Dynamic Modulation of HIV-1 Integrase Structure and Function by Cellular Lens Epithelium-derived Growth Factor (LEDGF) Protein

Abstract

The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra-and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.Integration of the reverse-transcribed RNA genome into a host chromosome is an obligatory step for HIV-1 3 replication (reviewed in Ref. 1). This process is catalyzed by the retroviral enzyme integrase (IN) in two reaction steps. In the first step, which is called 3Ј-processing and takes place shortly after the cDNA is made, IN hydrolyzes a GT dinucleotide from each end of the viral DNA. In the second step, IN catalyzes concerted integration of the processed viral DNA ends into chromosomal DNA. The sites of attack on the two target DNA strands are separated by 5 bp, which leads to dissociation of the small double-stranded DNA fragment between the attachment sites. The subsequent repair of the intermediate species by cellular enzymes completes the integration reaction. HIV-1 IN consists of three distinct structural and functional domains. The N-terminal domain (NTD) (residues 1-50) contains conserved pairs of histidine and cysteine residues that bind zinc (2, 3), which contributes to IN multimerization and its catalytic function (4, 5). The catalytic core domain (CCD) (residues 51-212) contains three acidic residues, Asp-64, Asp-116, and Glu-152, which play a key role in coordinating active site divalent metal ions (6, 7). The C-terminal domain (CTD) (residues 213-288) also contributes to functional IN multimerization (8, 9). Results of structural biology studies revealed each individual domain as a dimer (3,6,7,10,11) and more recent two-domain crystal structures comprised of the CCD and CTD (12) or NTD and CCD (13)...

BET proteins promote efficient murine leukemia virus integration at transcription start sites

“…The in vitro-concerted integration and homogeneous time-resolved fluorescence (HTRF)-based strand-transfer assays were carried out as reported previously (29,43), with minor changes. See SI Materials and Methods for more details.…”

Biochemical functions of integrase-binding domain of lens epithelium-derived growth factor

“…Previously, Pandey et al (2007) reported that the full-size LEDGF modestly enhances the production of SC assembly in the presence of tetramer form of IN and low molar ratio of LEDGF. Formation of the SC might depend on various factors such as concentration of substrate, ratio of HIV-1 IN versus donor substrate, and reaction buffer components which contains the cofactor that can both stimulate or inhibit the concert integration (Pandey et al 2007;Raghavendra & Engelman 2007: McKee et al 2008). …”

“…In the presence of LEDGF/p75, HIV-1 IN and viral DNA form tetramer and then transport to nucleus and initiate strand transfer reaction (McKee et al 2008). LEDGF/p75 protects the viral protein from degrading by proteosomal enzymes in human cell (Maertens et al 2003;Emiliani et al 2005).…”

“…In the second step, the 3′-end processed cDNA gets inserted into host DNA by retroviral IN, which is called integration or strand transfer activity (Craigie 2001). Finally, the repair process mediated by the cellular enzymes fills the unpaired nucleotides at the 5′-ends of viral DNA called gap repair activity (Kim et al 1999a;Craigie 2001;McKee et al 2008).…”

“…Lens epithelium-derived growth factor (LEDGF/p75) is a cellular protein which has already known to interact HIV-1 IN and function as cellular cofactor for HIV-1 integration and replication (McKee et al 2008;De Luca et al 2011). In the presence of LEDGF/p75, HIV-1 IN and viral DNA form tetramer and then transport to nucleus and initiate strand transfer reaction (McKee et al 2008).…”