RNA-binding protein CPEB1 remodels host and viral RNA landscapes.

Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.

Tethered Function Assays as Tools to Elucidate the Molecular Roles of RNA-Binding Proteins.

Dynamic regulation of RNA molecules is critical to the survival and development of cells. Messenger RNAs are transcribed in the nucleus as intron-containing pre-mRNAs and bound by RNA-binding proteins, which control their fate by regulating RNA stability, splicing, polyadenylation, translation, and cellular localization. Most RBPs have distinct mRNA-binding and functional domains; thus, the function of an RBP can be studied independently of RNA-binding by artificially recruiting the RBP to a reporter RNA and then measuring the effect of RBP recruitment on reporter splicing, stability, translational efficiency, or intracellular trafficking. These tethered function assays therefore do not require prior knowledge of the RBP's endogenous RNA targets or its binding sites within these RNAs. Here, we provide an overview of the experimental strategy and the strengths and limitations of common tethering systems. We illustrate specific examples of the application of the assay in elucidating the function of various classes of RBPs. We also discuss how classic tethering assay approaches and insights gained from them have been empowered by more recent technological advances, including efficient genome editing and high-throughput RNA-sequencing.

Camelid reporter gene imaging: a generic method for in vivo cell tracking.

BACKGROUND: To combine the sensitivity of bioluminescent imaging (BLI) with the 3D and quantitative properties of pinhole single-photon emission computed tomography (SPECT)/micro-computed tomography (CT) (phSPECT/micro-CT), we generated stable cell lines that express a yellow-fluorescent protein (YFP) and Gaussia luciferase (GLuc) fusion protein (YFP/GLuc). For in vivo phSPECT detection of this YFP/GLuc protein, a nanobody, targeted against yellow and green fluorescent proteins (anti-YFP-Nb), was site specifically labelled with (99m)Tc. METHODS: Human embryonic kidney cells (HEK293T) were cultured and passaged every 3 days. 10E5 cells were transduced with YFP/GLuc-containing vector: both membrane-targeted (MT-YFP/GLuc) and non-targeted (YFP/GLuc) fusion proteins were developed. These vectors were compared against a SKOV-3 cell line stably expressing green fluorescent-firefly luciferase (GFP/Fluc) and HEK293T cells expressing red fluorescent protein in combination with a Gaussia luciferase (Red/GLuc). Transduction efficiencies were scored by fluorescence microscopy, and transduced cells were enriched by fluorescence-activated cell sorting (FACS). GLuc and FLuc functionality was tested in vitro by list-mode BLI. Subsequently, cells were transplanted subcutaneously in athymic (nu/nu) mice (MT-YFP/GLuc: n = 4, YFP/GLuc: n = 6, GFP/FLuc: n = 6, Red/GLuc: n = 4). Labelling efficiency of anti-YFP-Nb was measured using instant thin layer chromatography. One week after transplantation, (99m)Tc-labelled anti-YFP-Nb was injected intravenously and pinhole (ph) SPECT/micro-CT was performed, followed by in vivo BLI. RESULTS: Cells showed high levels of fluorescence after transduction. The cells containing the MT-YFP/GLuc were positive on fluorescence microscopy, with the fluorescent signal confined to the cell membrane. After cell sorting, transduced cells were assayed by BLI and showed a significantly higher light output both in vitro and in vivo compared with non-transduced HEK293T cells. The anti-YFP-Nb labelling efficiency was 98%, and subsequent phSPECT/micro-CT demonstrated visible cell binding and significantly higher transplant-to-muscle ratio for both the MT-YFP/GLuc and YFP/GLuc transplanted cells, compared with the GFP/FLuc and Red/GLuc group. CONCLUSION: This study provides a proof of principle for a nanobody-based cell tracking method, using a YFP/GLuc fusion protein and anti-YFP-Nb in a model of subcutaneously transplanted transduced HEK293T cells.

Inhibition of firefly luciferase by general anesthetics: effect on in vitro and in vivo bioluminescence imaging.

UNLABELLED: Bioluminescence imaging is routinely performed in anesthetized mice. Often isoflurane anesthesia is used because of its ease of use and fast induction/recovery. However, general anesthetics have been described as important inhibitors of the luciferase enzyme reaction. AIM: To investigate frequently used mouse anesthetics for their direct effect on the luciferase reaction, both in vitro and in vivo. MATERIALS AND METHODS: isoflurane, sevoflurane, desflurane, ketamine, xylazine, medetomidine, pentobarbital and avertin were tested in vitro on luciferase-expressing intact cells, and for non-volatile anesthetics on intact cells and cell lysates. In vivo, isoflurane was compared to unanesthetized animals and different anesthetics. Differences in maximal photon emission and time-to-peak photon emission were analyzed. RESULTS: All volatile anesthetics showed a clear inhibitory effect on the luciferase activity of 50% at physiological concentrations. Avertin had a stronger inhibitory effect of 80%. For ketamine and xylazine, increased photon emission was observed in intact cells, but this was not present in cell lysate assays, and was most likely due to cell toxicity and increased cell membrane permeability. In vivo, the highest signal intensities were measured in unanesthetized mice and pentobarbital anesthetized mice, followed by avertin. Isoflurane and ketamine/medetomidine anesthetized mice showed the lowest photon emission (40% of unanesthetized), with significantly longer time-to-peak than unanesthetized, pentobarbital or avertin-anesthetized mice. We conclude that, although strong inhibitory effects of anesthetics are present in vitro, their effect on in vivo BLI quantification is mainly due to their hemodynamic effects on mice and only to a lesser extent due to the direct inhibitory effect.

