Specific recognition of phage clones selected during biopanning by mAb VRC01.

<p>(A) Lane 1, phage clone carrying C4 peptide; lane 2, phage clone carrying C1 peptide; Lane 3, phage clone with no random peptide insert (negative control). Phage titer (in plaque forming units per ml) is labeled to left the dot blot. (B) Spot intensity related to the symbols.</p

Inhibition of virus-neutralizing activity of monoclonal antibody VRC01 by synthesized peptides.

<p>The luciferase activity from virus-only infection was set to 100%. Red dashed line denotes luciferase activity produced by virus with the addition of VRC01 (IC90 = 6.26 μg/ml) without peptides. Black columns correspond to 1:5000 antibody to peptide ratio, grey columns correspond to 1:1000 antibody to peptide ratio). Asterisk indicates statistical significance from the virus/VRC01 control (p ≤ 0.05).</p

Models of interaction between fragments of the peptides which were able to rescue viral infectivity and VRC01-gp120 complex.

<p>Antibody showed as a surface (gray color), secondary structure of gp120 is colored in blue, amino acid residues of SGGDLEI gp120 region is labeled with red, fragments of peptides E1 (SWPEL), E2 (TAPEL), E10 (TTFDI) and E11 (SIADL) are shown as a colored sticks (A, B, C and D, respectively).</p

Alignment of the peptides from the phage clones showing the highest binding strength with VRC01 in dot blot with fragment 365–371 of gp120.

<p>Amino acid residues that correspond to the consensus sequence are marked by grey.</p

Dynamics and regulation of nuclear import and nuclear movements of HIV-1 complexes

<div><p>The dynamics and regulation of HIV-1 nuclear import and its intranuclear movements after import have not been studied. To elucidate these essential HIV-1 post-entry events, we labeled viral complexes with two fluorescently tagged virion-incorporated proteins (APOBEC3F or integrase), and analyzed the HIV-1 dynamics of nuclear envelope (NE) docking, nuclear import, and intranuclear movements in living cells. We observed that HIV-1 complexes exhibit unusually long NE residence times (1.5±1.6 hrs) compared to most cellular cargos, which are imported into the nuclei within milliseconds. Furthermore, nuclear import requires HIV-1 capsid (CA) and nuclear pore protein Nup358, and results in significant loss of CA, indicating that one of the viral core uncoating steps occurs during nuclear import. Our results showed that the CA-Cyclophilin A interaction regulates the dynamics of nuclear import by delaying the time of NE docking as well as transport through the nuclear pore, but blocking reverse transcription has no effect on the kinetics of nuclear import. We also visualized the translocation of viral complexes docked at the NE into the nucleus and analyzed their nuclear movements and determined that viral complexes exhibited a brief fast phase (<9 min), followed by a long slow phase lasting several hours. A comparison of the movement of viral complexes to those of proviral transcription sites supports the hypothesis that HIV-1 complexes quickly tether to chromatin at or near their sites of integration in both wild-type cells and cells in which LEDGF/p75 was deleted using CRISPR/cas9, indicating that the tethering interactions do not require LEDGF/p75. These studies provide novel insights into the dynamics of viral complex-NE association, regulation of nuclear import, viral core uncoating, and intranuclear movements that precede integration site selection.</p></div

CA-CypA interactions, but not reverse transcription, regulate nuclear import.

