Lens Epithelium-derived Growth Factor Research Articles

Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase

Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors.

The Structure-Specific Recognition Protein 1 Associates with Lens Epithelium-Derived Growth Factor Proteins and Modulates HIV-1 Replication

The lens epithelium-derived growth factor p75 (LEDGF/p75) is a chromatin-bound protein essential for efficient lentiviral integration. Genome-wide studies have located LEDGF/p75 inside actively transcribed genes where it mediates lentiviral integration. Although its role in HIV-1 integration is clearly established, the role of LEDGF/p75-associated proteins in HIV-1 infection remains unexplored. Using protein–protein interaction assays, we demonstrated that LEDGF/p75 complexes with a chromatin-remodeling complex facilitates chromatin transcription (FACT), a heterodimer of the structure-specific recognition protein 1 (SSRP1) and the human homolog of suppressor of Ty 16 (hSpt16). Detailed analysis of the interaction of LEDGF/p75 with the FACT complex indicates that LEDGF/p75 interacts with SSRP1 in an hSpt16-independent manner that requires the PWWP domain of LEDGF proteins and the HMG domain of SSRP1. Functional characterizations demonstrate a LEDGF/p75-independent role of SSRP1 in the regulation of HIV-1 replication. shRNA-mediated partial knockdown of SSRP1 reduces HIV-1 infection, but not Murine Leukemia Virus, in human CD4+ T cells. Similarly, SSRP1 knockdown affects infection by HIV-1-derived viruses that express genes from the viral LTR but not from an internal immediate-early CMV promoter, suggesting a role of SSRP1 in LTR-driven gene expression but not in viral DNA integration. Together, our data demonstrate for the first time the association of LEDGF proteins with the FACT complex and give further support to a role of SSRP1 in HIV-1 infection.

Will LEDGIN molecules be able to play a role in a cure for HIV infection?

Will LEDGIN molecules be able to play a role in a cure for HIV infection?

Correction: Lens Epithelium-Derived Growth Factor Is an Hsp70-2 Regulated Guardian of Lysosomal Stability in Human Cancer.

Correction: Lens Epithelium-Derived Growth Factor Is an Hsp70-2 Regulated Guardian of Lysosomal Stability in Human Cancer.

Abstract B06: Repositioning HIV-based small molecule inhibitors of the stress oncoprotein LEDGF/p75 to overcome prostate cancer resistance to taxane chemotherapy

