Interferon-Dependent Engagement of Eukaryotic Initiation Factor 4B via S6 Kinase (S6K)- and Ribosomal Protein S6K-Mediated Signals▿

Although the roles of Jak-Stat pathways in type I and II interferon (IFN)-dependent transcriptional regulation are well established, the precise mechanisms of mRNA translation for IFN-sensitive genes remain to be defined. We examined the effects of IFNs on the phosphorylation/activation of eukaryotic translation initiation factor 4B (eIF4B). Our data show that eIF4B is phosphorylated on Ser422 during treatment of sensitive cells with alpha IFN (IFN-α) or IFN-γ. Such phosphorylation is regulated, in a cell type-specific manner, by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) and results in enhanced interaction of the protein with eIF3A (p170/eIF3A) and increased associated ATPase activity. Our data also demonstrate that IFN-inducible eIF4B activity and IFN-stimulated gene 15 protein (ISG15) or IFN-γ-inducible chemokine CXCL-10 protein expression are diminished in S6k1/S6k2 double-knockout mouse embryonic fibroblasts. In addition, IFN-α-inducible ISG15 protein expression is blocked by eIF4B or eIF3A knockdown, establishing a requirement for these proteins in mRNA translation/protein expression by IFNs. Importantly, the generation of IFN-dependent growth inhibitory effects on primitive leukemic progenitors is dependent on activation of the S6K/eIF4B or RSK/eIF4B pathway. Taken together, our findings establish critical roles for S6K and RSK in the induction of IFN-dependent biological effects and define a key regulatory role for eIF4B as a common mediator and integrator of IFN-generated signals from these kinases

A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus

<div><p>To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy <i>in vitro</i> and/or in non-human primate studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an <i>in vitro</i> model of <i>Ebola Zaire</i> replication with transcription-competent virus like particles (trVLPs), requiring only level 2 biosafety containment, we compared the activities of the type I interferons (IFNs) IFN-α and IFN-ß, a panel of viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF)), and the estrogen receptor modulator, toremifene (TOR), in inhibiting viral replication in dose-response and time course studies. We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß inhibited viral replication 24 hours post-infection (IC₅₀ 0.038μM and 0.016μM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50–62%) or in combination (87%). They exhibited lower IC₅₀ (0.98–6.2μM) compared with FPV (36.8μM), when administered 24 hours post-infection. Unexpectedly, CDF had a narrow therapeutic window (6.25–25μM). When dosed >50μM, CDF treatment enhanced viral infection. IFN-ß exhibited strong synergy with 3TC (97.3% inhibition) or in triple combination with 3TC and AZT (95.8% inhibition). This study demonstrates that IFNs and viral polymerase inhibitors may have utility in EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity, confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for novel therapeutic options, in particular combination therapies, which could prevent and treat Ebola infection and potentially reduce drug resistance.</p></div

IFNs, toremifene and nucleoside analogs reduce trVLP replication and transcription.

<p>293 T cells were transfected with support plasmids and infected with trVLP. 24 hours post-infection, cells were treated with the indicated drugs at their IC₅₀ doses, determined from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004364#pntd.0004364.g002" target="_blank">Fig 2I</a>. At 48hrs post-infection, total RNA was extracted, reverse transcribed, then quantified by qPCR. Relative fold-change in -ve sense vRNA transcripts (A) and +ve sense cRNA and mRNA (B) was compared with infected, untreated cells (solvent,+ control). Technical duplicates were examined by qPCR, and means are the average of three biological replicates. Error bars are the standard error around the mean.</p

IFNs, toremifene, and nucleoside analogs inhibit trVLP replication.

<p>293 T cells were either left untreated (-), transfected with the mini-genome and the nucleocapsid plasmids NP, VP30, VP35 and T7 (-L), or transfected with the mini-genome and all expression plasmids to permit Ebola mini-genome entry and replication (+). (A-F) Cells were treated with the indicated drugs at the indicated doses, at each of the time points shown. Luciferase activity was measured 4 days post-trVLP infection. Values shown are the means of 4 biological replicates and are representative of 2 independent experiments. Error bars are the standard error of the mean. All drug treatment outcomes were statistically compared with the (+) control group. See also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004364#pntd.0004364.s001" target="_blank">S1 Fig</a>.</p

Synergistic 2 and 3 drug combinations against trVLP-LUC infection inhibit fully infectious ZEBOV-GFP.

