Agata Levay

Influenza virus targets the mRNA export machinery and the nuclear pore complex

The NS1 protein of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 functions to impair innate and adaptive immunity by inhibiting host signal transduction and gene expression, but its mechanisms of action remain to be fully elucidated. We show here that NS1 forms an inhibitory complex with NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5, which are key constituents of the mRNA export machinery that interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex. Increased levels of NXF1, p15, or Rae1 revert the mRNA export blockage induced by NS1. Furthermore, influenza virus down-regulates Nup98, a nucleoporin that is a docking site for mRNA export factors. Reduced expression of these mRNA export factors renders cells highly permissive to influenza virus replication, demonstrating that proper levels of key constituents of the mRNA export machinery protect against influenza virus replication. Because Nup98 and Rae1 are induced by interferons, down-regulation of this pathway is likely a viral strategy to promote viral replication. These findings demonstrate previously undescribed influenza-mediated viral–host interactions and provide insights into potential molecular therapies that may interfere with influenza infection.

Plasmodium Circumsporozoite Protein Promotes the Development of the Liver Stages of the Parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.

Reduced Sox9 Function Promotes Heart Valve Calcification Phenotypes In Vivo

Rationale: Calcification of heart valve structures is the most common form of valvular disease and is characterized by the appearance of bone-like phenotypes within affected structures. Despite the clinical significance, the underlying etiology of disease onset and progression is largely unknown and valve replacement remains the most effective treatment. The SRY-related transcription factor Sox9 is expressed in developing and mature heart valves, and its function is required for expression of cartilage-associated proteins, similar to its role in chondrogenesis. In addition to cartilage-associated defects, mice with reduced sox9 function develop skeletal bone prematurely; however, the ability of sox9 deficiency to promote ectopic osteogenic phenotypes in heart valves has not been examined. Objective: This study aims to determine the role of Sox9 in maintaining connective tissue homeostasis in mature heart valves using in vivo and in vitro approaches. Methods and Results: Using histological and molecular analyses, we report that, from 3 months of age, Sox9fl/+;Col2a1-cre mice develop calcific lesions in heart valve leaflets associated with increased expression of bone-related genes and activation of inflammation and matrix remodeling processes. Consistently, ectopic calcification is also observed following direct knockdown of Sox9 in heart valves in vitro. Furthermore, we show that retinoic acid treatment in mature heart valves is sufficient to promote calcific processes in vitro, which can be attenuated by Sox9 overexpression. Conclusions: This study provides insight into the molecular mechanisms of heart valve calcification and identifies reduced Sox9 function as a potential genetic basis for calcific valvular disease.

Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity

Recent studies have underscored the importance of memory T cells in mediating protective immunity against pathogens and cancer. Pharmacological inhibition of regulators that mediate T cell differentiation promotes the differentiation of activated CD8(+) T cells into memory cells. Nonetheless, pharmacological agents have broad targets and can induce undesirable immunosuppressive effects. Here, we tested the hypothesis that aptamer-targeted siRNA inhibition of mTOR complex 1 (mTORC1) function in CD8(+) T cells can enhance their differentiation into memory T cells and potentiate antitumor immunity more effectively than the pharmacologic inhibitor rapamycin. To specifically target activated cells, we conjugated an siRNA targeting the mTORC1 component raptor to an aptamer that binds 4-1BB, a costimulatory molecule that is expressed on CD8(+) T cells following TCR stimulation. We found that systemic administration of the 4-1BB aptamer-raptor siRNA to mice downregulated mTORC1 activity in the majority of CD8(+) T cells, leading to the generation of a potent memory response that exhibited cytotoxic effector functions and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, while treatment with the general mTORC1 inhibitor rapamycin also enhanced antigen-activated CD8(+) T cell persistence, the cytotoxic effector functions of the reactivated memory cells were reduced and the alloreactivity of DCs was diminished. Consistent with the immunological findings, mice treated with rapamycin, but not with 4-1BB aptamer-raptor siRNA, failed to reject a subsequent tumor challenge.