Abstract Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related deaths in men. In addition, African American (AA) men have a higher incidence of PCa with more aggressive tumors and increased mortality compared to other ethnic groups. The rise in the U.S. aging male population and the health disparities associated with this cancer type, are expected to make PCa a formidable public health and fiscal challenge in the next few decades. PCa mortality is driven by the ability of advanced prostate tumors to metastasize to vital organs and develop resistance against established anti-androgen therapies and chemotherapeutic drugs. Despite recent developments in the treatment of advanced PCa, the high mortality and the racial disparities associated with this disease still persist. For this reason, there is still an urgent medical need for innovative therapeutic approaches to reduce the burden of PCa mortality and its associated disparities. Our laboratory is at the forefront of research on the contribution of the lens epithelium-derived growth factor of 75 kD (LEDGF/p75) to PCa cancer aggressiveness. This protein is a stress response transcription coactivator that is overexpressed in PCa cells and clinical tumors, transcriptionally upregulates stress survival proteins in cancer cells, and promotes resistance to Docetaxel (DTX), the first line chemotherapeutic drug used to treat advanced PCa. Interestingly, LEDGF/p75 also plays a critical role in facilitating the integration of human immunodeficiency virus 1 (HIV-1) into active host chromatin by interacting with the HIV-integrase (HIV-IN). This biological function has made LEDGF/p75 an attractive druggable target, and for this purpose a series of novel and potent small molecule inhibitors (SMI) have been designed and studied to disrupt the interaction between HIV-IN and the C-terminal region of LEDGF/p75, which we previously implicated in its pro-survival activity. We hypothesize that repositioning these SMI for PCa treatment in combination with DTX is an innovative approach to re-sensitize DTX resistant PCa cells to chemotherapy. We observed that PC3 and DU145 cells selected for DTX resistance expressed high levels of LEDGF/p75, and were also selectively resistant to the taxanes Cabazitaxel (CTX) and Paclitaxel (PTX), but not to the classical apoptosis inducer TRAIL. Immunoblotting analysis indicated that LEDGF/p75 is cleaved and inactivated by caspases during TRAIL-induced cell death, whereas the protein remained intact in taxane treated cells. RNA interference-mediated knockdown of LEDGF/p75 in DTX-resistant cell lines sensitized them to taxane therapy. This provided proof of principle that functional inactivation of LEDGF/p75 could restore chemosensitivity. We then screened a panel of over 100 HIV-based candidate LEDGF/p75 SMI for their cytotoxicity against DTX-resistant and -sensitive PCa cells, in the presence and absence of DTX. These experiments yielded a number of inhibitors that re-sensitized the resistant cells to taxane treatment. We are currently expanding these studies to a panel of racially diverse PCa cell lines to determine the efficacy of this approach in multiple PCa cell types. Taken together, our results suggest that LEDGF/p75 is a druggable target that could potentially be functionally inactivated by repositioned HIV-based inhibitors in combination with established chemotherapeutic regimens to circumvent PCa chemoresistance and reduce the mortality disparities associated with the aggressive form of this disease. Citation Format: Leslimar Rios-Colon, Tino Wilson Sanchez, Catherine C. Elix, Ivana Alicea, Anamika Basu, Christina Du Ross, Nouri Neamati, Carlos A. Casiano. Repositioning HIV-based small molecule inhibitors of the stress oncoprotein LEDGF/p75 to overcome prostate cancer resistance to taxane chemotherapy. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B06.

A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin

Integration is vital to retroviral replication and influences the establishment of the latent HIV reservoir. HIV-1 integration favors active genes, which is in part determined by the interaction between integrase and lens epithelium-derived growth factor (LEDGF)/p75. Because gene targeting remains significantly enriched, relative to random in LEDGF/p75 deficient cells, other host factors likely contribute to gene-tropic integration. Nucleoporins 153 and 358, which bind HIV-1 capsid, play comparatively minor roles in integration targeting, but the influence of another capsid binding protein, cleavage and polyadenylation specificity factor 6 (CPSF6), has not been reported. In this study we knocked down or knocked out CPSF6 in parallel or in tandem with LEDGF/p75. CPSF6 knockout changed viral infectivity kinetics, decreased proviral formation, and preferentially decreased integration into transcriptionally active genes, spliced genes, and regions of chromatin enriched in genes and activating histone modifications. LEDGF/p75 depletion by contrast preferentially altered positional integration targeting within gene bodies. Dual factor knockout reduced integration into genes to below the levels observed with either single knockout and revealed that CPSF6 played a more dominant role than LEDGF/p75 in directing integration to euchromatin. CPSF6 complementation rescued HIV-1 integration site distribution in CPSF6 knockout cells, but complementation with a capsid binding mutant of CPSF6 did not. We conclude that integration targeting proceeds via two distinct mechanisms: capsid-CPSF6 binding directs HIV-1 to actively transcribed euchromatin, where the integrase-LEDGF/p75 interaction drives integration into gene bodies.

FRET Reveals an Interplay of the HIV-1 Integrase Subunits during its Journey from the Cytoplasm into the Nucleus

