LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro

Transcriptional co-activator LEDGF/p75 is the major cellular interactor of HIV-1 integrase (IN), critical to efficient viral replication. In this work, a series of INs from the Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Spumavirus and Lentivirus retroviral genera were tested for interaction with the host factor. None of the non-lentiviral INs possessed detectable affinity for LEDGF in either pull-down or yeast two-hybrid assays. In contrast, all lentiviral INs examined, including those from bovine immunodeficiency virus (BIV), maedi-visna virus (MVV) and equine infectious anemia virus (EIAV) readily interacted with LEDGF. Mutation of Asp-366 to Asn in LEDGF ablated the interaction, suggesting a common mechanism of the host factor recognition by the INs. LEDGF potently stimulated strand transfer activity of divergent lentiviral INs in vitro. Unprecedentedly, in the presence of the host factor, EIAV IN almost exclusively catalyzed concerted integration, whereas HIV-1 IN promoted predominantly half-site integration, and BIV IN was equally active in both types of strand transfer. Concerted BIV and EIAV integration resulted in 5 bp duplications of the target DNA sequences. These results confirm that the interaction with LEDGF is conserved within and limited to Lentivirus and strongly argue that the host factor is intimately involved in the catalysis of lentiviral DNA integration

Structure-based prediction of insertion-site preferences of transposons into chromosomes

Mobile genetic elements with the ability to integrate genetic information into chromosomes can cause disease over short periods of time and shape genomes over eons. These elements can be used for functional genomics, gene transfer and human gene therapy. However, their integration-site preferences, which are critically important for these uses, are poorly understood. We analyzed the insertion sites of several transposons and retroviruses to detect patterns of integration that might be useful for prediction of preferred integration sites. Initially we found that a mathematical description of DNA-deformability, called V(step), could be used to distinguish preferential integration sites for Sleeping Beauty (SB) transposons into a particular 100 bp region of a plasmid [G. Liu, A. M. Geurts, K. Yae, A. R. Srinivassan, S. C. Fahrenkrug, D. A. Largaespada,J. Takeda, K. Horie, W. K. Olson and P. B. Hackett (2005) J. Mol. Biol., 346, 161–173 ]. Based on these findings, we extended our examination of integration of SB transposons into whole plasmids and chromosomal DNA. To accommodate sequences up to 3 Mb for these analyses, we developed an automated method, ProTIS(©), that can generate profiles of predicted integration events. However, a similar approach did not reveal any structural pattern of DNA that could be used to predict favored integration sites for other transposons as well as retroviruses and lentiviruses due to a limitation of available data sets. Nonetheless, ProTIS(©) has the utility for predicting likely SB transposon integration sites in investigator-selected regions of genomes and our general strategy may be useful for other mobile elements once a sufficiently high density of sites in a single region are obtained. ProTIS analysis can be useful for functional genomic, gene transfer and human gene therapy applications using the SB system

Comparison of metal-dependent catalysis by HIV-1 and ASV integrase proteins using a new and rapid, moderate throughput assay for joining activity in solution

<p>Abstract</p> <p>Background</p> <p>HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag.</p> <p>Results</p> <p>A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the Mg⁺⁺- and Mn⁺⁺-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV-1 IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn⁺⁺than Mg⁺⁺. Although the pH optima for these two enzymes are similar with Mn⁺⁺, they differ significantly in the presence of Mg⁺⁺, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn⁺⁺.</p> <p>Conclusion</p> <p>A simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10–30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV-1 and ASV IN proteins.</p

Transcriptional provirus silencing as a crosstalk of de novo DNA methylation and epigenomic features at the integration site

The autonomous transcription of integrated retroviruses strongly depends on genetic and epigenetic effects of the chromatin at the site of integration. These effects are mostly suppressive and proviral activity can be finally silenced by mechanisms, such as DNA methylation and histone modifications. To address the role of the integration site at the whole-genome-scale, we performed clonal analysis of provirus silencing with an avian leucosis/sarcoma virus-based reporter vector and correlated the transcriptional silencing with the epigenomic landscape of respective integrations. We demonstrate efficient provirus silencing in human HCT116 cell line, which is strongly but not absolutely dependent on the de novo DNA methyltransferase activity, particularly of Dnmt3b. Proviruses integrated close to the transcription start sites of active genes into the regions enriched in H3K4 trimethylation display long-term stability of expression and are resistant to the transcriptional silencing after over-expression of Dnmt3a or Dnmt3b. In contrast, proviruses in the intergenic regions tend to spontaneous transcriptional silencing even in Dnmt3a−/− Dnmt3b−/− cells. The silencing of proviruses within genes is accompanied with DNA methylation of long terminal repeats, whereas silencing in intergenic regions is DNA methylation-independent. These findings indicate that the epigenomic features of integration sites are crucial for their permissivity to the proviral expression

