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Structural insights into the retroviral DNA integration apparatus
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Structural insights into the retroviral DNA integration apparatus Peter Cherepanov, Goedele N Maertens and Stephen Hare Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 30 viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes. Address Division of Infectious Diseases, Imperial College London, St. Mary’s Campus, Norfolk Place, London W2 1PG, UK Corresponding author: Cherepanov, Peter ([email protected])
Current Opinion in Structural Biology 2011, 21:249–256 This review comes from a themed issue on Macromolecular assemblages Edited by Felix Rey and Wes Sundquist Available online 1st February 2011 0959-440X/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2010.12.005
Introduction Before or, more commonly, following entry into a host cell, the retroviral diploid RNA genome is converted into a DNA form by the action of reverse transcriptase. The resulting double stranded linear DNA molecule is transported into the nucleus and inserted into a host cell chromosome. The key DNA strand cutting and joining reactions associated with this process are catalyzed by integrase (IN) (for detailed reviews on the early steps of retroviral replication and IN functions, see [1,2]). Firstly, IN carries out 30 -processing, typically cleaving a dinucleotide from both 30 ends of the viral DNA to expose 30 hydroxyls attached to invariant CA dinucleotides (Figure 1a and b). Following entry into the nucleus, IN inserts the recessed 30 ends of the viral DNA molecule into opposing strands of host cell chromosomal DNA, www.sciencedirect.com
across the major groove and separated by 4–6 bp (Figure 1d and e; the size of the stagger depends on the retroviral species). Host cell enzymes repair the resulting single-stranded gaps and 50 overhangs that initially flank integrated viral DNA to finally establish a stable provirus (Figure 1f and g). Retroviral INs belong to the functionally diverse superfamily of DDE(D) nucleotidyltransferases, whose other notable members include RNaseH as well as MuA, Tn5, and Mos1 transposases [3–6,7,8]. The active sites of these enzymes typically contain three essential carboxylates that coordinate a pair of divalent metal (Mg2+ or Mn2+) cations. The DDE(D) family members share a common mechanism of metal ion-dependent phosphodiester bond cleaving and joining via SN2 nucleophilic substitution at the scissile phosphodiester, using a water molecule or a 30 hydroxyl group as a nucleophile, respectively (reviewed in [9,10]). To carry out its function, retroviral IN must act upon two types of DNA substrates: a pair of viral DNA ends and chromosomal target DNA (tDNA). Although INs recognize viral DNA substrates in a sequence-specific manner [11,12], they are not very selective with respect to the target. Nonetheless, interesting patterns transpired upon analyses of large numbers of retroviral integration sites (reviewed in [13]). Firstly, each retrovirus has a weak, symmetric integration site sequence consensus [14,15]. Secondly, retroviruses display genus-specific biases towards genomic features, such as transcription units, CpG islands and epigenetic marks [16]. At the genomic level, the retroviral integration site preferences are thought to depend on host factors. Thus, the interaction between IN and LEDGF, a cellular chromatin-associated protein, targets HIV-1 integration to active transcription units [17,18] (reviewed in [19,20]). By contrast, the local integration site sequence consensus appears to primarily depend on IN itself [18,21,22]. To date, a wide range of small molecule inhibitors of HIV-1 IN capable of blocking viral replication have been reported (reviewed in [23]). The best-validated class of IN inhibitors are bioisosteres of the early diketo acid scaffold described by Hazuda et al. [24]. These compounds bind at the IN active site and specifically interfere with its strand transfer activity [25,26]. One such IN strand transfer inhibitor (INSTI), raltegravir [27], has been in clinical use since late 2007. HIV-1 IN comprises N-terminal, catalytic core and Cterminal domains (NTD, CCD and CTD, respectively), Current Opinion in Structural Biology 2011, 21:249–256
250 Macromolecular assemblages
Figure 1
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A schematic of the retroviral DNA integration process and its inhibition by INSTIs. (a) The ends of viral DNA are synapsed by a multimer of IN (large blue oval, with small red ovals representing a pair of active sites). 30 -Processing probably takes place within such a synaptic complex, resulting in the longlived intermediate (b). Binding of an INSTI to the intasome traps it in an inactive state (c). Upon nuclear entry, the intasome binds tDNA, forming the target capture complex (d), and strand transfer ensues. The post-catalytic nucleoprotein complex is known as the strand transfer complex (STC, e). Following disassembly of the STC, the gapped intermediate (f) is repaired to produce the stable provirus (g). The figure was adapted from [20].
each of which is essential for the biologically relevant enzymatic activities [28]. The domains, connected by flexible linkers within the full-length protein, are all capable of homodimerization. These canonical domains can be readily identified in all retroviral INs. Over the past 16 years, a collection of structures containing various fragments of INs from HIV-1 and divergent retroviruses were reported (for a detailed recent review see [29]). Unfortunately, the partial structures did not reveal a consistent conformation for the interdomain linkers, or the ordered active site, thwarting confident modeling of IN in its functional state. Recently, a series of structures of prototype foamy virus (PFV) IN in complex with its DNA substrates have been determined [22,30]. A high level of amino acid sequence identity within HIV and PFV IN active sites, made it possible to use the latter as a convenient proxy for structural studies of INSTIs [30,31].
Nucleoprotein complexes involved in retroviral DNA integration In the context of viral infection, IN operates within large nucleoprotein assemblies. The long-lived intermediate containing viral DNA with processed 30 ends is referred to as the pre-integration complex (PIC) [32,33]. Although in vivo integration probably relies on a variety of co-factors, it is possible to faithfully reproduce this process using Current Opinion in Structural Biology 2011, 21:249–256
purified recombinant IN and DNA substrates. The development of in vitro systems allowed biochemical characterization of some of the key HIV-1 integration intermediates [34–36], and moreover their visualization using atomic force microscopy [37]. The intasome, a highly stable synaptic complex that contains a pair of viral DNA ends and a tetramer of IN, appears to be the minimal nucleoprotein complex capable of concerted integration (i.e. integration of pairs of viral DNA ends) in vitro [35,36]. Upon engaging tDNA, in a target capture complex (TCC), the intasome will carry out strand transfer, forming the post-catalytic strand transfer complex (STC, Figure 1d and e) [34–36]. The intasome is the target for INSTIs, which compete with tDNA binding and thereby prevent formation of the TCC (Figure 1c) [25].
