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Nuclear architecture dictates HIV-1 integration site selection

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Abstract

Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome1,2,3,4. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This region contains a series of cellular genes, which are preferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associated domains5 and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellular Nup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome. These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.

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Figure 1: Localization of HIV RIGs at the nuclear periphery.
Figure 2: Integrated, transcriptionally active HIV-1 is found at the nuclear periphery.
Figure 3: HIV RIGs are transcriptionally active genes that are excluded from the LADs.

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Acknowledgements

This work was supported by grants from the Italian National Research Programme on AIDS of the Istituto Superiore di Sanità, Italy, to M.G. and M.L. and from the Young Investigator Grant RF2007-16 of the Italian Ministry of Health to M.L. The authors are grateful to S. Kerbavcic for editorial assistance.

Author information

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Authors

Contributions

B.M., B.L., K.L. and M.L. performed the immuno-DNA FISH and ChIP experiments; A.K.-F., B.M., S.P., M.L. and M.G. analysed the data; A.K.-F., B.M. and S.P. performed the bioinformatics analysis; H.A. and M.L. performed the experiments using infectious virus; L.M. generated and analysed integrase-defective HIV-1 molecular clones; R.L. contributed to studies in primary cells from patients with HIV; A.R. and F.M. generated lentiviral vectors and analysed integration into CD4+ T cells and CD34+ bone marrow cells; M.L. and M.G. conceived and supervised the experiments and wrote the paper with help from the other authors.

Corresponding authors

Correspondence to Mauro Giacca or Marina Lusic.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 HIV-1 RIGs.

a, Probability of recurrence of a random set of genes in different lists of HIV-1 integration sites. The histogram shows the distribution of the number of genes present at least twice in the six HIV-1 integration site lists considered; 1 × 109 independent drawings were evaluated. The distribution peaks around 20 genes, with a maximum observed of 50. The number of RIGs detected experimentally in at least two lists was instead 156 (P < 1 × 10−9). b, Human chromosome map showing the localization of 156 HIV RIGs. Genes found in four, three and two HIV-1 integration lists are highlighted in red, orange and black, respectively. Hotter genomic regions, favoured for HIV-1 integration as described in ref. 1, are highlighted in blue and indicated by a star.

Extended Data Figure 2 Distribution of RIGs or individual integration sites all over the loci analysed by FISH.

The scheme describes the distribution of RIGs (bold) or simple integration sites (regular) around the locus analysed by FISH in Fig. 1; RIGs are in the left panel and hotter zones are in the right panel. As indicated on the side, the total number of RIGs/integrants was calculated within 1, 5 or 10 Mb from the locus analysed by FISH. In total, considering all the RIGs and hotter zones, there are 44 other RIGs/integrants within a window of 1 Mb, 116 within 5 Mb and 169 within 10 Mb around the analysed locus.

Extended Data Figure 3 FISH analysis.

a, Representative images of three-dimensional immuno-DNA FISH of HIV-1 DNA (green) in human primary HIV-1 macrophages stained for mAb414 (red), with relative distribution of FISH signals according to the three concentric zones. b, FISH of HIV-1 DNA (green) in the HIV-1 infected U937 monocytic cell line. c, Representative images of three-dimensional immuno-DNA FISH of lentiviral vector pLV-THM (green) in Jurkat cells. d, Representative images of three-dimensional immuno-DNA FISH of the promoter-less lentiviral vector pCCL–18GFP (green) in Jurkat cells. e, Representative images of three-dimensional immuno-DNA FISH of a gammaretroviral vector (green) in Jurkat cells. For all panels, the graphs are organized as described in the main text.

Extended Data Figure 4 Reconstitution of Nup153 by transfection of an siRNA-resistant plasmid coding for eGFP–Nup153.

a, Scheme of the experiment performed in Jurkat cells. eGFP–Nup153b, Western blot showing Nup153 protein level at the moment of infection. siNT, not targeting siRNA. c, Real-time Alu-PCR in Jurkat cells 2 days after infection with HIV-1NL4.3. Values are mean and s.e.m. of three experiments after normalization over Jurkat transfected with a control, non-targeting siRNA (siNT). d, Representative images of three-dimensional immuno-DNA FISH of HIV-1 DNA (red) in Jurkat cells transfected first with the eGFP–Nup153

Extended Data Figure 5 ChIP-seq profiles for HIV RIGs, cold genes and controls.

ae, Profiles of chromatin modifications around the TSS for HIV RIGs (red) and cold genes (green) compared with highly active (black) and silent (blue) genes in activated CD4+ T cells. Each panel reports results for a specific modification, as indicated.

