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Title: A Novel Co-Crystal Structure Affords the Design of Gain-of-Function Lentiviral Integrase Mutants in the Presence of Modified PSIP1/LEDGF/p75}
Article:
Abstract:
Lens epithelium derived growth factor( LEDGF), also known as PC4 and SFRS1 interacting protein 1( PSIP1) and transcriptional co-activator p75, is the cellular binding partner of lentiviral integrase( IN) proteins. LEDGF accounts for the characteristic propensity of Lentivirus to integrate within active transcription units and is required for efficient viral replication. We now present a crystal structure containing the N-terminal and catalytic core domains( NTD and CCD) of HIV-2 IN in complex with the IN binding domain( IBD) of LEDGF. The structure extends the known IN–LEDGF interface, elucidating primarily charge–charge interactions between the NTD of IN and the IBD. A constellation of acidic residues on the NTD is characteristic of lentiviral INs, and mutations of the positively charged residues on the IBD severely affect interaction with all lentiviral INs tested. We show that the novel NTD–IBD contacts are critical for stimulation of concerted lentiviral DNA integration by LEDGF in vitro and for its function during the early steps of HIV-1 replication. Furthermore, the new structural details enabled us to engineer a mutant of HIV-1 IN that primarily functions only when presented with a complementary LEDGF mutant. These findings provide structural basis for the high affinity lentiviral IN–LEDGF interaction and pave the way for development of LEDGF-based targeting technologies for gene therapy.
Introduction:
Integration of reverse transcribed viral cDNA into the host cell genome is an essential step in the retroviral life cycle. This process is catalyzed by integrase( IN), a virus-derived enzyme, which carries out two separate reactions acting on both cDNA termini( reviewed in[1],[2]). Firstly, 3′-processing takes place in the cytoplasm of the host cell, in which a di- or trinucleotide is hydrolytically removed from each cDNA end, exposing 3′-hydroxyl groups of invariant CA dinucleotides. The enzyme remains attached to both viral cDNA ends within a higher order pre-integration complex( PIC). The PIC is transported into the nucleus and, upon locating a suitable chromatin environment, the second reaction, strand transfer, ensues. During this step, the pair of hydroxyl groups produced during 3′-processing nick and join to opposing strands of the cellular DNA, four to six base pairs apart, depending on the retroviral genus. To complete the process, cellular enzymes repair the integration site, resulting in a stable provirus flanked by short duplications of the target DNA sequence. Retroviral INs share a conserved three domain organization, each containing a central catalytic core domain( CCD), flanked by N- and C-terminal domains( NTD and CTD)[3]–[5]. The CCD spans the most conserved region of IN and bears close structural homology to prokaryotic transposases[6]. The enzyme active site is comprised of three invariant acidic residues( the D, DX35E motif) that coordinate a pair of Mg2+ cations during catalysis[7],[8]. The NTD forms a three-helical bundle, which folds around a zinc atom coordinated by His and Cys residues of an HHCC motif[9],[10]. The CTD features an SH3-like fold, is rich in basic residues and is likely involved in DNA binding[11],[12]. Despite Herculean efforts directed towards characterization of this key antiviral drug target, the structure of a full-length retroviral IN remains elusive. The active form of retroviral IN is a tetramer[13]–[15], and a plausible tetramer model for the apoenzyme was proposed based on a crystal structure of a two-domain fragment of HIV-1 IN containing its NTD and CCD( INNTD+CCD)[16]. Lentiviral DNA integration critically depends on lens epithelium-derived growth factor( LEDGF)( reviewed in[17]). LEDGF tightly associates with chromatin and has been implicated in regulation of cellular gene expression, epigenetic chromatin modifications and apoptosis[18]–[20]. The host factor directly binds HIV-1, HIV-2, as well as other lentiviral INs and dramatically stimulates their strand transfer activity[21]–[24]. LEDGF tethers lentiviral IN to host chromatin in the nucleus[24]–[27] and plays a critical role in directing PICs to active genes during integration[28]–[32]. LEDGF contains a pair of small structural domains: an ∼92 residue PWWP domain at its N-terminus, responsible for binding to an as yet unidentified component of chromatin, and the IN binding