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)

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a DNA-binding, transcriptional co-activator that participates in HIV-1 integration site targeting. Using complementary approaches, we determined the mechanisms of LEDGF/p75 DNA-binding in vitro and chromatin-association in living cells. The binding of highly-purified, recombinant protein was assayed by surface plasmon resonance (SPR) and electrophoretic mobility gel shift. Neither assay revealed evidence for sequence-specific DNA-binding. Residues 146-197 spanning the nuclear localization signal (NLS) and two AT-hook motifs mediated non-specific DNA-binding, and DNA-binding deficient mutants retained the ability to efficiently stimulate HIV-1 integrase activity in vitro. Chromatin-association was assessed by visualizing the localization of EGFP fusion proteins in interphase and mitotic cells. Although a conserved N-terminal PWWP domain was not required for binding to condensed mitotic chromosomes, its deletion subtly affected the nucleoplasmic distribution of the protein during interphase. A dual AT-hook mutant associated normally with chromatin, yet when the mutations were combined with NLS changes or deletion of the PWWP domain, chromatin-binding function was lost. As the PWWP domain did not readily bind free DNA in vitro, our results indicate that chromatin-association is primarily affected through DNA-binding, with the PWWP domain likely contributing a protein interaction to the overall affinity of LEDGF/p75 for human chromatin.
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PMID:A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin in vivo. 1654 78

Autoantibodies against DFS70/LEDGF, which is also known as an important partner of HIV-1 integrase, are found in 10% of healthy Japanese people, but in only approximately 2% of patients with systemic autoimmune disease (SAD). We wished to characterize the association of HLA class II alleles with the presence of autoantibodies against this molecule. MHC class II genes (DR, DQ, and DP alleles) were analyzed by the polymerase chain reaction-sequence specific primer method in 24 individuals with anti-DFS70 antibodies. The frequencies of HLA-DRB1*0410, -DQB1*0402, and -DPB1*0301 were increased in anti-DFS70 Ab-positive patients, while HLA-DQB1*0302 was decreased compared to Japanese controls. All anti-DFS70 Ab-positive individuals expressed at least one HLA-DQB1 allele with an aspartic acid at residue 57. The immunogenetic background of Japanese individuals with anti-DFS70 antibodies differs from that of patients with SAD. HLA class II genes influence the production of anti-DFS70 antibodies among individuals with various clinical manifestations.
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PMID:HLA-associated production of anti-DFS70/LEDGF autoantibodies and systemic autoimmune disease. 1671 78

To replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host cell. Integration is not random in the host genome but favors particular regions, and preferences differ among retroviruses. Several mechanisms might play a part in this favored integration targeting: (i) open chromatin might be preferentially accessible for viral DNA integration; (ii) DNA replication during cell division might facilitate access of integration complexes to favored sites; and (iii) cellular proteins bound to the host chromosome might tether integration complexes to favored regions. This review summarizes recent advances in understanding the mechanisms of retroviral integration, focusing on LEDGF/p75--the first cellular protein shown to have a role in directing HIV DNA integration. Studies on LEDGF/p75 indicate that it directs HIV integration site selection by a tethering interaction, whereas the chromatin accessibility or cell cycle models are less well supported. Understanding viral integration will help improve the safety of retrovirus-based vectors used in gene therapy.
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PMID:Retroviral DNA integration: HIV and the role of LEDGF/p75. 1673 94

Depletion of the transcriptional co-activator LEDGF/p75 by RNA interference alters the genome-wide pattern of HIV-1 integration, reducing integration into active genes, reducing integration into LEDGF/p75-regulated genes, and increasing integration into G+C-rich sequences. LEDGF/p75 is also able to act as a molecular tether linking HIV-1 integrase protein to chromatin, a phenomenon likely to underlie the integration site distribution effects. The LEDGF/p75 integrase-binding domain has been established but the domain or domains responsible for the chromatin-binding component of tethering are unknown. Here, we identify and characterize these domains. Complementary methods were used to assess condensed and uncondensed chromatin, and to determine the stringency of chromatin binding. Immuno-localization analyses revealed that an N-terminal PWWP domain and its beta-barrel substructure are needed for binding to metaphase chromatin. However, the PWWP domain is insufficient to transfer metaphase chromatin binding to green fluorescent protein, which requires addition of a downstream charged region (CR1). Biochemical analysis showed that full-length LEDGF/p75 resists Triton X-100 extraction from chromatin. To transfer Triton-resistant chromatin binding to green fluorescent protein, PWWP-CR1 is necessary but not sufficient. Further inclusion of a tandem pair of AT-hooks in combination with at least one of two identified downstream charged regions (CR2 or CR3) is needed. Deletion of just the PWWP or the AT-hook domain from full-length LEDGF/p75 reduced Triton-resistant chromatin binding, while deletion of both elements abolished it, underscoring their dominant and cooperative role. The results establish a molecular mechanism for LEDGF/p75-mediated tethering of HIV-1 integrase to chromatin.
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PMID:Identification and characterization of the chromatin-binding domains of the HIV-1 integrase interactor LEDGF/p75. 1679 62

