Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.
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PMID:Overexpression of the lens epithelium-derived growth factor/p75 integrase binding domain inhibits human immunodeficiency virus replication. 1698 86

Transcriptional co-activator LEDGF/p75 is the major cellular interactor of HIV-1 integrase (IN), critical to efficient viral replication. In this work, a series of INs from the Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Spumavirus and Lentivirus retroviral genera were tested for interaction with the host factor. None of the non-lentiviral INs possessed detectable affinity for LEDGF in either pull-down or yeast two-hybrid assays. In contrast, all lentiviral INs examined, including those from bovine immunodeficiency virus (BIV), maedi-visna virus (MVV) and equine infectious anemia virus (EIAV) readily interacted with LEDGF. Mutation of Asp-366 to Asn in LEDGF ablated the interaction, suggesting a common mechanism of the host factor recognition by the INs. LEDGF potently stimulated strand transfer activity of divergent lentiviral INs in vitro. Unprecedentedly, in the presence of the host factor, EIAV IN almost exclusively catalyzed concerted integration, whereas HIV-1 IN promoted predominantly half-site integration, and BIV IN was equally active in both types of strand transfer. Concerted BIV and EIAV integration resulted in 5 bp duplications of the target DNA sequences. These results confirm that the interaction with LEDGF is conserved within and limited to Lentivirus and strongly argue that the host factor is intimately involved in the catalysis of lentiviral DNA integration.
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PMID:LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro. 1715 50

An integrase dimer can process and integrate a single HIV-1 DNA end in vitro, whereas a tetramer is required to integrate two ends. LEDGF/p75 can potently stimulate integrase activity, but its effects on half- versus full-site integration have not been investigated. Stimulation of half-site but inhibition of full-site integration is revealed here. LEDGF/p75 seems to interfere with integrase oligomerization, but does not inhibit the catalytic activity of pre-assembled complexes. We therefore speculate that LEDGF/p75 function is restricted to a point in the viral lifecycle that occurs after the formation of the preintegration synaptic complex, for example, as a chromatin-associated tethering factor.
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PMID:LEDGF/p75 interferes with the formation of synaptic nucleoprotein complexes that catalyze full-site HIV-1 DNA integration in vitro: implications for the mechanism of viral cDNA integration. 1725 58

Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75-integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase-LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites.
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PMID:Virus evolution reveals an exclusive role for LEDGF/p75 in chromosomal tethering of HIV. 1739 62

Proteins are involved in various equilibria that play a major role in their activity or regulation. The design of molecules that shift such equilibria is of great therapeutic potential. This fact was demonstrated in the cases of allosteric inhibitors, which shift the equilibrium between active and inactive (R and T) states, and chemical chaperones, which shift folding equilibrium of proteins. Here, we expand these concepts and propose the shifting of oligomerization equilibrium of proteins as a general methodology for drug design. We present a strategy for inhibiting proteins by "shiftides": ligands that specifically bind to an inactive oligomeric state of a disease-related protein and modulate its activity by shifting the oligomerization equilibrium of the protein toward it. We demonstrate the feasibility of our approach for the inhibition of the HIV-1 integrase (IN) protein by using peptides derived from its cellular-binding protein, LEDGF/p75, which specifically inhibit IN activity by a noncompetitive mechanism. The peptides inhibit the DNA-binding of IN by shifting the IN oligomerization equilibrium from the active dimer toward the inactive tetramer, which is unable to catalyze the first integration step of 3' end processing. The LEDGF/p75-derived peptides inhibit the enzymatic activity of IN in vitro and consequently block HIV-1 replication in cells because of the lack of integration. These peptides are promising anti-HIV lead compounds that modulate oligomerization of IN via a previously uncharacterized mechanism, which bears advantages over the conventional interface dimerization inhibitors.
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PMID:Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium. 1748 11

LEDGF/p75 directly interacts with lentiviral integrase proteins and can modulate their enzymatic activities and chromosomal association. A novel genetic knockout model was established that allowed us for the first time to analyze HIV-1 integration in the absence of LEDGF/p75 protein. Supporting a crucial role for the cofactor in viral replication, HIV-1 vector integration and reporter gene expression were significantly reduced in LEDGF-null cells. Yet, integrase processed the viral cDNA termini normally and maintained its local target DNA sequence preference during integration. Preintegration complexes extracted from knockout cells moreover supported normal levels of DNA strand transfer activity in vitro. In contrast, HIV-1 lost its strong bias toward integrating into transcription units, displaying instead increased affinity for promoter regions and CpG islands. Our results reveal LEDGF/p75 as a critical targeting factor, commandeering lentiviruses from promoter- and/or CpG island-proximal pathways that are favored by other members of Retroviridae. Akin to yeast retrotransposons, disrupting the lentiviral targeting mechanism significantly perturbs overall integration.
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PMID:LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. 1763 82

