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Screening for inhibitors of HIV gp120-CD4 binding using an enzyme-linked immunoabsorbent assay
M Gilbert1, L Brigido, W E Müller
1Department of Hematology-Oncology, University of Washington, Seattle.
Insights
This study developed an assay to measure HIV-1 gp120 binding to CD4 cells, identifying inhibitors like aurintricarboxylic acid and dextran sulfate. The assay helps understand how these compounds block viral entry and syncytium formation.
Area of Science:
- Virology
- Immunology
- Biochemistry
Background:
- The binding of human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120 to the CD4 receptor is a critical step for viral infection and cell fusion.
- Understanding this interaction is key to developing antiviral therapies.
Purpose of the Study:
- To develop and validate an enzyme-linked immunoassay (ELISA) for quantifying HIV-1 gp120 binding to CD4.
- To assess the efficacy and mechanisms of action of potential inhibitors of the gp120-CD4 interaction.
Main Methods:
- An enzyme-linked immunoassay (ELISA) was employed using solid-phase CD4 to measure HIV-1 gp120 binding.
- Various inhibitors, including monoclonal antibodies, small molecules (aurintricarboxylic acid), polysaccharides (dextran sulfate), and lectins, were tested.
- The effect of gp120 deglycosylation and non-glycosylated gp120 on CD4 binding was also evaluated.
Main Results:
- Monoclonal antibodies targeting the gp120 binding site on CD4 effectively blocked gp120 binding.
- Aurintricarboxylic acid and dextran sulfate inhibited gp120-CD4 interactions by binding to CD4.
- Human polyclonal antibodies to gp120 blocked binding, but tested monoclonal antibodies did not.
- Lectins, such as mannose binding protein, inhibited binding by interacting with gp120.
- Enzymatic deglycosylation had minimal impact, but non-glycosylated gp120 showed significantly reduced binding to CD4.
Conclusions:
- The developed ELISA is a robust system for measuring inhibitors of HIV-1 gp120-CD4 binding.
- This assay facilitates the determination of the mechanism of action for identified inhibitors.
- Findings highlight the importance of gp120 glycosylation and identify specific molecular targets for therapeutic intervention.
Abstract:
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.
