Human immunodeficiency virus type-1 (HIV-1) envelope
glycoprotein gp140 interacts with its specific receptors on the surface of the target cells leading to cellular activation through various signaling pathways. The effect of blocking the
chemokine repertoire in human brain microvascular endothelial cells in
HIV dementia (HAD) disease has not been reported. Characterizing the nature of
HIV-1 envelope protein gp140 (T-tropic, HXBc2) receptor binding conditions to HBMEC is critical to gain insight into the
HIV dementia, and eventually to rationally design the agents to block envelope
protein receptor interactions. HIV-1
gp140 oligomers were purified and separated to monomers, dimers, and trimers. The binding conditions of
gp140 to HBMEC
chemokine receptor, CXCR4, were optimized with an aim of understanding the structural interactions in HAD. Analysis of the interaction between HIV-1
gp140 and CXCR4 of HBMEC by saturation binding, cross-competition analysis with radiolabeled SDF and
gp140, revealed a strong interaction, specificity between HIV-1
gp140 and CXCR4. Our binding data demonstrate that
HIV-1 envelope protein gp140 enters cells by
protein receptor mediated interactions that are regulated by the conformational state of the
gp140 at physiological environment (pH and temperature). The CXCR4 antibody 12G5 inhibited SDF-1 binding to HBMEC indicating the specificity of
gp140 binding to HBMEC. Scatchard analysis revealed the presence of approximately 70250
gp140 binding sites per cell with a K(d) of 4.5 nM. Cross-competition experiments using labeled SDF-1 and
gp140 revealed that both unlabeled SDF-1 and
gp140 are capable of displacing their radiolabeled counterparts. The binding assay conditions and radioligand binding assay are highly valuable to identify and design better HIV inhibitors for HAD.