Large double copy vectors are functional but show a size-dependent decline in transduction efficiency.

The development of SIN-lentivectors has paved the way for the double copy vectors (DCV), which substitute the deletion in the 3'LTR with either transgenic or insulator sequences. However, the limits of this approach remain unclear. Previous results have demonstrated that transduction efficiencies of DCV carrying large insulator inserts in their 3'LTRs were impaired in a size-dependent manner. We wondered if this was also true for promoter-transgene inserts and whether they remained functional upon integration into the genome. Therefore, we designed a series of DCV with increasing 3'LTR sizes containing different promoter-transgene combinations. Transduced cells were scored for both transduction efficiency and insert functionality. We found that the transduction efficiencies indeed were impaired in a size-dependent way. Efficiency with inserts below 1 kb linearly decreased with size, while sizes between 1 and 2 kb showed a further decrease to a minimum of 5% of an original "empty" SIN-vector. However, we did not find an LTR size that completely abolished transduction. Moreover, we demonstrated that all inserts remained functional regardless the promoter-transgene combination used. Therefore, we conclude from our data that DCV indeed remain functional, but transduction efficiencies drop radically when inserts larger than 1 kb are being used.

IGF-1 suppresses Bim expression in multiple myeloma via epigenetic and posttranslational mechanisms.

Insulin-like growth factor-1 (IGF-1) is an important growth and survival factor in multiple myeloma (MM). Here, we demonstrate that IGF-1 induces significant down-regulation of the proapoptotic BH3-only protein Bim in MM cells. Reduced Bim levels by RNA interference (RNAi) protected cells from drug-induced cell death. The IGF-1-mediated down-regulation of Bim was the result of (1) reduced transcription by activation of the Akt pathway and inactivation of the transcription factor FoxO3a, (2) increased proteasome-mediated degradation of the Bim extra-long protein by activation of the mitogen-activated protein kinase pathway, and (3) epigenetic regulation of both the Bim and the FoxO3a promoter. Treatment of cells with the histone deacetylase inhibitor LBH589 resulted in a clear up-regulation in the expression of Bim. Furthermore, the methylation inhibitor 5-aza-2'deoxycytidine (decitabine) significantly increased the effects of LBH589. On IGF-1 treatment, the Bim promoter region was found to be unmethylated, whereas chromatin immunoprecipitation analysis of the IGF-1-treated cells showed both a reduced histone H3 tail Lys9 (H3K9) acetylation and an increased H3K9 dimethylation, which contributed actively to its silencing. These data identify a new mechanism in the IGF-1-dependent survival of MM cells and emphasize the need for IGF-1-targeted drug therapy.

Enhancing the T-cell stimulatory capacity of human dendritic cells by co-electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA.

The effectiveness of the dendritic cell (DC) vaccination protocols that are currently in use could be improved by providing the DCs with a more potent maturation signal. We therefore investigated whether the T-cell stimulatory capacity of human monocyte-derived DCs could be increased by co-electroporation with different combinations of CD40L, CD70, and constitutively active toll-like receptor 4 (caTLR4) encoding mRNA. We show that immature DCs electroporated with CD40L and/or caTLR4 mRNA, but not those electroporated with CD70 mRNA, acquire a mature phenotype along with an enhanced secretion of several cytokines/chemokines. Moreover, these DCs are very potent in inducing naive CD4(+) T cells to differentiate into interferon-gamma (IFN-gamma)-secreting type 1 T helper (Th1) cells. Further, we assessed the capacity of the electroporated DCs to activate naive HLA-A2-restricted MelanA-specific CD8(+) T cells without the addition of any exogenous cytokines. When all three molecules were combined, a >500-fold increase in MelanA-specific CD8(+) T cells was observed when compared with immature DCs, and a >200-fold increase when compared with cytokine cocktail-matured DCs. In correlation, we found a marked increase in cytolytic and IFN-gamma/tumor necrosis factor-alpha (TNF-alpha) secreting CD8(+) T cells. Our data indicate that immature DCs genetically modified to express stimulating molecules can induce tumor antigen-specific T cells in vitro and could prove to be a significant improvement over DCs matured with the methods currently in use.

Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D: -luciferin: effect on intensity, time kinetics and repeatability of photon emission.

INTRODUCTION: In vivo bioluminescence imaging (BLI) is a promising technique for non-invasive tumour imaging. D: -luciferin can be administrated intraperitonealy or intravenously. This will influence its availability and, therefore, the bioluminescent signal. The aim of this study is to compare the repeatability of BLI measurement after IV versus IP administration of D: -luciferin and assess the correlation between photon emission and histological cell count both in vitro and in vivo. MATERIALS AND METHODS: Fluc-positive R1M cells were subcutaneously inoculated in nu/nu mice. Dynamic BLI was performed after IV or IP administration of D: -luciferin. Maximal photon emission (PE(max)) was calculated. For repeatability assessment, every acquisition was repeated after 4 h and analysed using Bland-Altman method. A second group of animals was serially imaged, alternating IV and IP administration up to 21 days. When mice were killed, PE(max) after IV administration was correlated with histological cell number. RESULTS: The coefficients of repeatability were 80.2% (IV) versus 95.0% (IP). Time-to-peak is shorter, and its variance lower for IV (p < 0.0001). PE(max) was 5.6 times higher for IV. A trend was observed towards lower photon emission per cell in larger tumours. CONCLUSION: IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden.