Selection of a CXCR4-using variant above the 454 sequencing error threshold during persistent viremia suppression in Subject 26.

<p><b><i>Panel A, antiretroviral treatment history, virological and immunological evolution</i></b><b>.</b> Continuous line, HIV-1 RNA levels; dashed line, CD4+ counts; horizontal bars, time period during which a given antiretroviral drug was prescribed. Vertical lines indicate the timepoints when 454 sequencing was performed. LPVr, lopinavir/ritonavir; AZT, zidovudine; ddI, didanosine; RAL, raltegravir. <b><i>Panel B, maximum likelihood nucleotide-based phylogenetic tree</i></b> including V3-loop haplotypes present at a frequency ≥0.6% in the virus population in plasma (triangles), PBMCs before therapy initiation (circles) and PBMCs after persistent viremia suppression (squares). The tree is rooted at the most frequent plasma sequence before antiretroviral treatment initiation. Filled symbols show predicted CXCR4-using viruses; open symbols show predicted CCR5-using viruses. Symbol size increases proportionally to the V3-loop haplotype frequency in the virus population in 10% intervals. Node reliability was tested using 1000 bootstraps; bootstrap values ≥50% are shown. The V3-loop aminoacid sequence translation is shown next to each taxon. Aminoacid changes relative to the predominant sequence in plasma are highlighted in bold and underlined. Gaps correspond to aminoacid indeterminations. A Geno2Pheno _[coreceptor] false positive rate (FPR) equal or lower than 10% was used to define CXCR4 use. The actual false positive rate of each sequence is shown. *Sequence #2 was identical to one detected in 0.04% of PBMC-associated viruses, below the error threshold, before treatment initiation.</p

Prevalence of CXCR4-using viruses using different tropism assays and settings.

<p>Bar plot showing the mean and 95% confidence intervals of the prevalence of subjects with CXCR4-using viruses using different tropism assays and settings. The Geno2Pheno_[coreceptor] clinical model was only used in pre-treatment bulk sequences derived from plasma RNA; otherwise, the clonal model was used. ESTA, Enhanced-Sensitivity Trofile™ Assay; FPR, Geno2Pheno_[coreceptor] false positive rate used to assign tropism; MT-2, Direct cocultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells. * p-value<0.05, two-sided exact binomial tst.</p

Accuracy of Tropism Assays Relative to the Enhanced-Sensitivity Trofile™ Assay.^{^a}

a<p>Values are mean percentages (95% confidence interval of the mean), calculated assuming a binomial distribution of the data.</p>b<p><i>G2P FPR</i>, Geno2Pheno_[coreceptor] false positive rate used for population and 454 sequencing to assign CXCR4 use . The Geno2Pheno_[coreceptor] clonal model was always used.</p>c<p><i>MT-2</i>, Direct cocultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells.</p>d<p><i>PPV</i>, Positive Predictive Value.</p>e<p><i>NPV</i>, Negative Predictive Value.</p>f<p>“Accuracy” is defined as: (True positives + True negatives)/Total.</p

Longitudinal tropism testing results per subject.^{^a^,^b^,^c}

a<p><i>ESTA</i>, <i>Enhanced-Sensitivity Trofile™ Assay</i>; <i>Pop Seq</i>, population sequencing of the V3-loop; <i>454</i>, 454 sequencing of the V3-loop; <i>MT2</i>, direct co-cultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells. HIV-1 RNA levels are in copies/mL; CD4+ cell counts are in cells/mm³.</p>b<p>Population and 454 sequencing data shown here used the Geno2Pheno_[coreceptor] false positive rate cut-off providing highest accuracy when assigning HIV-1 tropism, i.e.: 20% and 10%, respectively. Based on internal error controls, only V3 forms present in ≥0.6% of viruses were considered for tropism prediction with 454 sequencing.</p>c<p>Tests detecting CXCR4-using HIV are reported as “<i>dual-mixed, DM</i>” for ESTA, <i>“X4”</i> for population sequencing, “<i>percent of X4 viruses”</i> for 454, and “<i>syncytium-inducing, SI</i>” for MT-2 assays; for clarity, viruses only using CCR5 are shown as dashes; NA, tropism test result not available due to lack of amplification.</p

