Anne Brelot

Identification of Residues of CXCR4 Critical for Human Immunodeficiency Virus Coreceptor and Chemokine Receptor Activities

CXCR4 is a G-coupled receptor for the stromal cell-derived factor (SDF-1) chemokine, and a CD4-associated human immunodeficiency virus type 1 (HIV-1) coreceptor. These functions were studied in a panel of CXCR4 mutants bearing deletions in the NH2-terminal extracellular domain (NT) or substitutions in the NT, the extracellular loops (ECL), or the transmembrane domains (TMs). The coreceptor activity of CXCR4 was markedly impaired by mutations of two Tyr residues in NT (Y7A/Y12A) or at a single Asp residue in ECL2 (D193A), ECL3 (D262A), or TMII (D97N). These acidic residues could engage electrostatical interactions with basic residues of the HIV-1 envelope protein gp120, known to contribute to the selectivity for CXCR4. The ability of CXCR4 mutants to bind SDF-1 and mediate cell signal was consistent with the two-site model of chemokine-receptor interaction. Site I involved in SDF-1 binding but not signaling was located in NT with particular importance of Glu14 and/or Glu15 and Tyr21. Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp187), TMII (Asp97), and TMVII (Glu288). The first residues (2-9) of NT also seem required for SDF-1 binding and signaling. A deletion in the third intracellular loop abolished signaling, probably by disrupting the coupling with G proteins. The identification of CXCR4 residues involved in the interaction with both SDF-1 and HIV-1 may account for the signaling activity of gp120 and has implications for the development of antiviral compounds.

Antigenically Distinct Conformations of CXCR4

ABSTRACT The major human immunodeficiency virus type 1 (HIV-1) coreceptors are the chemokine receptors CCR5 and CXCR4. The patterns of expression of the major coreceptors and their use by HIV-1 strains largely explain viral tropism at the level of entry. However, while virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by a number of factors, including receptor concentration, affinity between envelope gp120 and receptors, and potentially receptor conformation. Indeed, seven-transmembrane domain receptors, such as CCR5, can exhibit conformational heterogeneity, although the significance for virus infection is uncertain. Using a panel of monoclonal antibodies (MAbs) to CXCR4, we found that CXCR4 on both primary and transformed T cells as well as on primary B cells exhibited considerable conformational heterogeneity. The conformational heterogeneity of CXCR4 explains the cell-type-dependent ability of CXCR4 antibodies to block chemotaxis to stromal cell-derived factor 1α and to inhibit HIV-1 infection. In addition, the MAb most commonly used to study CXCR4 expression, 12G5, recognizes only a subpopulation of CXCR4 molecules on all primary cell types analyzed. As a result, CXCR4 concentrations on these important cell types have been underestimated to date. Finally, while the factors responsible for altering CXCR4 conformation are not known, we found that they do not involve CXCR4 glycosylation, sulfation of the N-terminal domain of CXCR4, or pertussis toxin-sensitive G-protein coupling. The fact that this important HIV-1 coreceptor exists in multiple conformations could have implications for viral entry and for the development of receptor antagonists.

Determination of Coreceptor Usage of Human Immunodeficiency Virus Type 1 from Patient Plasma Samples by Using a Recombinant Phenotypic Assay

ABSTRACT We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populations with a different coreceptor (CCR5 or CXCR4) usage specificity present in each plasma sample. This procedure detected mixtures of CCR5- and CXCR4-exclusive virus populations as well as dualtropic viral variants, in variable proportions. Sequence analysis of dualtropic clones indicated that changes in the V3 loop are necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5. This convenient technique can greatly assist the study of virus evolution and compartmentalization in infected individuals.

Cooperation of the V1/V2 and V3 Domains of Human Immunodeficiency Virus Type 1 gp120 for Interaction with the CXCR4 Receptor

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry is triggered by the interaction of the gp120 envelope glycoprotein with a cellular chemokine receptor, either CCR5 or CXCR4. We have identified different mutations in human CXCR4 that prevent efficient infection by one HIV-1 strain (NDK) but not another (LAI) and sought to define these strain-dependent effects at the gp120 level. The lack of activity toward the NDK strain of the HHRH chimeric CXCR4 in which the second extracellular loop (ECL2) derived from the rat CXCR4 and of CXCR4 with mutations at an aspartic acid in ECL2 (D193A and D193R) was apparently due to the sequence of the third variable loop (V3) of gp120, more precisely, to its C-terminal part. Indeed, substitution of the LAI V3 loop or only its C-terminal part in the NDK gp 120 context was sufficient to restore usage of the HHRH, D193A, and D193R receptors. The same result was achieved upon mutation of a single lysine residue of the NDK V3 loop to alanine (K319A) but not to arginine (K319R). These results provide a strong case for a direct interaction between the gp120 V3 loop and the ECL2 domain of CXCR4. By contrast, V3 substitutions had no effect on the inability of NDK to infect cells via a mutant CXCR4 in which the amino-terminal extracellular domain (NT) is deleted. In experiments with a set of chimeric NDK-LAI gp120s, the V1/V2 region from LAI gp120 was both necessary and sufficient for usage of the NT-deleted CXCR4. Different variable domains of gp120 can therefore cooperate for a functional interaction with CXCR4.

Identification of a Postendocytic Sorting Sequence in CCR5

The chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family (GPCR), is used by human immunodeficiency virus type 1 (HIV-1) with a R5 tropism as an entry receptor in addition to CD4. It is a key target for an antiviral action aiming at inhibiting the HIV-1 entry process. Only few data are available today regarding the mechanism involved in the intracellular trafficking process of CCR5. Understanding how CCR5 cell surface expression is regulated is particularly important with regard to HIV-1 entry inhibition. We set out to investigate whether CCR5 molecular determinants were involved in the postendocytic recycling and degradative pathways. We constructed progressive deletion mutants of the C-terminal domain of CCR5 that we stably expressed in HEK293 cells. All of the deletion mutants were expressed at the cell surface and were functional HIV-1 receptors. The deletion mutants were internalized after stimulation, but they lost their ability to recycle to the plasma membrane. They were rerouted toward a lysosomal degradative pathway. We identified here a sequence of four amino acids, present at the extreme C terminus of CCR5, that is necessary for the recycling of the internalized receptor, independently of its phosphorylation. A detailed analysis of this sequence indicated that the four amino acids acted as a postsynaptic density 95/discs-large/zona occludens (PDZ) interacting sequence. These results show that the CCR5 cytoplasmic domain bears a sequence similar to the “recycling signals” previously identified in other GPCRs. Drugs able to disrupt the recycling pathway of CCR5 may constitute promising tools for therapeutic treatment.

On the dimerization of CCR5

To the editor: In the February 2004 issue of Nature Immunology, Hernanz-Falcon et al. reported that the combination of two point mutations (I52V and V150A) in the first and fourth transmembrane domains of the CCR5 chemokine receptor disrupted its ability to form dimers, as shown by both biochemistry and resonance energy transfer–based approaches1. The authors also showed that the formation of oligomers of CCR5 is essential for triggering signaling but is dispensable for its correct expression on the cell surface and its ability to bind chemokines. The properties of this form of CCR5 (CCR5mut) offered unique perspectives regarding the mechanism and function of seven-transmembrane receptor dimerization. They were also of considerable interest for studies of human immunodeficiency virus 1 (HIV-1) cell entry, because CCR5 is the principal CD4-associated HIV-1 coreceptor, and dimerization has been proposed to modulate its activity2. The availability of CCR5mut therefore allowed us to address the coreceptor activity of a strictly monomeric form of CCR5. We have expressed wild-type CCR5 and CCR5mut in CD4+ target cells and found no differences in their ability to mediate HIV-1 entry or fusion with cells expressing HIV-1 envelope proteins, in agreement with their similar cell surface receptor expression (Supplementary Table 1 online). Notably, incubation with the chemokine MIP-1β promoted efficient endocytosis of CCR5mut, confirming that the mutations did not impair ligand binding (Supplementary Table 1 online). Based on the findings of Hernanz-Falcon et al., these results would indicate that putative CCR5mut monomers remain functional HIV-1 coreceptors. To confirm this conclusion, we directly addressed the oligomeric status of CCR5mut by coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments, which have been used to demonstrate that dimerization of CCR5 is a constitutive process3. The association of epitope-tagged forms of CCR5mut was demonstrated by coimmunoprecipitation of lysates of HEK 293 cells and was comparable to that of wild-type CCR5 (Supplementary Fig. 1 online). BRET assays of the same cells showed that wild-type CCR5 and CCR5mut formed homoor heterodimers with a similar efficiency in intact cells but did not associate with an unrelated seven-transmembrane receptor (Supplementary Fig. 1 online). These experiments indicated that the mutations I52V and V150A do not impair the formation of CCR5 dimers, which is in disagreement with the report of HernanzFalcon et al. At present the precise reason for these discrepancies cannot be ascertained, but it could relate to the use of different techniques to detect dimers. The numerous causes for potential misinterpretations have been reviewed4,5. The loss of FRET signal, which was considered by Hernanz-Falcon et al. as a strong evidence of impaired dimerization, can be found after conformation changes affecting the spatial orientation of donor and acceptor without disturbing dimerization. Thus, it could be envisioned that the mutations I52V and V150A influence the conformation of CCR5 in a relatively subtle way, thereby affecting its coupling to the cell signaling machinery, but do not abrogate its dimerization. Although the issue of possible functional differences between CCR5 (or other seven-transmembrane receptors) monomers or dimers remains important, it may well be elusive, because a growing number of observations5 indicates that early dimerization of seven-transmembrane receptors is a prerequisite for their proper targeting to the cell surface.

