17 research outputs found
Validation and Characterization of co-factors of HIV integration
Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). Since the discovery of HIV in 1981 more than 30 million people have died of AIDS. An estimated 36.7 million people are living with HIV worldwide and approximate 2.1 million people were newly infected in 2015.
Current therapy is a combination of anti-viral drugs targeting different steps of the virus’ lifecycle; entry into the target cell, reverse transcription of viral RNA, integration of the proviral DNA, and proteolytic cleavage of viral proteins during maturation of viral particles. Combination therapy is able to suppress viral replication below the limit of detection. However, the continuing emergence of resistant viral strains urges continuous development of new antiretroviral drugs and warrants the search for new targets to impair the viral life cycle.
Because of its limited genetic make-up, HIV‑1 relies heavily on the cellular machinery to complete its life cycle. In all steps of the HIV‑1 life cycle, cellular proteins assist in its replication. Identification and validation of these virus-host interactions will lead to identification of new antiviral targets. These new drugs can be target against the viral proteins but also to cellular proteins. A disadvantage of targeting cellular proteins could be potential cellular toxicity. We believe that a systematic search for viral cofactors and the insight in their cellular and viral function will shed light on the mechanistic differences between retroviruses and will also lead to the identification of new antiviral targets.
Our research group is especially interested in cellular cofactors of HIV Integrase and their cellular function. An example of an IN cofactor is the lens epithelium-derived growth factor/p75 (LEDGF/p75). LEDGF/p75 was identified as a binding partner of IN in 2003 and since then it has been independently validated by several research groups as an essential cofactor of HIV and a possible new target for antiretroviral drugs. Antiviral compounds targeting the interaction between LEDGF/p75 and IN have since been described extensively and strengthens the approach of targeting host protein-viral protein interactions as antiviral therapy. In the general introduction of this thesis cofactors of HIV IN are described with special attention for LEDGF/p75 and TRN-SR2. The development of small molecule protein-protein interaction inhibitors against the LEDGF/p75-IN interaction are discussed.
Understanding the mechanism of action of LEDGF/p75 in the cell requires knowledge of its interaction partners in the living cell. To date only a few cellular interacting partners of LEDGF/p75 have been identified. These include the PC4 transcription factor, menin/MLL, the Cdc7/ASK, PogZ, the myc interacting protein JPO2 and IWS1. Studying the role of those interactions and the search for new interacting partners will improve our knowledge of the cellular properties of LEDGF/p75 and will aid us in designing better small molecules targeting the LEDGF/p75 HIV‑1 IN interaction. In previous work from our group we have shown that LEDGF/p75 moves in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. After interaction with HIV‑1 integrase, kinetics of LEDGF/p75 shift to 75-fold higher affinity for chromatin (chromatin stalling). In chapter three of this thesis we investigated whether chromatin stalling is a common feature of the LEDGF/p75 chromatin interaction. In particular we studied the interaction of LEDGF/p75 with its cellular binding partner JPO2 and the effect of JPO2 on the LEDGF/p75 chromatin interaction. We employed continuous photobleaching, fluorescence correlation and cross-correlation spectroscopy to investigate in vivo chromatin binding of JPO2. In the absence of LEDGF/p75, JPO2 performs chromatin scanning inherent to transcription factors. However, while 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 (SID) of JPO2 and the C-terminal domain of LEDGF/p75 comprising the integrase binding domain (IBD), 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.
In chapter four of this thesis we investigated MeCP2 (methyl-CpG binding protein 2), a new cellular interaction partner of LEDGF/p75. The interaction between MeCP2 and LEDGF/p75 was characterised and its influence on the transcriptional activity of LEDGF/p75 was investigated. LEDGF/p75 is a transcription co-activator that promotes resistance to cell death induced by increased oxidative stress and chemotherapy and has been implicated in human cancer, inflammatory and autoimmune conditions. To gain insights into the mechanisms by which LEDGF/p75 protect cancer cells against environmental stress, we initiated an analysis of its interactions with other transcription factors and the influence of these interactions on the activation of stress genes. We report in chapter four that both LEDGF/p75 and its short splice variant LEDGF/p52 interact with MeCP2. These interactions were established by several complementary approaches: transcription factor protein arrays, pull down and AlphaScreen assays, co-immunoprecipitation, and nuclear co-localization by confocal microscopy. MeCP2 was found to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1 domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). Co-expression of LEDGF/p75 and MeCP2 significantly enhanced MeCP2-induced Hsp27pr transactivation in PC3 prostate cancer cells but not in U2OS bone cells. However, knockdown of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediated Hsp27pr transactivation, suggesting that LEDGF/p75 may regulate MeCP2 transcriptional activity. Interestingly, LEDGF/p52 repressed MeCP2-induced Hsp27pr activity. Our results suggest that the LEDGF/p75-MeCP2 interaction differentially influences Hsp27pr activation depending on the cell context.