Lens epithelium-derived growth factor/dense fine speckles 70 kDa protein (LEDGF/DFS70) is a transcriptional cofactor, a transcriptional activator, survival factor, and HIV-1 transporter. It is also a major autoantigen in patients with atopic dermatitis (AD), because autoantibodies to this protein are found in approximately 30% of AD patients. To better understand the role of autoantibodies and autoantigens in the pathogenesis of AD, we examined the distribution of LEDGF/DFS70 in the epidermis of normal human skin by light and electron microscopic immunocytochemistry. Increased amounts of LEDGF/DFS70 were located in the nuclei of cells in the basal layer, whereas the cytoplasm of cells in the granular layer stained for LEDGF/DFS70 by light microscopy. Using immunoelectron microscopy, we observed the accumulation of LEDGF/DFS70 in keratohyalin granules (KGs) in the cytoplasm of cells in the granular layer. In addition, Ig heavy chain-binding protein/glucose-regulated protein, 78-kDa (Bip/GRP78), a stress sensing protein in the endoplasmic reticulum, colocalized with LEDGF/DFS70 in the KGs. These results suggest that LEDGF/DFS70 is predominantly located in the nucleus of the basal epidermal cells and translocates into the cytoplasm during differentiation. Once in the cytoplasm, LEDGF/DFS70 accumulates in the KGs in the granular layer. Finally, LEDGF/DFS70, a "nuclear" autoantigen in AD, may play a functional role in the KGs.
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PMID:LEDGF/DFS70, a major autoantigen of atopic dermatitis, is a component of keratohyalin granules. 1685 21

LEDGF/p75 binding-defective IN mutant viruses were previously characterized as replication-defective, yet RNAi did not reveal an essential role for the host factor in HIV-1 replication. Correlative analyses of protein binding and viral fitness were expanded here by targeting 12 residues at the IN-LEDGF/p75 binding interface. Whereas many of the resultant viruses were defective, the majority of the INs displayed wild-type in vitro integration activities. Though an overall trend of parallel loss of LEDGF/p75 binding and HIV-1 infectivity was observed, a strict correlation was not. His-tagged IN(A128Q), derived from a phenotypically wild-type virus, failed to pull-down LEDGF/p75, but IN(A128Q) was effectively recovered in a reciprocal GST pull-down assay. Under these conditions, IN(H171A), also derived from a phenotypically wild-type virus, interacted less efficiently than a previously described interaction-defective mutant, IN(Q168A). Thus, the relative affinity of the in vitro IN-LEDGF/p75 interaction is not a universal predictor of IN mutant viral fitness.
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PMID:Structure-based mutagenesis of the integrase-LEDGF/p75 interface uncouples a strict correlation between in vitro protein binding and HIV-1 fitness. 1695 83

The mechanisms controlling retroviral integration have been the topic of intense interest, in part because of adverse clinical events that occurred during retrovirus-mediated human gene therapy. Here we investigate the use of artificial tethering interactions to constrain retroviral integration site selection in an in vitro model. During normal infection, HIV DNA integration is favored in active cellular transcription units. One component of the targeting mechanism is the cellular LEDGF/p75 protein. LEDGF/p75 binds tightly to HIV integrase (IN) protein, and depletion of LEDGF/p75 from target cells results in reduced integration in transcription units, suggesting integration targeting by a tethering mechanism. We constructed and analyzed fusions of LEDGF/p75 or its IN-binding domain (IBD) to the DNA-binding domain of phage lambda repressor protein (lambdaR). In the presence of the lambdaR-LEDGF/p75 fusions, increased strand transfer by IN was seen in target DNA near lambdaR-binding sites in vitro . These data support the idea that a direct interaction between LEDGF/p75 and IN can mediate targeting via a tethering mechanism, and provide proof of concept for the idea that protein-protein interactions might be engineered to constrain integration site selection during human gene therapy.
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PMID:Modulating target site selection during human immunodeficiency virus DNA integration in vitro with an engineered tethering factor. 1697 64

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


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