Early enterovirus translation deficits extend viral RNA replication and elicit sustained MDA5-directed innate signaling.

Multiple pattern recognition receptors sense vRNAs and initiate downstream innate signaling: endosomal Toll-like receptors (TLRs) 3, 7, and 8 and cytoplasmic RIG-I-like receptors (RLRs) RIG-I, and MDA5. They engage distinct signaling scaffolds: mitochondrial antiviral signaling protein (RLR), MyD88, and TLR-adaptor interacting with SLC15A4 on the lysosome (TLR7 and TLR8) and toll/IL-1R domain-containing adaptor inducing IFN (TLR3). By virtue of their unusual vRNA structure and direct host cell entry path, the innate response to EVs uniquely is orchestrated by MDA5.

PKR Binds Enterovirus IRESs, Displaces Host Translation Factors, and Impairs Viral Translation to Enable Innate Antiviral Signaling.

For RNA virus families except , viral RNA sensing includes Toll-like receptors and/or RIG-I. Picornavirus RNAs, whose 5' termini are shielded by a genome-linked protein, are predominately recognized by MDA5. This has important ramifications for adaptive immunity, as MDA5-specific patterns of type-I interferon (IFN) release are optimal for CD4T cell T1 polarization and CD8T cell priming.

Enterovirus 2A Cleavage of the YTHDF mA Readers Implicates YTHDF3 as a Mediator of Type I Interferon-Driven JAK/STAT Signaling.

Enteroviruses (EV) deploy two proteases that mediate viral polyprotein cleavage and host cell manipulation. Here, we report that EV 2A proteases cleave all three members of the YTHDF protein family, cytosolic -methyladenosine (mA) "readers" that regulate target mRNA fate. YTHDF protein cleavage occurs very early during infection, before viral translation is detected or cytopathogenic effects are observed.

Harnessing virus tropism for dendritic cells for vaccine design.

Dendritic cells (DCs) are pivotal stimulators of T cell responses. They provide essential signals (epitope presentation, proinflammatory cytokines, co-stimulation) to T cells and prime adaptive immunity. Therefore, they are paramount to immunization strategies geared to generate T cell immunity.

Synergistic antitumor effects of 9.2.27-PE38KDEL and ABT-737 in primary and metastatic brain tumors.

Standard treatment, unfortunately, yields a poor prognosis for patients with primary or metastatic cancers in the central nervous system, indicating a necessity for novel therapeutic agents. Immunotoxins (ITs) are a class of promising therapeutic candidates produced by fusing antibody fragments with toxin moieties. In this study, we investigated if inherent resistance to IT cytotoxicity can be overcome by rational combination with pro-apoptotic enhancers.

Ribosomal RACK1:Protein Kinase C βII Modulates Intramolecular Interactions between Unstructured Regions of Eukaryotic Initiation Factor 4G (eIF4G) That Control eIF4E and eIF3 Binding.

The receptor for activated C kinase (RACK1), a conserved constituent of eukaryotic ribosomes, mediates phosphorylation of eukaryotic initiation factor 4G1(S1093) [eIF4G1(S1093)] and eIF3a(S1364) by protein kinase C βII (PKCβII) (M. I. Dobrikov, E.

Ribosomal RACK1:Protein Kinase C βII Phosphorylates Eukaryotic Initiation Factor 4G1 at S1093 To Modulate Cap-Dependent and -Independent Translation Initiation.

Eukaryotic ribosomes contain the high-affinity protein kinase C βII (PKCβII) scaffold, receptor for activated C kinase (RACK1), but its role in protein synthesis control remains unclear. We found that RACK1:PKCβII phosphorylates eukaryotic initiation factor 4G1 (eIF4G1) at S1093 and eIF3a at S1364. We showed that reversible eIF4G(S1093) phosphorylation is involved in a global protein synthesis surge upon PKC-Raf-extracellular signal-regulated kinase 1/2 (ERK1/2) activation and in induction of phorbol ester-responsive transcripts, such as cyclooxygenase 2 (Cox-2) and cyclin-dependent kinase inhibitor (p21), or in 5' 7-methylguanosine (mG) cap-independent enterovirus translation.

Regulation of Hypoxia-Inducible Factor 1α during Hypoxia by DAP5-Induced Translation of PHD2.

