21 research outputs found

    A novel interaction between dengue virus nonstructural protein 1 and the NS4A-2K-4B precursor is required for viral RNA replication but not for formation of the membranous replication organelle

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    <div><p>Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the <i>β-ladder</i> domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 “<i>Wing</i>” domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 –NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a <i>trans</i>-complementation assay, we show that both <i>cis</i> and <i>trans</i> acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication.</p></div

    Thogoto virus ML protein inhibits IFN-α/β production by interacting with TFIIB.

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    <p>A) Volcano plot of proteins enriched in ML vs. M pulldown in HEK293 cells and identified by AP-LC-MS/MS. HA-tagged M or ML proteins were overexpressed in 4 biological replicates. B) Schematic representation of ML protein and its mutants not binding TFIIB or CAPN15. C) IP of GST-tagged M or ML (wt and mut) and co-IP of FLAG-tagged CAPN15 and TFIIB transiently overexpressed in HEK293 cells. Western blot is a representative of two independent experiments with similar results. D) IFN-α/β levels after infection with THOV wt, ΔML or mutant ML(SW) 24 h.p.i. SN from infected HeLa cells were applied to 293T Mx1-luc. *—p value < 0.05, NS–non-significant. Bar graph shows mean with SD of three technical replicates and is a representative of four independent experiments with similar results. Significance was estimated with Kruskal-Wallis test with Dunn’s multiple comparison post-test.</p

    TFIIB depletion preferentially affects a subset of inflammatory cytokines and antiviral effector genes.

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    <p>A) Viability of HeLa cells after TFIIB knockdown at indicated time points. HeLa cells were treated with indicated siRNAs for 24, 48 and 72 hours and knockdown was validated by Western blot analysis. Cell viability was assessed by MTT assay. The bar graph shows mean and SD of three technical replicates and is a representative of two independent experiments with similar results. B) 2D-scatter plot of transcriptome analysis of HeLa cells before and after TFIIB knockdown mock-treated or stimulated with TNF-α in three biological replicates. HeLa cells were electroporated with Scrambled or TFIIB-targeting siRNAs. In 24 hours they were left untreated or stimulated with TNF-α (20 ng/ml) for 2 hours. Total RNA was extracted and analysed by RNA-seq. X axis shows log2 fold changes between mock-treated and TNF-α-treated cells with non-targeting siRNA (green dots), Y axis represents log2 fold changes between TNF-α-treated Scrambled-transfected cells and TNF-treated siTFIIB-transfected cells (blue dots). Genes upregulated by TNF-α in non-targeted cells and downregulated by TFIIB knockdown are shown in red. Genes upregulated by TNF-α in non-targeted cells and not regulated by TFIIB knockdown are shown in black. Numbers are shown in the scheme. Differentially regulated genes were showing at least 2-fold change with a q value < 0.05. C) qPCR analysis of genes regulated by TNF-α stimulation and affected (red) or non-affected (black) by TFIIB knockdown in HeLa cells. Bar graphs show representative genes. Scatter plots show all genes analysed. Dashed lines are showing the direction of change for affected (red) and non-affected (black) genes. Log2 fold changes are shown relative to Scrambled-transfected mock-treated cells. D) qPCR analysis of genes regulated by IFN-α stimulation. E) qPCR analysis of genes regulated by EGF stimulation. For all stimuli HeLa cells were electroporated with indicated siRNAs and in 16 hours stimulated with indicated ligands for 4 hours with subsequent RNA extraction. qPCR analysis was performed in two technical replicates in two independent experiments.</p

    Transcriptome analysis of THOV-induced changes suggests broad but selective effect of ML.

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    <p>A) RNA synthesis rate in THOV infected Vero cells. Newly synthesized RNA was labelled with [3H]-5-Uridine at 1, 10 and 14 hours post infection. Presented is total RNA fraction as mean and SD from three technical replicates. B) qPCR analysis of IFITs, THOV NP transcript and GAPDH expression in HeLa cells after infection with THOV-wt, THOV-ΔML and THOV-SW for 16 hours. Presented are means and SD from three independent infection experiments. C) 2D-scatter plot of transcriptome analysis of HeLa cells infected with THOV-wt, THOV-ΔML and THOV-SW for 16 hours in three biological replicates. X axis represents log2 fold changes between mock and THOV-wt (green dots), Y axis shows log2 fold changes between mock and THOV-ΔML (blue dots). Changes occurring in wt and ΔML infections are shown in orange. Unchanged genes are shown in grey. Numbers are presented in a pie chart. Differentially regulated genes were showing at least 2-fold change with a q value < 0.05. D) GO term over-representation analysis of differentially upregulated genes by THOV ΔML and THOV ML (SW/AA) compared to THOV wt performed with InnateDB analysis tool. S–signalling, R–regulation, TF–transcription factor.</p

    ML or TFIIB-depletion-mediated regulation of innate immune response and general transcription.

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    <p>When the cells are stimulated with a ligand, expression of a responsive gene can be activated by de novo recruitment of Pol II, which requires assembly of PIC (mode 1), or by releasing paused Pol II into the gene body (mode 2). In the presence of ML or after TFIIB depletion, expression of genes regulated by mode 1 (mostly cytokines and antiviral effector molecules) is severely impaired, while genes regulated by mode 2 (signalling components and housekeeping genes) continue to be expressed normally.</p

    ML sequesters TFIIB from the nucleus, but does not cause its degradation.

