CD4-chemokine receptor hybrids in human immunodeficiency virus type 1 infection

Most human immunodeficiency virus (HIV strains require both CD4 and a chemo kine receptor for entry into a host cell. In order to analyze how the HIV-1 envelope glycoprotein interacts with these cellular molecules, we construc ted single-molecule hybrids of CD4 and chemokine receptors and expressed th ese constructs in the mink cell line Mv-1-lu. The two N-terminal (2D) or al l four (4D) extracellular domains of CD4 were linked to the N terminus of t he chemokine receptor CXCR4. The CD4(2D)CXCR4 hybrid mediated infection by HIV-1(LAI) to nearly the same extent as the wild-type molecules, whereas CD 4(4D)CXCR4 was less efficient. Recombinant SULA1 protein competed more effi ciently with the CXCR4-specific monoclonal antibody 12G5 for binding to CD4 (2D)CXCR4 than for binding to CD4(4D)CXCR4. Stromal cell-derived factor 1 ( SDF-1) blocked HIV-1, infection of cells expressing CD4(2D)CXCR4 less effic iently than for cells expressing wild-type CXCR4 and CD4, whereas down-modu lation of CXCR4 by SDF-1 was similar for hybrids and wild-type CXCR4. In co ntrast, the bicyclam AMD3100, a nonpeptide CXCR4 ligand that did not down-m odulate the hybrids, blocked hybrid-mediated infection at least as potently as for wild-type CXCR4. Thus SDF-1, but not the smaller molecule AMD3100, may interfere at multiple points with the binding of the surface unit (SU)- CD4 complex to CXCR4, a mechanism that the covalent linkage of CD4 to CXCR4 impedes. Although the CD4-CXCR4 hybrids yielded enhanced SU interactions w ith the chemokine receptor moiety, this did not overcome the specific corec eptor requirement of different HIV-1 strains: the X4 virus HIV-1(LAI) and t he X4R5 virus HIV-1(89.6) unlike the R5 strain HIV-1(SF162) infected Mv-1-l u cells expressing the CD4(2D)CXCR4 hybrid, but none could use hybrids of C D4 and the chemokine receptor CCR2b, CCR5, or CXCR2. Thus single-molecule h ybrid constructs that mimic receptor-coreceptor complexes can be used to di ssect coreceptor function and its inhibition.