High-throughput docking for the identification of new influenza A virus polymerase inhibitors targeting the PA-PB1 protein-protein interaction

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Ribosome synthesis and MAPK activity modulate ionizing radiation-induced germ cell apoptosis in Caenorhabditis elegans

Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment

Double-blind, placebo-controlled, dose-escalating study evaluating the safety and immunogenicity of an epitope-specific chemically defined nanoparticle RSV vaccine

Simple Summary V-306 is a synthetic virus-like particle-based vaccine candidate displaying multiple respiratory syncytial virus (RSV) F site II protein mimetics (FsIIm) as an antigenic epitope. This first-in-human, double-blind, placebo-controlled, dose-escalating study in healthy young women showed that it was safe and induced an increase in immunoglobulin G specific of FsIIm. This did not translate into an increase in RSV-neutralizing antibody titers, which were already high at baseline. Background: V-306 is a virus-like particle-based vaccine candidate displaying respiratory syncytial virus (RSV) F site II protein mimetics (FsIIm) as an antigenic epitope. Methods: This was a randomized, placebo-controlled, double-blind, dose-escalating, first-in-human study, conducted in 60 women aged 18-45 years. Twenty subjects per cohort (15 vaccine and five placebo) received two V-306 intramuscular administrations on Days 0 and 56 at 15 mu g, 50 mu g, or 150 mu g. Safety and immunogenicity were assessed after each vaccination and for 1 year in total. Results: V-306 was safe and well tolerated at all dose levels, with no increase in reactogenicity and unsolicited adverse events between the first and second administrations. At 50 mu g and 150 mu g, V-306 induced an increase in FsIIm-specific immunoglobulin G (IgG) titers, which lasted at least 4 months. This did not translate into an increase in RSV-neutralizing antibody titers, which were already high at baseline. No increase in the anti-F protein-specific IgG titers was observed, which were also high in most subjects at baseline due to past natural infections. Conclusions: V-306 was safe and well-tolerated. Future modifications of the vaccine and assay conditions will likely improve the results of vaccination

SV40 at its entry triggers the upregulation of a number of integrins on the cell surface.

<p>(A) Cell surface N-glycoproteins that are significantly altered in cell surface abundance upon exposure to SV40 are visualized with a network view. The glycoproteins whose abundance was either increased (yellow) or decreased (blue) during exposure to SV40 (and which either did not change during exposure to VSV, or changed in the opposite direction compared to SV40) are depicted as nodes in the network. The different shades represent different degrees of relative abandunce (log2 values). The remaining nodes in the network are the hits from the RNAi screen (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055799#pone-0055799-g002" target="_blank">Figure 2A</a>), which either increased (green) or decreased (red) SV40 infection upon siRNA knockdown. For the common hits in the CSC and RNAi screens, the node border represents the RNAi phenotype (ITGA6, ITGB6 and CD47 were CSC-hits but gave no RNAi phenotype when tested). The grey connecting lines between nodes illustrate protein interactions, which were assessed using the STRING database with a combined score of at least 0.9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055799#pone.0055799-Szklarczyk1" target="_blank">[45]</a>, and were visualized using Cytoscape (<a href="http://www.cytoscape.org" target="_blank">www.cytoscape.org</a>) and the Cerebral plugin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055799#pone.0055799-Barsky1" target="_blank">[46]</a>.</p

Cell adhesion-signaling components are required for SV40 infection. Integrins, in addition to GM1 lipids, are required for SV40 binding and infection.

