La protein is required for efficient translation driven by encephalomyocarditis virus internal ribosomal entry site.
(17/642)
Translation of internal ribosomal entry site (IRES)-dependent mRNAs is mediated by RNA-binding proteins as well as canonical translation factors. In order to elucidate the roles of RNA-binding proteins in IRES-dependent translation, the role of polypyrimidine tract-binding protein (PTB) and La protein in encephalomyocarditis virus (EMCV) IRES-dependent translation was investigated. PTB was required for efficient EMCV IRES-driven translation but, intriguingly, an excess of PTB suppressed it. Such a translational suppression by surplus PTB was relieved by addition of La protein. A possible role for La protein in IRES-dependent translation is discussed. (+info)
Activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways.
(18/642)
Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH(2)-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i. e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections. (+info)
High doses of digitalis increase the myocardial production of proinflammatory cytokines and worsen myocardial injury in viral myocarditis: a possible mechanism of digitalis toxicity.
(19/642)
Results of recent studies suggest that proinflammatory cytokines cause myocardial contractile dysfunction, and that the drugs used to treat heart failure modulate the production of cytokines. This study was designed to examine the effects of digoxin in a murine model of heart failure induced by viral myocarditis. Four-week-old inbred DBA/2 mice were inoculated intraperitoneally with encephalomyocarditis virus (EMCV). Digoxin was given orally in doses of 0.1, 1 or 10 mg/kg daily from the day of virus inoculation. Interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha production in the heart were measured on day 5 after EMCV inoculation by enzyme-linked immunosorbent assay. The 14-day mortality tended to be increased in mice treated with 1 mg/kg, and was significantly increased in the group treated with 10 mg/kg per day. Myocardial necrosis and cellular infiltration on day 6 were significantly more severe in the high-dose digoxin group than in the control group. In the animals treated with 1 mg/kg digoxin, IL-1beta was significantly higher than in the control group. Intracardiac TNF-alpha levels were increased in a dose-dependent manner. These results suggest that digoxin worsens viral myocarditis, and that its use in high doses should be avoided in patients suffering from heart failure due to viral myocarditis. (+info)
Substitutions in the aspartate transcarbamoylase domain of hamster CAD disrupt oligomeric structure.
(20/642)
Aspartate transcarbamoylase (ATCase; EC 2.1.3.2) is one of three enzymatic domains of CAD, a protein whose native structure is usually a hexamer of identical subunits. Alanine substitutions for the ATCase residues Asp-90 and Arg-269 were generated in a bicistronic vector that encodes a 6-histidine-tagged hamster CAD. Stably transfected mammalian cells expressing high levels of CAD were easily isolated and CAD purification was simplified over previous procedures. The substitutions reduce the ATCase V(max) of the altered CADs by 11-fold and 46-fold, respectively, as well as affect the enzyme's affinity for aspartate. At 25 mM Mg(2+), these substitutions cause the oligomeric CAD to dissociate into monomers. Under the same dissociating conditions, incubating the altered CAD with the ATCase substrate carbamoyl phosphate or the bisubstrate analogue N-phosphonacetyl-L-aspartate unexpectedly leads to the reformation of hexamers. Incubation with the other ATCase substrate, aspartate, has no effect. These results demonstrate that the ATCase domain is central to hexamer formation in CAD and suggest that the ATCase reaction mechanism is ordered in the same manner as the Escherichia coli ATCase. Finally, the data indicate that the binding of carbamoyl phosphate induces conformational changes that enhance the interaction of CAD subunits. (+info)
Age-related changes in susceptibility of rat brain slice cultures including hippocampus to encephalomyocarditis virus.
(21/642)
Replication of the D variant of encephalomyocarditis virus (EMC-D) and its cytopathic effects were studied in the brain slice cultures including hippocampus (hippocampal slice) obtained from postnatal 1-, 4-, 7-, 14-, 28-and 56-day-old Fischer 344 rats. At 0, 12, 24, 36 and 48 h after infection, virus titres of the slices and culture media were assayed. Viral replication was observed in cultures from 1-to 28-day-old rats, and the highest titre was recorded in the slice and culture medium from the youngest rat. The peak of virus titre decreased with age and no distinct viral replication was observed in the cultures from 56-day-old rats. Light microscopy revealed that degenerative and necrotic changes appeared in the infected hippocampal slices from 1- to 28-day-old rats, and the changes became less prominent with age. In situ hybridization and indirect immunofluorescence staining showed that positive signals of viral RNA and antigen were prominent in younger rats and decreased with age. These results suggest that an age-related decrease in the susceptibility of rat brain to EMC-D is less related to the maturation of the immune system but possibly to that of the neurone. (+info)
Analysis of the c-myc IRES; a potential role for cell-type specific trans-acting factors and the nuclear compartment.
(22/642)
The 5' UTR of c -myc mRNA contains an internal ribo-some entry segment (IRES) and consequently, c -myc mRNAs can be translated by the alternative mechanism of internal ribosome entry. However, there is also some evidence suggesting that c -myc mRNA translation can occur via the conventional cap-dependent scanning mechanism. Using both bicistronic and monocistronic mRNAs containing the c- myc 5' UTR, we demonstrate that both mechanisms can contribute to c- myc protein synthesis. A wide range of cell types are capable of initiating translation of c- myc by internal ribosome entry, albeit with different efficiencies. Moreover, our data suggest that the spectrum of efficiencies observed in these cell types is likely to be due to variation in the cellular concentration of non-canonical translation factors. Interestingly, the c -myc IRES is 7-fold more active than the human rhinovirus 2 (HRV2) IRES and 5-fold more active than the encephalomyocarditis virus (EMCV) IRES. However, the protein requirements for the c -myc IRES must differ significantly from these viral IRESs, since an unidentified nuclear event appears to be a pre-requisite for efficient c -myc IRES-driven initiation. (+info)
Cell growth regulatory and antiviral effects of the P69 isozyme of 2-5 (A) synthetase.
(23/642)
Among the many interferon-induced proteins that carry out multiple cellular functions of interferons is the family of enzymes called 2'-5' oligoadenylate synthetases. We examined the anticellular and antiviral activities of a specific member of that family, the P69 isozyme. P69 was expressed in human cells by transfection and shown to be localized primarily in the endoplasmic reticulum. For further studies, permanent cell lines expressing different levels of P69 or an enzymatically inactive mutant were isolated. Constitutive P69 expression caused inhibition of replication of encephalomyocarditis virus but not of vesicular stomatitis virus, Sendai virus, or reovirus. Increasing levels of P69 expression also caused increasing perturbations in cell growth properties. There was increasing accumulations of the P69-expressing cells in the G1 phase of the cell cycle; cell-doubling time was increased by P69 expression; and there were many multinucleated cells in the P69-expressing line, indicating a defect in cytokinesis. (+info)
Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection.
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P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm. (+info)