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Published in final edited form as: Future Virol. 2012 Jul;7(7):679–686. doi: 10.2217/FVL.12.56

The molecular biology of HIV integrase

Robert Craigie 1
PMCID: PMC3458710  NIHMSID: NIHMS406040  PMID: 23024700

Abstract

Integration of viral DNA into cellular DNA is an essential step in the replication cycle of HIV and other retroviruses. The first antiviral drugs that target integrase, the viral enzyme that catalyzes DNA integration, have recently been approved and more are in the pipeline. These drugs bind to an intermediate in DNA integration called the intasome, in which a pair of viral DNA ends are synapsed by a tetramer of integrase, rather than free integrase enzyme. We discuss the biochemical mechanism of integration, which is now quite well understood, and recent progress towards obtaining atomic-resolution structures of HIV intasomes in complex with inhibitors. Such structures are ultimately required to understand the detailed mechanism of inhibition and the mechanisms by which mutations in integrase confer resistance. The path from early biochemical studies to therapeutic inhibitors of integrase highlights the value of basic science in fighting human diseases.

Keywords: HIV-1, intasome, integrase, integration, retrovirus

Retroviruses integrate a DNA copy of the viral genome into host DNA as an obligatory step in the replication cycle [1, 2]. Integration can occur at essentially any location in the genome, but certain regions of chromatin are preferred [3]. The integrated viral DNA is stably maintained and replicated along with cellular DNA through cycles of cell division. This presents a challenge to the treatment of retroviral infections. Although great strides have been made in antiviral therapy for HIV, the integrated virus persists in long-lived cells and eradication is an elusive goal [4]. The key enzyme that integrates retroviral DNA into the host genome is the virally encoded integrase protein. The first clues that a viral protein mediates DNA integration came from genetic studies [5]. Mutations were identified in the viral pol gene that allowed viral DNA to be synthesized at normal levels by reverse transcription, but this DNA failed to integrate. This part of the pol gene encodes the protein we now call integrase that is cleaved from a polyprotein precursor by the viral protease. The first antiviral drugs that target integrase have been recently developed [6], and these drugs complement the established protease and reverse transcriptase inhibitors.

Retroviral DNA is synthesized by reverse transcription within the cytoplasm of the infected cell. This DNA forms part of a large nucleoprotein complex, termed the preintegration complex (PIC), that is derived from the core of the infecting virion [7]. The PIC is transported to the nucleus and the viral DNA is integrated into cellular DNA by integrase. PICs are poorly defined because their low abundance in extracts of infected cells prevents direct analysis by biophysical methods. HIV PICs have been reported to contain the viral proteins integrase, nucleocapsid, matrix, reverse transcriptase and Vpr [8–15], in addition to a number of cellular proteins. PICs efficiently integrate their viral DNA into a target DNA in vitro with all the hallmarks of integration in vivo.

PICs derived from virus-infected cells efficiently integrate their viral DNA into a target DNA in vitro [11, 16, 17]. The DNA cutting and joining steps of retroviral DNA integration (Figure 1) were determined by analysis of the integration intermediate formed in vitro with PICs as the source of viral DNA and plasmid DNA as the target for integration [18, 19]. In the first step, 3′ end processing, two nucleotides are cleaved from each 3′ end of the initially bluntended DNA. This exposes the CAOH 3′ ends that mark the junction between viral and target DNA upon integration. In the next step, DNA strand transfer, the hydroxyl groups at the 3′ ends of the viral DNA attack a pair of phosphodiester bonds in the target DNA. The sites of attack are separated by five nucleotides on each DNA strand in the case of HIV. The result is an integration intermediate in which the 3′ ends of the viral DNA are covalently attached to target DNA. The 3′ ends of the target DNA and the 5′ ends of the viral DNA, two nucleotides of which are unpaired, are not joined. Cellular enzymes are required to complete the integration process by repairing the integration intermediate. The required steps are removal of the two unpaired nucleotides at 5′ end of the viral DNA, filling in of the single strand gaps, and ligation.

