Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion

The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp 3.49(125) and Arg 6.32(225) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr 2.56(82) , in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr 2.56(82) mutants were fully stabilized in active conformations. The Thr 2.56(82) Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr 2.56(82) Lys mutation with an Arg 6.32(225) Gln mutation partially reversed the decrease in expression. Mutants with Thr 2.56(82) Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr 2.65(82) Pro substitution exhibited full co-receptor function. Our results suggest that the Thr 2.65(82) Lys and Thr 2.65(82) Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion

Filamin A Binds to CCR2B and Regulates Its Internalization

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures

Independent and combined effects of improved water, sanitation, and hygiene, and improved complementary feeding, on child stunting and anaemia in rural Zimbabwe: a cluster-randomised trial.

BACKGROUND: Child stunting reduces survival and impairs neurodevelopment. We tested the independent and combined effects of improved water, sanitation, and hygiene (WASH), and improved infant and young child feeding (IYCF) on stunting and anaemia in in Zimbabwe. METHODS: We did a cluster-randomised, community-based, 2 × 2 factorial trial in two rural districts in Zimbabwe. Clusters were defined as the catchment area of between one and four village health workers employed by the Zimbabwe Ministry of Health and Child Care. Women were eligible for inclusion if they permanently lived in clusters and were confirmed pregnant. Clusters were randomly assigned (1:1:1:1) to standard of care (52 clusters), IYCF (20 g of a small-quantity lipid-based nutrient supplement per day from age 6 to 18 months plus complementary feeding counselling; 53 clusters), WASH (construction of a ventilated improved pit latrine, provision of two handwashing stations, liquid soap, chlorine, and play space plus hygiene counselling; 53 clusters), or IYCF plus WASH (53 clusters). A constrained randomisation technique was used to achieve balance across the groups for 14 variables related to geography, demography, water access, and community-level sanitation coverage. Masking of participants and fieldworkers was not possible. The primary outcomes were infant length-for-age Z score and haemoglobin concentrations at 18 months of age among children born to mothers who were HIV negative during pregnancy. These outcomes were analysed in the intention-to-treat population. We estimated the effects of the interventions by comparing the two IYCF groups with the two non-IYCF groups and the two WASH groups with the two non-WASH groups, except for outcomes that had an important statistical interaction between the interventions. This trial is registered with ClinicalTrials.gov, number NCT01824940. FINDINGS: Between Nov 22, 2012, and March 27, 2015, 5280 pregnant women were enrolled from 211 clusters. 3686 children born to HIV-negative mothers were assessed at age 18 months (884 in the standard of care group from 52 clusters, 893 in the IYCF group from 53 clusters, 918 in the WASH group from 53 clusters, and 991 in the IYCF plus WASH group from 51 clusters). In the IYCF intervention groups, the mean length-for-age Z score was 0·16 (95% CI 0·08-0·23) higher and the mean haemoglobin concentration was 2·03 g/L (1·28-2·79) higher than those in the non-IYCF intervention groups. The IYCF intervention reduced the number of stunted children from 620 (35%) of 1792 to 514 (27%) of 1879, and the number of children with anaemia from 245 (13·9%) of 1759 to 193 (10·5%) of 1845. The WASH intervention had no effect on either primary outcome. Neither intervention reduced the prevalence of diarrhoea at 12 or 18 months. No trial-related serious adverse events, and only three trial-related adverse events, were reported. INTERPRETATION: Household-level elementary WASH interventions implemented in rural areas in low-income countries are unlikely to reduce stunting or anaemia and might not reduce diarrhoea. Implementation of these WASH interventions in combination with IYCF interventions is unlikely to reduce stunting or anaemia more than implementation of IYCF alone. FUNDING: Bill & Melinda Gates Foundation, UK Department for International Development, Wellcome Trust, Swiss Development Cooperation, UNICEF, and US National Institutes of Health.The SHINE trial is funded by the Bill & Melinda Gates Foundation (OPP1021542 and OPP113707); UK Department for International Development; Wellcome Trust, UK (093768/Z/10/Z, 108065/Z/15/Z and 203905/Z/16/Z); Swiss Agency for Development and Cooperation; US National Institutes of Health (2R01HD060338-06); and UNICEF (PCA-2017-0002)

Env-directed membrane fusion mediated by wild type and mutant CCR5 receptors.

a<p>ND, not determined because maximum fusion was too low to allow determination of EC₅₀.</p><p>HOS-CD4-Luc cells expressing wild type or mutant CCR5 receptor constructs were co-cultured with increasing concentrations of HEK 293 cells expressing HIV Env and the HIV transactivator, tat, and luciferase activity was measured. Data are means ± SEM of at least five experiments performed in triplicate.</p

IP production, expression and competition binding of CCR5 receptors with mutations of Thr^2.56(82) and Arg^6.32(225).