The structure of the PFV intasome HIV-1 IN and a handful of other retroviral INs characterized early on are notorious for their unfavorable biochemical properties. Fortunately, the large family of Retroviridae provides an abundant pool of orthologous proteins, which are structurally conserved, yet highly divergent at the amino acid sequence level. Comparative analyses of a variety of retroviral INs in our laboratory identified the ortholog from prototype foamy virus (PFV) as a soluble enzyme, highly proficient in synapsing short www.sciencedirect.com
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oligonucleotide mimics of viral DNA ends in vitro [21]. Symmetric intasome complexes, assembled using fulllength PFV IN and oligonucleotide mimics of pre-processed U5 viral DNA ends, yielded diffracting crystals. The structure, initially solved at 2.8 A˚ [30] and later refined at resolutions of up to 2.0 A˚ [31], revealed a tetramer of IN and a pair of synapsed viral DNA ends (Figure 2). As predicted based on partial structures [38,39], the tetramer is a dimer-of-dimers with two functionally and structurally distinct pairs of IN subunits. The inner subunits (colored cyan and green in Figure 2) form an extensive dimer–dimer interface and are responsible for all interactions with viral DNA and catalysis. The outer IN subunits (yellow in Figure 2) do not interact with each other or the viral DNA and seem to play a supporting role. The CCD–CCD interface between the inner and outer IN chains is similar to that observed in partial IN
Figure 2
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structures. The NTD and CTD domains of the outer subunits are disordered in the crystals and their functions are currently unknown. Within the intasome, the protein–DNA interactions are very extensive, and each viral DNA end interacts with both inner IN subunits, contributing to the stability of the intasome (Figure 2). The reactive strands of the viral DNA ends (magenta) enter the IN tetramer through the dimer–dimer interface, inserting their 30 ends into the active sites of the inner IN subunits (Figure 2a). The nontransferred strands (orange) are threaded between layers of the protein structure, each sandwiched between the CCD and the interdomain linkers of the inner subunits (Figure 2b). This topology explains why IN requires its recognition site to be close to the end of its DNA substrate, and appears incompatible with a pre-formed IN tetramer being an intermediate in the intasome assembly pathway. Plausibly, an initial interaction of viral DNA ends and IN CCDs could trigger formation of the synaptic interface. Similar, functional multimerization of MuA and Tn5 transposases depends on DNA binding [6,40]. The mechanism of intasome assembly will be an important subject of further investigations. Interestingly, while recombinant PFV IN is predominantly monomeric in solution [21,41], HIV-1 IN tends to form tetramers and higher order multimers even at low mM concentrations [39,42,43]. This propensity for DNAindependent multimerization might account for the difficulties in obtaining sizable quantities of the HIV-1 intasome. IN tetramers are stabilized by inter-dimer NTD–CCD interactions, which are also involved in the intasome assembly [30,38,39], although the functional significance of the DNA-independent IN tetramerization remains unclear.
The IN active site and strand transfer inhibitors
Current Opinion in Structural Biology
The structure of the PFV intasome (PDB ID 3OY9), with IN chains shown as cartoons (a) or space-fill (b) representations. The intasome is viewed along (a) or normal to (b) the crystallographic two-fold axis. The inner IN chains are colored green and cyan; the outer chains are yellow. The reactive and non-transferred strands of the viral DNA ends are magenta and orange, respectively. Active site carboxylates are shown as red sticks; large and small gray spheres are Mn2+ and Zn2+ ions, respectively. Locations of the IN domains (N-terminal extension domain (NED), NTD, CCD and CTD) are indicated in panel a. The outer IN chains were not resolved beyond their CCDs in the available PFV intasome structures. www.sciencedirect.com
The region between b5 and a4 within the CCD of IN, historically referred to as the active site loop, is typically disordered in partial IN structures. The disorder often extends to the beginning of a4, including the invariant Glu of the DDE active site motif. The loop adopts a stable conformation within the PFV intasome structure due to intimate interactions with the viral DNA end. Residues Pro214-Gly218 (corresponding to Pro145-Gly149 in HIV1) form a 310 helix (h2), which packs against the invariant adenine base of viral DNA and separates the reactive and non-transferred strands (Figure 3a). The carboxylates comprising the DDE motif are in close proximity to the reactive 30 hydroxyl of the viral DNA, collectively forming an active site capable of binding a pair of Mn2+ ions (metal A and B; Figure 3a). In the presence of Mn2+, the reactive 30 hydroxyl group of viral DNA is coordinated by metal B. The positions of the catalytic carboxylates, the catalytic metal ions and the 30 nucleotide are remarkably similar to Current Opinion in Structural Biology 2011, 21:249–256
252 Macromolecular assemblages
Figure 3
those within the active sites of Tn5 and Mos1 transpososomes (Figure 3b), despite limited resemblance in the overall architectures of the nucleoprotein complexes (Figures 2, 3c and d).
(a) G T A
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HIV-1 and PFV INs share a high degree of amino acid sequence identity within their active sites. Furthermore, the active site residues ordered within partial crystal structures of divergent retroviral INs superpose extremely well [21]. It is not surprising therefore that INSTIs are effective against INs from a wide range of retroviral species, including PFV [21,44,45]. When PFV intasome crystals were soaked in the presence of raltegravir or other INSTIs, the compounds invariably bound at the active site, interacting with the catalytic metal ions via triads of strategically positioned heteroatoms (Figure 4a and b) [30,31]. The planar hydrophobic groups of the inhibitors intercalate between the 310 helix of the active site loop and the invariant CG base pair of the viral DNA, displacing the viral 30 deoxyadenosine together with its crucial 30 hydroxyl group from the active site. Superposition of various INSTIs in their intasome-binding conformations highlights the consensus features of the pharmacophore (Figure 4b). Because the intasome contacts are so similar among the INSTIs [31], teasing out the cryptic differences between them will require quantitative energy calculations.
