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