During the early stages of the viral replication cycle, HIV-1 travels from the cytoplasm across the nuclear pore into the nucleus to finally integrate its viral cDNA into the host genome. We recently developed a FRET-based assay well suited for the study of the oligomerization of fluorescent labeled HIV integrase (IN) inside individual viral complexes during the early stages of the replication cycle. Although the exact oligomerization of HIV-1 IN is not known, the oligomeric state of IN is tightly coupled to its function. A dimer is needed for 3' processing reaction, while at least a tetramer is needed for the strand transfer reaction. Moreover, this tetrameric form of IN is stabilized in vitro by the involvement of a human cofactor, the lens epithelium-derived growth factor p75 (LEDGF/p75). We show that IN oligomers inside nuclear complexes differ from those in the cytoplasmic complexes in that they possess a higher FRET signal and that this increased FRET signal in the nucleus is dependent on a direct interaction with LEDGF/p75. Next, we studied the influence of LEDGINs, known to bind the same binding pocket as LEDGF/p75 does. LEDGINs target the LEDGF/p75-IN interaction and allosterically modulates the IN multimerization. We previously showed that this enhanced multimerization already takes place at the level of a single viral particle and now show that this multimeric form is retained in single viral complexes both in the cytoplasm and the nucleus. In conclusion, the FRET-based assay offers an opportunity to study the IN oligomerization at the level of a single viral complex and reveals an interplay between different IN subunits during the viral replication cycle.

Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination.

We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage.

Recent Advances in the Development of Small-Molecular Inhibitors Target HIV Integrase-LEDGF/p75 Interaction.

Lens epithelium-derived growth factor (LEDGF/p75) plays an essential role in the HIV-1 replication. It acts by tethering integrase (IN) into the host cellular chromatin. Due to its significance of the IN-LEDGF/p75 interaction affords a novel therapeutic approach for the design of new classes of antiretroviral agents. To date, many small molecules have been found to be the inhibitors of INLEDGF/ p75 interaction. This review summarizes recent advances in the development of potential structure-based IN-LEDGF/p75 interaction inhibitors. The work will be helpful to shed light on the antiretroviral drug development pipeline in the next future.

Embryonic Lethality Due to Arrested Cardiac Development in Psip1/Hdgfrp2 Double-Deficient Mice.

Hepatoma-derived growth factor (HDGF) related protein 2 (HRP2) and lens epithelium-derived growth factor (LEDGF)/p75 are closely related members of the HRP2 protein family. LEDGF/p75 has been implicated in numerous human pathologies including cancer, autoimmunity, and infectious disease. Knockout of the Psip1 gene, which encodes for LEDGF/p75 and the shorter LEDGF/p52 isoform, was previously shown to cause perinatal lethality in mice. The function of HRP2 was by contrast largely unknown. To learn about the role of HRP2 in development, we knocked out the Hdgfrp2 gene, which encodes for HRP2, in both normal and Psip1 knockout mice. Hdgfrp2 knockout mice developed normally and were fertile. By contrast, the double deficient mice died at approximate embryonic day (E) 13.5. Histological examination revealed ventricular septal defect (VSD) associated with E14.5 double knockout embryos. To investigate the underlying molecular mechanism(s), RNA recovered from ventricular tissue was subjected to RNA-sequencing on the Illumina platform. Bioinformatic analysis revealed several genes and biological pathways that were significantly deregulated by the Psip1 knockout and/or Psip1/Hdgfrp2 double knockout. Among the dozen genes known to encode for LEDGF/p75 binding factors, only the expression of Nova1, which encodes an RNA splicing factor, was significantly deregulated by the knockouts. However the expression of other RNA splicing factors, including the LEDGF/p52-interacting protein ASF/SF2, was not significantly altered, indicating that deregulation of global RNA splicing was not a driving factor in the pathology of the VSD. Tumor growth factor (Tgf) β-signaling, which plays a key role in cardiac morphogenesis during development, was the only pathway significantly deregulated by the double knockout as compared to control and Psip1 knockout samples. We accordingly speculate that deregulated Tgf-β signaling was a contributing factor to the VSD and prenatal lethality of Psip1/Hdgfrp2 double-deficient mice.

