Affinity switching of the LEDGF/p75 IBD interactome is governed by kinase-dependent phosphorylation.

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

Correction to: HIV-1 IN/Pol recruits LEDGF/p75 into viral particles.

An amendment to this paper has been published and can be accessed via the original article.

Correction to: LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions.

An amendment to this paper has been published and can be accessed via the original article.

LEDGF/p75 is dispensable for hematopoiesis but essential for MLL-rearranged leukemogenesis.

Mixed lineage leukemia (MLL) represents a genetically distinct and aggressive subset of human acute leukemia carrying chromosomal translocations of the MLL gene. These translocations result in oncogenic fusions that mediate aberrant recruitment of the transcription machinery to MLL target genes. The N-terminus of MLL and MLL-fusions form a complex with lens epithelium-derived growth factor (LEDGF/p75; encoded by the PSIP1 gene) and MENIN. This complex contributes to the association of MLL and MLL-fusion multiprotein complexes with the chromatin. Several studies have shown that both MENIN and LEDGF/p75 are required for efficient MLL-fusion-mediated transformation and for the expression of downstream MLL-regulated genes such as HOXA9 and MEIS1 In light of developing a therapeutic strategy targeting this complex, understanding the function of LEDGF/p75 in normal hematopoiesis is crucial. We generated a conditional Psip1 knockout mouse model in the hematopoietic compartment and examined the effects of LEDGF/p75 depletion in postnatal hematopoiesis and the initiation of MLL leukemogenesis. Psip1 knockout mice were viable but showed several defects in hematopoiesis, reduced colony-forming activity in vitro, decreased expression of Hox genes in the hematopoietic stem cells, and decreased MLL occupancy at MLL target genes. Finally, in vitro and in vivo experiments showed that LEDGF/p75 is dispensable for steady-state hematopoiesis but essential for the initiation of MLL-mediated leukemia. These data corroborate the MLL-LEDGF/p75 interaction as novel target for the treatment of MLL-rearranged leukemia.

Host factors for retroviral integration site selection.

To achieve productive infection, retroviruses such as HIV stably integrate their reverse transcribed RNA genome into a host chromosome. Each retroviral family preferentially integrates near a unique subset of genomic features. HIV integrase (IN) is targeted to the body of active transcription units through interaction with lens epithelium-derived growth factor (LEDGF/p75). We describe the successful effort to develop inhibitors of the interaction between IN and LEDGF/p75, referred to as LEDGINs. Gammaretroviruses display a distinct integration pattern. Recently, BET (bromo- and extraterminal domain) proteins were identified as the LEDGF/p75 counterparts that target the integration of gammaretroviruses. The identification of the chromatin-readers LEDGF/p75 and BET as cellular cofactors that orchestrate lentiviral or gammaretroviral integration opens new avenues to developing safer viral vectors for gene therapy.

Protein-protein and protein-chromatin interactions of LEDGF/p75 as novel drug targets.

Lens epithelium-derived growth factor p75 (LEDGF/p75), a transcriptional co-activator, plays an important role in tethering protein complexes to the chromatin. Through this tethering function LEDGF/p75 is implicated in a diverse set of human diseases including HIV infection and mixed lineage leukemia, an aggressive form of cancer with poor prognosis. Here we provide an overview of recent progress in resolving protein-protein and protein-chromatin interaction mechanisms of LEDGF/p75. This review will focus on two well-characterized domains, the PWWP domain and the integrase binding domain (IBD). The PWWP domain interacts with methylated lysine 36 in histone H3, a marker of actively transcribed genes. The IBD interacts with the IBD binding motif, available in cellular binding partners of LEDGF/p75. Each domain forms an interesting new target for drug discovery.

Retroviral integration: Site matters: Mechanisms and consequences of retroviral integration site selection.

Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co-opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine-grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success.

Dynamics of the ternary complex formed by c-Myc interactor JPO2, transcriptional co-activator LEDGF/p75, and chromatin.