Death-associated protein 5 (DAP5) is an atypical isoform of the translation initiation scaffolds eukaryotic initiation factor 4GI (eIF4GI) and eIF4GII (eIF4GI/II), which recruit mRNAs to ribosomes in mammals. Unlike eIF4GI/II, DAP5 binds eIF2β, a subunit of the eIF2 complex that delivers methionyl-tRNA to ribosomes. We discovered that DAP5:eIF2β binding depends on specific stimuli, e.

Mitogen-activated protein kinase-interacting kinase regulates mTOR/AKT signaling and controls the serine/arginine-rich protein kinase-responsive type 1 internal ribosome entry site-mediated translation and viral oncolysis.

Unlabelled: Translation machinery is a major recipient of the principal mitogenic signaling networks involving Raf-ERK1/2 and phosphoinositol 3-kinase (PI3K)-mechanistic target of rapamycin (mTOR). Picornavirus internal ribosomal entry site (IRES)-mediated translation and cytopathogenic effects are susceptible to the status of such signaling cascades in host cells. We determined that tumor-specific cytotoxicity of the poliovirus/rhinovirus chimera PVSRIPO is facilitated by Raf-ERK1/2 signals to the mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) and its effects on the partitioning/activity of the Ser/Arg (SR)-rich protein kinase (SRPK) (M.

Oncolytic polio virotherapy of cancer.

Recently, the century-old idea of targeting cancer with viruses (oncolytic viruses) has come of age, and promise has been documented in early stage and several late-stage clinical trials in a variety of cancers. Although originally prized for their direct tumor cytotoxicity (oncolytic virotherapy), recently, the proinflammatory and immunogenic effects of viral tumor infection (oncolytic immunotherapy) have come into focus. Indeed, a capacity for eliciting broad, sustained antineoplastic effects stemming from combined direct viral cytotoxicity, innate antiviral activation, stromal proinflammatory stimulation, and recruitment of adaptive immune effector responses is the greatest asset of oncolytic viruses.

Mitotic phosphorylation of eukaryotic initiation factor 4G1 (eIF4G1) at Ser1232 by Cdk1:cyclin B inhibits eIF4A helicase complex binding with RNA.

During mitosis, global translation is suppressed, while synthesis of proteins with vital mitotic roles must go on. Prior evidence suggests that the mitotic translation shift involves control of initiation. Yet, no signals specifically targeting translation initiation factors during mitosis have been identified.

Inhibition of hepatitis C viral RNA-dependent RNA polymerase by α-P-boranophosphate nucleotides: exploring a potential strategy for mechanism-based HCV drug design.

Improved treatments for chronic HCV infections remain a challenge, and new chemical strategies are needed to expand the current paradigm. The HCV RNA polymerase (RdR(P)) has been a target for antiviral development. For the first time we show that the boranophosphate (BP) modification increases the substrate efficiency of ATP analogs into HCV NS5BΔ55 RdRP-catalyzed RNA.

Dynamic regulation of the translation initiation helicase complex by mitogenic signal transduction to eukaryotic translation initiation factor 4G.

Eukaryotic translation initiation factor 4F (eIF4F), comprising the cap-binding protein eIF4E, the helicase eIF4A, and the central scaffold eIF4G, is a convergence node for a complex signaling network that controls protein synthesis. Together with eIF3 and eIF4A/4B, eIF4G recruits ribosomal subunits to mRNAs and facilitates 5' untranslated region unwinding. Mammalian eIF4G contains three HEAT domains and unstructured regions involved in protein-protein interactions.

Oncolytic poliovirus against malignant glioma.

In cancerous cells, physiologically tight regulation of protein synthesis is lost, contributing to uncontrolled growth and proliferation. We describe a novel experimental cancer therapy approach based on genetically recombinant poliovirus that targets an intriguing aberration of translation control in malignancy. This strategy is based on the confluence of several factors enabling specific and efficacious cancer cell targeting.

Phosphorylation of eukaryotic translation initiation factor 4G1 (eIF4G1) by protein kinase C{alpha} regulates eIF4G1 binding to Mnk1.

Signal transduction through mitogen-activated protein kinases (MAPKs) is implicated in growth and proliferation control through translation regulation and involves posttranslational modification of translation initiation factors. For example, convergent MAPK signals to Mnk1 lead to phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), which has been linked to malignant transformation. However, understanding the compound effects of mitogenic signaling on the translation apparatus and on protein synthesis control remains elusive.