domain( IBD, residues 347–429) within its C-terminal portion[33]–[35]. The CCD and NTD of IN were both implicated in LEDGF binding: while the CCD is minimally sufficient, the NTD is required for high affinity binding[27],[36]. Deletion of the HIV-1 IN NTD, or a mutation destabilizing zinc coordination within this domain( His-12 to Asn), greatly reduced the interaction with LEDGF[27]. A close homolog of LEDGF, hepatoma derived growth factor-related protein 2( HRP2), contains conserved PWWP and IBD-like domains. Although HRP2 is able to interact with HIV-1 IN and stimulate its enzymatic activity in vitro[33], it remains to be established whether it plays a role in lentiviral integration. The structure of the LEDGF IBD, composed of a pair of α-helical hairpins, has been determined both separately and in complex with the HIV-1 IN CCD[34],[36]. In the INCCD:LEDGFIBD complex, Ile-365 of LEDGF inserts into a hydrophobic pocket at the IN dimer interface. The interaction is further bolstered by additional hydrophobic interactions between IN residue Trp-131 and LEDGF Phe-406 and Val-408. Asp-366 of LEDGF plays an important role in protein-protein recognition, forming a pair of essential hydrogen bonds with the main chain amides of IN residues Glu-170 and His-171[36]. Mutation of LEDGF Asp-366 to Asn ablated the interaction with all lentiviral INs tested so far, indicating a common mechanism of recognition[22]. In this work we extend the known lentiviral IN-LEDGF interface to include contacts between the IN NTD and the IBD of LEDGF. This part of the protein-protein interface is essential for high affinity binding and stimulation of concerted DNA integration, and allows designs of complementary pairs of IN and LEDGF mutants for practical uses in gene therapy.
Discussion:
In this work we extended the known lentiviral IN-LEDGF interface to include the interactions between the NTD of IN and the IBD of LEDGF. Since there is no evidence that the CTD of IN or LEDGF regions outside of the IBD are involved in the interaction, the contacts observed in our crystal structure may very well represent the entire IN-host factor interface. These novel structural details will invariably aid the development of inhibitors of the IN-LEDGF interaction. Intriguingly, both crystal forms of the HIV-2 INNTD+CCD:LEDGFIBD complex contained closed trimers of the IN2LEDGF substructure held together primarily by IN-IN contacts. Based on the three-fold symmetry of this assembly, we tentatively speculate that it could reflect packing arrangement of IN molecules within retroviral capsids, which too feature three-fold symmetry[45]. In both crystals the closed trimers further associated into a spherical particle containing twenty four IN chains, with their C-termini projecting inwards and the N-termini outwards. It remains to be determined if the higher order multimers of the IN2LEDGF substructure are biologically relevant. Such evidence could come, for example, by observing similar multimers in crystals of a divergent retroviral IN. Although we have not detected analogous large-sized complexes in solution, the calculated concentration of IN within retroviral capsids is very high[46], presenting an environment where it may very well adopt a paracrystalline state. Mounting experimental evidence suggests that the active form of retroviral IN is a tetramer[13]–[15]. Based on a crystal structure of the HIV-1 INNTD+CCD fragment, Craigie and colleagues proposed a plausible model for the synaptic IN tetramer( dimer of dimers)[16]. Notably, the positions of the IN NTDs relative to the CCD dimer and the supporting NTD-CCD contacts observed in our HIV-2 INNTD+CCD:LEDGFIBD complex were seen in the earlier structure, where the NTDs mediated contacts between IN dimers[16]. One significant difference is that in the tetramer model, the NTD occupying the position primed for the interaction with the IBD is donated by the other IN dimer[16]( Figure S3A and S3B). Such domain swapping is quite common, representing one of the mechanisms for homomeric protein-protein interactions[47]. However, it is important to note an ambiguity of the NTD assignments in the HIV-1 INNTD+CCD structure, which lacked appreciable electron density for the NTD-CCD linkers[16]. Nevertheless, the NTD-CCD interface observed in the two independent structures is almost certainly biologically relevant. If IN dimers do indeed swap their NTDs during tetramerization, the LEDGF binding platform would include a CCD dimer and an NTD from a separate IN dimer( Figure S3C). Alternatively, LEDGF binding to an IN