Abstract
Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome1,2. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome–nucleosome interface involving both gyres of nucleosomal DNA and one H2A–H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A–H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.
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Accession codes
Primary accessions
Electron Microscopy Data Bank
Gene Expression Omnibus
Data deposits
The cryo-EM electron density map has been deposited in the Electron Microscopy Data Bank under accession number EMD-2992. Integration sites have been deposited in the NCBI Gene Expression Omnibus under accession number GSE67730.
References
Li, M., Mizuuchi, M., Burke, T. R., Jr & Craigie, R. Retroviral DNA integration: reaction pathway and critical intermediates. EMBO J. 25, 1295–1304 (2006).
Hare, S., Gupta, S. S., Valkov, E., Engelman, A. & Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010).
Maertens, G. N., Hare, S. & Cherepanov, P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010).
Montaño, S. P., Pigli, Y. Z. & Rice, P. A. The mu transpososome structure sheds light on DDE recombinase evolution. Nature 491, 413–417 (2012).
Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389, 251–260 (1997).
Pryciak, P. M., Sil, A. & Varmus, H. E. Retroviral integration into minichromosomes in vitro. EMBO J. 11, 291–303 (1992).
Pryciak, P. M. & Varmus, H. E. Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection. Cell 69, 769–780 (1992).
Pruss, D., Bushman, F. D. & Wolffe, A. P. Human immunodeficiency virus integrase directs integration to sites of severe DNA distortion within the nucleosome core. Proc. Natl Acad. Sci. USA 91, 5913–5917 (1994).
Müller, H. P. & Varmus, H. E. DNA bending creates favored sites for retroviral integration: an explanation for preferred insertion sites in nucleosomes. EMBO J. 13, 4704–4714 (1994).
Wang, G. P., Ciuffi, A., Leipzig, J., Berry, C. C. & Bushman, F. D. HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic modifications. Genome Res. 17, 1186–1194 (2007).
Roth, S. L., Malani, N. & Bushman, F. D. Gammaretroviral integration into nucleosomal target DNA in vivo. J. Virol. 85, 7393–7401 (2011).
Baller, J. A., Gao, J., Stamenova, R., Curcio, M. J. & Voytas, D. F. A nucleosomal surface defines an integration hotspot for the Saccharomyces cerevisiae Ty1 retrotransposon. Genome Res. 22, 704–713 (2012).
Hare, S., Maertens, G. N. & Cherepanov, P. 3′-processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012).
Yin, Z., Lapkouski, M., Yang, W. & Craigie, R. Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration. Protein Sci. 21, 1849–1857 (2012).
Lowary, P. T. & Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276, 19–42 (1998).
Davey, C. A., Sargent, D. F., Luger, K., Maeder, A. W. & Richmond, T. J. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution. J. Mol. Biol. 319, 1097–1113 (2002).
Dyda, F. et al. Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science 266, 1981–1986 (1994).
Shaytan, A. K., Landsman, D. & Panchenko, A. R. Nucleosome adaptability conferred by sequence and structural variations in histone H2A–H2B dimers. Curr. Opin. Struct. Biol. 32, 48–57 (2015).
Müllers, E. et al. Novel functions of prototype foamy virus Gag glycine- arginine-rich boxes in reverse transcription and particle morphogenesis. J. Virol. 85, 1452–1463 (2011).
Trobridge, G. D. et al. Foamy virus vector integration sites in normal human cells. Proc. Natl Acad. Sci. USA 103, 1498–1503 (2006).
Nowrouzi, A. et al. Genome-wide mapping of foamy virus vector integrations into a human cell line. J. Gen. Virol. 87, 1339–1347 (2006).
Guelen, L. et al. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453, 948–951 (2008).
Deal, R. B., Henikoff, J. G. & Henikoff, S. Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 328, 1161–1164 (2010).
Schwabish, M. A. & Struhl, K. Asf1 mediates histone eviction and deposition during elongation by RNA polymerase II. Mol. Cell 22, 415–422 (2006).
Gupta, K. et al. Solution conformations of prototype foamy virus integrase and its stable synaptic complex with U5 viral DNA. Structure 20, 1918–1928 (2012).
Valkov, E. et al. Functional and structural characterization of the integrase from the prototype foamy virus. Nucleic Acids Res. 37, 243–255 (2009).
Hare, S. et al. Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance. Proc. Natl Acad. Sci. USA 107, 20057–20062 (2010).
Schnitzler, G. R. Isolation of histones and nucleosome cores from mammalian cells. Curr. Protoc. Mol. Biol. Chapter 21, Unit 21. (2001).
