274 Role of N-linked Glycosylation of CXCR4 in SDF-1 Binding and HIV-1 Infection J. Wang*1, G. J. Babcock1, H. Choe2, M. Farzan3, J. Sodroski1, D. Gabuzda1 1Dana-Farber Cancer Inst, Boston, MA; 2Children's Hosp, Boston, MA; and 3Partners AIDS Res Ctr, Cambridge, MA
Background: CXCR4 is a primary co-receptor for HIV-1, and also plays critical roles in immune responses and development. To better understand structural determinants of CXCR4 that play an important role in HIV-1 pathogenesis, we investigated cell-type dependent modifications of CXCR4 and their effects on SDF-1 and gp120 binding and HIV-1 infection.
Methods: Deglycosylated CXCR4-proteoliposomes and stable Cf2 cells expressing wild-type or mutant CXCR4 with a single point mutation at asparagine (N11) (X4-N11Q) were generated and used for studies on HIV-1 gp120 binding, calcium signaling, and HIV-1 entry.
Results: CXCR4 was modified in a cell-type dependent manner. Glycosylation at N11 was the major detectable post-translational modification. X4 and R5 HIV-1 gp120 bound more efficiently to deglycosylated than glycosylated CXCR4-proteoliposomes in a CD4-dependent manner. Similar results were observed in tunicamycin-treated Cf2-CXCR4 cells. Both X4 and R5 gp120 showed increased CD4-dependent binding to unglycosylated (X4-N11Q) compared to glycosylated (wild-type) CXCR4 expressed on cells. Furthermore, cells expressing CD4 and X4-N11Q allowed entry of R5-tropic HIV-1. In contrast, SDF-1 showed decreased binding and weaker calcium signaling in cells expressing X4-N11Q. Deletion of 24 aa in the N-terminus of CXCR4 diminished SDF-1 binding, but had no significant effect on binding of X4 or R5 gp120, and reduced the efficiency of HIV-1 entry.
Conclusions: These results suggest that the CXCR4 N-terminus (aa 2~25) contains critical binding sites for SDF-1, and plays an important role in HIV-1 entry. N-glycosylation at N11 enhances the ability of CXCR4 to bind SDF-1, but inhibits its ability to bind gp120 and mediate R5 HIV-1 entry. The finding that unglycosylated CXCR4 can mediate entry of R5 HIV-1 provides evidence for a conserved structure shared by CXCR4 and CCR5 that is important for Env co-receptor binding.
