Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75 (original) (raw)
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Identification of the LEDGF/p75 Binding Site in HIV-1 Integrase
Journal of Molecular Biology, 2007
Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3′ processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.
LEDGF/p75 Is Essential for Nuclear and Chromosomal Targeting of HIV-1 Integrase in Human Cells
Journal of Biological Chemistry, 2003
We have reported that human immunodeficiency virus type 1 (HIV-1) integrase (IN) forms a specific nuclear complex with human lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75) protein. We now studied the IN-LEDGF/p75 interaction and nuclear import of IN in living cells using fusions of IN and LEDGF/p75 with enhanced green fluorescent protein and far-red fluorescent protein HcRed1. We show that both the N-terminal zinc binding domain and the central core domains of IN are involved in the interaction with LEDGF/p75. Both domains are essential for nuclear localization of IN as well as for the association of IN with condensed chromosomes during mitosis. However, upon overexpression of LEDGF/p75, the core domain fragment of IN was recruited to the nuclei and mitotic chromosomes with a distribution pattern characteristic of the full-length protein, indicating that it harbors the main determinant for interaction with LEDGF/p75. Although the C-terminal domain of IN was dispensable for nuclear/chromosomal localization, a fusion of the C-terminal IN fragment with enhanced green fluorescent protein was found exclusively in the nucleus, with a diffuse nuclear/nucleolar distribution, suggesting that the C-terminal domain may also play a role in the nuclear import of IN. In contrast to LEDGF/p75, its alternative splice variant, p52, did not interact with HIV-1 IN in vitro and in living cells. Finally, RNA interference-mediated knock-down of endogenous LEDGF/ p75 expression abolished nuclear/chromosomal localization of IN. We conclude, therefore, that the interaction with LEDGF/p75 accounts for the karyophilic properties and chromosomal targeting of HIV-1 IN.
Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75
Nature Structural & Molecular Biology, 2005
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 ␣-helices. Based on folding topology, the IBD is structurally related to a diverse family of ␣-helical proteins that includes eukaryotic translation initiation factor eIF4G and karyopherin-. 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.
HIV-1 Integrase Forms Stable Tetramers and Associates with LEDGF/p75 Protein in Human Cells
Journal of Biological Chemistry, 2002
We studied human immunodeficiency virus, type 1 (HIV-1) integrase (IN) complexes derived from nuclei of human cells stably expressing the viral protein from a synthetic gene. We show that in the nuclear extracts IN exists as part of a large distinct complex with an apparent Stokes radius of 61 Å, which dissociates upon dilution yielding a core molecule of 41 Å.
Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry
Scientific Reports, 2016
Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies. Although active nuclear import is a hallmark in the replication cycle of lentiviruses such as the human immunodeficiency virus type 1 (HIV-1), nuclear entry is one of the least understood steps 1-4. After reverse transcription of the viral RNA into double stranded DNA, the pre-integration complex (PIC) is formed as an assembly of the viral DNA (vDNA) and cellular and viral proteins. Prior to integration, the PIC has to cross the natural barrier of the nuclear membrane through nuclear pore complexes (NPCs) which serve as selective entry gates 5. Recent evidence suggests that uncoating of the HIV capsid (CA) core occurs close to the nuclear membrane although some CA molecules may accompany the PIC into the nucleus 6-9. Genome-wide siRNA screens identified the nucleoporins Nup153 and Nup358 (RANBP2) as host cofactors of HIV nuclear import 10-13. Nup358 binds CA 14 and is believed to act as a docking station for the HIV PIC 10,14. Nup153 is located in the nuclear basket; interactions between its FG repeats and either viral integrase (IN) or CA are in line with a role during nuclear entry 10,15,16. Besides nucleoporins, importin α /β , importin 7 and Transportin-SR2 (TRN-SR2, TNPO3) have been proposed to be involved in nuclear import of the PIC 1,17-20. A role for the HIV DNA flap in nuclear import has been proposed as well 21,22. HIV-1 IN mediates the insertion of the viral cDNA in two consecutive steps: 3′ processing and strand transfer 23. IN catalytic activity is highly dependent on a dynamic equilibrium of IN multimers; evidence indicates that 3′ processing requires at least a dimer whereas at least a tetramer is needed for concerted integration 24-28. In line with this, the prototype foamy virus (PFV) intasome has been shown to consist of an IN tetramer 29. Concerted integration of the HIV cDNA occurs into active transcription sites 30,31 and is guided by the host factor LEDGF/p75 32-34. LEDGF/p75 contains an N-terminal chromatin/DNA binding moiety (residues 1-325) and a C-terminal integrase binding domain (IBD, residues 347-429) 35,36. The pivotal role of LEDGF/p75 in HIV-1 replication was revealed via mutagenesis, RNAi-mediated depletion, transdominant overexpression of the IBD of LEDGF/p75 and cellular knockout studies 32,33,37-43 .