Figure 1. DNA cutting and joining steps of retroviral DNA integration.

Figure 1

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(A) The viral DNA synthesized by reverse transcription is initially blunt ended. (B) The 3′ end processing reaction removes two nucleotides from each 3′ end. (C) Next, in the DNA strand transfer reaction, the 3′ hydroxyls at the ends of the viral DNA attack a pair of phosphodiester bonds in the target DNA; in the case of HIV, the sites of attack are separated by five nucleotides on the two target DNA strands. (D) The result is the integration intermediate, in which the 3′ ends of the viral DNA are joined to the 5′ ends of the target DNA at the site of integration. The integration intermediate is then repaired by cellular enzymes to complete the integration process.

Biochemistry of DNA integration

Retroviral integrases share a common biochemical mechanism of integration. Although we focus on the HIV enzyme, important advances have come from parallel studies of HIV and closely related retroviruses, notably avian sarcoma and leukosis virus, Moloney murine leukemia virus and, more recently, prototype foamy virus (PFV). Purified retroviral integrases, in the presence of a divalent metal ion, catalyze both 3′ end processing and DNA strand transfer in vitro with oligonucleotide DNA substrates that mimic the ends of the viral DNA (Figure 2) [20–22]. This simplified reaction system revealed that the chemical mechanism of DNA integration is one-step transesterification [23]; when chiral phosphorothioate is substituted for phosphate in the target DNA, the chirality is inverted in the integration product. This contrasts with DNA recombination enzymes that form a covalent intermediate between the enzyme and DNA.

Figure 2. In vitro catalytic activities of HIV integrase.

Figure 2

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(A) Integrase catalyzes 3′ end processing on an oligonucleotide DNA substrate mimicking the ends of HIV DNA. The GT dinucleotide is released, exposing the conserved CA that is to be joined to the target DNA. (B) Integrase also catalyzes DNA strand transfer, in which the 3′ end of the viral DNA attacks a phosphodiester bond in the target DNA, covalently joining the viral to target DNA. Integration can occur at essentially any location in the target DNA. Note that, in vitro with oligonucleotide DNA substrates, most of the reaction products result from joining only one viral DNA end into one strand of target DNA, rather than concerted integration of a pair of viral DNA ends as occurs in vivo.

Mutagenesis studies of several retroviral integrases showed that mutations generally affected 3′ end processing and DNA strand transfer in parallel [24–27], suggesting a common active site for these two reactions, a hypothesis that was later confirmed by structural studies. Although these two reactions at first sight appear to be quite different, they share the same chemical mechanism. In the 3′ end processing reaction the nucleophile is water, whereas in the DNA strand transfer reaction it is the 3′ hydroxyl at the ends of the viral DNA [23].

Although integrase efficiently catalyzes the chemical reactions of integration in vitro with oligonucleotide substrates, there are limitations to the most simplified systems. Firstly, the reaction takes place in an aggregated state as evidenced by pelleting of DNA substrate, product and protein with low-speed centrifugation [28]. This is often unappreciated and there are many conclusions in the literature based on kinetic analyses that are not readily applicable to the system because unreacted substrates and reaction products form mixed heterogeneous aggregates. Secondly, the vast majority of products result from the integration of only a single viral DNA end into one strand of target DNA, rather than the concerted integration of a pair of viral DNA ends [29] (compare the concerted integration of a pair of viral DNA ends shown in Figure 1 with the insertion of a single viral DNA end shown in Figure 2B). The reaction system resembles a ‘halfsite’ reaction that carries out correct chemistry, but lacks the full fidelity of integration catalyzed by PICs in vitro and in vivo.

Recently, improved in vitro reaction systems have been developed that catalyze concerted integration of a pair of viral DNA ends [30–34]. Under these reaction conditions, specific complexes are formed between integrase and DNA substrate that mimic the tight association of integrase with viral DNA in the PIC [35–37]. A tetramer of integrase stably bridges a pair of viral DNA ends to form a stable synaptic complex or intasome. Once formed, intasomes catalyze concerted integration with an efficiency approaching 100%. 3′ end processing occurs within the intasome, and strand transfer of the two viral DNAs into the target occurs sequentially, with a significant temporal separation between the joining of the two viral DNA ends to the target DNA. The resulting integration intermediate remains tightly associated with integrase in a complex termed the strand transfer complex (STC). It is likely that cellular enzymes are required to actively disassemble the STC before it can be repaired to complete integration.

