Ligand-directed post-endocytic sorting of CCR5 is not dependent on G protein signaling.

<p>CHO CCR5 cells growing on glass coverslips were incubated overnight with Pertussis toxin (100 ng/mL), then washed and stimulated with 100 nM chemokines as indicated in the presence of anti-CCR5 antibody 3A9 (green) at 37°C. Cells were then acid washed (15 min 4°C) to remove cell surface antibody, fixed, permeabilized, and labelled for either the ERC marker Rab11 (5P14-RANTES) or the TGN marker TGN38 (PSC-RANTES) (red). After staining with DAPI nucleic acid stain (blue), cells were analysed by confocal microscopy. Maximum intensity projections are shown, scale bar = 20 μm. Throughout the experiment the ERC and TGN markers (red) remain either in a discrete supranuclear spot (ERC) or at a perinuclear site (ring-shaped staining around the nuclei, TGN). Initially, CCR5 (green) is localized at the cell surface, but after 10 min incubation with either ligand it translocates to the supranuclear spot (visibly colocalizing with the ERC marker used in the cells treated with 5P14-RANTES). After 120 min incubation, CCR5 on cells treated with PSC-RANTES subsequently relocates from the ERC to accumulate in a perinuclear site (ring-shaped staining around the nuclei, visibly colocalizing with the TGN marker), while CCR5 in cells treated with 5P14-RANTES remains colocalized with the ERC marker.</p

Ligand-induced GRK phosphorylation of CCR5.

<p>CHO-CCR5 cells growing on coverslips were treated with the indicated chemokines (100 nM) for the indicated times, then washed, fixed, permeabilized and labeled with a monoclonal antibody specific for CCR5 phosphorylated on Serine 349 (green) and DAPI nucleic acid stain (blue). Maximal intensity projections are shown. Scale bar = 20 μm.</p

Spatial and temporal resolution of arrestin2-CCR5 association.

<p><b>A</b> CHO-CCR5 cells stably transfected with arrestin2-GFP were treated with chemokine analogs (100 nM) as indicated and the redistribution of arrestin2-GFP was followed by live fluorescence microscopy. Images captured prior to (0 min) and after ligand treatment (6 min) are shown. <b>B</b> CHO-CCR5 cells stably transfected with arrestin2-GFP (green), preincubated with rhodamine-labeled anti-CCR5 antibody (red), were washed and then incubated 90 min at 37°C with chemokine analogs (100 nM) prior to image capture. Maximal intensity projections are shown. Scale bar = 20 μm.</p

Antibodies used in this study.

<p>*where possible, stable public identifiers from the Antibody Registry (<a href="http://www.antibodyregistry.org/" target="_blank">www.antibodyregistry.org</a>) are provided; HRP; Horseradish peroxidase.</p><p>Antibodies used in this study.</p

G protein signaling of chemokine analogs on CHO-CCR5 cells.

<p><b>A.</b> G_i/o protein signaling activity of chemokines at the indicated doses was determined by measuring reduction in forskolin-stimulated cAMP levels. Results shown (Relative Light Units, RLU) are the mean of duplicate readings, with error bars indicating the range. <b>B.</b> G protein signaling activity of chemokines (100 nM) was determined by measuring reduction in forskolin-stimulated cAMP levels. Where indicated CHO-CCR5 cells were pre-incubated (100 ng/mL, overnight) with Pertussis toxin (PTX). Results shown (Relative Light Units, RLU) are the mean of duplicate readings, with error bars indicating the range.</p

Ligand-directed post-endocytic sorting of CCR5.

<p><b>A.</b> CHO CCR5 cells growing on coverslips were pre-treated (60 min, 4°C) with 100 nM chemokines and anti-CCR5 antibody 3A9 (green), then washed and incubated at 37°C for the indicated times. Cells were then acid washed to remove cell surface antibody, then fixed, permeabilized, and labeled for the ERC marker Rab11 (red) and DAPI (blue, nuclear staining) prior to analysis by confocal scanning microscopy are shown, scale bar = 20 μm. Throughout the experiment the ERC marker (red) remains in a discrete supranuclear spot that is visble in the center of the nuclei (blue) in the maximum intensity projections. Initially, CCR5 (green) is localized at the cell surface, but after 10 min incubation with either ligand it translocates to colocalize with the ERC marker. After 120 min incubation, CCR5 on cells treated with PSC-RANTES subsequently relocates from the ERC to accumulate in a perinuclear site (ring-shaped staining around the nuclei), while CCR5 in cells treated with 5P14-RANTES remains colocalized with the ERC marker. <b>B.</b> Individual Z-slice images from an identical experiment in which cells incubated with the indicated chemokines for 180 min were also labeled for either the ERC marker, Rab11 or the TGN marker, TGN38 (red). Slices in which the marked compartment is most abundant (through the middle of the nucleus for TGN, just above the nucleus for ERC) were chosen. While in cells treated with PSC-RANTES for 180 min, CCR5 colocalizes with TGN38 and does not colocalize with Rab11, in cells treated with 5P14-RANTES for 180 CCR5 colocalizes with Rab11 and does not colocalize with TGN38 scale bar = 20 μm.</p

