Sindbis virus translation is inhibited by a PKR/RNase L-independent effector induced by alpha/beta interferon priming of dendritic cells.

The tropism of Sindbis virus (SB) for cells of the dendritic cell (DC) lineage and the virulence of SB in vivo are largely determined by the efficacy of alpha/beta interferon (IFN-alpha/beta)-mediated antiviral responses. These responses are essentially intact in the absence of PKR and/or RNase L (K. D. Ryman, L. J. White, R. E. Johnston, and W. B. Klimstra, Viral Immunol. 15:53-76, 2002). In the present studies, we investigated the nature of antiviral effects and identity of antiviral effectors primed by IFN-alpha/beta treatment of bone marrow-derived DCs (BMDCs) generated from mice deficient in PKR and RNase L (TD). IFN-alpha/beta priming exerted significant antiviral activity at very early stages of SB replication and most likely inhibited the initial translation of infecting genomes. The early effect targeted cap-dependent translation as protein synthesis from an SB-like and a simple RNA were inhibited by interferon treatment, but an encephalomyocarditis virus internal ribosome entry site-driven element exhibited no inhibition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 was defective after virus infection of TD cells, suggesting other mechanisms of translation inhibition. To identify components of these alternative antiviral pathway(s), we have compared global gene regulation in BMDCs derived from normal 129 Sv/Ev, IFNAR1-/-, and TD mice following infection with SB or treatment with IFN-alpha/beta. Candidate effectors of alternative antiviral pathways were those genes induced by virus infection or IFN-alpha/beta treatment in 129 Sv/Ev and TD-derived BMDC but not in virus-infected or IFN-alpha/beta-treated IFNAR1-/- cells. Statistical analyses of gene array data identified 44 genes that met these criteria which are discussed.

Characterization of the two eIF4A-binding sites on human eIF4G-1.

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1.2 x 10(5) m(-1) s(-1), 2.1 x 10(-3) s(-1), and 17 nm for the central site and 5.1 x 10(3) m(-1) s(-1), 1.7 x 10(-3) s(-1), and 330 nm for the COOH-terminal site.

Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1.

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. The central region of eIF4G binds the ATP-dependent RNA helicase eIF4A, the 40 S binding factor eIF3, and RNA. In the present work, we have further characterized the binding properties of the central region of human eIF4G. Both titration and competition experiments were consistent with a 1:1 stoichiometry for eIF3 binding. Surface plasmon resonance studies showed that three recombinant eIF4G fragments corresponding to amino acids 642-1560, 613-1078, and 975-1078 bound eIF3 with similar kinetics. A dissociation equilibrium constant of approximately 42 nm was derived from an association rate constant of 3.9 x 10(4) m(-1) s(-1) and dissociation rate constant of 1.5 x 10(-3) s(-1). Thus, the eIF3-binding region is included within amino acid residues 975-1078. This region does not overlap with the RNA-binding site, which suggests that eIF3 binds eIF4G directly and not through an RNA bridge, or the central eIF4A-binding site. Surprisingly, the binding of eIF3 and eIF4A to the central region was mutually cooperative; eIF3 binding to eIF4G increased 4-fold in the presence of eIF4A, and conversely, eIF4A binding to the central (but not COOH-terminal) region of eIF4G increased 2.4-fold in the presence of eIF3.

Interaction of the universal mRNA-binding protein, p50, with actin: a possible link between mRNA and microfilaments.

We have shown previously that p50 is the most abundant protein associated with a variety of eukaryotic mRNAs and exhibits about 98% amino acid sequence identity to mammalian Y-box binding transcription factors. The dual function of p50 in the cell as a regulator of both transcription and translation has been suggested. To gain insight into the role of p50 in these processes, we performed the yeast two-hybrid screen to identify p50 molecular partners. Here we report the identification of actin as a p50-interacting protein. Coimmunoprecipitation of p50 and actin from HeLa extracts as well as in vitro binding studies indicate specificity and a high affinity for the interaction between p50 and actin. Interestingly, p50 binding to actin is affected by mRNA; binding was observed at a low p50/mRNA ratio and was greatly reduced at higher ratios. Since the p50/mRNA ratio appears to be important for mRNA translatability, we speculate that p50 can regulate the attachment of mRNA to the actin network depending on its translational activity. Using immunofluorescence, we show that p50 binds to actin filaments in permeabilized cells and causes actin fibers to bundle in vitro. Together, these findings support the view that p50 may play an important role in mRNA transport, anchoring, and localization on actin filaments in the cell.

Light microscopic analysis of ligand-induced actin filament suprastructures.

In this study, we describe a simple light microscopic assay which allows to rapidly determine the ligand-induced organization of actin filaments into specific suprastructures, such as web-like arrangements or bundles. The validity of this assay is demonstrated by accompanying low shear (falling ball) viscometry. While the visually identified webs demonstrated viscosity values significantly higher than the F-actin control, the bundles were characterized by viscosities distinctly lower than that of the control. In addition, we show that at least in some cases, the type of actin suprastructure formed depends on the molar ratio between the ligand and actin filaments. The assay should be useful to assess the conditions under which a particular ligand leads to a specific actin filament organization, to determine quickly the biological activity of recombinant proteins or isolated actin-binding domains, and to define new F-actin ligands.

[Evaluation of health status of agricultural machine operators and agriculture students]. / Otsenka zdorov'ia mekhanizatorov sel'skogo khoziaistva i uchashchikhsia SPTU.

To lower morbidity with transitory disablement, to better primary prophylaxis and health preservation for machine operators and professional training students, a system of therapeutic and sanitary measures is to be put into practice. Those measures include improved organization of medical care, better hygienic conditions, safe work and healthy lifestyle of the examinees.

[Phosphorylation of proteins of mRNP-particles of rabbit reticulocytes and their possible role in translation regulation]. / Fosforilirovanie belkov mRNP-chastits retikulotsitov krolika i ego vozmozhnaia rol' v reguliatsii transliatsii.

Protein kinase activity was found associated with free mRNP particles of rabbit reticulocytes. This enzyme was able to transfer phosphate group(s) from ATP to at least nine proteins of mRNP. Rabbit reticulocytes postmitochondrial supernatant proves the existence of protein phosphatase activity specifically dephosphorylating some mRNP proteins. The role of mRNP proteins phosphorylation--dephosphorylation in translation process is discussed.