dimer would lock one NTD in the orientation primed for tetramerization( Figure S3D). In either case, upon binding, the co-factor would enhance the thermodynamic stability of the tetramer. It is a tetramer of IN that mediates synapsis of a pair of donor DNA molecules[15] and, concordantly, the NTD-IBD contacts uncovered here are specifically required for concerted DNA integration. The model can also explain the surprising ability of LEDGF to inhibit HIV-1 concerted integration under some in vitro conditions[42]. LEDGF binding to a dimer of IN would fixate either one or both NTDs, preventing them from functionally interacting with a second IN dimer( Figure S3). Thus, the concentration of IN, LEDGF and donor DNA substrate in the reaction mixture, as well as the order of component addition significantly influence the outcome of the reaction( Ref.[43] and data not shown). Of note, one recent study suggested that IN must exist in a lower multimeric state, likely a dimer, before interacting with the donor DNA for proper synaptic complex formation[48]. The enhanced concerted integration assay described herein will be very useful in future biochemical and structural studies of HIV-1 IN. Furthermore, other lentiviral INs, and in particular EIAV IN, carry out very efficient LEDGF-dependent concerted integration utilizing oligonucleotide donor DNA substrates under similar reaction conditions( data not shown). Using complementary approaches we demonstrated that the NTD-IBD interface is important for the functional Lentivirus-host interaction. Compared to the lock-and-key CCD-IBD interface, the contacts involving the NTD are based on charge-charge interactions and therefore lend themselves to complementary reverse-charge engineering. Since LEDGF has been shown to target lentiviral integration to active transcription units[28]–[30], it has been speculated that modified versions of the host factor could be used to control integration site selection. Safety of retroviral vectors would be greatly improved if they could be directed towards specific pre-determined loci and away from proto-oncogenes[49]. In one recent work, a fusion of the DNA binding domain of bacteriophage λ repressor and the IBD of LEDGF targeted HIV-1 integration nearby λ operator sequences in vitro[50]. One fundamental problem can thwart practical application of such approaches. When delivered into the cell, the targeting factor, associated with a limited number of chromosomal loci, will have to compete with a vast excess of endogenous LEDGF for the incoming preintegration complex. Knockout or knockdown of endogenous LEDGF would unlikely be a practical or safe solution, especially considering its emerging role in epigenetics[18]. Here we demonstrated that an HIV-1 IN mutant carrying two reverse charge mutations within the NTD gained the ability to functionally interact with a modified version of LEDGF, while remaining basically unresponsive to the WT protein. Although the efficiency of the current system is somewhat modest, our results present a proof of principle that it is possible to engineer a viable complementary pair of IN and LEDGF mutants that could allow future development and practical applications of LEDGF-based lentiviral vector technologies.
Retroviruses crucially rely on insertion of their genomes into a host cell chromosome , and this process is carried out by the viral enzyme integrase . HIV and other lentiviruses also depend on LEDGF , a cellular chromatin-associated protein , which binds their integrase proteins and tethers them to a human chromosome . The interaction between integrase and LEDGF can potentially be exploited for directing integration of lentiviral vectors in gene therapy applications , as well as for development of antiretroviral drugs . Herein , we present a three-dimensional structure of a protein–protein complex containing a fragment of HIV integrase and the integrase-binding domain of LEDGF . Our structure elucidates the hitherto unknown LEDGF–integrase interface involving the amino terminal portion of the viral enzyme . Using a range of complementary approaches , we further show that these novel protein–protein contacts are essential for the function of LEDGF in HIV integration . The novel structural details will be very useful for the development of HIV inhibitors that target the integrase–LEDGF interaction . Furthermore , they enabled us to design a mutant of HIV integrase that depends on a reverse-engineered mutant of LEDGF , providing an inroad to the design of LEDGF-based lentiviral vector targeting strategies .