FEBS Letters, 2008
A lens epithelium-derived growth factor (LEDGF)/ p75 peptide was evaluated for human immunodeficiency virus type 1 integrase (IN) inhibitory activity. The LEDGF/p75 peptide modestly inhibited IN catalysis and was dependent on IN-DNA assembly. The peptide was also effective at disrupting LEDGF/p75-IN complex formation. We next investigated the activity of the LEDGF/p75 peptide on IN mutant proteins that are unable to catalyze the DNA strand transfer reaction. The LEDGF/p75 peptide displayed an increased potency on these IN proteins, from 5-fold to greater than 10-fold, indicating the IN multimeric state greatly influences the peptide inhibitory effects. These results shed light on IN-DNA multimeric formation, and how this process influences the LEDGF/p75-IN interaction.
Virology Journal, 2010
Background During the early stage of HIV-1 replication, integrase (IN) plays important roles at several steps, including reverse transcription, viral DNA nuclear import, targeting viral DNA to host chromatin and integration. Previous studies have demonstrated that HIV-1 IN interacts with a cellular Lens epithelium-derived growth factor (LEDGF/p75) and that this viral/cellular interaction plays an important role for tethering HIV-1 preintegration complexes (PICs) to transcriptionally active units of host chromatin. Meanwhile, other studies have revealed that the efficient knockdown and/or knockout of LEDGF/p75 could not abolish HIV infection, suggesting a LEDGF/p75-independent action of IN for viral DNA chromatin targeting and integration, even though the underlying mechanism(s) is not fully understood. Results In this study, we performed site-directed mutagenic analysis at the C-terminal region of the IN catalytic core domain responsible for IN/chromatin binding and IN/LEDGF/p75 int...
Journal of Cell Science, 2005
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 (146RRGRKRKAEKQ156) was found by deletion mapping and was shown to be transferable to pyruvate kinase. Four central basic ...
Small molecules targeting the interaction between HIV-1 integrase and LEDGF/p75 cofactor
Bioorganic & Medicinal Chemistry, 2010
The search of small molecules as protein-protein interaction inhibitors represents a new attractive strategy to develop anti-HIV-1 agents. We previously reported a computational study that led to the discovery of new inhibitors of the interaction between enzyme HIV-1 integrase (IN) and the nuclear protein lens epithelium growth factor LEDGF/p75. 1 Herein, we describe new findings about the binding site of LEDGF/p75 on IN employing a different computational approach. In this way further structural requirements, helpful to disrupt LEDGF/p75-IN binding, have been identified. The main result of this work was the exploration of a relevant hydrophobic region. So we planned the introduction of suitable and simple chemical modifications on our previously reported 'hit' and the new synthesized compounds were subjected to biological tests. The results obtained demonstrate that the hydrophobic pocket could play a key role in improving inhibitory efficacy thus opening new suggestions to design active ligands.
Differential Interaction of HIV1 Integrase and JPO2 with the C Terminus of LEDGF/p75
Journal of Molecular Biology, 2007
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.