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)

Human lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) protein was recently identified as a binding partner for HIV-1 integrase (IN) in human cells. In this work, we used biochemical and bioinformatic approaches to define the domain organization of LEDGF/p75. Using limited proteolysis and deletion mutagenesis we show that the protein contains a pair of evolutionarily conserved domains, assuming about 35% of its sequence. Whereas the N-terminal PWWP domain had been recognized previously, the second domain is novel. It is comprised of approximately 80 amino acid residues and is both necessary and sufficient for binding to HIV-1 IN. Strikingly, the integrase binding domain (IBD) is not unique to LEDGF/p75, as a second human protein, hepatoma-derived growth factor-related protein 2 (HRP2), contains a homologous sequence. LEDGF/p75 and HRP2 IBDs avidly bound HIV-1 IN in an in vitro GST pull-down assay and each full-length protein potently stimulated HIV-1 IN activity in vitro. LEDGF/p75 and HRP2 are predicted to share a similar domain organization and have an evident evolutionary and likely functional relationship.
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PMID:Identification of an evolutionarily conserved domain in human lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) that binds HIV-1 integrase. 1537 38

The transcriptional coactivator lens epithelium-derived growth factor (LEDGF)/p75 acts as a chromatin tethering factor for human immunodeficiency virus type 1 (HIV-1) integrase protein, determining its nuclear localization and its tight association with nuclear DNA. Here we identify a second function for the LEDGF/p75-integrase interaction. We observed that stable introduction of HIV-1 integrase (IN) transcription units into cells made stringently LEDGF/p75-deficient by RNAi resulted in much lower steady state levels of IN protein than introduction into LEDGF/p75 wild type cells. The same LEDGF/p75-dependent disparity was observed for feline immunodeficiency virus IN. However, IN mRNA levels were equivalent in the presence and absence of LEDGF/p75. A post-translational mechanism was confirmed when the half-life of HIV-1 IN protein was found to be much shorter in LEDGF/p75-deficient cells. Proteasome inhibition fully countered this extreme instability, increasing IN protein levels to those seen in LEDGF/p75 wild type cells and implicating proteasomal destruction as the main cause of IN instability. Consistent with these data, increased ubiquitinated HIV-1 IN was found in the LEDGF/p75 knock-down cells. Moreover, restoration of LEDGF/p75 to knocked down clones rescued HIV-1 IN stability. Subcellular fractionation showed that HIV-1 IN is exclusively cytoplasmic in LEDGF/p75-deficient cells, but mainly nuclear in LEDGF/p75 wild type cells, and that cytoplasmic HIV-1 IN has a shorter half-life than nuclear HIV-1 IN. However, using LEDGF proteins defective for nuclear localization and IN interaction, we further determined that protection of HIV-1 IN from the proteasome requires neither chromatin tethering nor nuclear residence. Protection requires only interaction with LEDGF/p75, and it is independent of the subcellular localization of the IN-LEDGF complex.
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PMID:Lens epithelium-derived growth factor/p75 prevents proteasomal degradation of HIV-1 integrase. 1547 59

Recently we described the interaction of human immunodeficiency virus type 1 (HIV-1)1 integrase (IN) with a cellular protein, lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75). We now present the study of the diffusion behavior of the three independent domains of IN and LEDGF/p75 using fluorescence correlation microscopy (FCM). We show that diffusion in the cell of the different enhanced green fluorescent protein (EGFP) fusion proteins is described by two components with different fractions and that the average parameters in the nucleus are comparable with those in the cytoplasm. In addition, we demonstrate that specific interaction between EGFP-fused HIV-1 IN and LEDGF/p75 results in a shift in diffusion coefficient (D). The opposite shift was observed in an IN-deletion mutant that does not exhibit LEDGF/p75 binding or in a LEDGF/p75 knock-down experiment using siRNA. We thus demonstrate that protein-protein interactions can be studied in living cells, using single-color FCM (scFCM).
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PMID:Measuring protein-protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type 1 integrase and LEDGF/p75. 1578 49

To investigate the basis for the LEDGF/p75 dependence of HIV-1 integrase (IN) nuclear localization and chromatin association, we used cell lines made stably deficient in endogenous LEDGF/p75 by RNAi to analyze determinants of its location in cells and its ability to interact with IN. Deletion of C-terminal LEDGF/p75 residues 340-417 preserved nuclear and chromatin localization but abolished the interaction with IN and the tethering of IN to chromatin. Transfer of this IN-binding domain (IBD) was sufficient to confer HIV-1 IN interaction to GFP. HRP-2, the only other human protein with an identifiable IBD domain, was found to translocate IN to the nucleus of LEDGF/p75(-) cells. However, in contrast to LEDGF/p75, HRP-2 is not chromatin bound and does not tether IN to chromatin. A single classical nuclear localization signal (NLS) in the LEDGF/p75 N-terminal region ((146)RRGRKRKAEKQ(156)) was found by deletion mapping and was shown to be transferable to pyruvate kinase. Four central basic residues in the NLS are critical for its activity. Strikingly, however, stable expression studies with NLS(+/-) and IBD(+/-) mutants revealed that the NLS, although responsible for LEDGF/p75 nuclear import, is dispensable for stable, constitutive nuclear association of LEDGF/p75 and IN. Both wild-type LEDGF/p75 and NLS-mutant LEDGF/p75 remain entirely chromatin associated throughout the cell cycle, and each tethers IN to chromatin. Thus, these experiments reveal stable nuclear sequestration of a transcriptional regulator by chromatin during the nuclear-cytosolic mixing of cell division, which additionally enables stable tethering of IN to chromatin. LEDGF/p75 is a multidomain adaptor protein that interacts with the nuclear import apparatus, lentiviral IN proteins and chromatin by means of an NLS, an IBD and additional chromatin-interacting domains.
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PMID:Identification of the LEDGF/p75 HIV-1 integrase-interaction domain and NLS reveals NLS-independent chromatin tethering. 1579 27

