Chemokine receptors are members of the seven transmembrane, G protein-coupled receptor superfamily, and in conjunction with CD4, act as coreceptors for human immunodeficiency virus type-1(HIV-1). Identified as the two principle coreceptors used by T cell line-tropic (X4 strain) and macrophagetropic (R5 strain) HIV-1 isolates, the chemokine receptors CXCR4 and CCR5 mediate direct fusion of the virus and target cell membranes. Chemokines specific for these HIV-1 coreceptors, such as the CXCR4- specific stromal cell-derived factor-1alpha (SDF-1alpha) and the CCR5-specific RANTES, MIP-1alpha and MIP-1beta, are potent inhibitors of HIV-1 infection. Antagonizing CXCR4 and CCR5 using chemokine-derived polypeptides or small-molecule compounds represents an attractive strategy for the development of anti-AIDS therapies. Growing evidence suggests that CXCR4 and CCR5 exist naturally as homodimers on the cell surface. As chemokines are known to function as monomers under normal physiological conditions, we hypothesize that covalently tethered SDF-1alpha and RANTES may bind more strongly to their cognate receptors than the monomeric forms due to the effect of molecular multivalency. This should, in principle, lead to inhibition of HIV-1 infection by tethered chemokine molecules at significantly lower molar concentrations than seen with monomeric proteins. Due to the prominent roles chemokine receptors play in HIV-1 transmission and pathogenesis, it is important that this hypothesis be tested. Such study will not only provide a definitive functional test for CXCR4 and CCR5 homodimerization, but more importantly, yield valuable insights into designing better receptor antagonists to block HIV-1 infection. Specific studies envisioned in this proposal are as follows: Specific Aim 1: Synthesize covalently tethered homodimeric SDF-1alpha and RANTES, respectively, with an inter-molecular linker peptide of varying length.Specific Aim 2: Test the hypothesis that tethered and linker-optimized SDF-1alpha and RANTES bind more strongly to CXCR4 and CCR5, respectively, than do monomers. Specific Aim 3: Test the hypothesis that tethered and linker-optimized SDF-1alpha and RANTES more potently inhibit X4 and R5 viruses, respectively, than do monomers. Our long-term objective is to delineate the structure function relationships for chemokines in HIV-1 inhibition and to develop chemokine-derived, novel antagonistic molecules for anti-AIDS therapies.