<p>(<b>A</b>) A schematic illustrating that the time of nuclear import consists of the time in the cytoplasm prior NE docking (time between infection and NE association) and NE residence time prior to nuclear import (time between NE association and nuclear import). The average time of NE association and nuclear import for the WT complexes (described below) are shown here for reference. <b>(B-D)</b> The time of nuclear import <b>(B)</b>, time in cytoplasm <b>(C)</b>, and NE residence time <b>(D)</b> for each viral complex that entered the nucleus is shown. For these experiments, the nuclear import of WT complexes in untreated cells, WT complexes in the presence of NVP, WT complexes in the presence of CsA, and P90A CA mutant complexes were detected manually from analysis of 10-hr long movies initiated 10 min after infection (1 frame/3 min). Nuclear import events for WT complexes in untreated cells also includes tracked viral complexes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006570#ppat.1006570.s002" target="_blank">S2 Table</a>). Numbers below sample name in <b>(B)</b> indicate the number of nuclear import events analyzed. For <b>(B-D)</b>, the average values ± SD are shown above each sample; black lines indicate median values; *, significantly different than WT and WT+NVP (<i>P</i> ≤ 0.05; Mann-Whitney test); n.s, not significant (<i>P</i> > 0.05; Mann-Whitney test). <b>(E-F)</b> Characterization of WT viral complexes labeled with A3F-YFP in infected cells treated without (WT) or cells treated with CsA (WT+CsA). HeLa cells treated with and without CsA were infected with VSV-G pseudotyped HIV-1 labeled with A3F-YFP and fixed 2 or 6 hrs post-infection (hpi). <b>(E)</b> The percentage of A3F-YFP⁺ signals that are CA⁺ and <b>(F)</b> relative CA signal intensity (average CA signal intensities associated with A3F-YFP signals in the cytoplasm at 2 hpi was set to 1). Error bars indicate the SD of 4 experiments; an average of ∼1400 particles from ∼100 cells were analyzed for each condition. *, <i>P</i> ≤ 0.05; n.s., not significant (<i>P</i> > 0.05; <i>t</i>-test). <b>(G)</b> Scatter plots of the NE residence times (hrs) and time in cytoplasm (hrs) for each viral complex described in <b>(B-C)</b>. Linear trend lines for each population was determined (solid lines); Pearson correlation values are shown for each population; n.s., indicates no significant correlation between NE residence time and time in cytoplasm (<i>P</i> > 0.05).</p

Analysis of intranuclear movements of A3F-YFP and IN-YFP labeled viral complexes in the presence or absence of LEDGF/p75.

<p><b>(A-D)</b> Generation of a LEDGF/p75 knockout HeLa cell line (HLKO) in which the integrase binding domain (IBD) of LEDGF/p75 was disrupted using CRISPR/cas9. <b>(A)</b> PSIP1 locus on chromosome 9, which codes for LEDGF/p75, is shown with exons (black boxes) labeled 1 to 16. Locations of the gRNA binding sites are shown as red triangles (a and b). Primers binding to the outside of the integrase binding domain (IBD), used for PCR amplification and identification of single cell clones, are labeled Lfor and Lrev. <b>(B)</b> PCR analysis of LEDGF/p75 knock out clone HLKO versus wild-type HeLa cells (WT). Expected band sizes are 1158 bp for undeleted IBD, versus ∼480 bp for deleted IBD. <b>(C)</b> WT or HLKO cell lysates were analyzed by western blot using antibodies that detect the C-terminal region of LEDGF/p75 or α-tubulin. <b>(D)</b> Relative infectivity of an HIV-1 luciferase-reporter virus (pHL) in WT or HLKO cells. The luciferase activity was determined 48 hrs after infection; luciferase activity in WT cells infected with 30 μl pHL virus was set to 100%. *, <i>P</i> ≤ 0.05; <i>t</i>-test. <b>(E)</b> Ensemble MSD analysis of the movements of intranuclear A3F-YFP and IN-YFP labeled HIV-1 complexes in WT and HLKO cells. WT or HLKO cells expressing POM121-mCherry were infected with A3F-YFP or IN-YFP labeled virus and ∼10 hr-long movies (1 frame/3 min) of cells with 1 or more nuclear viral complexes initiated ∼2 hrs after infection were acquired. Dotted lines are straight line fits using the first four time lags and extrapolated to the last time lag. The curves (solid lines) represent power law fits to the MSD values. The diffusion coefficients (× 10⁻⁴ μm²/sec) were calculated using the MSD values and are shown next to each curve; error bars represent 95% confidence intervals. <b>(F)</b> Average nuclear penetration distance for each viral complex in WT (n = 20) and HLKO cells (n = 20) described in <b>(E)</b>; the nuclear penetration distances for A3F-YFP and IN-YFP labeled viral complexes were not different (<i>P</i> > 0.05; Mann-Whitney test) and were combined for these experiments; black lines indicate median nuclear penetration distance; n.s., not significant (<i>P</i> > 0.05; Mann-Whitney test).</p

A model for regulation of nuclear import by the CA-CypA interaction and intranuclear movements of viral complexes.