Lens epithelium-derived growth factor (LEDGF/p75) is a transcriptional co-activator involved in targeting human immunodeficiency virus (HIV) integration and the development of MLL fusion-mediated acute leukemia. A previous study revealed that LEDGF/p75 dynamically scans the chromatin, and upon interaction with HIV-1 integrase, their complex is locked on chromatin. At present, it is not known whether LEDGF/p75-mediated chromatin locking is typical for interacting proteins. Here, we employed continuous photobleaching and fluorescence correlation and cross-correlation spectroscopy to investigate in vivo chromatin binding of JPO2, a LEDGF/p75- and c-Myc-interacting protein involved in transcriptional regulation. In the absence of LEDGF/p75, JPO2 performs chromatin scanning inherent to transcription factors. However, whereas the dynamics of JPO2 chromatin binding are decelerated upon interaction with LEDGF/p75, very strong locking of their complex onto chromatin is absent. Similar results were obtained with the domesticated transposase PogZ, another cellular interaction partner of LEDGF/p75. We furthermore show that diffusive JPO2 can oligomerize; that JPO2 and LEDGF/p75 interact directly and specifically in vivo through the specific interaction domain of JPO2 and the C-terminal domain of LEDGF/p75, comprising the integrase-binding domain; and that modulation of JPO2 dynamics requires a functional PWWP domain in LEDGF/p75. Our results suggest that the dynamics of the LEDGF/p75-chromatin interaction depend on the specific partner and that strong chromatin locking is not a property of all LEDGF/p75-binding proteins.

HIV-1 IN/Pol recruits LEDGF/p75 into viral particles.

BACKGROUND: The dynamic interaction between HIV and its host governs the replication of the virus and the study of the virus-host interplay is key to understand the viral lifecycle. The host factor lens epithelium-derived growth factor (LEDGF/p75) tethers the HIV preintegration complex to the chromatin through a direct interaction with integrase (IN). Small molecules that bind the LEDGF/p75 binding pocket of the HIV IN dimer (LEDGINs) block HIV replication through a multimodal mechanism impacting early and late stage replication including HIV maturation. Furthermore, LEDGF/p75 has been identified as a Pol interaction partner. This raised the question whether LEDGF/p75 besides acting as a molecular tether in the target cell, also affects late steps of HIV replication. RESULTS: LEDGF/p75 is recruited into HIV-1 particles through direct interaction with the viral IN (or Pol polyprotein) and is a substrate for HIV-1 protease. Incubation in the presence of HIV-1 protease inhibitors resulted in detection of full-length LEDGF/p75 in purified viral particles. We also demonstrate that inhibition of LEDGF/p75-IN interaction by specific mutants or LEDGINs precludes incorporation of LEDGF/p75 in virions, underscoring the specificity of the uptake. LEDGF/p75 depletion did however not result in altered LEDGIN potency. CONCLUSION: Together, these results provide evidence for an IN/Pol mediated uptake of LEDGF/p75 in viral particles and a specific cleavage by HIV protease. Understanding of the possible role of LEDGF/p75 or its cleavage fragments in the viral particle awaits further experimentation.

LEDGF/p75-independent HIV-1 replication demonstrates a role for HRP-2 and remains sensitive to inhibition by LEDGINs.

Lens epithelium-derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase (IN) that interacts with IN through its IN binding domain (IBD) and tethers the viral pre-integration complex to the host cell chromatin. Here we report the generation of a human somatic LEDGF/p75 knockout cell line that allows the study of spreading HIV-1 infection in the absence of LEDGF/p75. By homologous recombination the exons encoding the LEDGF/p75 IBD (exons 11 to 14) were knocked out. In the absence of LEDGF/p75 replication of laboratory HIV-1 strains was severely delayed while clinical HIV-1 isolates were replication-defective. The residual replication was predominantly mediated by the Hepatoma-derived growth factor related protein 2 (HRP-2), the only cellular protein besides LEDGF/p75 that contains an IBD. Importantly, the recently described IN-LEDGF/p75 inhibitors (LEDGINs) remained active even in the absence of LEDGF/p75 by blocking the interaction with the IBD of HRP-2. These results further support the potential of LEDGINs as allosteric integrase inhibitors.

LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions.

BACKGROUND: LEDGINs are novel allosteric HIV integrase (IN) inhibitors that target the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. They block HIV-1 integration by abrogating the interaction between LEDGF/p75 and IN as well as by allosterically inhibiting the catalytic activity of IN. RESULTS: Here we demonstrate that LEDGINs reduce the replication capacity of HIV particles produced in their presence. We systematically studied the molecular basis of this late effect of LEDGINs and demonstrate that HIV virions produced in their presence display a severe replication defect. Both the late effect and the previously described, early effect on integration contribute to LEDGIN antiviral activity as shown by time-of-addition, qPCR and infectivity assays. The late effect phenotype requires binding of LEDGINs to integrase without influencing proteolytic cleavage or production of viral particles. LEDGINs augment IN multimerization during virion assembly or in the released viral particles and severely hamper the infectivity of progeny virions. About 70% of the particles produced in LEDGIN-treated cells do not form a core or display aberrant empty cores with a mislocalized electron-dense ribonucleoprotein. The LEDGIN-treated virus displays defective reverse transcription and nuclear import steps in the target cells. The LEDGIN effect is possibly exerted at the level of the Pol precursor polyprotein. CONCLUSION: Our results suggest that LEDGINs modulate IN multimerization in progeny virions and impair the formation of regular cores during the maturation step, resulting in a decreased infectivity of the viral particles in the target cells. LEDGINs thus profile as unique antivirals with combined early (integration) and late (IN assembly) effects on the HIV replication cycle.

Lens epithelium-derived growth factor/p75 qualifies as a target for HIV gene therapy in the NSG mouse model.

Lens epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF(325-530)) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD(4)(+) T-cells were transduced with lentiviral vectors encoding LEDGF(325-530), the interaction-deficient mutant LEDGF(325-530)D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF(325-530) overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD(4)(+) T-cells expressing LEDGF(325-530) in comparison with engraftment with LEDGF(325-530)D366N cells. Four weeks after transplantation with LEDGF(325-530)D366N cells, 70% of the CD(4)(+) cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF(325-530) cells only a 20% decrease in CD(4)(+) cells was measured. Liver and spleen sections of LEDGF(325-530) mice contained less HIV than LEDGF(325-530)D366N mice as measured by p24 antigen detection. LEDGF(325-530) overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD(4)(+) T-cells.

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering.

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein-protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

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.

HRP-2 determines HIV-1 integration site selection in LEDGF/p75 depleted cells.

BACKGROUND: Lens epithelium-derived growth factor (LEDGF/p75) is a cellular co-factor of HIV-1 integrase (IN) that tethers the viral pre-integration complex to the host cell chromatin and determines the genome wide integration site distribution pattern of HIV-1. Recently, we demonstrated that HIV-1 replication was reduced in LEDGF/p75 knockout (KO) cells. LEDGF/p75 KO significantly altered the integration site preference of HIV-1, but the pattern remained distinct from a computationally generated matched random control set (MRC), suggesting the presence of an alternative tethering factor. We previously identified Hepatoma-derived growth factor related protein 2 (HRP-2) as a factor mediating LEDGF/p75-independent HIV-1 replication. However, the role of HRP-2 in HIV-1 integration site selection was not addressed. FINDINGS: We studied the HIV-1 integration site distribution in the presence and absence of LEDGF/p75 and/or HRP-2, and in LEDGF/p75-depleted cells that overexpress HRP-2. We show that HRP-2 functions as a co-factor of HIV-1 IN in LEDGF/p75-depleted cells. Endogenous HRP-2 only weakly supported HIV-1 replication in LEDGF/p75 depleted cells. However, HRP-2 overexpression rescued HIV-1 replication and restored integration in RefSeq genes to wild-type levels. Additional HRP-2 KD in LEDGF/p75-depleted cells reduces integration frequency in transcription units and shifts the integration distribution towards random. CONCLUSIONS: We demonstrate that HRP-2 overexpression can compensate for the absence of LEDGF/p75 and indicate that the residual bias in integration targeting observed in the absence of LEDGF/p75 can be ascribed to HRP-2. Knockdown of HRP-2 upon LEDGF/p75 depletion results in a more random HIV-1 integration pattern. These data therefore reinforce the understanding that LEDGF/p75 is the dominant HIV-1 IN co-factor.