D128 β1
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Tn5 Current Opinion in Structural Biology
The active site of PFV IN engaged with a viral DNA end (a). An inner IN chain of the intasome is shown as cartoons, with DNA and side chains of active site residues Asp128, Asp185 and Glu221 as sticks. Colors of cartoons and carbon atoms are specific to the protein and DNA chains, conserved from Figure 2, except for the IN residues Pro211-Gly218, displayed in dark blue. Secondary structure elements of the IN CCD [4,30], carboxylates of the DDE motif, invariant retroviral DNA nucleotides (50 -CA-30 /50 -TG-30 ) and Mn2+ ions (A and B) are indicated. (b) Conservation of the DDE(D) recombinase active site structure. The active sites of Tn5 (PDB ID 1MUS) and Mos1 (PDB ID 3HOT) transposomes and PFV intasome (PDB ID 3OY9) were superposed based on the Ca atoms of the active site carboxylates. Selected portions of the protein structures are shown as cartoons and the 30 -nucleotides as sticks. Cartoons, carbon and metal atoms of Tn5, Mos1, and PFV structures are colored green, pink, and cyan, respectively (c, d). Current Opinion in Structural Biology 2011, 21:249–256
The crystallographic studies of the PFV intasome explained how divergent INSTIs bind within the active site and even shed some light on the mechanism of the major raltegravir-resistance mutations [30,31]. Importantly, the PFV intasome crystal structures allow relatively straightforward modeling of the HIV-1 intasome in its drug-free and drug-bound forms [46]. Such models will be very useful in further development of the INSTIs and understanding the HIV drug resistance mutations. Although the PFV intasome is a convenient model for structural studies of INSTIs, it certainly is not ideal, as the level of amino acid identity between PFV and HIV-1 INs is low outside of their active sites, where mutations contributing to INSTI resistance often occur [47]. We hope that the homology models will aid the design of improved HIV-1 IN constructs to enable determination of an experimental structure.
Engagement of chromosomal DNA and strand transfer: the point of no return Co-crystallization of the PFV intasome with tDNA constructs resulted in visualization of both pre-catalytic (TCC) and post-catalytic (STC) nucleoprotein complexes at resolutions of 3.0 and 2.8 A˚, respectively [22]. As predicted based on the intasome structure, Comparison of the Mos1 (c) and Tn5 (d) transposome structures. Protein chains are shown as cartoons (cyan and green). Reactive and nontransferred DNA strands are magenta and orange, respectively. www.sciencedirect.com
Structural insights into the retroviral DNA integration apparatus Cherepanov, Maertens and Hare 253
Figure 4
Figure 5
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The mode of INSTI binding at the IN active site. (a) A molecule of raltegravir (RAL, yellow) bound at the active site (PDB ID 3OYA). Red arrow indicates displacement of the viral DNA 30 hydroxyl group upon drug binding. (b) Superposition of nine drug molecules of various scaffolds in their bound conformations (PDB IDs 3OYA-3OYG and 3L2W). The INSTIs are shown as sticks with yellow carbon atoms. Red and green ovals indicate positions of the INSTI metal chelating and halobenzyl features, respectively. The PFV IN active site carboxylates and the corresponding residues in HIV-1 are indicated in black and gray, respectively.
tDNA binds along the groove dividing the symmetric complex (Figure 5a). A severe deformation of tDNA, accompanied by a dramatic expansion of its major groove, allows insertion of the scissile phosphodiesters into the intasome active sites separated by 27 A˚. By contrast, the intasome itself does not undergo significant changes upon tDNA capture. The bending of tDNA is focused on a single dinucleotide step at the center of the integration site, leading to a complete unstacking of the consecutive base pairs (Figure 5b and c). Concordantly, www.sciencedirect.com
β1
E221 β2
α1
Current Opinion in Structural Biology
Capture of tDNA by the intasome and strand transfer. (a) Structure of the PFV TCC (PDB ID 3OS1), viewed along the crystallographic two-fold axis and in the orientation conserved from Figure 2a. IN chains are shown in space-fill mode; tDNA strands are shown as red and dark gray cartoons. Positions of Arg329 and Ala188 are indicated. (b, c) Conformations of the DNA strands within TCC (PDB ID 3OS2, b) and STC (PDB ID 3OS0, c). A negative roll angle of 608, leading to unstacking of the base pairs at the center of the integration site is indicated. (d) Active site mechanics during strand transfer. Structures of the PFV TCC, STC and Mn2+-bound intasome (PDB IDs 3OS1, 3OS0 and 3OY9, respectively) were superposed based on the Ca atoms of the active site carboxylates. Displayed are tDNA strands from the TCC and STC structures with carbon atoms in light and dark gray; reactive viral DNA strands from the intasome and STC in light and dark magenta; metal ions from the intasome and STC in light and dark gray, respectively. Red dotted line indicates the direction of in-line nucleophilic attack by the 30 hydroxyl group on the scissile tDNA phophodiester. Orange arrow indicates the displacement of the target phosphodiester in the STC relative to its position in the TCC.
Current Opinion in Structural Biology 2011, 21:249–256
254 Macromolecular assemblages
PFV integration sites are naturally enriched with more flexible pyrimidine–purine dinucleotides at the central positions [22]. The path of the scissile strand of tDNA within the IN active site clashes with the observed binding location of INSTIs, explaining the competition between tDNA and INSTIs for binding to the intasome [25]. While effects of chromatin structure on integration are not fully understood, retroviral INs can utilize nucleosomal DNA as a target [48–52]. In the PFV TCC and STC structures, the kinking of the tDNA duplex at the center of the integration site is considerably more extreme than the average DNA bending within the nucleosome structure [53]. It is not surprising therefore that when supplied with chromatinized tDNA in vitro, HIV-1 IN is biased towards sites where nucleosomal DNA is most severely deformed, with the widest major groove exposure [50]. Yet, differences in tDNA bending requirements of retroviruses that integrate with staggers other than 4 bp can be foreseen. Thus, if a similar overall tDNA deformation is required for HIV-1 integration, which targets phosphodiesters separated by 5 bp in tDNA, the expected twofold symmetry of the TCC would necessitate the deformation to be distributed over two base pair steps rather than one, resulting in a smoother tDNA bend. As in the original intasome crystals, the outer IN chains are not resolved beyond their CCDs. The network of interactions between the intasome and tDNA include eight rigid hydrogen bonds between IN main chain amides and the phosphodiester backbone, which serve to ensure precise positioning of the tDNA with respect to the active sites. Strong sequence selectivity for tDNA would limit the pool of suitable integration sites and thus would be disadvantageous for a virus. As expected, direct contacts between tDNA bases and IN are few and are not optimized for sequence selectivity. The most intimate of these involves the symmetric pair of Arg329 residues, which insert their side chains into the expanded major groove of tDNA. Additionally, Ala188 within the short a2 helix contacts the minor groove (Figure 5a). Mutations at these IN positions have drastic effects on the strand transfer activity and/or the integration site selection [22,54,55]. The combination of the indirect recognition (tDNA flexibility) and direct weak interactions with tDNA bases appears to account for the observed PFV integration site consensus [22]. The PFV TCC and STC crystal structures can now be used as a starting base for the rational design of tDNA sequence-specific INs, which may allow the development of safer retroviral vectors targeted to predefined loci of human genome [56].
