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

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.

Lens epithelium-derived growth factor fusion proteins redirect HIV-1 DNA integration

“…Controlling integration site selection in cells from patients will take additional work, but recent progress developing a modified LEDGF-IBD/HIV-1 IN pair whose interactions differ significantly from wild-type seems to be a promising step. This type of approach has two obvious advantages: (i) the vectors will not efficiently infect cells that express only normal LEDGF, and (ii) it will be possible to redirect HIV-1 DNA integration without eliminating endogenous LEDGF (13).…”

“…A crystal structure shows that the integrase binding domain (IBD), located in the C-terminal portion of LEDGF, makes important contacts with both the catalytic core and N-terminal domains of IN (13). The N-terminal portion of LEDGF contains a PWWP domain and AT hooks that bind chromatin and DNA, respectively (14) (Fig.…”

“…We show that replacing the PWWP domain and AT hooks of LEDGF with other chromatin binding domains (CBDs) supports viral integration and redirects the viral DNA integration to sites in the genome that reflect the chromatin binding patterns of the CBDs. The ability to redirect HIV-1 DNA integration may help solve one of the problems associated with using retroviral vectors in gene therapy: the unintentional activation of host cell genes by the nearby insertion of vector DNA (13,24).…”

Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif

“…Structural comparison of the complexes of the IBD with the IBM of MLL1 (ref. 14) or HIV-2 IN 48 shows considerable overlap of both parts of the interface (Fig. 6a,b).…”

“…The IN NTD-LEDGF/p75 interface is necessary for lentiviral integration 48 . In particular, LEDGF/p75 residues K401, R404 and R405 have a crucial role in HIV infectivity 48 . Their counterparts, corresponding to E10 and E13 of HIV-2 IN, are conserved among all lentiviral integrases (Fig.…”