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. 2015 Mar 6:6:6483.
doi: 10.1038/ncomms7483.

Chromatin organization at the nuclear pore favours HIV replication

Mickaël Lelek  1 , Nicoletta Casartelli  2 , Danilo Pellin  3 , Ermanno Rizzi  4 , Philippe Souque  5 , Marco Severgnini  4 , Clelia Di Serio  3 , Thomas Fricke  6 , Felipe Diaz-Griffero  6 , Christophe Zimmer  1 , Pierre Charneau  5 , Francesca Di Nunzio  5
Affiliations

Chromatin organization at the nuclear pore favours HIV replication

Mickaël Lelek et al. Nat Commun. .

Abstract

The molecular mechanisms that allow HIV to integrate into particular sites of the host genome are poorly understood. Here we tested if the nuclear pore complex (NPC) facilitates the targeting of HIV integration by acting on chromatin topology. We show that the integrity of the nuclear side of the NPC, which is mainly composed of Tpr, is not required for HIV nuclear import, but that Nup153 is essential. Depletion of Tpr markedly reduces HIV infectivity, but not the level of integration. HIV integration sites in Tpr-depleted cells are less associated with marks of active genes, consistent with the state of chromatin proximal to the NPC, as analysed by super-resolution microscopy. LEDGF/p75, which promotes viral integration into active genes, stabilizes Tpr at the nuclear periphery and vice versa. Our data support a model in which HIV nuclear import and integration are concerted steps, and where Tpr maintains a chromatin environment favourable for HIV replication.

