Mutagenesis of CXCR4 identifies important domains for human immunodeficiency virus type 1 X4 isolate envelope-mediated membrane fusion and virus entry and reveals cryptic coreceptor activity for R5 isolates

CXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates . Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelop e glycoprotein (Env)-expressing cells. The delineation of the critical regi ons involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of corecep tor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mut agenesis of the extracellular domains of CXCR4 coupled with a highly sensit ive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a pan el of 41 different CXCR4 mutants, we have identified several charged residu es that appear important for coreceptor activity for X4 Ems; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alan ine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Ems. In additio n, substitution of alanine for any of the four extracellular cysteines alon e resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure, Our data sup port the notion that all four cysteines are involved in disulfide bond form ation. We have also identified substitutions which greatly enhance or conve rt CXCR4's coreceptor activity to support R5 Env-mediated fusion (N11A, R30 A, D187A, and D193A), and together our data suggest the presence of conserv ed extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direc t further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.