The insertion of a DNA copy of its RNA genome into a chromosome of the host cell is mediated by the viral integrase with the help of mostly uncharacterized cellular cofactors. We have recently described that the transcriptional co-activator LEDGF/p75 strongly interacts with HIV-1 integrase. Here we show that interaction of HIV-1 integrase with LEDGF/p75 is important for viral replication. Using multiple approaches including two-hybrid interaction studies, random and directed mutagenesis, we could demonstrate that HIV-1 virus harboring a single mutation that disrupts integrase-LEDGF/p75 interaction, resulted in defective HIV-1 replication. Furthermore, we found that LEDGF/p75 tethers HIV-1 integrase to chromosomes and that this interaction may be important for the integration process and the replication of HIV-1.
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PMID:Integrase mutants defective for interaction with LEDGF/p75 are impaired in chromosome tethering and HIV-1 replication. 1585 67

Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase (IN) in human cells. We have determined the NMR structure of the integrase-binding domain (IBD) in LEDGF and identified amino acid residues essential for the interaction. The IBD is a compact right-handed bundle composed of five alpha-helices. Based on folding topology, the IBD is structurally related to a diverse family of alpha-helical proteins that includes eukaryotic translation initiation factor eIF4G and karyopherin-beta. LEDGF residues essential for the interaction with IN were localized to interhelical loop regions of the bundle structure. Interaction-defective IN mutants were previously shown to cripple replication although they retained catalytic function. The initial structure determination of a host cell factor that tightly binds to a retroviral enzyme lays the groundwork for understanding enzyme-host interactions important for viral replication.
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PMID:Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75. 1589 93

HIV DNA integration is favored in active genes, but the underlying mechanism is unclear. Cellular lens epithelium-derived growth factor (LEDGF/p75) binds both chromosomal DNA and HIV integrase, and might therefore direct integration by a tethering interaction. We analyzed HIV integration in cells depleted for LEDGF/p75, and found that integration was (i) less frequent in transcription units, (ii) less frequent in genes regulated by LEDGF/p75 and (iii) more frequent in GC-rich DNA. LEDGF is thus the first example of a cellular protein controlling the location of HIV integration in human cells.
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PMID:A role for LEDGF/p75 in targeting HIV DNA integration. 1631 5

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) functions in cells within the context of high molecular weight preintegration complexes (PICs). Lens epithelium-derived growth factor (LEDGF) transcriptional coactivator/p75 and hepatoma-derived growth factor related protein 2 (HRP2) tightly bind to HIV-1 IN and stimulate its integration activity in vitro. Here, we show that each recombinant host cell factor efficiently reconstitutes the in vitro activity of HIV-1 PICs disrupted for functional integration by pre-treatment with high concentrations of salt. Mutational analysis reveals that both the IN-binding and DNA-binding activities of LEDGF/p75 contribute to functional PIC reconstitution. We also investigate a role(s) for these proteins in HIV-1 infection by using short-interfering RNA. HIV-1 infection was essentially unaffected in HeLa-P4 cells depleted for LEDGF/p75, HRP2, or both proteins. We conclude that cells knocked-out for LEDGF/p75 and/or HRP2 will be useful genetic tools to address the roles of these host cell factors in HIV-1 replication.
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PMID:Biochemical and genetic analyses of integrase-interacting proteins lens epithelium-derived growth factor (LEDGF)/p75 and hepatoma-derived growth factor related protein 2 (HRP2) in preintegration complex function and HIV-1 replication. 1633 83

To achieve productive infection, the reverse transcribed cDNA of human immunodeficiency virus type 1 (HIV-1) is inserted in the host cell genome. The main protein responsible for this reaction is the viral integrase. However, studies indicate that the virus is assisted by cellular proteins, or co-factors, to achieve integration into the infected cell. The barrier-to-autointegration factor (BAF) might prevent autointegration. Its ability to bridge DNA and the finding that the nuclear lamina-associated polypeptide-2alpha interacts with BAF suggest a role in nuclear structure organization. Integrase interactor 1 was found to directly interact with HIV-1 integrase and to activate its DNA-joining activity, and the high mobility group chromosomal protein A1 might approximate both long terminal repeat (LTR) ends and facilitate integrase binding by unwinding the LTR termini. Furthermore, the lens-epithelium-derived growth factor (LEDGF; also known as p75) seems to tether HIV-1 integrase to the chromosomes. Although a direct role in integration has only been demonstrated for LEDGF/p75, to date, each validated cellular co-factor for HIV-1 integration could constitute a promising new target for antiviral therapy.
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PMID:Cellular co-factors of HIV-1 integration. 1640 35

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


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