<p>The CA-CypA interaction regulates the nuclear import of viral complexes by stabilizing the viral core, which thereby delays the time of docking at the nuclear pore complex (NPC; longer time in cytoplasm) and translocation of viral complexes from the NE to the nucleus (longer NE residence time). Disruption of the CA-CypA interaction results in faster uncoating, NE docking, and nuclear import. After import, the viral complexes exhibit a brief phase of fast mobility as they move away from the nuclear point of entry, followed by a long phase of slow mobility, during which they become tethered to chromatin. Blue circles, CA; red circles, CypA; green circles, A3F-YFP or IN-YFP.</p

Kinetics of HIV-1 complex-NE association.

<p>(<b>A</b>) Experimental protocol. HeLa cells stably expressing POM121-mCherry were infected with VSV-G pseudotyped HIV-1 labeled with A3F-YFP. A z-stack centered near the equatorial region of the nucleus was acquired every 1 min for 2 hrs; movies were initiated from 45 min to 6 hrs post-infection. <b>(B)</b> Examples of A3F-YFP complexes (white triangles) that co-localize with the POM121-mCherry signal. Scale bar, 5 μm. <b>(C)</b> NE residence time analysis of A3F-YFP labeled HIV-1 complexes. The residence time for each HIV-1 complex that contacted the NE for at least 1 min (two consecutive frames) was determined and is plotted using 10 min bins. The percentage of these particles with NE residence times ≤ 20 min and > 20 min is shown; 194 particles from 23 cells were analyzed. <b>(D)</b> Relative infectivity of an HIV-1 GFP-reporter virus containing wild-type CA (WT), K203A, or E128A/R132A CA mutations (WT set to 100%; left). The relative infectivity of WT virus when the target cells are treated with control or Nup358 siRNA (control siRNA set to 100%; right) is shown. The percentage of GFP⁺ cells was determined by flow cytometry 48 hrs after infection. Error bars indicate the SD of 3–5 experiments. <b>(E)</b> The number of HIV-1 complexes that stably associate with the NE for > 20 min in the 2-hr long movies (per cell) is shown for WT, K203A, E128A/R132A, WT (no VSV-G) and for WT virus in cells treated with control siRNA or Nup358 siRNA. Error bars indicate standard error of the mean (SEM) of 19–27 cells. *, <i>P</i> ≤ 0.05, <i>t</i>-test.</p

Translocation of HIV-1 complexes from the cytoplasm to the nucleus.

<p>(<b>A</b>) An A3F-YFP-labeled HIV-1 complex that was stably-associated with the NE (labeled by POM121-mCherry) for ∼3 hours prior to nuclear entry (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006570#ppat.1006570.s015" target="_blank">S7 Movie</a>). The z-slice in which the particle was located (as determined by 3D tracking) was extracted for each time point and then a maximum intensity projection of frames of the YFP and mCherry channels from the entire 10 hr time-lapse movie is shown. Scale bars, 5 μm (left) and 2 μm (right). <b>(B)</b> A 2D projection of the 3D track for the particle described in <b>(A)</b>. <b>(C)</b> An IN-YFP-labeled HIV-1 complex that was stably-associated with the NE for ∼1.5 hours prior to nuclear entry (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006570#ppat.1006570.s016" target="_blank">S8 Movie</a>) and <b>(D)</b> 2D projection of the 3D track for the particle described in <b>(C)</b>. For <b>(B)</b> and <b>(D)</b>, arrow indicates the first frame in which the HIV-1 complex was observed in the nucleus. <b>(E)</b> Composite images of the YFP and mCherry channels for the frame before and 6 frames after nuclear import for the IN-YFP labeled HIV-1 complex described in <b>(C)</b>. Scale bar, 1 μm. (<b>F</b>) The mobility of HIV-1 complexes before and after nuclear import. The average distance (μm) the particle moved per 3-min frame was determined for each A3F-YFP or IN-YFP complex prior to import, the 3 frames immediately following import (<9 min after particle was first detected in nucleus), and all subsequent frames. Error bars indicate SD of 7 import events for A3F-YFP complexes and 14 import events for IN-YFP complexes. <b>(G)</b> A3F-YFP and IN-YFP remain stably associated with viral complexes through nuclear import. The relative YFP intensities before (average YFP intensities for the 5 frames before import) and after nuclear import (average YFP intensities for the 5 frames after import) for each particle is shown; the YFP intensities before import were set to 1. *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.01; n.s., not significant (<i>P</i> > 0.05), <i>t</i>-test.</p