<p>(A) A targeted siRNA screen reveals several structural and signaling components of cell adhesion to regulate the SV40 infectious route. A set of four siRNAs against 263 genes was applied in A431 human epithelial cells and virus infection was assessed by the presence of nuclear large T-antigen. Low-resolution imaging and image processing with the CellProfiler analysis software were subsequently performed. A Support Vector Machine (SVM)-based classification method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055799#pone.0055799-Rm1" target="_blank">[47]</a> was then used to determine percentage of infection upon siRNA treatment. The table shows the genes that reduced (red shades) or enhanced (green shades) SV40 infection with different strength when knocked down. The values in the boxes represent the number of different siRNAs that gave a similar phenotype. (B) Epistasis analysis between Cav1, GRAF1, and Ezrin. A431 cells were treated with siRNA against each one of these genes or combinations of two. Two or three siRNAs were used per gene. Cells were subsequently treated with SV40 and infection levels were assessed by the presence of nuclear T-antigen. The graph shows values pooled from the individual infection indices. p-values: 1.3×10⁻⁴ (Ezrin-siRNA, Cav1-siRNA), 0.39 (Ezrin-siRNA, GRAF1-siRNA). (C) Blocking integrin α2 function with an antibody reduces SV40 infection, similar to siRNA-mediated knock down. A431 cells were pre-incubated with 0.02 µg/µL of blocking antibody 20 min prior to infection (p-values 1×10^−4–7×10⁻⁴). (D) siRNA against integrins α2 and β1 reduces binding of SV40 at the surface of A431 cells. Binding was performed at cold for 2 h and binding capacity was determined by immunoblotting for the presence of the major capsid protein VP1 in cell extracts. Signal intensity was quantified by the ImageJ software and standard deviation corresponds to two independent experiments. (E) SV40 binds onto the surface of various cell lines with different intensity; GM1-deficient cells retain the ability to bind SV40. Quantification of signal intensity from two independent experiments was performed as in (D). (F) SV40-like particles (VLPs) can bind cells that lack its native receptor GM1 in a dose-dependent manner. (G) SV40 can bind cells that lack its native receptor GM1 via integrins. GM95 cells were treated with siRNA against integrin α2 and SV40 binding was determined by the abundance of VP1 protein, as described in (D). (H) Integrins can serve as binding sites for SV40. Integrin α2β1 was immunoprecipitated from A431 cells pretreated for 2 h with SV40 in the cold, and the VP1 protein was detected in the immunocomplex by immunoblotting (red box). Transferrin receptor was used as a negative control.</p

Inhibition of RhoA via GRAF1 promotes Ezrin inactivation and SV40 infection.

<p>(A) Constitutively active RhoA leads to increased levels of phosphorylated Ezrin that persist upon SV40 treatment. Inactive RhoA abrogates basal levels of p-ERM. A431 cells were transfected with RhoA-G14V-GFP or RhoA-T19N-GFP before SV40 was applied for 15 min and fixed cells were stained with a p-ERM antibody. (B) Expression of an inactive RhoA mutant form positively correlates with SV40 infection. Large populations of A431 cells were transfected with RhoA-G14V, RhoA-T19N and wild-type RhoA GFP constructs and subjected to SV40. Cells that were both transfected (GFP signal) and infected (T-antigen signal) were scored and compared with the expected number emerging from a random occurrence of the two signals. Positive log2 ratio values represent a positive correlation, demonstrating a stimulation of infection, whereas negative values denote anti-correlation, demonstrating an inhibition of infection (p-value 4.2×10⁻⁴). (C) Inhibition of GRAF1 function leads to increased levels of p-ERM. siRNA was applied onto A431 cells followed by addition of SV40 for 15 min. Levels of p-ERM were assessed using immunofluorescence in fixed cells. (D) RhoA is inactivated 10 min after SV40 treatment. Inhibition of PI3K and PDK1 with wortmannin or the PDK1 inhibitor, respectively, abolished the SV40-induced reduction in RhoA activity. Cells that had undergone the indicated treatment were subjected to RhoA-GTP immunoprecipitation, which was subsequently detected with a RhoA antibody using immunoblotting. The graph shows the quantification of the RhoA-GTP signal in SV40 exposed cells, as expressed in % reduction compared to the non-treated cells, and normalized against total RhoA and tubulin (quantification based on two different experiments). (E) PDK1 and GRAF1 act both upstream of RhoA to signal to ERM proteins. A431 cells were subjected to the following conditions before being scored for the presence or absence of p-ERM signal using immunofluorescence: transfection with RhoA-WT-GFP, RhoA-G14V-GFP or RhoA-T19N-GFP constucts, incubation with the PDK1 inhibitor for 1.5 h, siRNA treatment against GRAF1, or a combination of RhoA-T19N-GFP expression and the PDK1 inhibitor or GRAF1 siRNA. Acquired confocal images were processed with ImageJ to quantify the number of p-ERM-expressing cells. Inhibition of PDK1 function or silencing of GRAF1 led to partial or no restoration of the fraction of p-ERM-positive RhoA-T19N-expressing cells (asterisks), respectively. Values shown are the average of 2–4 independent experiments ± standard deviation. (F) Representative image used to extract the values shown in (E). The white line outlines manually segmented cells, whereas green and red depict RhoA-T19N-GFP transfected cells and p-ERM-positive cells, respectively. Dapi-stained nuclei are shown in blue.</p

Proposed model of the signaling events upon SV40 infectious entry.

<p>(A) The various players participating in the integrin-mediated signaling through host SV40 engagement. The respective time intervals during which these signaling events occur are also depicted.</p

Activated Ras/MAPK pathway enhances the apoptotic DNA damage response and restores irradiation-induced germ cell death in <i>rpoa-2(op259)</i> mutants.