placed for in-line nucleophilic attack on the scissile tDNA phosphodiester, coordinated between metals A and B (Figure 5d). This geometry is indeed consistent with two-metal catalysis [5,9]. The net chemical bond energy does not change in the course of a transesterification reaction, making it an inherently reversible process. Yet, retroviral integration and prokaryotic transposition would be highly inefficient if a frequent reversal of strand transfer were permitted. Although the overall conformations of the DNA chains within TCC and STC structures are quite similar (Figure 5b and c), the positions of the scissile phosphodiester in the TCC and the phosphodiester joining viral and tDNA strands in the STC do not match. While the former is placed in the IN active site, the latter is shifted by 2.3 A˚ and thus ejected from the active site (Figure 5d). The configuration of the STC cannot support a reverse reaction, which would involve a nucleophilic attack by the 30 hydroxyl of the tDNA on the newly formed phosphodiester. Although crystal structures do not provide dynamic information, it seems likely that the subtle change is driven by the strain in the tDNA conformation, which is relieved upon scission of the target phosphodiester bond. The conformations of the tDNA base pairs flanking the integration site in the STC are more consistent with the energetically favorable B form than those in the TCC. Intriguingly, Tn10 transposase also bends tDNA within its TCC and STC [57]. The transposon inserts with a 9-bp duplication of the target sequence and, therefore, engages phosphodiesters separated by almost 35 A˚ in a B-form DNA duplex. Hence, tDNA bending could be a common feature of DDE(D) recombinases, used not only to gain access to the scissile phosphodiesters in tDNA, but also to introduce a directional bias into the transesterification reaction. The available crystal structures of the PFV intasome, TCC and STC do not hint at any mechanistic linkage between the active sites of the inner IN subunits. Therefore, what we know as ‘concerted integration’ is probably a result of two independent strand transfer events, occurring stochastically and not necessarily simultaneously. Owing to a combination of design and chance, the PFV TCC and STC crystallized as perfectly symmetric assemblies [22]. In a more general case, as the intasome encounters asymmetric tDNA in vitro or chromatin in vivo, one of its active sites may well be in a more favorable environment to carry out strand transfer than the other. Indeed, concerted HIV-1 IN integration appears to occur sequentially in vitro [35].
Perspectives Superposition of the TCC and Mn2+-bound intasome structures allows modeling of the IN active site in a state committed for strand transfer. In this model, the 30 hydroxyl group of viral DNA bound to metal ion B is Current Opinion in Structural Biology 2011, 21:249–256
The recent crystal structures of the PFV IN nucleoprotein complexes [22,30] have revealed a great amount of detail about the mechanism of retroviral DNA integration and also opened exciting avenues for further investiwww.sciencedirect.com
Structural insights into the retroviral DNA integration apparatus Cherepanov, Maertens and Hare 255
gations. The structures are already helping to understand the cryptic mechanisms of HIV-1 resistance to INSTIs [30,31] and will hopefully aid in the determination of an experimental structure of the HIV-1 or a closely related lentiviral intasome. Among many open questions are how the intasome engages chromatin, how the resulting product complex is disassembled, and how this process is coupled to post-integration DNA repair. We are also optimistic that PFV TC/STC structures will guide the design of tDNA sequence-specific INs, which would be extremely helpful in reducing the chance of side effects in gene therapy applications.
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Acknowledgements
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We thank A. Engelman for critical reading of the manuscript, D. Haniford and M.D. Cummings for helpful discussions. Our work is funded by UK Medical Research Council grants G0900116 and G1000917.
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10. Nowotny M: Retroviral integrase superfamily: the structural perspective. EMBO Rep 2009, 10:144-151.
28. Engelman A, Bushman FD, Craigie R: Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex. EMBO J 1993, 12:3269-3275.
11. Esposito D, Craigie R: Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein– DNA interaction. EMBO J 1998, 17:5832-5843.
29. Jaskolski M, Alexandratos JN, Bujacz G, Wlodawer A: Piecing together the structure of retroviral integrase, an important target in AIDS therapy. FEBS J 2009, 276:2926-2946.
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30. Hare S, Gupta SS, Valkov E, Engelman A, Cherepanov P: Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 2010, 464:232-236. This breakthrough paper contains the initial description of the PFV intasome structure and the mechanism of action of the IN strand transfer inhibitors (PDB IDs 3L2Q-3L2W). 31. Hare S, Vos AM, Clayton RF, Thuring JW, Cummings MD, Cherepanov P: Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance. Proc Natl Acad Sci USA 2010, 107(46):20057-20062. Herein, Hare et al. present a set of crystal structures of the PFV intasome with an expanded set of HIV IN inhibitors and suggests a mechanism for the key Raltegravir resistance mutations (PDB IDs 3OY9-3OYN). 32. Miller MD, Farnet CM, Bushman FD: Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition. J Virol 1997, 71:5382-5390. 33. Farnet CM, Haseltine WA: Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J Virol 1991, 65:1910-1915. 34. Pandey KK, Bera S, Zahm J, Vora A, Stillmock K, Hazuda D, Grandgenett DP: Inhibition of human immunodeficiency virus type 1 concerted integration by strand transfer inhibitors which recognize a transient structural intermediate. J Virol 2007, 81:12189-12199. 35. Li M, Mizuuchi M, Burke TR Jr, Craigie R: Retroviral DNA integration: reaction pathway and critical intermediates. EMBO J 2006, 25:1295-1304. 36. Li M, Craigie R: Nucleoprotein complex intermediates in HIV-1 integration. Methods 2009, 47:237-242. 37. Kotova S, Li M, Dimitriadis EK, Craigie R: Nucleoprotein intermediates in HIV-1 DNA integration visualized by atomic force microscopy. J Mol Biol 2010, 399:491-500. This paper describes visualization and characterization of HIV-1 IN intasome and strand transfer complexes at low resolution. 38. Wang JY, Ling H, Yang W, Craigie R: Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein. EMBO J 2001, 20:7333-7343. 39. Hare S, Di Nunzio F, Labeja A, Wang J, Engelman A, Cherepanov P: Structural basis for functional tetramerization of lentiviral integrase. PLoS Pathog 2009, 5:e1000515. 40. Baker TA, Mizuuchi K: DNA-promoted assembly of the active tetramer of the Mu transposase. Genes Dev 1992, 6:2221-2232. 41. Delelis O, Carayon K, Guiot E, Leh H, Tauc P, Brochon JC, Mouscadet JF, Deprez E: Insight into the integrase-DNA recognition mechanism. A specific DNA-binding mode revealed by an enzymatically labeled integrase. J Biol Chem 2008, 283:27838-27849. 42. Deprez E, Tauc P, Leh H, Mouscadet JF, Auclair C, Brochon JC: Oligomeric states of the HIV-1 integrase as measured by timeresolved fluorescence anisotropy. Biochemistry 2000, 39:9275-9284. 43. McKee CJ, Kessl JJ, Shkriabai N, Dar MJ, Engelman A, Kvaratskhelia M: Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein. J Biol Chem 2008, 283:31802-31812.
