A Novel Co-Crystal Structure Affords the Design of Gain-of-Function Lentiviral Integrase Mutants in the Presence of Modified PSIP1/LEDGF/p75

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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. 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.