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Figures

Figure 1
Figure 1. Depletion of Nup153 and Tpr followed by reconstitution of the nuclear basket by complementation and in vitro CA–NC binding assay.
(a,b) We used the pTrip.GFP.H1shRNA vector to knockdown (KD) the expression of Nup153 and Tpr in HeLa P4CCR5 cells. Viral particles produced using the pTrip.GFP.H1shRNA vector containing the specific shRNA against Nup153 and Tpr were used to transduce HeLa P4CCR5 at the indicated multiplicity of infection (MOI). Control cells were transduced with the empty pTrip.GFP.H1shRNA vector. The efficiency of the KD was monitored by western blotting using antibodies against the endogenous Nup153 proteins and Tpr. As a loading control, samples were also blotted using antibodies against actin and lamin A/C. (c) Nup153-depleted cells at MOI 50 were complemented with a plasmid, pC2GFP, GFP-Nup153w/o FG and GFP-Nup153. The efficiency of the complementation was monitored by western blotting using antibodies against the endogenous Tpr, GFP and actin. (d, e) Nup153 and Tpr -depleted cells were challenged with HIV-1 containing luciferase as a reporter of infection. HIV-1-Luc was normalized by p24 as described in Methods. Infectivity was determined 48 h p.i. by measuring luciferase activity normalized to the amount of protein. 24 h p.i. total genomic DNA from infected cells was used to measure 2LTR circles by real-time PCR normalized to actin. The percentage of 2LTR circles with respect to the control is shown in the histograms of Nup153 and Tpr-depleted cells at MOI 50 and 100, respectively. The s.d. of three independent experiments are shown. (f) The binding of GFP-Nup153 and GFP-Tpr proteins to in vitro assembled HIV-1 capsid–nucleocapsid (CA–NC) complexes was performed by transfecting human 293T cells with plasmids expressing GFP-Nup153, GFP-Tpr, GFP and Trim5αrh-GFP. The assay is described in Methods. Quantification of BOUND versus INPUT fractions of three independent experiments was plotted with their respective standard deviations on the lower panels.
Figure 2
Figure 2. Involvement of Tpr in HIV-1 replication.
(a) HeLa P4CCR5 cells were depleted for Tpr using different doses of LVshRNA against Tpr and (b) infectivity was measured 48 h p.i. by flow cytometry (c) Jurkat cells were depleted for Tpr using different doses of LVshRNA against Tpr, as shown in the western blot and (d) infectivity was measured 48 h p.i. by luciferase activity normalized to the amount of protein. (e,f,g) Viral late reverse transcription (LRT) levels were analysed at 7 h p.i., nuclear import and integration were analysed at 24h p.i. by 2LTRs and Alu-PCR, respectively. Infections carried out in the presence of Nevirapine at 5 μM led to undetectable levels of LRT, 2LTR circles and Alu-PCR. Similar results were obtained in three independent experiments and s.d. are shown.
Figure 3
Figure 3. Tpr is specifically involved in HIV-1 infection.
(a) Three out of ~10 final clones obtained by limiting dilution were selected for the expression of Tpr by western blotting and (b) MLV-Luc and HIV-1-Luc were used in parallel to infect the control clone (obtained with an empty LV) and clones 2, 5 and 6. (c) Tat was transfected in all clones to evaluate LacZ expression by β-galactosidase assay normalized to the amount of protein. Clones were thawed and frozen multiple times and yielded similar results (s.d. are shown). (d–f) Transcriptome profiles comparison: scatter plots show RPKM values observed in ~36,000 annotated in control sample (x axis) and Tpr KD (y axis). Scatter plots of Pearson correlation calculated using the RNA-seq log2 RPKM values of Tpr depleted versus control for (d) HeLa cells, (e) Jurkat cells and (f) clones.
Figure 4
Figure 4. Increase of intensity of Tpr at the nuclear periphery by LEDGF/p75 overexpression and vice versa.
The 293 T cells were cotransfected with GFP-Tpr and (a) HA alone or (b) HA-Nup153 or (c) HA-LEDGF/p75. (d) As control we cotransfected GFP-Nup98 with HA-LEDGF/p75. HA fused proteins were detected using a monoclonal anti-HA antibody and a secondary antibody conjugated with Cy3. (e) RT–PCR, mean of ratio between relative mRNA of PSIP1 and of Tpr genes by actin in Tpr depleted versus control HeLa and Jurkat cells is shown in the histogram. (f) Flow cytometry analysis show the percentage of GFP-Tpr-positive cells when cotransfected with HA, HA-LEDGF/p75 and HA-Nup153. (g) Relative increase of Nup GFP-positive cells on overexpression of LEDGF/p75 relative to the endogenous level of LEDGF/p75 based on flow cytometry data. S.d. of three independent experiments are shown.
Figure 5
Figure 5. Short-range association between integration sites and genomic features.
(a, b) Histograms show the distribution of absolute genomic distances to specific features of HIV-1 integration sites, with bin size of 200. The genomic features are Histone methylation H3K36me3 and H3K4me3 in a 2 kb window centred on integration sites. The distributions are shown for control cells (orange bars), Tpr KD cells (light blue bars) and as predicted for random integration (grey bars). (c) Frequency of ISs from histone methylation H3K36me3 and (d) H3K4me3, as function of genomic distance, plotted with bins of 100 bp in a ±50 kb window centred on integration sites in control cells (left, orange) and Tpr KD cells (right, blue). Dashed curves correspond to kernel density estimations of the distance distribution for control cells (red), Tpr KD (blue) and random integration (grey).
Figure 6
Figure 6. Super-resolution imaging of active chromatin underneath NPCs in control and Tpr depleted HeLa cells.
(a) STORM image (visualized as smoothed histogram of computed positions) of Nup153-A568 (green) and H3K36me3-Cy5 (red) in control cells (left) and Tpr KD cells (right). The top left corner of the figure shows the low resolution image that would have been obtained with a conventional (diffraction limited) microscope to appreciate the gain in resolution. The yellow spots outside of the nuclei are multicolour fluorescent beads used to correct sample drift and chromatic shifts. (b) STORM image (visualized as scatter plots of computed positions): zoom on a part of left image in panel a showing rectangles oriented across the NE and passing either through NPCs (solid boxes) or in between neighbouring NPCs (dashed boxes). The positions of boxes are defined manually based on the green (NPC or NE) channel only. These boxes are used to quantify the distribution of H3K36me3 in panel c. (c) Density profiles of H3K36me3 as function of distance from the NE, computed from the rectangular boxes as shown in b. Solid curves correspond to boxes passing through NPCs, dashed curves to boxes passing in between neighbouring NPCs. Black curves correspond to control cells, red curves to Tpr KD HeLa P4CCR5 cells. The profiles were computed as described in Methods from 122 NPCs and 305 positions between NPCs in Tpr KD cells versus 140 NPCs and 265 positions between NPCs in control cells; au, arbitrary units.
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