HIV-1 entry and how to block it.

Our understanding of the process by which the HIV enters cells and initiates its replication has considerably evolved over the past few years. Among the most spectacular achievements are the identification of novel cellular factors behaving as viral receptors the elucidation at least partial of the spatial structure of the viral envelope proteins and the discovery of compounds efficiently blocking the cell entry process. Knowledge has been growing at an unprecedented pace allowing questions left open since the HIV disease emerged some 20 years ago to meet their answer. In this brief article we shall attempt to summarize the recent evolution of this field of HIV research and discuss the promises of antiviral strategies based on the novel findings. But we also would like to show that a number of important questions remain unsolved. The issues that will be discussed represent a selection perhaps arbitrary within the research field of HIV entry for which both recent and excellent reviews are available [1-3]. We focus on the type 1 virus (HIV-1) for which there is considerably more information than for the related human (HIV-2) simian or feline immunodeficiency viruses. (excerpt)

CCR5 adopts three homodimeric conformations that control cell surface delivery

Distinct dimeric conformations of the HIV-1 co-receptor CCR5 govern its appearance at the cell surface. CCR5 delivery to the cell surface The chemokine receptor CCR5 is a class A GPCR and co-receptor for HIV-1 infection. How class A GPCRs dimerize and how it affects their function are unclear. Jin et al. analyzed the structures of related receptors and identified CCR5 residues that mediated homodimerization. Cross-linking and energy transfer experiments and monitoring the release of receptors from the endoplasmic reticulum identified two dimeric CCR5 conformations that directed its delivery to the plasma membrane. A third CCR5 homodimer was stabilized by maraviroc, a clinically used inhibitor that binds to CCR5 and inhibits its interaction with HIV-1. These data increase our understanding of class A GPCR dimerization and provide insight into the mechanisms of inhibiting HIV-1 entry. Biophysical methods and x-ray crystallography have revealed that class A G protein–coupled receptors (GPCRs) can form homodimers. We combined computational approaches with receptor cross-linking, energy transfer, and a newly developed functional export assay to characterize the residues involved in the dimerization interfaces of the chemokine receptor CCR5, the major co-receptor for HIV-1 entry into cells. We provide evidence of three distinct CCR5 dimeric organizations, involving residues of transmembrane helix 5. Two dimeric states corresponded to unliganded receptors, whereas the binding of the inverse agonist maraviroc stabilized a third state. We found that CCR5 dimerization was required for targeting the receptor to the plasma membrane. These data suggest that dimerization contributes to the conformational diversity of inactive class A GPCRs and may provide new opportunities to investigate the cellular entry of HIV-1 and mechanisms for its inhibition.