HIV-1 entry requires target-cell expression of CD4 and a chemokine co-receptor. We have previously shown that the amino-terminus of the principal HIV-1 co-receptor CCR5 contains four tyrosines which are modified by sulfate. These sulfotyrosines, and the sulfate groups themselves, make critical contributions to HIV-1 entry and to chemokine association. In addition, we have shown that HIV-1- infected individuals can express neutralizing antibodies that recognize the co-receptor-binding region of gp120 and which are similarly modified in their heavy chain CDR3 region by tyrosine sulfation. Again, these sulfate groups directly participate in gp120 recognition. The co-receptor-binding region is among the most conserved on HIV-1 gp120; some antibodies that recognize the co-receptor-binding region are so broadly neutralizing that they also recognize the co-receptor-binding regions of HIV-2 and SIV gp120. Moreover, as recent structural studies show, the sulfate-binding region of gp120 is conserved in isolates that utilize CXCR4. This region is therefore a potentially important target of anti-viral small molecules, peptides, and antibodies. To the extent that these anti-viral therapeutics closely emulate CCR5, viral escape should impose a high fitness cost. To date, however, peptides based on the CCR5 amino-terminus have proved disappointing as therapeutics. It is technically difficult and expensive to synthesize tyrosine-sulfated peptides, and nearly impossible to make those containing more than two sulfate groups. Also, other regions of CCR5 bind gp120, and, as we show in this proposal, the CCR5 amino-terminus includes residues that do not contact gp120 and which interfere with binding of free peptide to gp120. We also show that peptides derived from the CDR3 regions of tyrosine-sulfated neutralizing antibodies more effectively block HIV-1 infection than do CCR5-based peptides, suggesting that further improvement of tyrosine-sulfated CCR5 mimetics may be possible. Very recently, the laboratory of Peter Schultz, a collaborator on this proposal, has developed a system for bacterial expression of tyrosine-sulfated proteins and peptides. This is an important development: bacterial expression makes feasible phage-based selection and the low-cost production of larger, disulfide-bonded peptides containing more than two sulfotyrosines. In the current proposal, we will use system and phage display technology to generate tyrosine-sulfated CCR5 mimetics selected for breadth and potency of HIV-1 neutralization. These studies will provide useful structural information on the interaction of sulfotyrosines with gp120, and will generate candidate therapeutics targeting a highly conserved region of the HIV-1 envelope glycoprotein. Sulfotyrosines at the amino-terminus of CCR5, the principal HIV-1 co-receptor, mediate association with the HIV-1 envelope glycoprotein and viral entry. Recent advances in bacterial expression of proteins and peptides containing sulfotyrosines permit use of phage-based selection systems to develop novel CCR5 mimetics. In this proposal, we will use structure-guided mutagenesis and phage display technology to identify soluble peptides that closely resemble CCR5, for structural studies and as candidate therapeutics. ? ? ? ? ?