Dyer, P. N. et al. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol. 375, 23–44 (2004).
Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).
Heymann, J. B. & Belnap, D. M. Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18 (2007).
van Heel, M., Harauz, G., Orlova, E. V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996).
Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).
Hohn, M. et al. SPARX, a new environment for Cryo-EM image processing. J. Struct. Biol. 157, 47–55 (2007).
de la Rosa-Trevín, J. M. et al. Xmipp 3.0: an improved software suite for image processing in electron microscopy. J. Struct. Biol. 184, 321–328 (2013).
Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Heinkelein, M. et al. Improved primate foamy virus vectors and packaging constructs. J. Virol. 76, 3774–3783 (2002).
Stirnnagel, K. et al. Differential pH-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles. Retrovirology 9, 71 (2012).
Stange, A., Luftenegger, D., Reh, J., Weissenhorn, W. & Lindemann, D. Subviral particle release determinants of prototype foamy virus. J. Virol. 82, 9858–9869 (2008).
Rasheed, S., Nelson-Rees, W. A., Toth, E. M., Arnstein, P. & Gardner, M. B. Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer 33, 1027–1033 (1974).
Matreyek, K. A. et al. Host and viral determinants for MxB restriction of HIV-1 infection. Retrovirology 11, 90 (2014).
Chatterjee, A. G. et al. Serial number tagging reveals a prominent sequence preference of retrotransposon integration. Nucleic Acids Res. 42, 8449–8460 (2014).
Schröder, A. R. et al. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110, 521–529 (2002).
Kent, W. J. BLAT—the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002).
Deyle, D. R. et al. A genome-wide map of adeno-associated virus-mediated human gene targeting. Nature Struct. Mol. Biol. 21, 969–975 (2014).
Meuleman, W. et al. Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence. Genome Res. 23, 270–280 (2013).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).
Acknowledgements
This work was supported by the European Union FP7 HIVINNOV consortium grant 305137 (to P.C.), the US National Institute of General Medical Sciences P50 grant GM082251-06 (to P.C.) and the US National Institutes of Health R01 grant AI070042-08 (to A.N.E.). Data collection was in part funded by the Netherlands Centre for Electron Nanoscopy (NeCEN) by grants from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (project 175.010.2009.001) and by the European Union’s Regional Development Fund through ‘Kansen voor West’ (project 21Z.014). We would like to thank L. Collinson, R. Carzaniga and Kirsty MacLellan-Gibson for EM access, R. Horton-Harpin for provision of HeLa cell pellets and assistance with tissue culture. We also thank F. Santoni, N. Sweeny and all our colleagues for helpful discussions.
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Authors and Affiliations
Contributions
D.P.M. analysed interactions of the PFV intasome and nucleosomes, discovered conditions to produce the stable intasome–nucleosome complex and prepared it for EM; L.R., D.P.M. and A.C. performed all EM work with the exception of cryo-EM grid preparation and screening, which was performed by L.R.; R.M. collected cryo-EM data; L.R. and A.C. determined the structure. S.H. designed the co-dependent K120E–D273K PFV IN pair; D.L. designed and provided wild-type PFV vector constructs; P.C. cloned PFV vector mutants; P.C. and P.L. carried out PFV infections; E.S. and A.N.E. developed the protocol and carried out sequencing of PFV integration sites; P.C. analysed integration site distributions.
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Extended data figures and tables
Extended Data Figure 1 PFV integration into recombinant mono-nucleosomes.
a, W601, D02, F02 and H04 nucleosome core particles (left) and W601 nucleosome with 30 bp tails mimicking linker DNA (W601L30; right) were separated by native PAGE and detected by staining with ethidium bromide. b, Major products of PFV integration into a nucleosome core particle. Concerted integration of intasomal oligonucleotides (blue lines) into discontinuous tDNA (black lines) produces pairs of strand transfer products containing viral DNA mimics joined to tDNA fragments via 4 bp gaps. nt, nucleotides. c, PFV integration into nucleosome core particles (W601) and extended nucleosomes (W601L30). Fluorescein-labelled intasomal DNA and reaction products were separated by PAGE and detected by fluorescence scanning. Migration positions of the strand transfer products obtained with W601L30 nucleosome shift relative to those with the W601 core particle by ∼30 bp. Thus, linker DNA does not appear to influence integration. d, Positions of integration events on D02 nucleosomal DNA before (left) or after (right) purification of the complex. The histograms show relative frequencies of integration events along the D02 DNA fragment into the top (blue bars) or bottom (pink bars) strands. The inset shows the nucleotide sequence at the preferred integration site; arrowheads indicate precise positions of the major integration events into the top and bottom strands of D02 DNA.
