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2a). flips back the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry and may guide development of vaccines and therapeutics. Introduction HIV-1 envelope glycoprotein (Env) fuses viral and cell membranes, allowing entry of the virus into host cells. Its precursor, gp160, trimerizes to (gp160)3, which is usually cleaved into two noncovalently associated fragments: gp120 (receptor-binding) and gp41 (fusion)1. Three copies of each fragment constitute the mature viral spike (gp120/gp41)3. Sequential binding of gp120 to primary receptor CD4 and a coreceptor (e.g. CCR5 or CXCR4) are believed to induce conformational changes that trigger likely dissociation of gp120 and refolding of gp411. Structural rearrangements in gp41 bring the two membranes together, promoting membrane fusion. We Inosine pranobex have detailed structural information for the conversation of HIV-1 Env with CD42-6, but no molecular picture for its interaction with a coreceptor, which continues to be the subject of speculative molecular modeling7-10 The chemokine receptors, CCR5 and CXCR4, were identified as the HIV-1 coreceptors in 199611. They are G protein-coupled receptors (GPCRs) with seven transmembrane-spanning segments (7TMs). Choice of coreceptor is the major determinant for viral tropism11. Viruses using CCR5 (R5 viruses) are generally responsible for viral transmission; those using CXCR4 (X4 viruses) or both (dual-tropic; R5X4 viruses) emerge later during disease progression12,13. Both CCR5 and CXCR4 have an extracellular N-terminal segment, three extracellular loops (ECL), three intracellular loops (ICL) and a cytoplasmic C-terminal tail. Crystal structures have been reported for a Inosine pranobex C-terminally truncated CXCR4 made up of stabilizing mutations and a T4 lysozyme fusion, in complex with different ligands, and for a similarly modified CCR5 made up of a rubredoxin fusion in complex with either an anti-HIV drug, maraviroc or a modified chemokine, [5P7]CCL57,10,14,15. These structures show a typical 7TM helical bundle topology seen for other GPCRs (Extended Data Fig. 1). Consistent with the so-called two-site model16, the N-terminal segment of CXCR4 or CCR5 forms chemokine recognition site 1 (CRS1) to interact with the globular core domain name of chemokine, while their 7TM bundle forms a binding pocket (chemokine recognition site 2; CRS2) that accommodates the N-terminus of the chemokine. While they elucidate chemokine receptor function, these structures do not explain how CCR5 and CXCR4 function as HIV-1 coreceptors. Mutagenesis studies have mapped the gp120 binding site to the N-terminal segment and ECL2 for CCR5; and to the N-terminal segment, ECL2, and ECL3 for CXCR417,18. The footprint of the coreceptor on gp120 probably includes the V3 loop and the bridging sheet, a structure accessible only after CD4 binding19,20. Inosine pranobex The N-terminus of the coreceptor may contact the gp120 bridging sheet, while the tip of the V3 loop may insert into the coreceptor CRS220. Interactions of V3 with CCR5 or CXCR4 have also been modeled by molecular dynamics simulations and free energy calculations7-10. Tyrosine sulfation near the N-terminus only enhances HIV-1 entry for CCR5, but not for CXCR421,22. The C-terminal tail, made up of palmitoylation and phosphorylation sites, is required for efficient cell signaling, but not for Inosine pranobex HIV-1 coreceptor function23. Chemokines such as, MIP-1, MIP-1, CCL5/RANTES and CXCL12/SDF-1 block gp120 binding and prevent viral contamination11. Results To understand how a coreceptor functions, we have decided Inosine pranobex the structure of an unmodified CCR5 in complex with a full-length HIV-1 gp120 and a 4-domain name soluble CD4 by single-particle cryo-electron microscopy (cryo-EM). Purification of the CCR5 complex and Rabbit Polyclonal to OR51B2 structure determination To produce functional CCR5, we generated HEK293T or Expi293F cell lines stably expressing wildtype human CCR5 by our published protocol24. CCR5 on these cells was fully active as a chemokine receptor (Extended Data Fig. 2a), and it also formed a tight complex with HIV-1 gp120 in the presence of soluble CD4 (Extended Data Fig. 2b). Moreover, these cells fused efficiently with HIV-1 Env-expressing cells only when activated by soluble CD4 (Extended Data Fig. 2c), suggesting that this expressed CCR5 is usually a fully functional HIV-1 coreceptor. Finally, neither gp120 alone nor the gp120-CD4 complex could activate the G-protein mediated signaling pathways.