Screening for inhibitors of HIV gp120-CD4 binding using an enzyme-linked immunoabsorbent assay.
Binding of the HIV-1 major viral surface glycoprotein, gp120, to the major cell receptor, CD4, is essential for HIV infection of the target cell and syncytium formation. An enzyme-linked immunoassay using solid phase CD4 was used to quantitate the binding of HIV-1 gp120 to CD4, and to assess the activity and mechanism of action of putative inhibitors of that reaction. Monoclonal antibodies to the gp120 binding site on CD4 (e.g., Leu3a) blocked gp120 binding, while monoclonal antibodies to other portions of CD4 (e.g. OKT4) did not. Both aurintricarboxylic acid and sulfonated polysaccharides (e.g., dextran sulfate) blocked CD4-gp120 interactions by binding to the CD4 component. Human polyclonal antibodies to gp120 also blocked gp120-CD4 binding, but none of the monoclonal antibodies tested (including several with neutralizing activity) were effective. In contrast, several lectins (including mannose binding protein) bound to gp120 and blocked CD4-gp120 interactions. Enzymatic deglycosylation of gp120 only minimally affected its CD4 binding capacity, while non-glycosylated gp120 (produced in Escherichia coli)-bound CD4 about 10-fold less well than fully-glycosylated material. The results demonstrate that this assay system can be used to measure the activity of inhibitors of CD4-gp120 binding, and to determine the mechanism of action of those inhibitors.
Gilbert
M
M
Department of Hematology-Oncology, University of Washington, Seattle.
Brigido
L
L
Müller
W E
WE
Hansen
J E
JE
Ezekowitz
R A
RA
Mills
J
J
D000998:Antiviral Agents; D015704:CD4 Antigens / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006023:Glycoproteins / Q000494:pharmacology; D015699:HIV Envelope Protein gp120 / Q000276:immunology / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D037102:Lectins / Q000494:pharmacology; D011134:Polysaccharides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results
D000998:Antiviral Agents; D015704:CD4 Antigens; D006023:Glycoproteins; D015699:HIV Envelope Protein gp120; D037102:Lectins; D011134:Polysaccharides; D011994:Recombinant Proteins