Extended Data Figure 2 Pull-down of native nucleosomes and naked DNA by biotinylated PFV intasome.
a, Mono-nucleosomes prepared by micrococcal nuclease digestion of HeLa cell chromatin were incubated with biotinylated intasomes under conditions of indicated ionic strength (190–340 mM NaCl). The intasomes used were wild type (WT), A188D or a hybrid intasome lacking the NTDs and CTDs on the outer subunits (indicated as ΔNTD/ΔCTD; see main text and Extended Data Fig. 6a–c for details of hybrid intasome design). The intasome–nucleosome complexes were isolated on streptavidin agarose and separated by SDS–PAGE. Proteins and nucleosomal DNA were detected by staining with Coomassie blue and GelRed, respectively. Two leftmost lanes contained 50% and 25% of input nucleosomes, as indicated. Migration positions of protein sizes standards (kDa) are shown to the left of the gel. b, Isolation of HeLa nucleosomes preferentially binding to the PFV intasome. Biotinylated wild-type or A188D intasomes were incubated with tenfold excess HeLa nucleosomes in the presence of 290 mM NaCl. Nucleosomal DNA recovered with wild-type intasome was cloned into a bacterial vector; the histogram depicts distribution of nucleosomal insert sizes obtained in this experiment. The inset shows separation of deproteinized nucleosomal DNA from 10% of input nucleosome material and from the fractions recovered with wild-type and A188D intasomes. c, Nucleotide sequences of three human DNA fragments (H04, F02 and D02) recovered with the intasome and used to assemble recombinant nucleosomes in this work. d, Naked W601 D02, F02 or H04 DNA was incubated with biotinylated wild-type or A188D intasomes in the presence of 190 or 240 mM NaCl, as indicated; DNA fractions recovered after pull-down on streptavidin beads were separated by PAGE and detected by staining with GelRed.
Extended Data Figure 3 Thermal denaturation of recombinant nucleosomes.
Derivative melt profiles of recombinant nucleosomes used in this study. The table in the inset shows experimentally determined melting temperatures.
Extended Data Figure 4 Overview of the cryo-EM data.
a, Representative micrograph of frozen hydrated intasome–nucleosome complex. b, Two-dimensional class averages (phase-flipped only; box size 26 nm). c, Euler angle distribution of all particles included in the final three-dimensional reconstruction. Sphere size relates to particle number. d, Gold standard Fourier-shell correlation and resolution using the 0.143 criterion. e, Three-dimensional volume of the intasome–nucleosome complex refined with RELION. f, Match between reference-free two-dimensional class averages and three-dimensional re-projections of the cryo-EM structure. Two-dimensional class averages of fully contrast transfer function (CTF)-corrected particles are matched with the re-projections of the refined three-dimensional structure before map sharpening (post-processing); 30–6 Å band-pass filter imposed. g, Overview of the three-dimensional classification and structure refinement. The initial negative stain structure was used for one round of structure refinement using a smaller cryo data set. The resulting map was used as a starting model for one round of three-dimensional classification (three classes) on a complete cryo data set. Particles from the two most populated three-dimensional classes were merged and used for one further round of three-dimensional classification (six classes). Each three-dimensional class was refined independently; the most populated three-dimensional class comprising 53,887 particles refined to 7.8 Å resolution.
Extended Data Figure 5 Nucleosomal DNA plasticity.
Overview of the intasome–nucleosome complex structure (left) and a magnified stereo view of nucleosomal DNA engaged within tDNA binding cleft of the intasome (right). DNA conformations as in free nucleosomes (Protein Data Bank accession 1KX5) and as tDNA in complex with the PFV intasome (3OS1) are shown in light and dark grey, respectively; the arrowhead shows approximate direction of the DNA deformation. Asterisks indicate nucleosomal DNA ends.
