Crystal structure of the Rous sarcoma virus intasome


Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome1. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers2,3,4,5, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

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Figure 1: Overall structure of the RSV intasome.
Figure 2: Proximal and distal IN dimers.
Figure 3: Comparison between the RSV and PFV intasomes.
Figure 4: Viral DNA contacts.
Figure 5: Target DNA.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession number 5EJK.


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We thank K. Kurahashi for generating many mutant IN expression plasmids, J. Kankanala and Z. Wang for synthesizing Pt-modified oligonucleotides, and J. Nix for help with X-ray data collection. X-ray data were collected at the Advanced Photon Source (APS) NE-CAT beamlines, which are supported by the National Institute of General Medical Sciences (P41 GM103403). APS is a US Department of Energy Office of Science User Facility operated by Argonne National Laboratory under contract DE-AC02-06CH11357. This research was supported by National Institutes of Health grants GM109770, AI087098 to H.A. and AI100682 to D.P.G.

Author information




Z.Y. designed strategy and developed protocols for producing and crystallizing the RSV intasome, and prepared all crystals. Z.Y., K.S. and S.Ba. collected X-ray diffraction data. K.S. analysed the data and determined the crystal structure. K.P. and S.Be. examined behaviours of the RSV intasome in solution and analysed mutant IN activities. D.P.G. and H.A. conceived the project and supervised the research. H.A. wrote the manuscript with important input from all authors.

Corresponding author

Correspondence to Hideki Aihara.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Amino-acid sequence alignment of RSV, HIV-1, and PFV INs.

The secondary structure elements for RSV IN are colour coded on the basis of the three IN domains similarly to Fig. 1a. The residue numbering at the top is for RSV IN. For each IN, the black dots mark every ten residues. This figure was made using ESPript41.

Extended Data Figure 2 DNA substrate used for assembling the RSV intasome.

a, The half-site (gapped duplex) substrate prepared by annealing three oligonucleotides dimerizes via the self-complementary six-base spacer sequence (underlined) to form a branched structure mimicking the product of the concerted integration reaction (Fig. 1b). b, DNA structure in the RSV intasome. Viral DNA nucleotides are numbered, and some of the structural elements of RSV IN involved in the viral DNA interactions are shown.

Extended Data Figure 3 RSV intasome crystal.

a, A crystal of the RSV intasome. b, X-ray diffraction pattern from a crystal not treated with metatungstate. c, X-ray diffraction pattern after the metatungstate-soaking (see Methods for details). d, e, Lattice contacts within the RSV intasome crystal. DNA strands are coloured as in Fig. 1b, while IN subunits from only one intasome are coloured. The unit cell is shown in green. The small blue spheres represent tungsten atoms. The view in e is perpendicular to the twofold screw (b) axis of the monoclinic lattice, which lies horizontally. f, Paired-refinement analysis40 to assess the resolution limit of the RSV intasome diffraction data. For each pairwise comparison, model refinements were run at two different resolution limits and the R-factors calculated for a common (lower) resolution cutoff were compared. Inclusion of data beyond 3.7 Å in the refinement compromised model quality.

Extended Data Figure 4 Biochemical characterization of RSV intasome.

a, Representative SEC profile for RSV intasome, overlaid with that for a mixture of molecular mass markers. The buffer condition was as mentioned in the methods. b, SEC profiles for RSV intasomes formed with IN of varying C termini. c, SEC–MALS analysis of RSV intasome. The intasome formed with RSV IN (1–269 amino acids) was separated by SEC in a modified condition containing 20 mM HEPES pH 7.5, 1.0 M NaCl, 5% glycerol, and 1.0 mM TCEP. The absolute molecular mass was determined by light scattering using in-line detectors described previously42. The mass profile for the intasome is shown in red across the peak. The molecular mass of RSV intasome was 240 ± 10 kDa (n = 4). A similar SEC–MALS analysis of the intasome formed with wild-type full-length RSV IN (1–286 amino acids) yielded a molecular mass of 268 kDa (n = 2, data not shown). The calculated mass of an intasome containing eight RSV IN (1–269 or 270) molecules is ~288 kDa. d, Chemical cross-linking analysis of RSV intasome. The RSV intasome and free IN (1–269 amino acids) were purified by SEC in the running buffer: 20 mM HEPES (pH 7.5), 1.0 M NaCl, 5% glycerol, and 1.0 mM TCEP. The peak fractions of the intasome and IN were cross-linked with the indicated amount of ethylene glycol bis-succinimidylsuccinate (EGS) as described previously42 and analysed by SDS–PAGE. Most cross-linked species within the intasome were larger than a tetramer. The highest oligomeric species observed is consistent with an octamer migrating at ~220 kDa. The molecular mass markers are in the far right lane. A NuPAGE 4–12% gradient gel with a MES-based SDS–PAGE running buffer was used.