High-resolution profiling of the LEDGF/p75 chromatin interaction in the ENCODE region.

Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a transcriptional coactivator involved in stress response, autoimmune disease, cancer and HIV replication. A fusion between the nuclear pore protein NUP98 and LEDGF/p75 has been found in human acute and chronic myeloid leukemia and association of LEDGF/p75 with mixed-lineage leukemia (MLL)/menin is critical for leukemic transformation. During lentiviral replication, LEDGF/p75 tethers the pre-integration complex to the host chromatin resulting in a bias of integration into active transcription units (TUs). The consensus function of LEDGF/p75 is tethering of cargos to chromatin. In this regard, we determined the LEDGF/p75 chromatin binding profile. To this purpose, we used DamID technology and focused on the highly annotated ENCODE (Encyclopedia of DNA Elements) regions. LEDGF/p75 primarily binds downstream of the transcription start site of active TUs in agreement with the enrichment of HIV-1 integration sites at these locations. We show that LEDGF/p75 binding is not restricted to stress response elements in the genome, and correlation analysis with more than 200 genomic features revealed an association with active chromatin markers, such as H3 and H4 acetylation, H3K4 monomethylation and RNA polymerase II binding. Interestingly, some associations did not correlate with HIV-1 integration indicating that not all LEDGF/p75 complexes on the chromosome are amenable to HIV-1 integration.

BET-Independent Murine Leukemia Virus Integration Is Retargeted In Vivo and Selects Distinct Genomic Elements for Lymphomagenesis.

Moloney murine leukemia virus (MLV) infects BALB/c mice and induces T-cell lymphoma in mice. Retroviral integration is mediated by the interaction of the MLV integrase (IN) with members of the bromodomain and extraterminal motif (BET) protein family (BRD2, BRD3, and BRD4). The introduction of the W390A mutation into MLV IN abolishes the BET interaction. Here, we compared the replication of W390A MLV to that of wild-type (WT) MLV in adult BALB/c mice to study the role of BET proteins in replication, integration, and tumorigenesis in vivo. Comparing WT and W390A MLV infections revealed similar viral loads in the blood, thymus, and spleen cells. Interestingly, W390A MLV integration was retargeted away from GC-enriched genomic regions. However, both WT MLV- and W390A MLV-infected mice developed T-cell lymphoma after similar latencies represented by an enlarged thymus and spleen and multiorgan tumor infiltration. Integration site sequencing from splenic tumor cells revealed clonal expansion in all WT MLV- and W390A MLV-infected mice. However, the integration profiles of W390A MLV and WT MLV differed significantly. Integrations were enriched in enhancers and promoters, but compared to the WT, W390A MLV integrated less frequently into enhancers and more frequently into oncogene bodies such as Notch1 and Ppp1r16b. We conclude that host factors direct MLV in vivo integration site selection. Although BET proteins target WT MLV integration preferentially toward enhancers and promoters, insertional lymphomagenesis can occur independently from BET, likely due to the intrinsically strong enhancer/promoter of the MLV long terminal repeat (LTR). IMPORTANCE In this study, we have shown that the in vivo replication of murine leukemia virus happens independently of BET proteins, which are key host determinants involved in retroviral integration site selection. This finding opens a new research line in the discovery of alternative viral or host factors that may complement the dominant host factor. In addition, our results show that BET-independent murine leukemia virus uncouples insertional mutagenesis from gene enhancers, although lymphomagenesis still occurs despite the lack of an interaction with BET proteins. Our findings also have implications for the engineering of BET-independent MLV-based vectors for gene therapy, which may not be a safe alternative.