Extended Data Figure 6 Hybrid intasomes: structure-based design and validation in vitro.
a, Views on the environment of Lys 120 and Asp 273 PFV IN residues within the intasome structure. Protein is shown as cartoons with side chains of selected amino acid residues shown as sticks; the cartoons and carbon atoms of the inner and outer IN chains are shown in green and light orange, respectively. Lys 120 of the outer and Asp 273 of the inner IN subunit are involved in a network of interactions; by contrast, Lys 120 of the inner and Asp 273 of the outer IN subunit are solvent-exposed. Consequently, IN mutants harbouring substitutions of Lys 120 or Asp 273 can only have a role in the inner or outer intasomal subunits, respectively; b, PFV IN mutants K120E and D273K are co-dependent for intasome assembly. Products of intasome assembly using wild-type (WT), D273K, K120E PFV IN or an equimolar mixture of D273K and K120E INs were separated by size-exclusion chromatography. Elution positions of the intasome, IN and free DNA are indicated. The assembly was successful with wild-type IN or with a mixture of the two IN mutants, but not with either of the IN variants separately. c, Validation of the hybrid intasome design. Left, possible types of strand transfer products obtained by reacting the intasome with circular DNA target (pGEM, black lines). Full-site integration (strand transfer involving both intasomal DNAs, dark blue lines) results in a linear concerted product, which may be targeted by further strand transfer events. Half-site integration (strand transfer involving a single intasomal DNA end) results in a circular branched single-end product. Right, strand transfer assays using mutant intasomes and circular pGEM DNA target. The intasomes were assembled using wild-type IN or a mixture of K120E and D273K mutants, as indicated on top of the gel. IN variants marked with a cross additionally incorporated the E221Q amino acid substitution that disables the enzyme active site. Reaction products were separated by agarose gel electrophoresis. Intasomes were used at indicated concentrations; the leftmost lane contained a mock sample, which received no intasome. Migration positions of the reaction products, intasomal DNA and unreacted supercoiled (s.c.) pGEM are indicated to the right of the gel. As predicted, the strand transfer function of the hybrid intasome strictly requires the active site from the K120E (inner) IN subunit, but not the D273K (outer) subunit. d, Strand transfer activity of mutant intasomes on naked plasmid DNA. Mutations indicated in orange or green were restricted to the outer or inner subunits of the hybrid PFV intasome, respectively.
Extended Data Figure 7 Infectivity of the mutant PFV vectors.
a, Schematic of the experiments. PFV vectors were produced in 293T cells transfected with DNA constructs encoding PFV GAG, POL and ENV, plus a GFP reporter transfer vector (pMD9). The virus, concentrated by centrifugation, was applied onto target HT1080 cells. Five days post-infection, the cells were analysed by FACS and/or used for isolation of genomic DNA and integration site sequencing. IN mutations were introduced into the packaging construct encoding POL (pcoP-POL). b, Validation of the hybrid intasome design in viral culture conditions. PFV GFP virus was produced using wild-type (WT), K120E, D273K POL packaging construct or a mixture of K120E and D273K mutants. The variants marked with a cross additionally contained a double point mutation inactivating the IN active site (D185N/E221Q). The graph and the western blot show mean relative infectivity and GAG contents (pr71 and p68) of the resultant viruses, respectively. All infectivity experiments were done at least in triplicate, with two or more independent virus preparations; error bars represent standard deviations. The K120E and D273K IN mutants are co-dependent for production of infectious PFV vector, and the functional active site of the K120E IN component is essential for production of infectious hybrid virus. c, Relative infectivity of the PFV vectors harbouring wild-type, K168E, or active-site-dead D185N/E221Q (indicated with a cross) IN. d, Relative infectivities of hybrid viruses produced using D273K/D185N/E221Q (indicated as D273K with a cross) and K120E, K120E/D185N/E221Q (cross), K120E/P135E, K120E/T240E, and K120E/P135E/T240E. The western blots below the graphs show GAG (pr71 and p68) contents of the respective PFV vector preps.
Extended Data Figure 8 Local nucleotide sequence biases at PFV integration sites.
Nucleotide sequence preferences at PFV integration sites in cellula (wild type (WT), K168E, hybrid control and P135E/T240E) or in vitro displayed in the form of sequence logos. The heights of the logos correspond to the maximum information content at each position (maximum information content being 2 bits per base). Position 0 corresponds to the target nucleotide joined to the processed U5 PFV end.
Extended Data Figure 9 Negative-stain EM analysis.
a, Representative micrograph. b, Reference-free class averages. c, Three-dimensional electron density map of the intasome–nucleosome complex with a docked intasome structure. Note that DNA density is not recovered with negative-stain EM.
Supplementary information
The PFV intasome - nucleosome complex at 7.8 Å resolution
This video displays the overall structure, intasome-nucleosome interface and the key components of the complex. (MOV 15144 kb)
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Maskell, D., Renault, L., Serrao, E. et al. Structural basis for retroviral integration into nucleosomes. Nature 523, 366–369 (2015). https://doi.org/10.1038/nature14495
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DOI: https://doi.org/10.1038/nature14495
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