PURIFICATION AND CHARACTERIZATION OF A SHIGA-TOXIN-A SUBUNIT-CD4 FUSION PROTEIN CYTOTOXIC TO HUMAN IMMUNODEFICIENCY VIRUS-INFECTED CELLS

Citation
Ay. Aljaufy et al., PURIFICATION AND CHARACTERIZATION OF A SHIGA-TOXIN-A SUBUNIT-CD4 FUSION PROTEIN CYTOTOXIC TO HUMAN IMMUNODEFICIENCY VIRUS-INFECTED CELLS, Infection and immunity, 63(8), 1995, pp. 3073-3078
Citations number
31
Categorie Soggetti
Immunology,"Infectious Diseases
Journal title
ISSN journal
00199567
Volume
63
Issue
8
Year of publication
1995
Pages
3073 - 3078
Database
ISI
SICI code
0019-9567(1995)63:8<3073:PACOAS>2.0.ZU;2-L
Abstract
In a previous paper, we reported that a chimeric toxin composed of the enzymatic domain of the Shiga toxin A polypeptide (StxAl) genetically fused to the human CD4 (hCD4) molecule selectively kills cells infect ed with human immunodeficiency virus type 1 (HIV-1). Although other hC D4-containing chimeras cytotoxic to HIV-infected cells have been devel oped, there is limited information regarding their receptor binding an d internalization. Therefore, the goals of this study were to purify t he StxA1-hCD4 fusion protein, identify the receptor(s), and investigat e the cytosolic trafficking route used by the chimeric toxin. Sufficie nt quantities of the StxA1-hCD4 hybrid were isolated for this investig ation by using the pET expression and purification system. Cos-1 cells were rendered sensitive to the StxA1-hCD4 chimera by transfection wit h the env gene, which encodes HIV-1 envelope glycoproteins. The entry and translocation pathway used by the StxA1-hCD4 hybrid toxin was inve stigated by assessing the protective capacities of chemical reagents w hich interfere with microfilament movement, acidification of endosomes , and the integrity df the Golgi apparatus. Our findings indicated tha t the chimera uses HIV-1 glycoprotein gp120, and perhaps gp41, as a re ceptor which directs its entry through receptor cycling. Uptake is pH independent, and the StxA1-hCD4 hybrid is apparently translocated to t he Golgi complex as with other bipartite toxins.