Abstract
CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1alpha, or MIP-1beta. This mAb inhibited most of the RANTES and MIP-1alpha chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1-derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.
Highlights
We sought to generate mAbs to CCR5 to inhibit the various functions of this molecule, and to understand how different CCR5 domains bind chemokines and HIV-1
2D7 showed an identical pattern of reactivity against human leukocytes, as previously noted for our other anti-CCR5 mAbs [19, 48]
We used the blocking mAb to determine the importance of CCR5 for T cell and monocyte responses to RANTES, macrophage inflammatory protein (MIP)-1␣, and MIP-1
Summary
Several CCR5/CCR2 chimeras (C25-01 to C25-14) were constructed by transferring restriction fragments flanked by the common BamHI, AfIII, ClaI, EcoRI, and XbaI sites between CCR5 and CCR2b. The construction and characterization of these chimeras have been previously described [35]. The constructs were transferred into a bicistronic vector [41], under dependence of the elongation factor 1a promoter, and transfected in Chinese hamster ovary-K1 cells as previously described [42]. The CD3ϩ blasts were generated using anti-CD3 antibody TR77 and maintained in medium supplemented with recombinant human IL-2 as previously described [19]. Other cell lines used included THP-1 and transfectants of the L1.2 murine pre–B cell lymphoma, expressing high levels of CCR5 [19, 26] or CXCR4. The different transfectants were monitored for expression of the relevant receptors, using specific mAbs [15, 19]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