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44. Rabaaoui S, Zouhiri F, Lancon A, Leh H, d’Angelo J, Wattel E: Inhibitors of strand transfer that prevent integration and inhibit human T-cell leukemia virus type 1 early replication. Antimicrob Agents Chemother 2008, 52:3532-3541. 45. Smith RA, Gottlieb GS, Miller AD: Susceptibility of the human retrovirus XMRV to antiretroviral inhibitors. Retrovirology 2010, 7:70. 46. Krishnan L, Li X, Naraharisetty HL, Hare S, Cherepanov P, Engelman A: Structure-based modeling of the functional HIV-1 intasome and its inhibition. Proc Natl Acad Sci USA 2010, 107:15910-15915. Using the crystal structure of PFV intasome as a template, Krishnan et al. assemble a model for the HIV-1 intasome and validate it using biochemical approaches. 47. Metifiot M, Marchand C, Maddali K, Pommier Y: Resistance to integrase inhibitors. Viruses 2010, 2:1347-1366. 48. Muller HP, Varmus HE: DNA bending creates favored sites for retroviral integration: an explanation for preferred insertion sites in nucleosomes. EMBO J 1994, 13:4704-4714. 49. Pryciak PM, Sil A, Varmus HE: Retroviral integration into minichromosomes in vitro. EMBO J 1992, 11:291-303. 50. Pruss D, Bushman FD, Wolffe AP: Human immunodeficiency virus integrase directs integration to sites of severe DNA distortion within the nucleosome core. Proc Natl Acad Sci USA 1994, 91:5913-5917. 51. Taganov KD, Cuesta I, Daniel R, Cirillo LA, Katz RA, Zaret KS, Skalka AM: Integrase-specific enhancement and suppression of retroviral DNA integration by compacted chromatin structure in vitro. J Virol 2004, 78:5848-5855. 52. Wang GP, Ciuffi A, Leipzig J, Berry CC, Bushman FD: HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic modifications. Genome Res 2007, 17:1186-1194. 53. Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ: Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997, 389:251-260. 54. Harper AL, Sudol M, Katzman M: An amino acid in the central catalytic domain of three retroviral integrases that affects target site selection in nonviral DNA. J Virol 2003, 77:3838-3845. 55. Konsavage WM Jr, Burkholder S, Sudol M, Harper AL, Katzman M: A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities. J Virol 2005, 79:4691-4699. 56. Lim KI, Klimczak R, Yu JH, Schaffer DV: Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties. Proc Natl Acad Sci USA 2010, 107:12475-12480. Herein, the authors use an elegant selection approach to modify IN of Moloney murine leukemia virus and create retroviral vectors with distinct integration site selection properties. 57. Pribil PA, Haniford DB: Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition. J Mol Biol 2003, 330:247-259.
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Structural insights into the retroviral DNA integration apparatus Peter Cherepanov, Goedele N Maertens and Stephen Hare Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 30 viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes. Address Division of Infectious Diseases, Imperial College London, St. Mary’s Campus, Norfolk Place, London W2 1PG, UK Corresponding author: Cherepanov, Peter ([email protected])
Current Opinion in Structural Biology 2011, 21:249–256 This review comes from a themed issue on Macromolecular assemblages Edited by Felix Rey and Wes Sundquist Available online 1st February 2011 0959-440X/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2010.12.005
Introduction Before or, more commonly, following entry into a host cell, the retroviral diploid RNA genome is converted into a DNA form by the action of reverse transcriptase. The resulting double stranded linear DNA molecule is transported into the nucleus and inserted into a host cell chromosome. The key DNA strand cutting and joining reactions associated with this process are catalyzed by integrase (IN) (for detailed reviews on the early steps of retroviral replication and IN functions, see [1,2]). Firstly, IN carries out 30 -processing, typically cleaving a dinucleotide from both 30 ends of the viral DNA to expose 30 hydroxyls attached to invariant CA dinucleotides (Figure 1a and b). Following entry into the nucleus, IN inserts the recessed 30 ends of the viral DNA molecule into opposing strands of host cell chromosomal DNA, www.sciencedirect.com
across the major groove and separated by 4–6 bp (Figure 1d and e; the size of the stagger depends on the retroviral species). Host cell enzymes repair the resulting single-stranded gaps and 50 overhangs that initially flank integrated viral DNA to finally establish a stable provirus (Figure 1f and g). Retroviral INs belong to the functionally diverse superfamily of DDE(D) nucleotidyltransferases, whose other notable members include RNaseH as well as MuA, Tn5, and Mos1 transposases [3–6,7,8]. The active sites of these enzymes typically contain three essential carboxylates that coordinate a pair of divalent metal (Mg2+ or Mn2+) cations. The DDE(D) family members share a common mechanism of metal ion-dependent phosphodiester bond cleaving and joining via SN2 nucleophilic substitution at the scissile phosphodiester, using a water molecule or a 30 hydroxyl group as a nucleophile, respectively (reviewed in [9,10]). To carry out its function, retroviral IN must act upon two types of DNA substrates: a pair of viral DNA ends and chromosomal target DNA (tDNA). Although INs recognize viral DNA substrates in a sequence-specific manner [11,12], they are not very selective with respect to the target. Nonetheless, interesting patterns transpired upon analyses of large numbers of retroviral integration sites (reviewed in [13]). Firstly, each retrovirus has a weak, symmetric integration site sequence consensus [14,15]. Secondly, retroviruses display genus-specific biases towards genomic features, such as transcription units, CpG islands and epigenetic marks [16]. At the genomic level, the retroviral integration site preferences are thought to depend on host factors. Thus, the interaction between IN and LEDGF, a cellular chromatin-associated protein, targets HIV-1 integration to active transcription units [17,18] (reviewed in [19,20]). By contrast, the local integration site sequence consensus appears to primarily depend on IN itself [18,21,22]. To date, a wide range of small molecule inhibitors of HIV-1 IN capable of blocking viral replication have been reported (reviewed in [23]). The best-validated class of IN inhibitors are bioisosteres of the early diketo acid scaffold described by Hazuda et al. [24]. These compounds bind at the IN active site and specifically interfere with its strand transfer activity [25,26]. One such IN strand transfer inhibitor (INSTI), raltegravir [27], has been in clinical use since late 2007. HIV-1 IN comprises N-terminal, catalytic core and Cterminal domains (NTD, CCD and CTD, respectively), Current Opinion in Structural Biology 2011, 21:249–256
250 Macromolecular assemblages
Figure 1
(a)
(b)
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OH OH OH
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A schematic of the retroviral DNA integration process and its inhibition by INSTIs. (a) The ends of viral DNA are synapsed by a multimer of IN (large blue oval, with small red ovals representing a pair of active sites). 30 -Processing probably takes place within such a synaptic complex, resulting in the longlived intermediate (b). Binding of an INSTI to the intasome traps it in an inactive state (c). Upon nuclear entry, the intasome binds tDNA, forming the target capture complex (d), and strand transfer ensues. The post-catalytic nucleoprotein complex is known as the strand transfer complex (STC, e). Following disassembly of the STC, the gapped intermediate (f) is repaired to produce the stable provirus (g). The figure was adapted from [20].
