Role of the PWWP domain of lens epithelium-derived growth factor (LEDGF)/p75 cofactor in lentiviral integration targeting

LEDGF/p75 is a chromatin-interacting, cellular cofactor of HIV integrase that dictates lentiviral integration site preference. In this study we determined the role of the PWWP domain of LEDGF/p75 in tethering and targeting of the lentiviral pre-integration complex, employing potent knockdown cell lines allowing analysis in the absence of endogenous LEDGF/p75. Deletion of the PWWP domain resulted in a diffuse subnuclear distribution pattern, loss of interaction with condensed chromatin, and failure to rescue proviral integration, integration site distribution, and productive virus replication. Substitution of the PWWP domain of LEDGF/p75 with that of hepatoma-derived growth factor or HDGF-related protein-2 rescued viral replication and lentiviral integration site distribution in LEDGF/p75-depleted cells. Replacing all chromatin binding elements of LEDGF/p75 with full-length hepatoma-derived growth factor resulted in more integration in genes combined with a preference for CpG islands. In addition, we showed that any PWWP domain targets SMYD1-like sequences. Analysis of integration preferences of lentiviral vectors for epigenetic marks indicates that the PWWP domain is critical for interactions specifying the relationship of integration sites to regions enriched in specific histone post-translational modifications.     

In Vitro DNA Tethering of HIV-1 Integrase by the Transcriptional Coactivator LEDGF/p75

Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.     

Structural basis for high-affinity binding of LEDGF PWWP to mononucleosomes

Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin.     

Identification and characterization of PWWP domain residues critical for LEDGF/p75 chromatin binding and human immunodeficiency virus type 1 infectivity

Lens epithelium-derived growth factor (LEDGF)/p75 functions as a bimodal tether during lentiviral DNA integration: its C-terminal integrase-binding domain interacts with the viral preintegration complex, whereas the N-terminal PWWP domain can bind to cellular chromatin. The molecular basis for the integrase-LEDGF/p75 interaction is understood, while the mechanism of chromatin binding is unknown. The PWWP domain is homologous to other protein interaction modules that together comprise the Tudor clan. Based on primary amino acid sequence and three-dimensional structural similarities, 24 residues of the LEDGF/p75 PWWP domain were mutagenized to garner essential details of its function during human immunodeficiency virus type 1 (HIV-1) infection. Mutating either Trp-21 or Ala-51, which line the inner wall of a hydrophobic cavity that is common to Tudor clan members, disrupts chromatin binding and virus infectivity. Consistent with a role for chromatin-associated LEDGF/p75 in stimulating integrase activity during infection, recombinant W21A protein is preferentially defective for enhancing integration into chromatinized target DNA in vitro. The A51P mutation corresponds to the S270P change in DNA methyltransferase 3B that causes human immunodeficiency, centromeric instability, and facial anomaly syndrome, revealing a critical role for this amino acid position in the chromatin binding functions of varied PWWP domains. Our results furthermore highlight the requirement for a conserved Glu in the hydrophobic core that mediates interactions between other Tudor clan members and their substrates. This initial systematic mutagenesis of a PWWP domain identifies amino acid residues critical for chromatin binding function and the consequences of their changes on HIV-1 integration and infection.     

LEDGF/p75 Proteins with Alternative Chromatin Tethers Are Functional HIV-1 Cofactors

LEDGF/p75 can tether over-expressed lentiviral integrase proteins to chromatin but how this underlies its integration cofactor role for these retroviruses is unclear. While a single integrase binding domain (IBD) binds integrase, a complex N-terminal domain ensemble (NDE) interacts with unknown chromatin ligands. Whether integration requires chromatin tethering per se, specific NDE-chromatin ligand interactions or other emergent properties of LEDGF/p75 has been elusive. Here we replaced the NDE with strongly divergent chromatin-binding modules. The chimeras rescued integrase tethering and HIV-1 integration in LEDGF/p75-deficient cells. Furthermore, chromatin ligands could reside inside or outside the nucleosome core, and could be protein or DNA. Remarkably, a short Kaposi''s sarcoma virus peptide that binds the histone 2A/B dimer converted GFP-IBD from an integration blocker to an integration cofactor that rescues over two logs of infectivity. NDE mutants were corroborative. Chromatin tethering per se is a basic HIV-1 requirement and this rather than engagement of particular chromatin ligands is important for the LEDGF/p75 cofactor mechanism.     

Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.     

HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.     

Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75

The stable insertion of the retroviral genome into the host chromosomes requires the association between integration complexes and cellular chromatin via the interaction between retroviral integrase and the nucleosomal target DNA. This final association may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To investigate this and better understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase, we used a combination of biochemical and chromosome-binding assays. Our study revealed that retroviral integrase has an intrinsic ability to bind and recognize specific chromatin regions in metaphase even in the absence of its cofactor. Furthermore, this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromosome regions. We also better determined the chromatin features recognized by each partner alone or within the functional intasome, as well as the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. Our data support a new chromatin-binding function of integrase acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate. This work also provides additional information about the behavior of retroviral integration complexes in metaphase chromatin and the mechanism of action of LEDGF/p75 in this specific context.     

FRET analysis reveals distinct conformations of IN tetramers in the presence of viral DNA or LEDGF/p75

A tetramer of HIV-1 integrase (IN) stably associates with the viral DNA ends to form a fully functional concerted integration intermediate. LEDGF/p75, a key cellular binding partner of the lentiviral enzyme, also stabilizes a tetrameric form of IN. However, functional assays have indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for productive concerted integration. Here, we employed Förster Resonance Energy Transfer (FRET) to monitor assembly of individual IN subunits into tetramers in the presence of viral DNA and LEDGF/p75. The IN–viral DNA and IN–LEDGF/p75 complexes yielded significantly different FRET values suggesting two distinct IN conformations in these complexes. Furthermore, the order of addition experiments indicated that FRET for the preformed IN–viral DNA complex remained unchanged upon its subsequent binding to LEDGF/p75, whereas pre-incubation of LEDGF/p75 and IN followed by addition of viral DNA yielded FRET very similar to the IN–LEDGF/p75 complex. These findings provide new insights into the structural organization of IN subunits in functional concerted integration intermediates and suggest that differential multimerization of IN in the presence of various ligands could be exploited as a plausible therapeutic target for development of allosteric inhibitors.     

Structural and Functional Role of INI1 and LEDGF in the HIV-1 Preintegration Complex

Integration of the HIV-1 cDNA into the human genome is catalyzed by the viral integrase (IN) protein. Several studies have shown the importance of cellular cofactors that interact with integrase and affect viral integration and infectivity. In this study, we produced a stable complex between HIV-1 integrase, viral U5 DNA, the cellular cofactor LEDGF/p75 and the integrase binding domain of INI1 (INI1-IBD), a subunit of the SWI/SNF chromatin remodeling factor. The stoichiometry of the IN/LEDGF/INI1-IBD/DNA complex components was found to be 4/2/2/2 by mass spectrometry and Fluorescence Correlation Spectroscopy. Functional assays showed that INI1-IBD inhibits the 3′ processing reaction but does not interfere with specific viral DNA binding. Integration assays demonstrate that INI1-IBD decreases the amount of integration events but inhibits by-product formation such as donor/donor or linear full site integration molecules. Cryo-electron microscopy locates INI1-IBD within the cellular DNA binding site of the IN/LEDGF complex, constraining the highly flexible integrase in a stable conformation. Taken together, our results suggest that INI1 could stabilize the PIC in the host cell, by maintaining integrase in a stable constrained conformation which prevents non-specific interactions and auto integration on the route to its integration site within nucleosomes, while LEDGF organizes and stabilizes an active integrase tetramer suitable for specific vDNA integration. Moreover, our results provide the basis for a novel type of integrase inhibitor (conformational inhibitor) representing a potential new strategy for use in human therapy.