<p>A) Apoptotic germ cell corpses at 24 hours post irradiation. <i>lip-1(lf)</i> and <i>let-60(gf)</i> increase the IR response of <i>rpoa-2(op259)</i> mutants. <i>lip-1</i> is epistatic to <i>rpoa-2(op259)</i> regarding the IR response; the response in <i>rpoa-2(op259); let-60(gf)</i> is intermediate between the single mutants. Error bars, SD; n≥40 for each condition. B) Simplified model of the EGFR/Ras/MAPK signal axis. Gain-of-function mutations in LET-60 or loss of the phosphatase LIP-1 increase MPK-1 activity. GAP-1 is a GTPase-activating protein acting as a negative regulator of LET-60; <i>gap-1(lf)</i> mutants show synthetic effects on vulval induction with other genes but have no strong phenotype on their own. C) Reduced kinase activity in the Ras/MAPK pathway mutant <i>mpk-1(ga111)</i> or in the p38 MAPK mutant <i>pmk-1(km25)</i> but not the Jnk mutant <i>jnk-1(gk7)</i> reduces germ cell apoptosis. Experiments performed at 20°C; error bars, SD; n = 20 animals per condition. D) <i>rpoa-2(op259)</i> suppresses excessive vulval induction of <i>let-60(n1046gf)</i>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003943#s4" target="_blank">Methods</a> for assessment of vulval induction index (VI) (reference value is 3.0 for wild type, corresponding to normal induction of one vulva). Error bars, SD of at least 20 animals.</p

Baseline levels of activated MPK-1 in the gonad are reduced in <i>rpoa-2(op259)</i> and do not increase upon irradiation.

<p>A) Gonads were extruded from non-irradiated (0 Gy) and irradiated (60 Gy) adult worms 4 hours after treatment, fixed, and immunostained for the doubly phosphorylated (activated) MPK-1. MPK-1 activity is high in the late meiotic pachytene region (white dashed lines) and is further increased following IR in wild-type worms, but not in <i>rpoa-2(op259)</i> mutants. Control staining included total MPK-1 with anti-ERK, and anti-dsDNA to detect an epitope other than protein for normalisation of fluorescence intensity. Size bar, 40 µm. B) Quantification of the anti-ppMPK signal intensity along the central axis (white streak) of the late meiotic pachytene region, starting distally to the first oocyte (white dashed reference line) and extending into the mid-pachytene region. Profiles were plotted for at least 8 worms per condition (thin grey lines for individual wild-type worms, thin green lines for individual <i>rpoa-2(op259)</i> mutants) and a spline was calculated from their overlay. Size bar, 25 µm.</p

The <i>rpoa-2(op259)</i> mutation reduces irradiation-induced germ cell apoptosis but not cell cycle arrest response or DNA damage repair.

<p>A) DIC images of adult worms 24 hours after irradiation. Basal (physiologic) as well as DNA damage-induced germ cell death is confined to the late meiotic pachytene region of the germ line tubes; corpses are visible as cellularised, refractile discs (arrowheads). Size bar, 15 µm. B) Apoptotic cell corpses in the germ lines of X-ray treated adult hermaphrodites. Dashed lines represent basal levels (0 Gy), straight lines the levels following irradiation (60 Gy). At least three independent experiments (with n = 20 animals per condition) were performed with wild-type and <i>rpoa-2(op259)</i> worms, and one with animals mutant for the 9-1-1 complex subunit HUS-1. Error bars, standard deviation (SD) of corpse number per gonad over all experiments. C, D) In contrast to the DNA damage response mutant <i>hus-1(op241)</i>, cell cycle arrest is induced normally in <i>rpoa-2(op259)</i>. C) Percentage of mitotic cells with nuclear enlargement following IR. Error bars, SD from at least 12 gonads (approx. 40–50 total mitotic cells per gonad). D) Gonads were dissected from <i>opIs257[P_rad-54::rad-54::yfp 3′UTR; unc-119(+)]; unc-119(ed3)</i> or <i>rpoa-2(op259)</i>; <i>opIs257; unc-119(ed3)</i> adults, stained with Hoechst, and scored for YFP::RAD-54 foci. Size bar, 10 µm. E, F) A subset of rRNA processing and translation initiation mutants have reduced IR-induced apoptosis. Apoptotic response to IR irradiation in the gonads of the rRNA processing mutants <i>pro-2(na27)</i> and <i>pro-3(ar226)</i> (E) and of the eIF4E homolog mutants <i>ife-1(ok1978)</i> and <i>ife-2(ok306)</i> (F). Dashed lines, 0 Gy, straight lines, 60 Gy; error bars, SD of the number of germ cell corpses per gonad over at least 3 experiments (n = 20 animals per data point and experiment).</p