<p>A, HEK-Gqi cells were transfected with the wild type (▪) or mutant CCR5 receptors Thr^2.56(82)Lys (•), Thr^2.56(82)Pro (▴), Thr^2.56(82)Lys/Arg^6.32(225)Gln (○) or Thr^2.56(82)Pro/Arg^6.32(225)Gln (Δ). Untransfected cells (□) were used as a negative control. Cells pre-labeled with [³H]<i>myo</i>-inositol were incubated with increasing concentrations of MIP-1β. Data are from a single experiment that is representative of at least three independent experiments performed in duplicate. B, HEK cells were transfected with wild type or mutant CCR5 receptors and stained with PE-2D7 for FACS analysis. Results are mean values ± SEM from at least three independent experiments performed in duplicate. C, HEK 293 cells were transiently transfected with wild type (▪) or mutant CCR5 receptors, Thr^2.56(82)Lys (•), Thr^2.56(82)Pro (▴), Thr^2.56(82)Lys/Arg^6.32(225)Gln (○) or Thr^2.56(82)Pro/Arg^6.32(225)Gln (Δ) and incubated with ¹²⁵I-MIP-1β and various concentrations of unlabelled MIP-1β. Cell-bound radioactivity was collected by filtration and counted. Data are from a single experiment, representative of at least three independent experiments performed in triplicate.</p

IP production and surface expression of wild type and mutant CCR5 receptors.

a<p>significantly different from wild type, p<0.05.</p><p>To assess constitutive- and ligand-stimulated IP production, HEK-Gqi cells transiently expressing wild type or mutant CCR5 receptors were labeled with [H³]-<i>myo</i>-inositol and incubated with buffer (Basal) or MIP-1β (10⁻⁷ M, Stimulated). To assess cell surface expression of receptors HEK 293 cells transiently transfected with wild type or mutant CCR5 constructs were incubated with PE-2D7 antibody before FACS analysis. Every experiment included wild type CCR5 and mock transfected cells. Data are means ± SEM calculated from at least three independent experiments performed in duplicate.</p

IP production and expression of wild type and mutant CCR5 receptors.

<p>HEK-Gqi cells were transiently transfected with wild type or mutant CCR5 receptors, labeled with [³H]<i>myo</i>-inositol and incubated without (basal) or with chemokine agonist, MIP-1β (10⁻⁷ M). Specific CPM denotes the CPM determined for receptor expressing-cells minus the CPM for vector-transfected cells. Data are from a representative experiment performed at least three times in duplicate. B, HEK 293 cells transiently transfected with wild type or mutant CCR5 receptors were stained with a PE-2D7 anti-CCR5 antibody and analyzed by FACS. Data are representative of at least three independent experiments performed in duplicate.</p

Venn diagram depicting ensembles of CCR5 receptor conformations stabilized by mutation of Thr^2.56(82).

<p>Triangles represent receptor conformations stabilized by mutation of Thr^2.56(82) to Lys or Pro. Circles represent receptor conformations that mediate G protein activation, receptor internalization or HIV Env-directed membrane fusion. Mutation of Thr^2.56(82) to Lys stabilizes an ensemble of receptor conformations that activate G protein-mediated signaling and conformations with increased susceptibility to internalization, but not conformations that support HIV Env dependent membrane fusion. The Thr^2.56(82)Pro mutation stabilizes an ensemble of receptor conformations that activate the G protein and conformations that support HIV-1 fusion, but it does not appear to increase population of receptor conformations that result in decreased membrane expression of CCR5.</p

Fusion activity of wild type and mutant CCR5 receptors.

<p>A, HOS cells stably expressing CD4 and the luciferase reporter gene were transiently transfected with wild type (▪) or mutant CCR5 receptors Thr^2.56(82)Lys (•), Thr^2.56(82)Pro (▴), Thr^2.56(82)Lys/Arg^6.32(225)Gln (○) or Thr^2.56(82)Pro/Arg^6.32(225)Gln (Δ). CCR5-expressing HOS-CD4-Luc cells were co-cultured overnight with HEK cells transiently expressing tat, rev and Env and luciferase activity was assessed. B, CCR5-expressing HOS-CD4-Luc cells were labeled with PE-2D7 and analyzed by FACS analysis. C, To compare fusion efficiency among mutant receptors that were expressed at different levels the fusion coefficient was derived by dividing the luciferase activity by the mean fluorescence of each construct.</p