Transportin-SR2 imports HIV into the nucleus.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) and other lentiviruses have the capacity to infect nondividing cells like macrophages. This requires import of the preintegration complex (PIC) through the nuclear pore. Although many cellular and viral determinants have been proposed, the mechanism leading to nuclear import is not yet understood. RESULTS: Using yeast two-hybrid and pull-down, we identified and validated transportin-SR2 (TRN-SR2) as a bona fide binding partner of HIV-1 integrase. We confirmed the biological relevance of this interaction by RNAi. Depletion of TRN-SR2 interfered with the replication of HIV-1 and HIV-2 but not MoMLV in HeLaP4 cells. Knockdown of TRN-SR2 in primary macrophages likewise interfered with HIV-1 replication. Using Q-PCR, we pinpoint this block in replication to the early steps of the viral lifecycle. A reduction in 2-LTR formation suggests a block in PIC nuclear import upon siRNA-mediated knockdown. Different lines of evidence clearly proved that the late steps of viral replication are not affected. In an in vivo nuclear-import assay using labeled HIV-1 particles, the defect in nuclear import after depletion of TRN-SR2 was directly visualized. In comparison with control cell lines, the great majority of siRNA-treated cells did not contain any PIC in the nucleus. CONCLUSION: Our data clearly demonstrate that TRN-SR2 is the nuclear-import factor of HIV.

Interplay between HIV entry and transportin-SR2 dependency.

BACKGROUND: Transportin-SR2 (TRN-SR2, TNPO3, transportin 3) was previously identified as an interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase and functions as a nuclear import factor of HIV-1. A possible role of capsid in transportin-SR2-mediated nuclear import was recently suggested by the findings that a chimeric HIV virus, carrying the murine leukemia virus (MLV) capsid and matrix proteins, displayed a transportin-SR2 independent phenotype, and that the HIV-1 N74D capsid mutant proved insensitive to transportin-SR2 knockdown. RESULTS: Our present analysis of viral specificity reveals that TRN-SR2 is not used to the same extent by all lentiviruses. The DNA flap does not determine the TRN-SR2 requirement of HIV-1. We corroborate the TRN-SR2 independent phenotype of the chimeric HIV virus carrying the MLV capsid and matrix proteins. We reanalyzed the HIV-1 N74D capsid mutant in cells transiently or stably depleted of transportin-SR2 and confirm that the N74D capsid mutant is independent of TRN-SR2 when pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Remarkably, although somewhat less dependent on TRN-SR2 than wild type virus, the N74D capsid mutant carrying the wild type HIV-1 envelope required TRN-SR2 for efficient replication. By pseudotyping with envelopes that mediate pH-independent viral uptake including HIV-1, measles virus and amphotropic MLV envelopes, we demonstrate that HIV-1 N74D capsid mutant viruses retain partial dependency on TRN-SR2. However, this dependency on TRN-SR2 is lost when the HIV N74D capsid mutant is pseudotyped with envelopes mediating pH-dependent endocytosis, such as the VSV-G and Ebola virus envelopes. CONCLUSION: Here we discover a link between the viral entry of HIV and its interaction with TRN-SR2. Our data confirm the importance of TRN-SR2 in HIV-1 replication and argue for careful interpretation of experiments performed with VSV-G pseudotyped viruses in studies on early steps of HIV replication including the role of capsid therein.