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
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.