Next to characterization of its cellular function of known integrase cofactors we continue the search for new cofactors in chapter 5. To go from hit identification to target validation two major milestones have to be reached. First, the interaction between HIV‑1 integrase and the specific cofactors has to be confirmed and characterised. Secondly, the cofactor should be important for viral replication and its function in viral replication needs to be determined. Before the start of this thesis new potential HIV‑1 integrase cofactors had been identified by a large scale co-IP of a Flag-tagged integrase stably expressed in 293T cells from a synthetic gene followed by identification of the binding partners by mass spectrometry. Next to HIV‑1 integrase we also analysed MLV and rous sarcoma virus integrases, which allowed comparison of the interactome of different viral integrases. Hits were cross-referenced to a genome wide siRNA screen for HIV and MLV single round replication, which enabled us to bring the list down to 6 potential cofactors for HIV IN. We employed single round HIV replication on cells with the specific cofactor knocked down which resulted in 50% reduction of transduction efficiency for PC2. This effect was also shown for multiple round infection without affecting cell growth. Although viral replication seemed to be hampered predominantly at the early steps of viral replication the effect of PC2 KD on the late steps of viral replication could be underestimated as a 50% increase in LTR-driven transcription was observed. Taken together our results qualify PC2 as a co-factor of HIV‑1 integration and therefore warrant further research. Its mode of action was shown to take place after RT, but before nuclear import.
PC2 not only interacted with HIV-1 IN but also with other retroviral integrases like MLV and RSV. Furthermore, endogenous PC2 colocalized with MLV-IN in the cytoplasm of HeLaP4 cells. This indicates that PC2 is not specific to HIV-1 IN but probably has a more general function in the lifecycle of retroviruses after RT and before import into the nucleus. Our results indicate that PC2 affects multiple stages of HIV replication. Future work will determine how important PC2 is for viral replication in CD4+ T‑cells and the interaction between PC2 and the virus will reveal whether this protein qualifies as a new antiviral target.status: publishe
Lentiviral integration and the role of the cellular cofactors LEDGF/p75 and transportin-SR2
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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.status: publishe
Modulation of amphetamine-induced dopamine release by group II metabotropic glutamate receptor agonist LY354740 in non-human primates studied with positron emission tomography
Pharmacological evidence suggests that schizophrenia is associated with increased stimulation of dopamine (DA) D2 receptors. Recently, several groups have demonstrated that amphetamine-induced DA release is increased in schizophrenia, providing direct evidence for dysregulation of DA systems in this condition. In healthy volunteers, pretreatment with the noncompetitive N-methyl-D-aspartate (NMDA) antagonist ketamine increases amphetamine-induced DA release to levels similar to those observed in patients with schizophrenia. Therefore, the dysregulation of DA function observed in schizophrenia might be secondary to NMDA hypofunction. In this study, the regulation of this response by glutamate (GLU) transmission was further characterized by using a metabotropic glutamate (mGlu) receptor group II agonist to inhibit GLU transmission. The amphetamine- (0.5 mg/kg intravenously (i.v.)) induced decrease in [11C]raclopride equilibrium-specific binding (V3″) was measured under control conditions and following pretreatment with the mGlu2/3 receptor agonist LY354740 (20 mg/kg i.v.) in four baboons. Amphetamine reduced [11C]raclopride V3″ by 28 ± 7% under control conditions. Following LY354740 pretreatment, amphetamine-induced reduction in [11C]raclopride V3″ was significantly enhanced (35 ± 7%, p = 0.002). The enhancement of the amphetamine-induced reduction in [11C]raclopride V3″ by LY354740 was not a simple additive effect, as LY354740 alone did not reduce [11C]raclopride V3″. In conclusion, the results of this study further document the involvement of GLU transmission in regulating the effect of amphetamine-induced DA release, and provide additional support to the hypothesis that the dysregulation of DA function revealed by the amphetamine challenge in schizophrenia might stem from a deficit in GLU transmission
Correction: Y-box-binding protein 1 supports the early and late steps of HIV replication.