<p>(A-B) 293 T cells were infected with ZEBOV-eGFP at an MOI of 0.1, then treated with 2 and 3 drug combinations as indicated, 24 hours post-infection, at their monotherapy IC₂₅ doses. GFP fluorescence was measured 3 days post-infection. Values are the means of 4 biological replicates, and error bars represent the standard error of the mean. Data are representative of 2 independent experiments. The combination index (CI) was plotted against the fractional inhibition (Fi) for each drug combination. (C-D) Plot of CIs for ZEBOV infections compared with CIs for trVLP infections.</p

IFNs, toremifene and nucleoside analogs administered 24hrs post-exposure inhibit Ebola-mini-genome replication.

<p>293 T cells were either left untreated (-), transfected with the mini-genome and the nucleocapsid plasmids NP, VP30, VP35 and T7 (-L), or transfected with the mini-genome and all expression plasmids to permit Ebola mini-genome entry and replication (+), as described in Materials and Methods. 24 hours post-trVLP infection, cells were either left untreated, or treated with the indicated drugs (A-I) Dose-response plots for each of the indicated drugs. Luciferase activity (black circles) or cell viability (white squares) was measured 4 days post-infection (3 days after drug treatment). Values are the means of 4 biological replicates and are representative of 2 independent experiments. Error bars are the standard error of the mean. See also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004364#pntd.0004364.s002" target="_blank">S2</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004364#pntd.0004364.s003" target="_blank">S3</a> Figs.</p

2 and 3 drug combinations synergistically inhibit Ebola trVLP infection.

<p>293 T cells were either left untreated, transfected with the mini-genome and the support plasmids NP, VP30, VP35, T7 but not L, or the mini-genome plus all the support plasmids. (A) Polygonogram of 28 two-drug combinations (at monotherapy IC₅₀ doses) administered at 24 hrs post-trVLP infection, then luciferase activity evaluated 3 days later. A thick red line represents strong synergy between two drugs (CI<1), a thin black line represents additive effects (CI = 1), and a thick blue line represents strong sub-additive (less than additive) between two drugs (CI>1). (B-I) Polygonograms of 56 three-drug combinations. The backbone drug in each triple combination is listed above the heptagon, and the synergism/sub-additive effect of the additional two drugs is represented within each heptagon. (J-K) Combination index (CI) vs. fractional inhibition (Fi) plots of the most synergistic and sub-additive double (J) and triple (K) drug combinations on trVLP luciferase activity. Dotted lines identify thresholds of synergy/additive effects. Values shown are the means of 2–4 biological replicates in 2 independent experiments. Error bars are the standard error of the mean. See also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004364#pntd.0004364.s005" target="_blank">S1</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004364#pntd.0004364.s006" target="_blank">S2</a> Tables.</p

Central Role of ULK1 in Type I Interferon Signaling

We provide evidence that the Unc-51-like kinase 1 (ULK1) is activated during engagement of the type I interferon (IFN) receptor (IFNR). Our studies demonstrate that the function of ULK1 is required for gene transcription mediated via IFN-stimulated response elements (ISRE) and IFNγ activation site (GAS) elements and controls expression of key IFN-stimulated genes (ISGs). We identify ULK1 as an upstream regulator of p38α mitogen-activated protein kinase (MAPK) and establish that the regulatory effects of ULK1 on ISG expression are mediated possibly by engagement of the p38 MAPK pathway. Importantly, we demonstrate that ULK1 is essential for antiproliferative responses and type I IFN-induced antineoplastic effects against malignant erythroid precursors from patients with myeloproliferative neoplasms. Together, these data reveal a role for ULK1 as a key mediator of type I IFNR-generated signals that control gene transcription and induction of antineoplastic responses