each of which is essential for the biologically relevant enzymatic activities [28]. The domains, connected by flexible linkers within the full-length protein, are all capable of homodimerization. These canonical domains can be readily identified in all retroviral INs. Over the past 16 years, a collection of structures containing various fragments of INs from HIV-1 and divergent retroviruses were reported (for a detailed recent review see [29]). Unfortunately, the partial structures did not reveal a consistent conformation for the interdomain linkers, or the ordered active site, thwarting confident modeling of IN in its functional state. Recently, a series of structures of prototype foamy virus (PFV) IN in complex with its DNA substrates have been determined [22,30]. A high level of amino acid sequence identity within HIV and PFV IN active sites, made it possible to use the latter as a convenient proxy for structural studies of INSTIs [30,31].
Nucleoprotein complexes involved in retroviral DNA integration In the context of viral infection, IN operates within large nucleoprotein assemblies. The long-lived intermediate containing viral DNA with processed 30 ends is referred to as the pre-integration complex (PIC) [32,33]. Although in vivo integration probably relies on a variety of co-factors, it is possible to faithfully reproduce this process using Current Opinion in Structural Biology 2011, 21:249–256
purified recombinant IN and DNA substrates. The development of in vitro systems allowed biochemical characterization of some of the key HIV-1 integration intermediates [34–36], and moreover their visualization using atomic force microscopy [37]. The intasome, a highly stable synaptic complex that contains a pair of viral DNA ends and a tetramer of IN, appears to be the minimal nucleoprotein complex capable of concerted integration (i.e. integration of pairs of viral DNA ends) in vitro [35,36]. Upon engaging tDNA, in a target capture complex (TCC), the intasome will carry out strand transfer, forming the post-catalytic strand transfer complex (STC, Figure 1d and e) [34–36]. The intasome is the target for INSTIs, which compete with tDNA binding and thereby prevent formation of the TCC (Figure 1c) [25].
The structure of the PFV intasome HIV-1 IN and a handful of other retroviral INs characterized early on are notorious for their unfavorable biochemical properties. Fortunately, the large family of Retroviridae provides an abundant pool of orthologous proteins, which are structurally conserved, yet highly divergent at the amino acid sequence level. Comparative analyses of a variety of retroviral INs in our laboratory identified the ortholog from prototype foamy virus (PFV) as a soluble enzyme, highly proficient in synapsing short www.sciencedirect.com
Structural insights into the retroviral DNA integration apparatus Cherepanov, Maertens and Hare 251
oligonucleotide mimics of viral DNA ends in vitro [21]. Symmetric intasome complexes, assembled using fulllength PFV IN and oligonucleotide mimics of pre-processed U5 viral DNA ends, yielded diffracting crystals. The structure, initially solved at 2.8 A˚ [30] and later refined at resolutions of up to 2.0 A˚ [31], revealed a tetramer of IN and a pair of synapsed viral DNA ends (Figure 2). As predicted based on partial structures [38,39], the tetramer is a dimer-of-dimers with two functionally and structurally distinct pairs of IN subunits. The inner subunits (colored cyan and green in Figure 2) form an extensive dimer–dimer interface and are responsible for all interactions with viral DNA and catalysis. The outer IN subunits (yellow in Figure 2) do not interact with each other or the viral DNA and seem to play a supporting role. The CCD–CCD interface between the inner and outer IN chains is similar to that observed in partial IN
Figure 2
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structures. The NTD and CTD domains of the outer subunits are disordered in the crystals and their functions are currently unknown. Within the intasome, the protein–DNA interactions are very extensive, and each viral DNA end interacts with both inner IN subunits, contributing to the stability of the intasome (Figure 2). The reactive strands of the viral DNA ends (magenta) enter the IN tetramer through the dimer–dimer interface, inserting their 30 ends into the active sites of the inner IN subunits (Figure 2a). The nontransferred strands (orange) are threaded between layers of the protein structure, each sandwiched between the CCD and the interdomain linkers of the inner subunits (Figure 2b). This topology explains why IN requires its recognition site to be close to the end of its DNA substrate, and appears incompatible with a pre-formed IN tetramer being an intermediate in the intasome assembly pathway. Plausibly, an initial interaction of viral DNA ends and IN CCDs could trigger formation of the synaptic interface. Similar, functional multimerization of MuA and Tn5 transposases depends on DNA binding [6,40]. The mechanism of intasome assembly will be an important subject of further investigations. Interestingly, while recombinant PFV IN is predominantly monomeric in solution [21,41], HIV-1 IN tends to form tetramers and higher order multimers even at low mM concentrations [39,42,43]. This propensity for DNAindependent multimerization might account for the difficulties in obtaining sizable quantities of the HIV-1 intasome. IN tetramers are stabilized by inter-dimer NTD–CCD interactions, which are also involved in the intasome assembly [30,38,39], although the functional significance of the DNA-independent IN tetramerization remains unclear.