Development of resistance to 5P12-RANTES (5P12).

<p><b>A.</b> Weekly increases in p24 capsid antigen during four successive rounds of selection: 5P12 1 (blue, weeks 1–15); 5P12 2 (green, weeks 13–25); 5P12 3 (purple, weeks 24–33); and 5P12 4 (brown, weeks 30–44). Control cultures with no inhibitor are shown in black filled circles. <b>B.</b> Increasing concentrations of 5P12 expressed as multiples of the 90% inhibitory concentration (IC₉₀ = 0.12 nM) for each of the four rounds of selection, with colors matching panel A. <b>C.</b> Replication of viruses from indicated weeks of selection on activated CD4⁺ T cells from a CCR5Δ32 homozygous donor. Values are mean p24 capsid antigen levels (± SE of triplicate cultures) after 7 days of culture. <b>D.</b> Viruses from the indicated weeks of 5P12-RANTES round 4 of selection (5P12 4) or control cultures with no inhibitors were used to infect activated CD4⁺ T cells from normal donors in the presence of the CXCR4 blocking agent AMD3100 (AMD). The percent inhibition by AMD3100 of p24 capsid antigen levels after 7 days of culture is plotted versus the week of virus isolation.</p

V3 sequences of control, maraviroc-resistant, and 5P12-RANTES-selected CC1/85 HIV-1 viruses.

a<p>Log₁₀ relative light units (RLU) in single cycle infection of NP-2.CD4.CCR5 cells mediated by envelope (<i>env</i>) clones with the indicated V3 sequence. Mean values for multiple <i>env</i> clones with the same V3 sequence, representative single values for individual clones. Note that <i>env</i> clones with the same V3 sequence may differ in sequence in other regions of envelope.</p

Env (gp160) sequence evolution to CXCR4 use.

<p><b>A.</b> A phylogenetic tree representing the <i>env</i> clones that evolved from CCR5 to CXCR4 use. The tree is rooted with one of two variants found in the starting CC1/85 virus population with a TNNTxN motif sequence at position 459–465 (HXB2 numbering) in C5 instead of NDTSGT. All <i>env</i> clones that developed CXCR4 use were derived from this founder sequence. The weeks at which the <i>env</i> clones were isolated is indicated by the symbol legend, and the V3 sequence is indicated by the color given in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone-0022020-g003" target="_blank">Fig. 3</a>. All <i>env</i> clones from week 36 and later of 5P12-RANTES selection were capable of using CXCR4 (see below), whereas only a subset of control <i>env</i> clones from week 44 or later were capable of entry via CXCR4. <b>B.</b> Entry data for <i>env</i> clones from weeks 36, 42, and 44 either from control cultures (open symbols) or 5P12-RANTES containing cultures (closed symbols, weeks depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone-0022020-g004" target="_blank">Fig. 4A</a>). The symbols are color coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone-0022020-g003" target="_blank">Figs. 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone-0022020-g004" target="_blank">4A</a>.</p

Calculation of virus replication cycles during selection.

1<p>AUC; area under curve for cumulative increase in capsid p24 antigen shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone-0022020-g002" target="_blank">Fig. 2A</a> for the indicated number of weeks (first to last).</p>2<p>Calculation of replication cycles assume 1 cycle/day for HIV-1 CC1/85 in control cultures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone.0022020-Roos1" target="_blank">[66]</a>, and is corrected for weeks of replication (i.e., allowing for pauses) and diminished p24 levels (% control) for cultures under 5P12-RANTES (5P12) selection.</p>3<p>Expected mutations are calculated based on the rate found by Abram <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone.0022020-Abram1" target="_blank">[54]</a> of 1.4×10⁻⁵ mutations/bp/cycle and the 2553 bp target envelope gene.</p>4<p>Observed mutations are the mean number of nucleotide mutations observed in all envelope molecular clones with confirmed entry via CXCR4.</p