Extended Data Figure 5 Selenium anomalous difference Fourier peaks confirming the model.

Anomalous difference Fourier maps calculated using the data collected on selenomethionine-labelled RSV intasome at the Se K-edge wavelength, contoured at 3.5σ (blue mesh) or 5.0σ (orange mesh). Methionine side chains are shown in sticks. a, A view covering the octameric RSV intasome. b, c, Close-up view of a proximal (b) and distal (c) IN dimer, respectively.

Extended Data Figure 6 Comparison between RSV and PFV intasomes.

a, b, Protein arrangement in the octameric RSV intasome. The inner and outer subunit of one proximal IN dimer is coloured in green and cyan, respectively, with the catalytic triad (DDE) of the inner subunit shown in red. The other proximal IN dimer is coloured similarly but in more pale colours. Arrows indicate the two proximal IN dimers. A distal IN dimer is coloured in slate and orange. DNA is omitted in a. b, Same view as Fig. 3c. c, Close-up view around the active site of the inner IN subunit in the RSV intasome. The DNA strands are coloured as in Fig. 1, and the catalytic triad residues (DDE) are shown in red sticks. d, e, Protein arrangement in the tetrameric PFV intasome2,3. The colour scheme follows that used for the proximal IN dimers of RSV IN in a and b. Arrows indicate the two IN dimers. DNA is omitted in d. e, Same view as Fig. 3f. f, Close-up view around the active site of the inner IN subunit in the PFV intasome (PDB accession number 3OS0 (ref. 3)). The colour scheme follows that in c.

Extended Data Figure 7 Composite omit maps.

Simulated annealing composite omit 2mFo − DFc density contoured at 1.0σ, shown for area within 3.5 Å from any protein or DNA atom in the final model. Various parts of the RSV intasome are shown in panels a-i. In a and b, electron densities around protein and DNA are coloured differently (blue and green, respectively).

Extended Data Figure 8 DNA conformations in the RSV and PFV intasomes.

a, b, DNA structure in the RSV (a) or PFV (b) intasome, alternatively referred to as the strand-transfer complex (STC). The PFV intasome model is PDB accession number 3OS0 (ref. 3). c, A comparison of DNA structures between the RSV and PFV intasomes (STCs). The integration product DNAs (RSV in cyan, PFV in red) superimposed at a viral DNA terminus are shown in three different view angles. Note the significant deviation in overall trajectory of the target DNA, and difference in the orientation of the second viral DNA molecule. The region spanning the two integration sites on opposing target DNA strands is 6 bp for RSV and 4 bp for PFV.

Extended Data Figure 9 Electron density for the central 6 bp of the target DNA.

The sigma-A weighted 2mFo − DFc map contoured at 1.5σ (a) or 2.5σ (b), overlaid with the final model for the central 6 bp region between the two integration sites.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains the uncropped gel image for Extended Data Figure 4d. (PDF 1491 kb)

The RSV intasome crystal structure

This video shows the overall structure of the RSV intasome and positioning of the three structural domains of IN within the intasome. (MP4 29418 kb)

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Yin, Z., Shi, K., Banerjee, S. et al. Crystal structure of the Rous sarcoma virus intasome. Nature 530, 362–366 (2016).

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