[This corrects the article DOI: 10.1371/journal.pone.0200080.].status: Published onlin
Y-box-binding protein 1 supports the early and late steps of HIV replication
The human immunodeficiency virus (HIV) depends on cellular proteins, so-called cofactors, to complete its replication cycle. In search for new therapeutic targets we identified the DNA and RNA binding protein Y-box-binding Protein 1 (YB-1) as a cofactor supporting early and late steps of HIV replication. YB-1 depletion resulted in a 10-fold decrease in HIV-1 replication in different cell lines. Dissection of the replication defects revealed that knockdown of YB-1 is associated with a 2- to 5-fold decrease in virion production due to interference with the viral RNA metabolism. Using single-round virus infection experiments we demonstrated that early HIV-1 replication also depends on the cellular YB-1 levels. More precisely, using quantitative PCR and an in vivo nuclear import assay with fluorescently labeled viral particles, we showed that YB-1 knockdown leads to a block between reverse transcription and nuclear import of HIV-1. Interaction studies revealed that YB-1 associates with integrase, although a direct interaction with HIV integrase could not be unambiguously proven. In conclusion, our results indicate that YB-1 affects multiple stages of HIV replication. Future research on the interaction between YB-1 and the virus will reveal whether this protein qualifies as a new antiviral target.status: publishe
The stress oncoprotein LEDGF/p75 interacts with the methyl CpG binding protein MeCP2 and influences its transcriptional activity
The lens epithelium derived growth factor p75 (LEDGF/p75) is a transcription co-activator that promotes resistance to oxidative stress- and chemotherapy-induced cell death. LEDGF/p75 is also known as the dense fine speckles autoantigen of 70 kD (DFS70), and has been implicated in cancer, HIV-AIDS, autoimmunity, and inflammation. To gain insights into mechanisms by which LEDGF/p75 protects cancer cells against stress, we initiated an analysis of its interactions with other transcription factors and the influence of these interactions on stress gene activation. We report here that both LEDGF/p75 and its short splice variant LEDGF/p52 interact with MeCP2, a methylation-associated transcriptional modulator, in vitro and in various human cancer cells. These interactions were established by several complementary approaches: transcription factor protein arrays, pull down and AlphaScreen® assays, co-immunoprecipitation, and nuclear co-localization by confocal microscopy. MeCP2 was found to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1 domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). LEDGF/p75 modestly enhanced MeCP2-induced Hsp27pr transactivation in U2OS cells, while this effect was more pronounced in PC3 cells. LEDGF/p52 repressed Hsp27pr activity in U2OS cells. Interestingly, siRNA-induced silencing of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediated Hsp27pr transactivation, whereas this effect was less pronounced in PC3 cells depleted of LEDGF/p75. These results suggest that the LEDGF/p75-MeCP2 interaction differentially influences Hsp27pr activation depending on the cellular and molecular context. These findings are of significance in understanding the contribution of this interaction to the activation of stress survival genes.status: publishe
Y-box-binding protein 1 supports the early and late steps of HIV replication
<div><p>The human immunodeficiency virus (HIV) depends on cellular proteins, so-called cofactors, to complete its replication cycle. In search for new therapeutic targets we identified the DNA and RNA binding protein Y-box-binding Protein 1 (YB-1) as a cofactor supporting early and late steps of HIV replication. YB-1 depletion resulted in a 10-fold decrease in HIV-1 replication in different cell lines. Dissection of the replication defects revealed that knockdown of YB-1 is associated with a 2- to 5-fold decrease in virion production due to interference with the viral RNA metabolism. Using single-round virus infection experiments we demonstrated that early HIV-1 replication also depends on the cellular YB-1 levels. More precisely, using quantitative PCR and an <i>in vivo</i> nuclear import assay with fluorescently labeled viral particles, we showed that YB-1 knockdown leads to a block between reverse transcription and nuclear import of HIV-1. Interaction studies revealed that YB-1 associates with integrase, although a direct interaction with HIV integrase could not be unambiguously proven. In conclusion, our results indicate that YB-1 affects multiple stages of HIV replication. Future research on the interaction between YB-1 and the virus will reveal whether this protein qualifies as a new antiviral target.</p></div