunit-CD4 fusion protein cytotoxic to human immunodeficiency virus-infected cells.
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.
al-Jaufy
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AY
Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
King
S R
SR
Jackson
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MP
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry*; D001483:Base Sequence; D020126:Brefeldin A; D015704:CD4 Antigens / Q000737:chemistry*; D002451:Cell Compartmentation / Q000187:drug effects; D002460:Cell Line; D002522:Chlorocebus aethiops; D002738:Chloroquine / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D003603:Cytotoxins / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D015658:HIV Infections / Q000628:therapy*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D022603:Shiga Toxins; D014709:Vero Cells
D001427:Bacterial Toxins; D015704:CD4 Antigens; D003517:Cyclopentanes; D003603:Cytotoxins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D022603:Shiga Toxins; D000643:Ammonium Chloride; D020126:Brefeldin A; D002738:Chloroquine