Host determinants of HTLV-1 integration site preference

Integration of the provirus into the host genome is an essential step in the establishment of a chronic retroviral infection. Retroviruses do not integrate at random. Each genus of retrovirus preferentially integrates in genomic sites associated with certain genomic features. This preference is thought to result from an interaction of the pre-integration complex with a specific host co-factor. Such an interaction has been well documented between HIV-1 integrase and lens epithelium-derived growth factor (LEDGF) and between MLV and members of the Bromoextraterminal Domain Protein (BET) family. HTLV-1 also has a reproducible preference to integrate near various genomic features; however, the putative host co-factor of HTLV-1 integrase remains unknown. We present evidence that demonstrates that the HTLV-1 integrase co-factor may be the protein phosphatase 2A (PP2A) complex. Two independent protein interaction screens (yeast-2-hybrid and co-immunoprecipitation) both revealed an interaction between HTLV-1 integrase and the PP2A holoenzyme. We used co-culture with HTLV-1 producer cell line MT2 to infect rat cell lines containing an inducible knockdown of PP2A. We found that PP2A-knockdown altered the integration profile of these de novo integration sites. In contrast to infection in PP2A-competent cells, integration sites in PP2A-knockdown cells lacked the characteristic proximity to genomic features associated with transcriptional activity (genes, transcription start sites, CpG islands) and to epigenetic marks associated with active chromatin. We are in the process of investigating how PP2A inhibitors and shRNAs targeting specific regulatory subunits of the PP2A holoenzyme influence integration site selection.

Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif.

Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.

Abstract 5411: Repositioning HIV-based small molecule inhibitors of the stress survival oncoprotein LEDGF/p75 to overcome taxane resistance in prostate cancer

Abstract Prostate cancer (PCa) is the second most commonly diagnosed cancer in men and the leading cause of cancer-related deaths in elderly males. Although early stage PCa is treatable, many patients eventually develop castration-resistant disease (mCRPC), characterized by metastasis and resistance to therapy. Furthermore, PCa also presents a major health disparity problem as evidenced by its disproportionately high incidence and mortality in African American men compared to other racial/ethnic groups. Reducing or eliminating these disparities in PCa mortality will require innovative therapeutic approaches to circumvent chemoresistance and increase patient survival. We have previously demonstrated that lens epithelium-derived growth factor of 75 kD (LEDGF/p75) is a stress survival protein that is overexpressed in PCa cells and clinical tumors, and promotes resistance to Docetaxel (DTX), the gold standard for mCRPC chemotherapy. LEDGF/p75 has also been extensively studied as a target to inhibit HIV-1 integration into host chromatin because of its role as a key cellular cofactor of HIV integrase (IN). A series of novel and potent small molecule inhibitors (SMI) of LEDGF/p75 were previously evaluated for their potential to disrupt its interaction with HIV-IN. We hypothesized that repositioning these SMI to target LEDGF/p75 in the context of PCa could be a promising strategy for overcoming taxane resistance. We observed that PC3 and DU145 mCRPC cells selected for DTX resistance expressed high levels of LEDGF/p75, and were also resistant to the taxanes Cabazitaxel (CTX) and Paclitaxel (PTX). This chemoresistance was selective since LEDGF/p75 did not protect cells against the classical apoptotic inducer TRAIL, which triggers caspase-mediated cleavage and inactivation of LEDGF/p75. RNAi-mediated knockdown of LEDGF/p75 in these cell lines sensitized them to taxane therapy. We then evaluated 130 LEDGF/p75 SMI for their cytotoxicity in DTX-resistant and sensitive PC3 and DU145 cells, in the presence and absence of DTX. These experiments yielded a number of inhibitors that induced cell death directly or re-sensitized resistant cells to taxanes only when combined with these drugs. Our results suggest that LEDGF/p75 is a druggable oncoprotein that could be targeted with repositioned HIV-based SMI to overcome PCa chemoresistance. Citation Format: Leslimar Rios Colon, Catherine Elix, Ivana Alicea, Anamika Basu, Christina Du Ross, Tino Sanchez, Nouri Neamati, Carlos Casiano. Repositioning HIV-based small molecule inhibitors of the stress survival oncoprotein LEDGF/p75 to overcome taxane resistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5411. doi:10.1158/1538-7445.AM2015-5411

Specificity of antinuclear autoantibodies recognizing the dense fine speckled nuclear pattern: Preferential targeting of DFS70/LEDGFp75 over its interacting partner MeCP2

