Overview of JC virus NCCR rearrangements (<i>i.e.</i> deletions and insertions) identified by Sanger sequencing and 454 sequencing.

<p>Comparison of the consensus NCCR as determined by Sanger sequencing (n = 61) revealed four distinct groups of samples: 1) archetype sequences with no DNA rearrangements compared to the reference NCCR (CY isolate), 2) samples harboring polymorphic deletions, 3) samples carrying polymorphic insertions and 4) sample harboring both a deletion and an insertion. 454 sequencing was further applied (n = 54) to identify JCV quasispecies. Healthy subject (HS) samples in which quasispecies were identified, the nature of the minority sequences contributing to the quasispecies and the percentage of viral NCCR sequences harboring the minority sequences are indicated. The sequence blocks (a to f) affected by the NCCR rearrangements are indicated between brackets. Of note, HS11 carries a 1 bp deletion at position 165 that in ∼1.5% of the viral population is part of a more extensive deletion. The numbering scheme is based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070950#pone.0070950-Frisque1" target="_blank">[12]</a>.</p

Validation of JC virus quasispecies by nested PCR.

<p>(A) A nested PCR approach was developed to validate the presence of the 28 bps deletion identified in ∼1.5% of the viral DNA sequences in HS11. (B) The nested PCR approach was applied on viral DNA extracted from urine from HS11. Urine aliquots donated at different time points were included: T0, T1, T2 and T3. Viral DNA extracted from HS6 T1 was included as a negative control, together with a no template control (NTC). The upper panel shows successful amplification of the consensus DNA fragment in all samples. In contrast, only in HS11 T1 a PCR fragment could be amplified when using the reverse primer spanning the deletion instead of the consensus reverse primer, confirming the presence of this deletion. L = 25 base pair DNA ladder (Invitrogen). (C) A similar nested PCR approach was used to demonstrate the existence of quasispecies in HS53. DNA fragments were generated when a primer set designed to amplify a consensus sequence from both HS53 and HS26 (lanes HS53 con and HS26 con) were used. When the reverse primer was replaced by a primer specifically targeting the sequence deleted in the quasispecies succesfull amplification was only detected in HS53 (HS53 del), but not in HS26 (HS26 del). NTC: no template control. L = 25 base pair DNA ladder (Invitrogen).</p

Phylogenetic analysis based on the VP1 coding sequence from healthy subjects.

<p>The full length VP1 coding sequence (1065 bp) was obtained for all healthy subjects for which also the non-coding control region (NCCR) was Sanger sequenced (n = 61). The relatedness of the HS samples to defined JC virus genotypes is illustrated in a phylogenetic tree. For each JCV genotype the following reference VP1 coding sequences were used (NCBI accession number between brackets; reference sequences taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070950#pone.0070950-Agostini1" target="_blank">[26]</a>): genotype 1A (AF015526), genotype 1B (AF015527), genotype 2A (AF015529), genotype 2B (AF015533), genotype 2C (AF015535), genotype 2D (AF015536), genotype 2E (AF281606), genotype 3A (U73500), genotype 3B (U73501), genotype 4 (AF015528), genotype 6 (AF015537), genotype 7 (U61771). Healthy subjects (HS) in which deletions or insertions were identified in the non-coding control region via Sanger sequencing are indicated by * (deletion) or + (insertion). HSs in which JCV quasispecies were identified by 454 sequencing are circled.</p

Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors

<div><p>The chemokine receptor CXCR4 is activated by its unique chemokine ligand CXCL12 and regulates many physiological and developmental processes such as hematopoietic cell trafficking. CXCR4 is also one of the main co-receptors for human immunodeficiency virus (HIV) entry. Dysfunction of the CXCL12/CXCR4 axis contributes to several human pathologies, including cancer and inflammatory diseases. Consequently, inhibition of CXCR4 activation is recognized as an attractive target for therapeutic intervention. In this regard, numerous agents modifying CXCR4 activity have been evaluated in <i>in vitro</i> experimental studies and pre-clinical models. Here, we evaluated a CXCL12 competition binding assay for its potential as a valuable initial screen for functional and competitive CXCR4 inhibitors. In total, 11 structurally diverse compounds were included in a side-by-side comparison of <i>in vitro</i> CXCR4 cell-based assays, such as CXCL12 competition binding, CXCL12-induced calcium signaling, CXCR4 internalization, CXCL12-guided cell migration and CXCR4-specific HIV-1 replication experiments. Our data indicated that agents that inhibit CXCL12 binding, <i>i</i>.<i>e</i>. the anti-CXCR4 peptide analogs T22, T140 and TC14012 and the small molecule antagonists AMD3100, AMD3465, AMD11070 and IT1t showed inhibitory activity with consistent relative potencies in all further applied CXCR4-related assays. Accordingly, agents exerting no or very weak receptor binding (i.e., CTCE-9908, WZ811, Me6TREN and gambogic acid) showed no or very poor anti-CXCR4 inhibitory activity. Thus, CXCL12 competition binding studies were proven to be highly valuable as an initial screening assay and indicative for the pharmacological and functional profile of competitive CXCR4 antagonists, which will help the design of new potent CXCR4 inhibitors.</p></div

Effect of different compounds on the CXCL12-induced CXCR4 impedance response.

<p>Relative AUC within the first 60 minutes after ligand addition (50 nM CXCL12) (black) and calcium response intensity (grey) induced by 50 nM CXCL12, both after pre-incubation with the indicated test compound. Values are calculated relative to the reference response i.e. CXCL12 response without compound pre-incubation. Compounds were pre-incubated for 1 h except for PTX and YM-254890 which were pre-incubated overnight. The mean and standard deviation from 3 independent experiments are shown. Within each experiment at least two technical replicates for each condition are averaged. The different compounds are listed with their targets in the text box underneath the figure. P-values were determined using one sample t-tests with null hypothesis stating that the response values after compound pre-incubation did not differ from the reference response value 100. A correction for multiple testing was performed based on the Benjamini-Hochberg procedure. * indicates significant differences between responses with compound pre-incubation and the reference response value 100 for FDR = 0.1. Calcium fluxes were measured using the FLIPR tetra system with the calcium-sensitive dye Fluo-2 AM whereas the impedance measurements were done using the xCELLigence device.</p

Impedance profile resulting from CXCL12-induced CXCR4 signaling.

<p><b>(a)</b> CXCL12-induced impedance response on U87.CD4.CXCR4 cells with (orange) and without (blue) one hour pre-incubation with the specific CXCR4 antagonist AMD3100. CXCL12-induced impedance response on U87.CD4 (yellow) and U87.CD4.CXCR7 cells (purple). For each time point the mean CI value and standard deviation of 3 technical repeats within the same experiment are shown. <b>(b)</b> Dose-dependent response on U87.CD4.CXCR4 cells elicited by 0.2 nM—150 nM CXCL12. The figure depicts one out of three independent datasets used to calculate the mean EC₅₀. The mean CI and standard deviation of 6 technical repeats are shown. Normalization and baseline-correction were done as described in the method section. For clarity, one in every 5 data points is plotted.</p