Inhibitors of HIV integrase

The in vitro reaction system with oligonucleotide DNA substrates laid the foundation for large-scale screening for inhibitors of integrase within the pharmaceutical industry. Progress was painstakingly slow and the first integrase inhibitor, raltegravir, was only approved by the US FDA in 2007 [6]. Several other integrase inhibitors are currently in late-stage clinical trials [38]. These inhibitors share the property of having low affinity for integrase alone, but high affinity for intasomes in which the active sites are engaged with a pair of viral DNA ends [39]. The implication is that DNA binding elicits a protein conformational change central to high-affinity drug binding and/or the DNA itself makes direct contacts with the inhibitor. By extension, knowledge of the structures of intasomes is required to understand the molecular mechanism of action, and structures of the protein alone are unlikely to be informative.

In addition to inhibitors of integrase that target the active site, compounds have been identified that inhibit integration in vitro by an entirely different mechanism. One promising class of compounds binds to the LEDGF/p75 (discussed below) cofactor binding site of integrase [40–42].

Structure of retroviral integrase nucleoprotein intermediates in DNA integration

Structural studies of most retroviral integrases have been frustrated by the poor solubility of the protein and the flexibility between domains. With the exception of PFV [43, 44], no structures of a full-length retroviral integrase have been determined. The structures of the catalytic domain of HIV and ASV integrase were determined almost simultaneously [45, 46]. They were essentially the same and confirmed the identity of the catalytic residues postulated based on mutagenesis studies. Otherwise, the structures were largely uninformative in retrospect. They were nearly spherical dimers with a pair of active sites diametrically opposed away from the dimer interface. The spacing is incompatible with the 5-base pairs staggered integration sites on a target DNA and suggested that a higher order multimer other than a dimer is required for the authentic integration reaction. The N-terminal domain is a helix bundle, stabilized by the coordination of a zinc ion to a pair of conserved His and Cys residues [47, 48]. Finally, the isolated C-terminal domain closely resembles an SH3 domain [49, 50]. Two-domain structures comprising the N-terminal plus catalytic domain and C-terminal plus catalytic domain have also been solved for several retroviral integrases (reviewed in [51, 52]). Strikingly, the relative positions of the domains are quite different among these structures, highlighting the flexibility of the linkers.

A major breakthrough in structural studies of retroviral integrase came when Cherepanov and colleagues solved the structure of the PFV intasome [43]. PFV integrase shares only limited sequence identity with HIV integrase and has an additional N-terminal domain and longer linkers between domains. Nevertheless it would be surprising if the PFV and HIV intasomes were radically different, given the functional and structural similarity of these proteins. PFV integrase is more amenable to biophysical studies than HIV integrase. First, it is more soluble, and second, unlike HIV integrase, it readily assembles intasomes on short oligonucleotide DNA substrates [53]. DNA is the glue that holds the PFV intasome together in a way that is reminiscent of the Tn5 transposase in complex with transposons [54]. It is therefore perhaps not surprising that all the models of the HIV intasome structure based on the partial structures of the protein alone were proved wrong. The organization of the PFV intasome is depicted in Figure 3. The catalytic domain dimer interface, present in all previous structures, is preserved. The integrase tetramer is a dimer of dimers, and each dimer contains an inner and outer subunit. All the DNA contacts are with the inner subunits. The N-terminal domain, C-terminal domain and the extra N-terminal extension domain of the outer PFV integrase subunits are disordered in the current structures. Structures of the PFV intasome in complex with the inhibitor raltegravir elucidate the basic inhibition mechanism. The inhibitor indeed intimately contacts the viral DNA ends, displacing the 3′ ends from the active site and making them unavailable for nucleophilic attack on the target DNA. The HIV intasome has been modeled based on the PFV structure and it is predicted to bind inhibitors in the same way [55]. The high degree of similarity in the immediate vicinity of the active site allows reliable modeling, but the proteins are too dissimilar to accurately model detailed molecular interactions further away. Structures of the HIV intasome will be required to fully understand how mutations confer resistance to drugs, because some are in residues not predicted to directly interact with the inhibitor [56]. The structure of the PFV STC has also been determined [44]. The target DNA is severely bent, explaining the preference for integration into distorted DNA [57].

