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)

After identifying the interaction between the transcriptional coactivator lens epithelium-derived growth factor (LEDGF/p75) and the human immunodeficiency virus type 1 (HIV-1) integrase (IN), we have now investigated the role of LEDGF/p75 during HIV replication. Transient small interfering RNA-mediated knockdown of LEDGF/p75 in HeLaP4 cells resulted in a three- to fivefold inhibition of HIV-1 (strain NL4.3) replication. Quantitative PCR was used to pinpoint the replication block to the integration step. Next, polyclonal and monoclonal HeLaP4-derived cell lines were selected with a stable knockdown of LEDGF/p75 mediated by a lentiviral vector (lentivector) encoding a short hairpin RNA (shRNA) targeting this protein. Cell lines stably transduced with a lentivector encoding an unrelated hairpin or a double-mismatch hairpin served as controls. Again, a two- to fourfold reduction of HIV-1 replication was observed. The extent of LEDGF/p75 knockdown closely correlated with the reduction of HIV-1 replication. After the back-complementation of LEDGF/p75 in the poly- and monoclonal knockdown cell lines using an shRNA-resistant expression plasmid, viral replication was restored to nearly wild-type levels. The Q168A mutation in integrase has been shown to interfere with the interaction with LEDGF/p75 without reducing the enzymatic activity. Transduction by HIV-1-derived lentivectors carrying the Q168A IN mutant was severely hampered, pointing again to a requirement for LEDGF/p75. Altogether, our data validate LEDGF/p75 as an important cellular cofactor for HIV integration and as a potential target for antiviral drug development.
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PMID:Transient and stable knockdown of the integrase cofactor LEDGF/p75 reveals its role in the replication cycle of human immunodeficiency virus. 1643 44

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

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

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

Human transcriptional coactivator p75/lens epithelium-derived growth factor (LEDGF) binds human immunodeficiency virus type 1 (HIV-1) integrase (IN). We studied the effects of LEDGF on the assembly and activity of HIV-1 synaptic complexes, which, upon association with a target, mediate concerted integration of viral DNA substrates in vitro. We found that while augmenting single-ended viral DNA integration into target DNA, the host factor was able to either stimulate or abrogate concerted integration in a concentration-dependent manner. LEDGF modestly stimulated (two- to threefold) concerted integration at low molar ratios to IN (<1). The modest stimulation was independent of solution conditions and several different viral DNA substrates. In solution, concerted integration was inhibited if the molar ratios of LEDGF to IN were >1, apparently due to the disruption of IN-IN interactions essential for the formation of active synaptic complexes prior to their association with a circular target. The isolated IN binding domain of LEDGF was sufficient to stimulate and inhibit concerted integration, as observed with full-length protein, albeit at lower efficiencies. Our data show that LEDGF differentially affects IN-DNA complexes mediating single-ended viral DNA integration and synaptic complexes mediating concerted integration. Synaptic complexes associated with target, termed strand transfer complexes, are resistant to disruption by high concentrations of LEDGF. The results suggest that LEDGF may influence HIV-1 integration in vivo.
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PMID:Transcriptional coactivator LEDGF/p75 modulates human immunodeficiency virus type 1 integrase-mediated concerted integration. 1726 86

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

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

Retroviral replication proceeds through a stable proviral DNA intermediate, and numerous host cell factors have been implicated in its formation. In particular, recent results have highlighted an important role for the integrase-interactor lens epithelium-derived growth factor (LEDGF)/p75 in lentiviral integration. Cells engineered to over-express fragments of LEDGF/p75 containing its integrase-binding domain but lacking determinants essential for chromatin association are refractory to HIV-1 infection. Furthermore, both the levels of HIV-1 integration and the genomic distribution of the resultant proviruses are significantly perturbed in cells devoid of endogenous LEDGF/p75 protein. A strong bias towards integration along transcription units is a characteristic feature of lentiviruses. In the absence of LEDGF/p75, HIV-1 in large part loses that preference, displaying concomitant integration surges in the vicinities of CpG islands and gene promoter regions, elements naturally targeted by other types of retroviruses. Together, these findings highlight that LEDGF/p75 is an important albeit not strictly essential cofactor of lentiviral DNA integration, and solidify a role for chromatin-associated LEDGF/p75 as a receptor for lentiviral preintegration complexes. By now one of the best characterized virus-host interactions, the integrase-LEDGF/p75 interface opens a range of opportunities for lentiviral vector targeting for gene therapy applications as well as for the development of novel classes of antiretroviral drugs.
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PMID:The lentiviral integrase binding protein LEDGF/p75 and HIV-1 replication. 1836 82


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