Extended Data Figure 6 Association of HIV-1 provirus with nucleoporins.

a, Positions of primers used for ChIP on the HIV-LTR (numbering is according to the TSS and nucleosomes are shown), the NPLOC4 RIG, the PTPRD cold gene, B48 and B13 genomic controls for DNA standardization. B48 maps within the human lamin B2 origin of DNA replication. b, Control ChIP data in CD4+ T cells infected with HIV-1NL4-3/E-R-, using total immunoglobulin-γ and an antibody against the unrelated Mcm2 cellular protein. For each analysed region, the amount of immunoprecipitated chromatin using the indicated antibodies was normalized according to the input amount of chromatin. Mean and s.e.m. from at least three independent experiments. P < 0.01. c, ChIP results in CD4+ T cells, 4 days after HIV-1 infection, using the indicated antibodies. The amount of immunoprecipitated chromatin was normalized according to input. Mean and s.e.m. from at least three independent experiments. P < 0.01,P < 0.05. d, ChIP results in CD4+ T cells, 4 days after infection with wild-type HIV-1 or the IN(D64E) mutant virus. For the PPR1 region, corresponding to the viral promoter, the amount of immunoprecipitated chromatin using the indicated antibodies (mAb414, Nup153 and Pol2) was calculated according to the input amount of chromatin, and then normalized over the B13 control genomic region. The graphs show the mean and s.e.m. from three independent experiments. P < 0.001; P < 0.05.

Extended Data Figure 7 HIV-1 transcriptional activation is concomitant with, and requires, nucleoporins binding to the provirus.

a, Quantitative reverse transcription PCR measurement of HIV-1 mRNA in mock- or TPA-treated J-Lat 15.4 cells. b, Three-dimensional immuno-DNA FISH of HIV-1 DNA (green) in J-Lat 15.4 cells stained for NPC (red) before and after TPA reactivation. c, Scheme of the experiment for the generation of a primary, cellular model of HIV-1 latency to study HIV-1 DNA localization in activated and resting primary CD4+ T cells. d, Quantitative reverse transcription PCR measurement of HIV-1 mRNA levels in primary infected CD4+ T cells before and after reactivation, normalized over the 18S housekeeping gene. Latent versus reactivated: P < 0.001. e, Three-dimensional immuno-DNA FISH of HIV-1 DNA (green) in latently infected CD4+ T cells stained for the NPC (red) before and after reactivation, with relative distribution of HIV-1 FISH signals according to the three concentric zones considered in this work. e, ChIP in control and TPA-stimulated J-Lat 15.4 cells, with the indicated antibodies. Mean and s.e.m. from at least three independent experiments. g, Immunoblot for Tpr (upper panel) and Nup153 (lower panel), 36 h after transfection of the indicated siRNAs (NT, non-targeting control). h, Levels of HIV-1 RNA in siRNA-treated J-Lat 15.4 cells after TPA activation. Mean and s.e.m. from three independent experiments. P < 0.05.

Extended Data Figure 8 Silencing of Tpr in HeLa cells and 15.4 J-Lat clones.

a, Scheme of the experiment to study HIV-1 integration in infected HeLa cells after Tpr silencing. b, Western blot showing Tpr protein level at the moment of infection, after treatment of HeLa cells with a non-targeting siRNA (siNT) or an siRNA targeting Tpr at two different doses. Values are mean and s.e.m. of three experiments after normalization over HeLa cells transfected with a control non-targeting siRNA. c, Real-time Alu PCR in HeLa cells infected with HIV-1NL4.3 and previously transfected with a non-targeting siRNA (siNT) or an siRNA targeting Tpr at two different doses. Values are mean and s.e.m. of three experiments after normalization over HeLa cells transfected with a control non-targeting siRNA. d, Luciferase activity assay in HeLa infected with HIV-1NL4.3 and previously transfected with a non-targeting siRNA (siNT) or an siRNA targeting Tpr at two different doses. Values are mean and s.e.m. of three experiments. Statistical significance: P < 0.001; P < 0.01. e, Real-time PCR quantification of IL-2 mRNA levels in J-Lat 15.4 cells. The following conditions were tested: untreated cells, plus TPA (4 h), transfection with non-targeting siRNA or an siRNA targeting Tpr for 24 h, followed by treatment with TPA (4 h). Values are mean and s.e.m. of three experiments after normalization over GAPDH. Transcription of interleukin-2 (IL-2) was not significantly altered upon Tpr downregulation.

Extended Data Figure 9 Model for HIV-1 integration site selection.

After entry into the nucleus through the nuclear pore, the viral DNA integrates into the active chromatin closest to the NPC (green zones), avoiding both LADs and the inner part of the nucleus (red zones).

Extended Data Table 1 List of HIV-1 integration sites considered in this work

Supplementary information

Supplementary Information

This file contains a list of HIV-1 recurrent integration genes (RIGs), hotter zones (HZs) and Controls. (PDF 99 kb)

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Marini, B., Kertesz-Farkas, A., Ali, H. et al. Nuclear architecture dictates HIV-1 integration site selection. Nature 521, 227–231 (2015). https://doi.org/10.1038/nature14226

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