Figure 3. Cartoon representation of the prototype foamy virus intasome structure.

Figure 3

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The integrase monomers in the intasome are distinguished by their colors. The CCD, NTD, CTD and NED are distinguished by shape. The pair of viral DNA ends are represented as helices. All the contacts between integrase and viral DNA are with the inner subunits. The CTD, NTD and NED of the outer subunits are disordered.

CCD: Catalytic core domain; CTD: C-terminal domain; NED: N-terminal extension domain; NTD: N-terminal domain.

Adapted with permission from [81].

Interaction of integrase with other proteins

Although integrase is both necessary and sufficient for integration in vitromany proteins have been reported to interact with integrase, and at least some of them are functionally relevant to the integration process. Roles that have been proposed include directing the PIC to particular regions of chromatin, import of the PIC across the nuclear envelope, and blocking integration of the viral DNA into itself (autointegration). A complete list of cellular factors is outside the scope of this review, but the following are a few examples of the role of cellular factors in integration. The topic has been reviewed in depth in [57–60].

The most studied cellular factor is LEDGF/p75 (reviewed in [61, 62]). This protein was first identified as an interacting partner in affinity screens [63, 64]. Early siRNA studies were inconclusive regarding the functional relevance of the interaction, probably because trace amounts are sufficient to support HIV replication. Later experiments with more complete knockdown and knockout cell lines clearly demonstrated that LEDGF stimulates HIV replication up to approximately 100-fold in cell culture [65–67]. Strikingly, PICs isolated from LEDGF/p75-knockout cells are fully competent for integration in vitro [66], demonstrating that LEDGF/p75 does not play a role in PIC assembly or intrinsic activity. How, then, does LEDGF/p75 exert its activity on integration in cells? A convincing body of evidence indicates that LEDGF/p75 tethers PICs to chromatin prior to catalysis of integration. HIV preferentially integrates into active transcription units [68]. This correlates well with the distribution of LEDGF/p75 in chromatin, where ‘LEDGF islands’ preferentially lie in transcription units [69]. Biochemical and structural studies indicate that LEDGF/p75 can serve as a bridge between PICs and chromatin. LEDGF/p75 contains an integrasebinding domain that binds tightly to integrase [70, 71]. The structure of the catalytic domain of HIV integrase complexed with the integrase-binding domain has been determined by X-ray crystallography [72] and small molecules that mimic the LEDGF/p75 integrase interaction have defined a novel class of allosteric integrase inhibitors [42, 73]. LEDGF/p75 also contains a PWWP chromatin-binding domain and an A/T hook domain that is likely involved in DNA binding [74, 75].

Treatment of Moloney murine leukemia virus PICs with high ionic strength, followed by size exclusion chromatography to remove small liberated factors, abolishes intermolecular integration in vitro [76]. Instead, the viral DNA uses itself as a target in a self-destructive reaction termed autointegration. Intermolecular integration is restored and autointegration is abolished by addition of an extract of uninfected cells. Using this as an assay, the protein factor BAF (or BANF1) was identified [77]. It is a highly conserved DNA bridging protein that compacts DNA [78]. It was hypothesized that BAF blocks autointegration by compacting the viral DNA, making it inaccessible as a target for integration. Such compaction of DNA by BAF was later confirmed by total internal reflection fluorescence microscopy experiments [79]. It is unclear if BAF also performs a similar function for HIV PICs. Because BAF is an essential protein, it is difficult, if not impossible, to directly test the effect of knockdown or knockout in vivo because of the indirect effects of perturbing normal cellular processes. This is a conundrum that has also hindered the investigation of the role of other essential cellular proteins in DNA integration.