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    <p>A) Confocal immunofluorescence analysis of HeLa Kyoto cells stably expressing GFP-TFIIB infected with THOV wt, THOV-ΔML or THOV-SW mutants for 24 hours at MOI 3. HeLa cells were treated as indicated, fixed and stained with GFP-DyLight488, THOV NP+rbAlexa546 and DAPI and subjected to confocal microscopy. Images are representative of two independent experiments with similar results. White bar – 10 μm. B) Confocal immunofluorescence analysis of HeLa Kyoto cells stably expressing GFP-TFIIB and transiently transfected with HA-M or HA-ML for 16 hours. HeLa cells were treated as indicated, fixed and stained with GFP-DyLight488, HA+msAlexa594 and DAPI and subjected to confocal microscopy. Images are representative of four independent experiments with similar results. White bar – 10 μm. C) Cytoplasmic-nuclear fractionation of Vero cells transiently transfected with HA-tagged M or ML for 24 hours. Depicted is endogenous TFIIB. Western blot is a representative of three experiments with similar results. D) Cytoplasmic-nuclear fractionation of Vero cells transiently transfected with FLAG-TFIIB and HA-tagged M or ML for 24 hours. Western blot is a representative of three experiments with similar results. E) Total protein intensity of TFIIB as defined by iBAQ, levels of newly synthesized TFIIB as determined from heavy intensities, and translation rates of identified proteins determined from H (newly synthesized)/L (total) ratios presented as box-whisker plots with whiskers showing 10–90 percentile. Protein levels were estimated in four biological replicates.</p

    Multiple but not all inducible promoters are repressed by ML.

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    <p>(A-C) Reporter assays in HEK293 cells, where Firefly luciferase under indicated promoters was transfected together with EF-1a Renilla and M/ML/ML-SW. 24 h.p.t. cells were treated with indicated stimuli for 16 hours and the luciferase activity was measured. Shown is fold induction (mean and SD) over untreated cells from three independent experiments performed in six technical replicates. *—p<0.05; **—p<0.01; ***—p<0.001; ns–not significant. D) qPCR analysis of HeLa FlipIn cells expressing stably integrated ML from Tet-On promoter. HeLa cells were left untreated or treated with Doxycycline for 24h and subsequently stimulated with EGF for 16 hours. Total RNA was extracted and expression of indicated genes measured by qPCR. Shown are expression levels in relation to the non-Doxycycline treated unstimulated condition. The bar graphs show mean +/- error from two technical replicates and are representative of two independent experiments with similar results.</p

    Genes subject to TFIIB regulation require <i>de novo</i> Pol II recruitment.

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    <p>A) Schematic representation of profile plots of the genes affected (red) and non-affected (black) by TFIIB knockdown in RNA-seq (top panel) and qPCR screen (bottom panel). B) Gene-averaged occupancy profiles of TFIIB, Pol II, NELF-A and DSIF for genes affected (red) or non-affected (black) by TFIIB depletion and for the genome background (blue). Embedded boxplots show the distribution of ChIP-Seq read coverage in the downstream promoter (Pol II, NELF, DSIF) or promoter (TFIIB) regions across genes. C) Distribution of pausing indices (pi) between affected and non-affected genes (pi>2 can be considered paused).</p

    IFN induction by NSV vRNAs depends on RIG-I and the 5′ triphosphate group.

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    <p>(A) Verification of knockdowns. Human 293T cells were treated with retroviral shRNA constructs directed against either RIG-I or MDA5, and cotransfected with expression constructs for HA-tagged MDA5 (left panels) or GFP-fused RIG-I (right panels). Western blot analysis using antibodies against the respective fusion tags is shown. Detection of cellular β-tubulin was used as an internal control. (B) Effect of shRNA knockdowns on IFN induction by viral RNAs, using the reporter constructs and RNA transfection protocols as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002032#pone-0002032-g001" target="_blank">Fig. 1A</a>. The negative control shRNA construct (CTRL) targets the heat shock 70 interacting protein and was tested to have no effect on IFN induction (data not shown). (C) Genomic RNAs from ZEBOV and NiV were either mock treated, treated with SAP, or treated with SAP in the presence of the phosphatase inhibitor EDTA. IFN-β reporter assays and RNA transfections were performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002032#pone-0002032-g001" target="_blank">Fig. 1A</a>. Mean values and standard deviations from 3 independent experiments are shown.</p

    vRNA binding by RIG-I.

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    <p>GFP-RIG-I expressed by 293T cells was coupled to protein G Sepharose beads via a GFP-specific antiserum. Beads were incubated with vRNAs of either RVFV, HTNV, CCHFV, or BDV. After extensive washing, RNAs were extracted from the precipitates and cDNA synthesis was performed using random hexanucleotide oligomers. An aliquot of 10% of the input RNA was kept as RT-PCR control (first lane). All precipitated RNAs were subjected to RT-PCR specific for sequences of RVFV (panel 1), HTNV (panel 2), CCHFV (panel 3), and BDV (panel 4). H<sub>2</sub>O was used as negative control.</p
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