HIV-1 envelope protein gp140 binding studies to human brain microvascular endothelial cells.
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.
Mendu
Damodara Rao
DR
Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Park 256, Baltimore, MD 21287, USA. protein.massspec@gmail.com
Katinger
Hermann
H
Sodroski
Joseph
J
Kim
Kwang Sik
KS
D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001921:Brain / Q000098:blood supply* / Q000166:cytology; D015704:CD4 Antigens / Q000276:immunology; D002478:Cells, Cultured; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D015700:HIV Envelope Protein gp41 / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding / Q000187:drug effects; D011869:Radioligand Assay; D019718:Receptors, CXCR4 / Q000276:immunology / Q000378:metabolism; D013696:Temperature; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D015704:CD4 Antigens; D015700:HIV Envelope Protein gp41; D019718:Receptors, CXCR4; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1