Ongoing work

The biochemical mechanism of DNA integration is quite well understood, but atomic-level structural details are incomplete. The structure of the HIV intasome remains elusive. The success with the PFV intasome structure is a giant step forward, but the protein is sufficiently different that the HIV intasome structure is required to properly understand the mechanism of drug resistance. Six of the domains are disordered in the PFV intasome structure. Are these domains required for function or are they dispensable? Additional structures of retroviral intasomes and biochemical studies are required to answer this question.

A major focus of ongoing research is on the role of interacting partners of HIV integrase. PICs are much too large to passively enter the nucleus, and so an active process must be involved. This field has been controversial and is not yet settled. Numerous claims have been made, some involving proteins that interact with integrase and some not. There is likely to be redundancy in the nuclear import pathways [80] and this may in part explain the apparent contradictions in the literature. The role of cellular proteins in targeting PICs to chromatin is generally accepted. The dual interaction of LEDGF/p75 with HIV integrase and chromatin is clearly a major factor in the case of HIV DNA integration. Are other proteins involved in targeting integration of HIV, and do other factors substitute for LEDGF/p75 for retroviruses with integrases that do not bind LEDGF? There is still much to learn.

Future perspective

The groundwork for studies on HIV integrase was laid with biochemical studies of closely related DNA transposition systems. It was not clear at the time that such basic science would contribute to the treatment of an emerging disease. The early biochemical studies of HIV integrase resulted in assays that were adapted by the pharmaceutical industry for high-throughput screening for integrase inhibitors. Almost two decades after initiating these efforts, the first antiviral drug that targets integrase was brought to market and more are in the pipeline. The story highlights the value of basic research to tackle future emerging diseases. It is now necessary to study the atomic details of how these drugs work and the mechanisms of drug resistance.

Executive summary.

DNA integration

  • ▪

    DNA integration is essential for the replication of HIV and other retroviruses and can be a target for antiretroviral drugs.

Integrase

  • ▪

    The viral enzyme integrase catalyzes the key DNA cutting and joining steps of integration.

Biochemistry

  • ▪

    A tetramer of integrase stably bridges the ends of the viral DNA in a nucleoprotein complex called the intasome. The chemical steps of integration take place within intasomes.

  • ▪

    Integrase cleaves two nucleotides from the 3′ ends of the viral DNA (3′ end processing).

  • ▪

    Integrase catalyzes nucleophilic attack of the 3′ hydroxyl group at the ends of the processed DNA on a pair of phosphodiester bonds in the target DNA (DNA strand transfer).

  • ▪

    Cellular enzymes complete integration by repairing the resulting integration intermediate.

Inhibitors

  • ▪

    The first integrase inhibitors were recently approved by the US FDA.

  • ▪

    These inhibitors have high affinity for intasomes, but low affinity for free integrase.

  • ▪

    The mechanism involves displacement of the 3′ ends of the viral DNA away from the active site, making them unavailable for nucleophilic attack on the target DNA.

Structure

  • ▪

    Knowledge of the structures of HIV intasomes is needed to understand the detailed mechanism of inhibitors and mutations that confer resistance.

  • ▪

    Only partial structures of HIV integrase without DNA have so far been determined.

  • ▪

    Prototype foamy virus is currently the only retrovirus for which intasome structures are available.

Future perspective

  • ▪
    We need to:
    • –
      Determine high-resolution structures of HIV intasomes in complex with inhibitors.
    • –
      Apply these structures to understand the mechanism of inhibition at the atomic level and how mutations in integrase confer resistance.
    • –
      Develop new classes of integrase inhibitors.

Acknowledgements

The author thanks A Engelman and W Yang for reading the manuscript.

This work was supported by the Intramural Program of NIDDK, NIH and by the AIDS Targeted Antiviral Program of the Office of the Director of the NIH.

Footnotes

Financial & competing interests disclosure

The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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