Human antinuclear autoantibodies (ANAs) targeting the dense fine speckled (DFS) nuclear protein DFS70, commonly known as lens epithelium derived growth factor p75 (LEDGFp75), present a clinical puzzle since their significance remains elusive. While their frequencies are low in ANA-positive autoimmune rheumatic diseases, they are relatively elevated in clinical laboratory referrals, diverse inflammatory conditions, and ‘apparently’ healthy individuals. We reported previously that DFS70/LEDGFp75 is an autoantigen in prostate cancer that closely interacts with another 70kD DFS nuclear protein, methyl CpG binding protein 2 (MeCP2). This led us to investigate if anti-DFS sera exclusively target DFS70/LEDGFp75 or also recognize MeCP2. Using several complementary autoantibody detection platforms and cellular/molecular approaches we evaluated 65 human sera producing anti-DFS autoantibodies. Our results show that these antibodies are highly specific for DFS70/LEDGFp75 and do not target MeCP2. Establishing the specificity of anti-DFS autoantibodies has implications for increasing our understanding of their biological significance and clinical utility.

The significance of autoantibodies to DFS70/LEDGFp75 in health and disease: integrating basic science with clinical understanding.

Antinuclear autoantibodies (ANAs) displaying the nuclear dense fine speckled immunofluorescence (DFS-IIF) pattern in HEp-2 substrates are commonly observed in clinical laboratory referrals. They target the dense fine speckled autoantigen of 70 kD (DFS70), most commonly known as lens epithelium-derived growth factor p75 (LEDGFp75). Interesting features of these ANAs include their low frequency in patients with systemic autoimmune rheumatic diseases (SARD), elevated prevalence in apparently healthy individuals, IgG isotype, strong trend to occur as the only ANA specificity in serum, and occurrence in moderate to high titers. These autoantibodies have also been detected at varied frequencies in patients with diverse non-SARD inflammatory and malignant conditions such as atopic diseases, asthma, eye diseases, and prostate cancer. These observations have recently stimulated vigorous research on their clinical and biological significance. Some studies have suggested that they are natural, protective antibodies that could serve as biomarkers to exclude a SARD diagnosis. Other studies suggest that they might be pathogenic in certain contexts. The emerging role of DFS70/LEDGFp75 as a stress protein relevant to human acquired immunodeficiency syndrome, cancer, and inflammation also points to the possibility that these autoantibodies could be sensors of cellular stress and inflammation associated with environmental factors. In this comprehensive review, we integrate our current knowledge of the biology of DFS70/LEDGFp75 with the clinical understanding of its autoantibodies in the contexts of health and disease.

533. A Clinically Relevant Approach for Retargeting Foamy Viral Vectors Into Heterochromatin

Retroviral (RV) vector mediated gene therapy offers immense potential for permanently treating a broad spectrum of genetic conditions and has shown efficacy in clinical trials. However, vector proviruses can dysregulate nearby host genes, referred to as genotoxicity, which can lead to oncogenic transformation. Studies on the integration profiles and the mechanisms responsible for targeting integration have been carried out, resulting in a better understanding of the safety of RV vectors. Integration is influenced by the pre-integration complex (PIC) and its interactions with host proteins. Retargeting RV vectors to safe locations in the genome is an important step in reducing potential genotoxic effects. Although the RV PIC and its interactions are poorly defined, studies have shown that it is possible to retarget RV vector integration. It has been demonstrated that lentiviral vector integration can be retargeted using artificial tethering factors. A modified cell line was produced that expressed lens epithelium–derived growth factor (LEDGF) fused to chromobox protein homolog 1 (CBX1) in place of the wild type LEDGF. The LEDGF-CBX1 fusion protein targeted lentiviral vector integration into methylated regions in the human genome. However, this strategy requires modifying a cell to express a fusion protein and is therefore not practical for clinical use. Other studies have also shown it is possible to retarget RV vector integration, but these studies resulted in reduced transgene expression or impaired vector titers. A clinically relevant method for retargeting integration to safer genomic locations is needed.Here we demonstrate an approach to retarget foamy viral (FV) vector integration into safer heterochromatic regions that does not require a modified cell line. Foamy viruses have a broad tropism, a large packaging capacity, and efficiently transduce hematopoietic stem cells, making them well suited for gene therapy. We describe a FV vector with a modified Pol which expresses an integrase-CBX1 (IN-CBX1) fusion protein and a modified Gag which expresses a mutated chromatin binding site (CBS) domain. CBX1 binds to trimethylated histone H3K9 (H3K9me3), so we hypothesized the IN-CBX1 fusion protein would target FV vector integration into heterochromatin. The integration sites of the modified FV vector (n = 1494) and the control FV vector were compared in normal human fibroblast cells. We analyzed the retargeting ability by comparing the integration sites to normal human fibroblast H3K9me3 ChIP-Seq data. Significant retargeting to H3K9me3 regions was observed with the modified FV vector (P 107 transducing units/ml). This study demonstrates that FV vectors can be efficiently retargeted using foamy integrase fusion proteins. Since this approach does not require engineering the target cells, it is promising for use in the clinic, including hematopoietic stem cell gene therapy.