Scleraxis Is Required for Cell Lineage Differentiation and Extracellular Matrix Remodeling During Murine Heart Valve Formation In Vivo

Heart valve structures, derived from mesenchyme precursor cells, are composed of differentiated cell types and extracellular matrix arranged to facilitate valve function. Scleraxis (scx) is a transcription factor required for tendon cell differentiation and matrix organization. This study identified high levels of scx expression in remodeling heart valve structures at embryonic day 15.5 through postnatal stages using scx-GFP reporter mice and determined the in vivo function using mice null for scx. Scx−/− mice display significantly thickened heart valve structures from embryonic day 17.5, and valves from mutant mice show alterations in valve precursor cell differentiation and matrix organization. This is indicated by decreased expression of the tendon-related collagen type XIV, increased expression of cartilage-associated genes including sox9, as well as persistent expression of mesenchyme cell markers including msx1 and snai1. In addition, ultrastructure analysis reveals disarray of extracellular matrix and collagen fiber organization within the valve leaflet. Thickened valve structures and increased expression of matrix remodeling genes characteristic of human heart valve disease are observed in juvenile scx−/− mice. In addition, excessive collagen deposition in annular structures within the atrioventricular junction is observed. Collectively, our studies have identified an in vivo requirement for scx during valvulogenesis and demonstrate its role in cell lineage differentiation and matrix distribution in remodeling valve structures.

Sec13 Shuttles between the Nucleus and the Cytoplasm and Stably Interacts with Nup96 at the Nuclear Pore Complex

ABSTRACT Sec13 is a constituent of the endoplasmic reticulum and the nuclear pore complex (NPC). At the endoplasmic reticulum, Sec13 is involved in the biogenesis of COPII-coated vesicles, whereas at the NPC its function is unknown. We show here, by yeast two-hybrid screenings and biochemical assays, that a region at the amino terminus of the human nuclear pore complex protein Nup96 interacts with the WD (Trp-Asp) repeat region of human Sec13. By using immunofluorescence and confocal and immunoelectron microscopy, we found that in interphase, Sec13 and Nup96 are localized at both sides of the NPC in addition to other intracellular sites. In mitosis, Sec13 was found dispersed throughout the cell, whereas a pool of Nup96 colocalized with the spindle apparatus. Photobleaching experiments showed that Sec13 shuttles between intranuclear sites and the cytoplasm, and a fraction of Sec13 is stably associated with NPCs. Cotransfection of Sec13 and the Sec13 binding site of Nup96 decreased the mobile pool of Sec13, demonstrating the interaction of Sec13 and Nup96 in vivo. Targeting studies showed that Sec13 is actively transported into the nucleus and contains a nuclear localization signal. These results indicate that Sec13 stably interacts with Nup96 at the NPC during interphase and that the shuttling of Sec13 between the nucleus and the cytoplasm may couple and regulate functions between these two compartments.

Targeting 4-1BB Costimulation to the Tumor Stroma with Bispecific Aptamer Conjugates Enhances the Therapeutic Index of Tumor Immunotherapy

Schrand and colleagues report the efficacy in five murine tumor models of an immunotherapeutic approach whereby systemic administration of tumor stroma-targeted 4-1BB aptamer conjugates, which target disseminated tumor lesions, elicits potent antitumor immunity with minimal dose-limiting toxicity. Despite the recent successes of using immune modulatory Abs in patients with cancer, autoimmune pathologies resulting from the activation of self-reactive T cells preclude the dose escalations necessary to fully exploit their therapeutic potential. To reduce the observed and expected toxicities associated with immune modulation, here we describe a clinically feasible and broadly applicable approach to limit immune costimulation to the disseminated tumor lesions of the patient, whereby an agonistic 4-1BB oligonucleotide aptamer is targeted to the tumor stroma by conjugation to an aptamer that binds to a broadly expressed stromal product, VEGF. This approach was predicated on the premise that by targeting the costimulatory ligands to products secreted into the tumor stroma, the T cells will be costimulated before their engagement of the MHC–peptide complex on the tumor cell, thereby obviating the need to target the costimulatory ligands to noninternalizing cell surface products expressed on the tumor cells. Underscoring the potency of stroma-targeted costimulation and the broad spectrum of tumors secreting VEGF, in preclinical murine tumor models, systemic administration of the VEGF-targeted 4-1BB aptamer conjugates engendered potent antitumor immunity against multiple unrelated tumors in subcutaneous, postsurgical lung metastasis, methylcholantrene-induced fibrosarcoma, and oncogene-induced autochthonous glioma models, and exhibited a superior therapeutic index compared with nontargeted administration of an agonistic 4-1BB Ab or 4-1BB aptamer. Cancer Immunol Res; 2(9); 867–77. ©2014 AACR.