Modeling Insertional Mutagenesis Using Gene Length and Expression in Murine Embryonic Stem Cells

Background. High-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs) is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of genetrapping events have not been tested on a genome-wide scale. Methodology/Principal Findings. In this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identifie

Deciphering the Code for Retroviral Integration Target Site Selection

Upon cell invasion, retroviruses generate a DNA copy of their RNA genome and integrate retroviral cDNA within host chromosomal DNA. Integration occurs throughout the host cell genome, but target site selection is not random. Each subgroup of retrovirus is distinguished from the others by attraction to particular features on chromosomes. Despite extensive efforts to identify host factors that interact with retrovirion components or chromosome features predictive of integration, little is known about how integration sites are selected. We attempted to identify markers predictive of retroviral integration by exploiting Precision-Recall methods for extracting information from highly skewed datasets to derive robust and discriminating measures of association. ChIPSeq datasets for more than 60 factors were compared with 14 retroviral integration datasets. When compared with MLV, PERV or XMRV integration sites, strong association was observed with STAT1, acetylation of H3 and H4 at several positions, and methylation of H2AZ, H3K4, and K9. By combining peaks from ChIPSeq datasets, a supermarker was identified that localized within 2 kB of 75% of MLV proviruses and detected differences in integration preferences among different cell types. The supermarker predicted the likelihood of integration within specific chromosomal regions in a cell-type specific manner, yielding probabilities for integration into proto-oncogene LMO2 identical to experimentally determined values. The supermarker thus identifies chromosomal features highly favored for retroviral integration, provides clues to the mechanism by which retrovirus integration sites are selected, and offers a tool for predicting cell-type specific proto-oncogene activation by retroviruses

Etiology of pulmonary hypertension in multiple myeloma: A case series and literature review

BackgroundMultiple myeloma is often complicated by pulmonary hypertension through a variety of mechanisms. These mechanisms include pulmonary hypertension (PH) due to concomitant cardiac amyloid, high output heart failure due to anemia or lytic bone lesions, chronic thromboembolic pulmonary hypertension (CTEPH), toxicity from medications to treat multiple myeloma, and congestive heart failure. This case series highlights the various mechanisms through which multiple myeloma patients develop pulmonary hypertension.ObjectivesTo identify the etiologies of pulmonary hypertension and their management among multiple myeloma patients treated at University of California San Diego.MethodsA retrospective chart review was performed to identify patients with multiple myeloma and pulmonary hypertension who were evaluated at the University of California San Diego between July 2013 and July 2021. Patients also required a right heart catheterization to be included. Demographics, comorbidities, clinical course, and etiology of pulmonary hypertension were obtained from chart review.ResultsThere were 11 patients included. Of the 11 patients described, two had PH due to cardiac amyloid, one had PH due to high output heart failure, one had PH due to CTEPH, two had pulmonary arterial hypertension due to medications (carfilzomib), and five had PH due to congestive heart failure. The right heart catheterization and echocardiogram findings of the various mechanisms of PH in multiple myeloma are described.ConclusionsPulmonary hypertension in multiple myeloma is a common finding that necessitates further evaluation. The initial evaluation should include an echocardiogram and thorough medication review. Further diagnostic testing should be guided by the patient's history and can include right heart catheterization, cardiac biopsy, ventilation-perfusion scan, and bone scan

Molecular mechanisms of anthracycline cardiovascular toxicity

Anthracyclines are effective chemotherapeutic agents, commonly used in the treatment of a variety of hematologic malignancies and solid tumors. However, their use is associated with a significant risk of cardiovascular toxicities and may result in cardiomyopathy and heart failure. Cardiomyocyte toxicity occurs via multiple molecular mechanisms, including topoisomerase II-mediated DNA double-strand breaks and reactive oxygen species (ROS) formation via effects on the mitochondrial electron transport chain, NADPH oxidases (NOXs), and nitric oxide synthases (NOSs). Excess ROS may cause mitochondrial dysfunction, endoplasmic reticulum stress, calcium release, and DNA damage, which may result in cardiomyocyte dysfunction or cell death. These pathophysiologic mechanisms cause tissue-level manifestations, including characteristic histopathologic changes (myocyte vacuolization, myofibrillar loss, and cell death), atrophy and fibrosis, and organ-level manifestations including cardiac contractile dysfunction and vascular dysfunction. In addition, these mechanisms are relevant to current and emerging strategies to diagnose, prevent, and treat anthracycline-induced cardiomyopathy. This review details the established and emerging data regarding the molecular mechanisms of anthracycline-induced cardiovascular toxicity