Molecular mapping of epitopes for interaction of HIV-1 as well as natural ligands with the chemokine receptors, CCR5 and CXCR4.
OBJECTIVE
Mapping coreceptor epitopes used by the prototypic R5 and X4 strains, HIV-1BaL and HIV-1IIIB, in comparison with epitopes involved in the activation and signaling induced by the natural ligands, RANTES and SDF-1beta.


METHODS
Receptor hybrids between CCR5 and CXCR4 were constructed.


METHODS
Using single-overlap and extension PCR, increasing portions of CCR5 were replaced with corresponding parts of CXCR4. Viral interaction with these constructs was monitored in infection experiments using stably transfected cell lines, and ligand-induced activation of cells transiently expressing the constructs was measured in terms of calcium fluxes.


RESULTS
SDF-1beta required an essentially complete CXCR4, whereas RANTES demanded both the N terminus and the first two extracellular loops of CCR5. HIV-1 infection experiments emphasized the importance of the CCR5 N terminus for infection with HIV-1BaL, whereas HIV-1IIIB was less demanding in its use of CXCR4.


CONCLUSIONS
This study, for the first time monitoring CCR5 and CXCR4 ligand activation and HIV-1 interaction concomitantly, indicates that ligands and virus use different receptor epitopes which, in turn, vary between the two receptors. One particular chimera (FC-4b), having its junctional region close to the conserved cysteine in ECL2, functioned as coreceptor for both HIV-1BaL and HIV-1IIIB, but was not activated with RANTES or SDF-1beta. The results provide a basis for tailoring drugs that block viral entry through the two major coreceptors without interfering with their physiological function.
Antonsson
Liselotte
L
Division of Molecular Neurobiology, Wallenberg Neuroscience Center, Lund University, Sweden.
Boketoft
Ake
A
Garzino-Demo
Alfredo
A
Olde
Björn
B
Owman
Christer
C
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018946:Chemokine CCL5 / Q000235:genetics; D054377:Chemokine CXCL12; D002678:Chimera / Q000235:genetics; D006224:Cricetinae; D016207:Cytokines / Q000235:genetics; D000939:Epitopes / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods; D015497:HIV-1 / Q000235:genetics*; D008024:Ligands; D051379:Mice; D041681:NIH 3T3 Cells; D019713:Receptors, CCR5 / Q000235:genetics*; D019718:Receptors, CXCR4 / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection
D018946:Chemokine CCL5; D054377:Chemokine CXCL12; D016207:Cytokines; D000939:Epitopes; D008024:Ligands; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D011993:Recombinant Fusion Proteins; D002118:Calcium