Population structure analysis of plasma and PBMC V3 forms detected by 454 sequencing.^{^a}

a<p>The intracompartment variability (<i>Π</i>) of each sample is measured with the best evolutionary model found with <i>Findmodel</i> (<a href="http://www.hiv.lanl.gov" target="_blank">www.hiv.lanl.gov</a>); it corresponds to the average number of nucleotide differences per site between sequences. Migration events with <i>p-value</i>, and <i>F_ST</i> with <i>p-value</i> are indicated for Slatkin-Madison population structure tests. <i>NA</i> indicates comparisons where the tests were not applicable. <i>NC</i> indicates that variability cannot be calculated because there is only one haplotype.</p>*<p>p-value between 0.05 and 0.01;</p>**<p>p-value<0.01 and 10⁻⁶.</p><p>Statistically significant p-values are colored; the color intensity is proportional to the p-value. Note that a complete dataset was not available for subjects 10 and 13, which were, therefore, not included in this analysis.</p

Analysis of Gag Polyprotein Processing

<p>Western blot analysis of cell lysates (left panel) or virus particles (right panel) obtained by ultracentrifugation of cell culture supernatants from 293T cells transfected with the respective recombinant virus plasmids in the absence of inhibitor or in the presence of 50 or 500 nM of RO033-4649. Molecular mass standards are depicted on the left, Gag-derived proteins are identified on the right. Protein detection was performed following incubation with a specific antibody against NC.</p

Determination of Phenotypic Drug Susceptibility of C-Terminal Gag Clones

<p>Representation of the fold increases in phenotypic drug susceptibility of the C-terminal Gag clone derived from IVS-1, containing the 436E+437T amino acid change as compared to wild-type HIV (HXB2). The black bars indicate the fold increase as determined in the multiple-cycle MTT drug susceptibility assay, and the gray bars indicate the fold increase as determined in the single-cycle PhenoSense drug susceptibility assay. Drug susceptibility to the PI RO033-4649, LPV, TPV, ATV, AMP, ritonavir (RTV), saquinavir (SQV), indinavir (IDV), and nelfinavir (NFV), and as a control to the RT inhibitor NVP was determined. RO033-4649 and TPV were not available at the time of PhenoSense assay testing.</p

Schematic Representation of the Distribution of all Amino Acid Changes Appearing during in vitro Selection Experiments Using RO033-4649

<p>Horizontal bars below the gene organization scheme represent the genomes of viruses from the three in vitro selection experiments: IVS-1, IVS-32, and IVS-34. Vertical lines illustrate the observed amino acid changes. The C-terminal portion of Gag was expanded to precisely map the nucleotide changes leading to amino acid changes (bold) in both translational reading frames (Gag and GagPol). In addition, the three different cleavage sites (NC/p1, NC/TFP, and TFP/p6^pol) are indicated. The control experiments, in which no drug was added during the in vitro selections, demonstrated no amino acid changes in the viral Gag and protease region.</p

Schematic Representation of the Viral Frameshift Region and Investigation of Frameshift Efficiency

<div><p>The RNA structure of the frameshift stimulatory signal is represented here [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040036#pmed-0040036-b030" target="_blank">30</a>,<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040036#pmed-0040036-b051" target="_blank">51</a>]. The nucleotide and amino acid changes (between brackets) as observed in our in vitro selection experiments are indicated.</p> <p>(A) The relative frameshift efficiency of wild-type HIV (HXB2) and the p1 constructs of IVS-1 (436E+437T), IVS-32 (437V), and IVS-34 (437T). The values of the frameshift efficiency are the means of four independent experiments, with bars representing the standard errors.</p> <p>(B) Analysis of differences in levels of HIV GagPol products (RT, p66, and p51) as compared to HIV Gag products (CA, p24) by Western blot analysis.</p></div