Boranophosphate siRNA-aptamer chimeras for tumor-specific downregulation of cancer receptors and modulators.

There is a need for novel, effective, and cell- and gene-specific therapeutics for cancer. Modified oligonucleotides can be used to modulate specifically and potently the expression of several genes that are upregulated in breast and prostate cancer and have been found to be causal to the tumor phenotype. Synergistic downregulation of these genes may be a potent therapeutic intervention.

Synthesis and properties of (alpha-P-borano)-nucleoside 5'-triphosphate analogues as potential antiviral agents.

The alpha-P-borano modification, where one of the alpha-phosphate oxygens is replaced by borane, of chain terminating nucleoside triphosphates are currently being tested in cell culture and are showing promise as effective viral polymerase inhibitors. The goal of this project is to combine the alpha-P-borano and Nanogel drug delivery technology to increase the antiviral potency of chain terminating sugar and base modified purine nucleosides versus the Hepatitis C Viral RNA dependent RNA polymerase (HCV RdRp). Here we show the synthesis of Cordycepin and 2'-O-methyl alpha-P-borano triphosphate via a one-pot phosphorochloridite synthesis under mild conditions.

Stereospecificity, substrate, and inhibitory properties of nucleoside diphosphate analogs for creatine and pyruvate kinases.

Antiviral alpha-P-borano substituted NTPs are promising chain terminators targeting HIV reverse transcriptase (RT). Activation of antiviral nucleoside diphosphates (NDPs) to NTPs may be carried out by pyruvate kinase (PK) and creatine kinase (CK). Herein, are presented the effects of nucleobase, ribose, and alpha-phosphate substitutions on substrate specificities of CK and PK.

Synthesis of alpha-P-modified nucleoside diphosphates with ethylenediamine.

This report describes a one-pot synthesis of alpha-P-borano-, alpha-P-thio-, and alpha-P-seleno-modified nucleoside diphosphate analogues that are otherwise difficult to obtain. The key step involves the intramolecular nucleophilic attack by an amino group in 5 to remove the gamma-phosphate. The absolute configurations of P-diastereomers were confirmed by analysis of their 1H NMR.

Synthesis of 5-ethynyl-2'-deoxyuridine-5'-boranomono phosphate as a potential thymidylate synthase inhibitor.

The 5-ethynyl-2'-deoxyuridine nucleoside and the 5'-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2'-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.

Affinity of (alpha-P-borano)-NTP analogs to rabbit muscle pyruvate kinase.

The binding affinity of (alpha-P-borano) and other NTP analogs to rabbit muscle pyruvate kinase (PK) was investigated using a fluorescence quenching approach to obtain structure-activity relationships for substrate specificity of nucleotide analogs.

Reading, writing, and modulating genetic information with boranophosphate mimics of nucleotides, DNA, and RNA.

The P-boranophosphates are efficient and near perfect mimics of natural nucleic acids in permitting reading and writing of genetic information with high yield and accuracy. Substitution of a borane (-BH3) group for oxygen in the phosphate ester bond creates an isoelectronic and isosteric mimic of natural nucleotide phosphate esters found in mononucleotides, i.e.

Incorporation of (alpha-P-borano)-2',3'-dideoxycytidine 5'-triphosphate into DNA by drug-resistant MMLV reverse transcriptase and Taq DNA polymerase.

The Rp-stereoisomer of 5'-(alpha-P-borano)triphosphates of 2'-deoxycytidine (Rp-dCTPalphaB) and 2',3'-dideoxycytidine (Rp-ddCTPalphaB) were synthesized. Their steady-state kinetics of incorporation by ddNTP-resistant enzymes, e.g.

RNase H activation by stereoregular boranophosphate oligonucleotide.

A stereoregular all-(Sp)-boranophosphate oligodeoxyribonucleotide (BH3(-)-ODN) 15-mer was synthesized using an enzymatic approach. The BH3(-)-ODN formed a hybrid with the complementary RNA 15-mer and induced RNase H hydrolysis of the RNA strand at ODN concentrations as low as 10 nM at 37 degrees C, but with a lower efficiency than that of its natural phosphodiester analogue.