The transcriptional co-activator lens epithelium-derived growth factor (LEDGF) has been shown to protect cells against environmental stress. The protein has been implicated in auto-immunity and cancer, and is present in cells as the p52 or p75 splice variant. Recently, LEDGF/p75, but not p52, was identified as the prominent interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase. This interaction of HIV-1 integrase with the C-terminal integrase-binding domain of LEDGF/p75 is crucial for HIV-1 replication. To gain insight into the cell biology of LEDGF/p75, we were interested in identifying cellular binding partners of its C-terminal domain. By yeast-two-hybrid screening with a CEMC7 cDNA-library, we were able to identify JPO2 as a binding partner of the C-terminal part of LEDGF/p75. The specific interaction between JPO2 and LEDGF/p75 was verified by pull-down, AlphaScreen, and co-immunoprecipitation. Competition assays using recombinant proteins show a mutually exclusive binding of either JPO2 or HIV-1 integrase to LEDGF/p75. However, differing mechanisms of binding were suggested by continuing interaction of JPO2 with some LEDGF/p75 mutants (I365A, D366A, F406A) that are totally defective for interaction with HIV-1 integrase. This finding is of significance for the development of specific inhibitors targeting only the interaction between LEDGF/p75 and HIV-1 integrase, without disturbing interaction with other cellular factors. Over-expression of JPO2 resulted in a modest but reproducible inhibition of HIV-1 replication, consistent with competition between integrase and JPO2 for binding to LEDGF/p75. Furthermore, JPO2 over-expression activated transcription from the HIV-1 LTR.
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PMID:Differential interaction of HIV-1 integrase and JPO2 with the C terminus of LEDGF/p75. 1766 26

Integration is an essential step in the retroviral lifecycle, and the lentiviral integrase binding protein lens epithelium-derived growth factor (LEDGF)/p75 plays a crucial role during human immunodeficiency virus type 1 (HIV-1) cDNA integration. In vitro, LEDGF/p75 stimulates HIV-1 integrase activity into naked target DNAs. Here, we demonstrate that this chromatin-associated protein also stimulates HIV-1 integration into reconstituted polynucleosome templates. Activation of integration depended on the LEDGF/p75-integrase interaction with either type of template. A differential requirement for the dominant DNA and chromatin-binding elements of LEDGF/p75 was however observed when using naked DNA versus polynucleosomes. With naked DNA, the complete removal of these N-terminal elements was required to abate cofactor function. With polynucleosomes, activation mainly depended on the PWWP domain, and to a lesser extent on nearby AT-hook DNA-binding motifs. GST pull-down assays furthermore revealed a role for the PWWP domain in binding to nucleosomes. These results are completely consistent with recent ex vivo studies that characterized the PWWP and integrase-binding domains of LEDGF/p75 as crucial for restoring HIV-1 infection to LEDGF-depleted cells. Our studies therefore establish novel in vitro conditions, highlighting chromatinized DNA as target acceptor templates, for physiologically relevant studies of LEDGF/p75 in lentiviral cDNA integration.
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PMID:Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro. 1817 27

The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by "shiftides": peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev-derived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein-protein interactions of IN may serve as a general valuable source for shiftide design.
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PMID:Peptides derived from HIV-1 Rev inhibit HIV-1 integrase in a shiftide mechanism. 1821 78

Human cellular protein LEDGF/p75 (lens epithelium-derived growth factor) is an important binding partner of human immunodeficiency virus type 1 (HIV-1) integrase (IN). Without LEDGF/p75, HIV-1 can not complete its life cycle. To study the detailed interactions between LEDGF/p75 and HIV-1 IN, and then obtain the hotspots at the binding interface, 13 ns molecular dynamics simulations were carried out here. One-hundred snapshots extracted from the last 4 ns trajectories were used for calculation of binding free energy and decomposition of the energy by residue. First, the structural changes and their dynamic interactions were investigated focused on the production stage. And then, the free energy was discussed. On the basis of the above results, it could be suggested that residues Gln168, Glu170, and Thr174 in chain A of IN, Thr125, and Trp131 in chain B of IN as well as Ile365, Asp366, Phe406, and Val408 in LEDGF/p75 were responsible for their binding. These results might be helpful for discovery and design of small molecules to interrupt the interaction between HIV-1 IN and LEDGF/p75.
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PMID:Insights into the interactions between HIV-1 integrase and human LEDGF/p75 by molecular dynamics simulation and free energy calculation. 1824 52


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