Involvement of the Second Extracellular Loop and Transmembrane Residues of CCR5 in Inhibitor Binding and HIV-1 Fusion: Insights into the Mechanism of Allosteric Inhibition

Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
Journal of Molecular Biology (Impact Factor: 4.33). 09/2008; 381(4):956-974. DOI: 10.1016/j.jmb.2008.06.041


C-C chemokine receptor 5 (CCR5), a member of G-protein-coupled receptors, serves as a coreceptor for human immunodeficiency virus type 1 (HIV-1). In the present study, we examined the interactions between CCR5 and novel CCR5 inhibitors containing the spirodiketopiperazine scaffolds AK530 and AK317, both of which were lodged in the hydrophobic cavity located between the upper transmembrane domain and the second extracellular loop (ECL2) of CCR5. Although substantial differences existed between the two inhibitors—AK530 had 10-fold-greater CCR5-binding affinity (Kd = 1.4 nM) than AK317 (16.7 nM)—their antiviral potencies were virtually identical (IC50 = 2.1 nM and 1.5 nM, respectively). Molecular dynamics simulations for unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. Indeed, AK530 and AK317, when bound to CCR5, disrupted these interhelix hydrogen bond interactions, a salient molecular mechanism enabling allosteric inhibition. Mutagenesis and structural analysis showed that ECL2 consists of a part of the hydrophobic cavity for both inhibitors, although AK317 is more tightly engaged with ECL2 than AK530, explaining their similar anti-HIV-1 potencies despite the difference in Kd values. We also found that amino acid residues in the β-hairpin structural motif of ECL2 are critical for HIV-1-elicited fusion and binding of the spirodiketopiperazine-based inhibitors to CCR5. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but also prohibits HIV-1 from utilizing the “inhibitor-bound” CCR5 for cellular entry—a mechanism of HIV-1's resistance to CCR5 inhibitors. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants.

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    • "This supports a specific interaction with a binding partner, such as CCR5. In fact, the region between the second extracellular loop of CCR5 (ECL2) and parts of transmembrane helices of CCR5 contain pockets lined by hydrophobic and aromatic residues [20]–[22]. Moreover, these pockets are targeted by the CCR5 blocking drugs such as Maraviroc [23], Aplaviroc [22], or Vicriviroc [24] which all have hydrophobic and aromatic functional groups in arrangements similar to the side chains of I/L14 and F/W22. "
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    ABSTRACT: The epidemiology of HIV-1 in China has unique features that may have led to unique viral strains. We therefore tested the hypothesis that it is possible to find distinctive patterns in HIV-1 genomes sampled in China. Using a rule inference algorithm we could indeed extract from sequences of the third variable loop (V3) of HIV-1 gp120 a set of 14 signature patterns that with 89% accuracy distinguished Chinese from non-Chinese sequences. These patterns were found to be specific to HIV-1 subtype, i.e. sequences complying with pattern 1 were of subtype B, pattern 2 almost exclusively covered sequences of subtype 01_AE, etc. We then analyzed the first of these signature patterns in depth, namely that L and W at two V3 positions are specifically occurring in Chinese sequences of subtype B/B' (3% false positives). This pattern was found to be in agreement with the phylogeny of HIV-1 of subtype B inside and outside of China. We could neither reject nor convincingly confirm that the pattern is stabilized by immune escape. For further interpretation of the signature pattern we used the recently developed measure of Direct Information, and in this way discovered evidence for physical interactions between V2 and V3. We conclude by a discussion of limitations of signature patterns, and the applicability of the approach to other genomic regions and other countries.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "The pcDNA6.2-CCR5 and pcDNA6.2-HIV-tat plasmids were constructed as described previously35. Briefly, the entire human CCR5 gene including a stop codon was amplified using pZeoSV-CCR536 as a template. "
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    ABSTRACT: The third variable region (V3) of HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism). However, which combinations of mutations in V3 confer a tropism shift is still unclear. A unique pattern of mutations in antiretroviral therapy-naive HIV-1 patient was observed associated with the HIV-1 tropism shift CCR5 to CXCR4. The insertion of arginine at position 11 and the loss of the N-linked glycosylation site were indispensable for acquiring pure CXCR4-tropism, which were confirmed by cell-cell fusion assay and phenotype analysis of recombinant HIV-1 variants. The same pattern of mutations in V3 and the associated tropism shift were identified in two of 53 other patients (3.8%) with CD4(+) cell count <200/mm(3). The combination of arginine insertion and loss of N-linked glycosylation site usually confers CXCR4-tropism. Awareness of this rule will help to confirm the tropism prediction from V3 sequences by conventional rules.
    Full-text · Article · Aug 2013 · Scientific Reports
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    • ", 2010 ; Tilton and Doms , 2010 ). Maraviroc , similar to other CCR5 antagonists such as vicriviroc ( VVC ) and aplaviroc ( APL ) ( Dragic et al. , 2000 ; Maeda et al. , 2008 ; Seibert et al. , 2006 ) , is therefore an allosteric inhibitor of HIV - 1 entry. Maraviroc is currently being used as an HIV - 1 antiretroviral therapy for both treatment - experienced and antiretroviral therapy ( ART ) - naïve adults who have no evidence of CXCR4 using virus in their plasma ( Gorry et al. , 2010 ). "
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    ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) resistance to CCR5 antagonists, including maraviroc (MVC), results from alterations in the HIV-1 envelope glycoproteins (Env) enabling recognition of antagonist-bound CCR5. Here, we characterized tropism alterations for CD4+ T-cell subsets and macrophages by Envs from two subjects who developed MVC resistance in vivo, which displayed either relatively efficient or inefficient recognition of MVC-bound CCR5. We show that MVC-resistant Env with efficient recognition of drug-bound CCR5 displays a tropism shift for CD4+ T-cell subsets associated with increased infection of central memory T-cells and reduced infection of effector memory and transitional memory T-cells, and no change in macrophage infectivity. In contrast, MVC-resistant Env with inefficient recognition of drug-bound CCR5 displays no change in tropism for CD4+ T-cell subsets, but exhibits a significant reduction in macrophage infectivity. The pattern of HIV-1 tropism alterations for susceptible cells may therefore be variable in subjects with MVC resistance.
    Full-text · Article · Apr 2013 · Virology
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