Abstract
CCR5 acts as the principal coreceptor during HIV-1 transmission and early stages of infection. Efficient HIV-1 entry requires a series of processes, many dependent on the conformational state of both viral envelope protein and cellular receptor. Monoclonal antibodies (MAbs) are able to identify different CCR5 conformations, allowing for their use as probes to distinguish CCR5 populations. Not all CCR5 MAbs are able to reduce HIV-1 infection, suggesting the use of select CCR5 populations for entry. In the U87.CD4.CCR5-GFP cell line, we used such HIV-1-restricting MAbs to probe the relation between localization, trafficking and G protein association for individual CCR5 conformations. We find that CCR5 conformations not only exhibit different localization and abundance patterns throughout the cell, but that they also display distinct sensitivities to endocytosis inhibition. Using chemokine analogs that vary in their HIV-1 inhibitory mechanisms, we also illustrate that responses to ligand engagement are conformation-specific. Additionally, we provide supporting evidence for the select sensitivity of conformations to G protein association. Characterizing the link between the function and dynamics of CCR5 populations has implications for understanding their selective targeting by HIV-1 and for the development of inhibitors that will block CCR5 utilization by the virus.
Highlights
C-Chemokine Receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that functions as the primary HIV-1 coreceptor during transmission and early stages of infection
Using a panel of CCR5 Monoclonal antibodies (MAbs) that were inhibitory to various HIV-1 isolates as probes for CCR5 conformations [31], we investigated whether the cellular distribution of such surface CCR5 populations was distinct
MAb 45531 was previously demonstrated to stain in clusters and bind to membrane regions high in cholesterol [31].We observed that MAb 45531, as well as MC-5, typically bound in select regions of the plasma membrane while all other MAbs were more abundant and uniformly distributed throughout the surface of U87.CD4.CCR5-GFP cells (Figure 1A-D)
Summary
C-Chemokine Receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that functions as the primary HIV-1 coreceptor during transmission and early stages of infection. Much emphasis has been placed on inhibiting HIV-1 utilization of surface CCR5 through the development of several CCR5 antagonists [7,8,9] One such antagonist is Maraviroc (MVC), a CCR5 inhibitor approved for the treatment of HIV-1 infection, which blocks receptor activation without masking the binding sites for chemokines and the HIV envelope glycoprotein [7,10,11]. Several groups have exploited the natural chemokine mechanisms of restriction by modifying the N-terminus of RANTES to produce CCR5 small-molecule inhibitors Such RANTES analogs have different effects on CCR5 internalization and signaling, and have been demonstrated to be potent HIV-1 inhibitors [14,15,16,17,18]
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