The IN active site and strand transfer inhibitors
Current Opinion in Structural Biology
The structure of the PFV intasome (PDB ID 3OY9), with IN chains shown as cartoons (a) or space-fill (b) representations. The intasome is viewed along (a) or normal to (b) the crystallographic two-fold axis. The inner IN chains are colored green and cyan; the outer chains are yellow. The reactive and non-transferred strands of the viral DNA ends are magenta and orange, respectively. Active site carboxylates are shown as red sticks; large and small gray spheres are Mn2+ and Zn2+ ions, respectively. Locations of the IN domains (N-terminal extension domain (NED), NTD, CCD and CTD) are indicated in panel a. The outer IN chains were not resolved beyond their CCDs in the available PFV intasome structures. www.sciencedirect.com
The region between b5 and a4 within the CCD of IN, historically referred to as the active site loop, is typically disordered in partial IN structures. The disorder often extends to the beginning of a4, including the invariant Glu of the DDE active site motif. The loop adopts a stable conformation within the PFV intasome structure due to intimate interactions with the viral DNA end. Residues Pro214-Gly218 (corresponding to Pro145-Gly149 in HIV1) form a 310 helix (h2), which packs against the invariant adenine base of viral DNA and separates the reactive and non-transferred strands (Figure 3a). The carboxylates comprising the DDE motif are in close proximity to the reactive 30 hydroxyl of the viral DNA, collectively forming an active site capable of binding a pair of Mn2+ ions (metal A and B; Figure 3a). In the presence of Mn2+, the reactive 30 hydroxyl group of viral DNA is coordinated by metal B. The positions of the catalytic carboxylates, the catalytic metal ions and the 30 nucleotide are remarkably similar to Current Opinion in Structural Biology 2011, 21:249–256
252 Macromolecular assemblages
Figure 3
those within the active sites of Tn5 and Mos1 transpososomes (Figure 3b), despite limited resemblance in the overall architectures of the nucleoprotein complexes (Figures 2, 3c and d).
(a) G T A
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HIV-1 and PFV INs share a high degree of amino acid sequence identity within their active sites. Furthermore, the active site residues ordered within partial crystal structures of divergent retroviral INs superpose extremely well [21]. It is not surprising therefore that INSTIs are effective against INs from a wide range of retroviral species, including PFV [21,44,45]. When PFV intasome crystals were soaked in the presence of raltegravir or other INSTIs, the compounds invariably bound at the active site, interacting with the catalytic metal ions via triads of strategically positioned heteroatoms (Figure 4a and b) [30,31]. The planar hydrophobic groups of the inhibitors intercalate between the 310 helix of the active site loop and the invariant CG base pair of the viral DNA, displacing the viral 30 deoxyadenosine together with its crucial 30 hydroxyl group from the active site. Superposition of various INSTIs in their intasome-binding conformations highlights the consensus features of the pharmacophore (Figure 4b). Because the intasome contacts are so similar among the INSTIs [31], teasing out the cryptic differences between them will require quantitative energy calculations.
D128 β1
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Tn5 Current Opinion in Structural Biology
The active site of PFV IN engaged with a viral DNA end (a). An inner IN chain of the intasome is shown as cartoons, with DNA and side chains of active site residues Asp128, Asp185 and Glu221 as sticks. Colors of cartoons and carbon atoms are specific to the protein and DNA chains, conserved from Figure 2, except for the IN residues Pro211-Gly218, displayed in dark blue. Secondary structure elements of the IN CCD [4,30], carboxylates of the DDE motif, invariant retroviral DNA nucleotides (50 -CA-30 /50 -TG-30 ) and Mn2+ ions (A and B) are indicated. (b) Conservation of the DDE(D) recombinase active site structure. The active sites of Tn5 (PDB ID 1MUS) and Mos1 (PDB ID 3HOT) transposomes and PFV intasome (PDB ID 3OY9) were superposed based on the Ca atoms of the active site carboxylates. Selected portions of the protein structures are shown as cartoons and the 30 -nucleotides as sticks. Cartoons, carbon and metal atoms of Tn5, Mos1, and PFV structures are colored green, pink, and cyan, respectively (c, d). Current Opinion in Structural Biology 2011, 21:249–256
The crystallographic studies of the PFV intasome explained how divergent INSTIs bind within the active site and even shed some light on the mechanism of the major raltegravir-resistance mutations [30,31]. Importantly, the PFV intasome crystal structures allow relatively straightforward modeling of the HIV-1 intasome in its drug-free and drug-bound forms [46]. Such models will be very useful in further development of the INSTIs and understanding the HIV drug resistance mutations. Although the PFV intasome is a convenient model for structural studies of INSTIs, it certainly is not ideal, as the level of amino acid identity between PFV and HIV-1 INs is low outside of their active sites, where mutations contributing to INSTI resistance often occur [47]. We hope that the homology models will aid the design of improved HIV-1 IN constructs to enable determination of an experimental structure.
Engagement of chromosomal DNA and strand transfer: the point of no return Co-crystallization of the PFV intasome with tDNA constructs resulted in visualization of both pre-catalytic (TCC) and post-catalytic (STC) nucleoprotein complexes at resolutions of 3.0 and 2.8 A˚, respectively [22]. As predicted based on the intasome structure, Comparison of the Mos1 (c) and Tn5 (d) transposome structures. Protein chains are shown as cartoons (cyan and green). Reactive and nontransferred DNA strands are magenta and orange, respectively. www.sciencedirect.com
Structural insights into the retroviral DNA integration apparatus Cherepanov, Maertens and Hare 253
Figure 4
Figure 5
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D128 (PFV) D64 (HIV-1) Current Opinion in Structural Biology
The mode of INSTI binding at the IN active site. (a) A molecule of raltegravir (RAL, yellow) bound at the active site (PDB ID 3OYA). Red arrow indicates displacement of the viral DNA 30 hydroxyl group upon drug binding. (b) Superposition of nine drug molecules of various scaffolds in their bound conformations (PDB IDs 3OYA-3OYG and 3L2W). The INSTIs are shown as sticks with yellow carbon atoms. Red and green ovals indicate positions of the INSTI metal chelating and halobenzyl features, respectively. The PFV IN active site carboxylates and the corresponding residues in HIV-1 are indicated in black and gray, respectively.
tDNA binds along the groove dividing the symmetric complex (Figure 5a). A severe deformation of tDNA, accompanied by a dramatic expansion of its major groove, allows insertion of the scissile phosphodiesters into the intasome active sites separated by 27 A˚. By contrast, the intasome itself does not undergo significant changes upon tDNA capture. The bending of tDNA is focused on a single dinucleotide step at the center of the integration site, leading to a complete unstacking of the consecutive base pairs (Figure 5b and c). Concordantly, www.sciencedirect.com
β1
E221 β2
α1
Current Opinion in Structural Biology
Capture of tDNA by the intasome and strand transfer. (a) Structure of the PFV TCC (PDB ID 3OS1), viewed along the crystallographic two-fold axis and in the orientation conserved from Figure 2a. IN chains are shown in space-fill mode; tDNA strands are shown as red and dark gray cartoons. Positions of Arg329 and Ala188 are indicated. (b, c) Conformations of the DNA strands within TCC (PDB ID 3OS2, b) and STC (PDB ID 3OS0, c). A negative roll angle of 608, leading to unstacking of the base pairs at the center of the integration site is indicated. (d) Active site mechanics during strand transfer. Structures of the PFV TCC, STC and Mn2+-bound intasome (PDB IDs 3OS1, 3OS0 and 3OY9, respectively) were superposed based on the Ca atoms of the active site carboxylates. Displayed are tDNA strands from the TCC and STC structures with carbon atoms in light and dark gray; reactive viral DNA strands from the intasome and STC in light and dark magenta; metal ions from the intasome and STC in light and dark gray, respectively. Red dotted line indicates the direction of in-line nucleophilic attack by the 30 hydroxyl group on the scissile tDNA phophodiester. Orange arrow indicates the displacement of the target phosphodiester in the STC relative to its position in the TCC.