DFS70/LEDGFp75: An Enigmatic Autoantigen at the Interface between Autoimmunity, AIDS, and Cancer

Clinical and diagnostic laboratories often encounter patient sera containing antinuclear antibodies (ANAs) that produce a nuclear dense fine speckled immunofluorescence pattern on HEp-2 cells. These autoantibodies usually target the dense fine speckled protein of 70 kDa (DFS70), commonly known as lens epithelium-derived growth factor p75 (LEDGFp75). Anti-DFS70/LEDGFp75 autoantibodies have recently attracted much interest because of their relatively common occurrence in sera from patients with positive ANA tests with no clinical evidence of systemic autoimmune rheumatic disease (SARD). Their presence has been documented primarily in patients with diverse non-SARD inflammatory conditions and “apparently healthy” individuals. While there is circumstantial evidence that depending on the context these autoantibodies could play protective, pathogenic, or sensor roles, their significance remains elusive. DFS70/LEDGFp75 has emerged during the past decade as a stress transcription co-activator relevant to HIV integration, cancer, and inflammation. It is not clear, however, what makes this protein the target of such a common autoantibody response. We suggest that a better understanding of DFS70/LEDGFp75 biology is key to elucidating the significance of its associated autoantibodies. Here, we discuss briefly our current understanding of this enigmatic autoantigen and potential scenarios leading to its targeting by the immune system.

SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

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

BackgroundThe 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.ResultsLEDGF/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.ConclusionTogether, 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0134-4) contains supplementary material, which is available to authorized users.

Novel near-infrared BiFC systems from a bacterial phytochrome for imaging protein interactions and drug evaluation under physiological conditions

Monitoring protein–protein interactions (PPIs) in live subjects is critical for understanding these fundamental biological processes. Bimolecular fluorescence complementation (BiFC) provides a good technique for imaging PPIs; however, a BiFC system with a long wavelength remains to be pursued for in vivo imaging. Here, we conducted systematic screening of split reporters from a bacterial phytochrome-based, near-infrared fluorescent protein (iRFP). Several new near-infrared phytochrome BiFC systems were built based on selected split sites including the amino acids residues 97/98, 99/100, 122/123, and 123/124. These new near-infrared BiFC systems from a bacterial phytochrome were verified as powerful tools for imaging PPIs under physiological conditions in live cells and in live mice. The interaction between HIV-1 integrase (IN) and cellular cofactor protein Lens epithelium-derived growth factor (LEDGF/p75) was visualized in live cells using the newly constructed iRFP BiFC system because of its important roles in HIV-1 integration and replication. Because the HIV IN-LEDGF/p75 interaction is an attractive anti-HIV target, drug evaluation assays to inhibit the HIV IN-LEDGF/p75 interaction were also performed using the newly constructed BiFC system. The results showed that compound 6 and carbidopa inhibit the HIV IN-LEDGF/p75 interaction in a dose-dependent manner under physiological conditions in the BiFC assays. This study provides novel near-infrared BiFC systems for imaging protein interactions under physiological conditions and provides guidance for splitting other bacterial phytochrome-like proteins to construct BiFC systems. The study also provides a new method for drug evaluation in live cells based on iRFP BiFC systems and supplies some new information regarding candidate drugs for anti-HIV therapies.