Collagen XIV is important for growth and structural integrity of the myocardium.

Collagen XIV is a fibril-associated collagen with an interrupted triple helix (FACIT). Previous studies have shown that this collagen type regulates early stages of fibrillogenesis in connective tissues of high mechanical demand. Mice null for Collagen XIV are viable, however formation of the interstitial collagen network is defective in tendons and skin leading to reduced biomechanical function. The assembly of a tightly regulated collagen network is also required in the heart, not only for structural support but also for controlling cellular processes. Collagen XIV is highly expressed in the embryonic heart, notably within the cardiac interstitium of the developing myocardium, however its role has not been elucidated. To test this, we examined cardiac phenotypes in embryonic and adult mice devoid of Collagen XIV. From as early as E11.5, Col14a1(-/-) mice exhibit significant perturbations in mRNA levels of many other collagen types and remodeling enzymes (MMPs, TIMPs) within the ventricular myocardium. By post natal stages, collagen fibril organization is in disarray and the adult heart displays defects in ventricular morphogenesis. In addition to the extracellular matrix, Col14a1(-/-) mice exhibit increased cardiomyocyte proliferation at post natal, but not E11.5 stages, leading to increased cell number, yet cell size is decreased by 3 months of age. In contrast to myocytes, the number of cardiac fibroblasts is reduced after birth associated with increased apoptosis. As a result of these molecular and cellular changes during embryonic development and post natal maturation, cardiac function is diminished in Col14a1(-/-) mice from 3 months of age; associated with dilation in the absence of hypertrophy, and reduced ejection fraction. Further, Col14a1 deficiency leads to a greater increase in left ventricular wall thickening in response to pathological pressure overload compared to wild type animals. Collectively, these studies identify a new role for type XIV collagen in the formation of the cardiac interstitium during embryonic development, and highlight the importance of the collagen network for myocardial cell survival, and function of the working myocardium after birth.

Mmp15 is a direct target of Snai1 during endothelial to mesenchymal transformation and endocardial cushion development

Cardiac valves originate from endocardial cushions (EC) formed by endothelial-to-mesenchymal transformation (EMT) during embryogenesis. The zinc-finger transcription factor Snai1 has previously been reported to be important for EMT during organogenesis, yet its role in early valve development has not been directly examined. In this study we show that Snai1 is highly expressed in endothelial, and newly transformed mesenchyme cells during EC development. Mice with targeted snai1 knockdown display hypocellular ECs at E10.5 associated with decreased expression of mesenchyme cell markers and downregulation of the matrix metalloproteinase (mmp) family member, mmp15. Snai1 overexpression studies in atrioventricular canal collagen I gel explants indicate that Snai1 is sufficient to promote mmp15 expression, cell transformation, and mesenchymal cell migration and invasion. However, treatment with the catalytically active form of MMP15 promotes cell motility, and not transformation. Further, we show that Snai1-mediated cell migration requires MMP activity, and caMMP15 treatment rescues attenuated migration defects observed in murine ECs following snai1 knockdown. Together, findings from this study reveal previously unappreciated mechanisms of Snai1 for the direct regulation of MMPs during EC development.

Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.