Current Opinion in Structural Biology 2011, 21:249–256
254 Macromolecular assemblages
PFV integration sites are naturally enriched with more flexible pyrimidine–purine dinucleotides at the central positions [22]. The path of the scissile strand of tDNA within the IN active site clashes with the observed binding location of INSTIs, explaining the competition between tDNA and INSTIs for binding to the intasome [25]. While effects of chromatin structure on integration are not fully understood, retroviral INs can utilize nucleosomal DNA as a target [48–52]. In the PFV TCC and STC structures, the kinking of the tDNA duplex at the center of the integration site is considerably more extreme than the average DNA bending within the nucleosome structure [53]. It is not surprising therefore that when supplied with chromatinized tDNA in vitro, HIV-1 IN is biased towards sites where nucleosomal DNA is most severely deformed, with the widest major groove exposure [50]. Yet, differences in tDNA bending requirements of retroviruses that integrate with staggers other than 4 bp can be foreseen. Thus, if a similar overall tDNA deformation is required for HIV-1 integration, which targets phosphodiesters separated by 5 bp in tDNA, the expected twofold symmetry of the TCC would necessitate the deformation to be distributed over two base pair steps rather than one, resulting in a smoother tDNA bend. As in the original intasome crystals, the outer IN chains are not resolved beyond their CCDs. The network of interactions between the intasome and tDNA include eight rigid hydrogen bonds between IN main chain amides and the phosphodiester backbone, which serve to ensure precise positioning of the tDNA with respect to the active sites. Strong sequence selectivity for tDNA would limit the pool of suitable integration sites and thus would be disadvantageous for a virus. As expected, direct contacts between tDNA bases and IN are few and are not optimized for sequence selectivity. The most intimate of these involves the symmetric pair of Arg329 residues, which insert their side chains into the expanded major groove of tDNA. Additionally, Ala188 within the short a2 helix contacts the minor groove (Figure 5a). Mutations at these IN positions have drastic effects on the strand transfer activity and/or the integration site selection [22,54,55]. The combination of the indirect recognition (tDNA flexibility) and direct weak interactions with tDNA bases appears to account for the observed PFV integration site consensus [22]. The PFV TCC and STC crystal structures can now be used as a starting base for the rational design of tDNA sequence-specific INs, which may allow the development of safer retroviral vectors targeted to predefined loci of human genome [56].
placed for in-line nucleophilic attack on the scissile tDNA phosphodiester, coordinated between metals A and B (Figure 5d). This geometry is indeed consistent with two-metal catalysis [5,9]. The net chemical bond energy does not change in the course of a transesterification reaction, making it an inherently reversible process. Yet, retroviral integration and prokaryotic transposition would be highly inefficient if a frequent reversal of strand transfer were permitted. Although the overall conformations of the DNA chains within TCC and STC structures are quite similar (Figure 5b and c), the positions of the scissile phosphodiester in the TCC and the phosphodiester joining viral and tDNA strands in the STC do not match. While the former is placed in the IN active site, the latter is shifted by 2.3 A˚ and thus ejected from the active site (Figure 5d). The configuration of the STC cannot support a reverse reaction, which would involve a nucleophilic attack by the 30 hydroxyl of the tDNA on the newly formed phosphodiester. Although crystal structures do not provide dynamic information, it seems likely that the subtle change is driven by the strain in the tDNA conformation, which is relieved upon scission of the target phosphodiester bond. The conformations of the tDNA base pairs flanking the integration site in the STC are more consistent with the energetically favorable B form than those in the TCC. Intriguingly, Tn10 transposase also bends tDNA within its TCC and STC [57]. The transposon inserts with a 9-bp duplication of the target sequence and, therefore, engages phosphodiesters separated by almost 35 A˚ in a B-form DNA duplex. Hence, tDNA bending could be a common feature of DDE(D) recombinases, used not only to gain access to the scissile phosphodiesters in tDNA, but also to introduce a directional bias into the transesterification reaction. The available crystal structures of the PFV intasome, TCC and STC do not hint at any mechanistic linkage between the active sites of the inner IN subunits. Therefore, what we know as ‘concerted integration’ is probably a result of two independent strand transfer events, occurring stochastically and not necessarily simultaneously. Owing to a combination of design and chance, the PFV TCC and STC crystallized as perfectly symmetric assemblies [22]. In a more general case, as the intasome encounters asymmetric tDNA in vitro or chromatin in vivo, one of its active sites may well be in a more favorable environment to carry out strand transfer than the other. Indeed, concerted HIV-1 IN integration appears to occur sequentially in vitro [35].
Perspectives Superposition of the TCC and Mn2+-bound intasome structures allows modeling of the IN active site in a state committed for strand transfer. In this model, the 30 hydroxyl group of viral DNA bound to metal ion B is Current Opinion in Structural Biology 2011, 21:249–256
The recent crystal structures of the PFV IN nucleoprotein complexes [22,30] have revealed a great amount of detail about the mechanism of retroviral DNA integration and also opened exciting avenues for further investiwww.sciencedirect.com
Structural insights into the retroviral DNA integration apparatus Cherepanov, Maertens and Hare 255
gations. The structures are already helping to understand the cryptic mechanisms of HIV-1 resistance to INSTIs [30,31] and will hopefully aid in the determination of an experimental structure of the HIV-1 or a closely related lentiviral intasome. Among many open questions are how the intasome engages chromatin, how the resulting product complex is disassembled, and how this process is coupled to post-integration DNA repair. We are also optimistic that PFV TC/STC structures will guide the design of tDNA sequence-specific INs, which would be extremely helpful in reducing the chance of side effects in gene therapy applications.
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Acknowledgements
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