Significance of the C-terminal amino acid residue in mengovirus RNA-dependent RNA polymerase.
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Replication of picornavirus genomes is accomplished by the virally encoded RNA-dependent RNA polymerase (RdRP). Although the primary structure of this enzyme exhibits a high level of conservation, there are several significant differences among different picornavirus genera. In particular, a comparative alignment indicates that the C-terminal sequences of cardiovirus RdRP (known also as 3D(pol)), are 1-amino-acid residue (arginine or tryptophan) longer than that of the enterovirus or rhinovirus enzymes. Here, it is shown that alterations of the last codon of the RdRP-encoding sequence of mengovirus RNA leading to deletion of the C-terminal Trp460 or its replacement by Ala or Phe dramatically impaired viral RNA replication and, in the former case, resulted in a quasi-infectious phenotype (i.e., the mutant RNA might generate a low yield of pseudorevertants acquiring a Tyr residue in place of the deleted Trp460). The replacement of Trp460 by His or Tyr did not appreciably alter the viral growth potential. Homology modeling of three-dimensional structure of mengovirus RdRP suggested that Trp460 may be involved in interaction between the thumb and palm domains of the enzyme. Specifically, Trp460 of the thumb may form a hydrogen bond with Thr219 and hydrophobically interact with Val216 of the palm. The proposed interactions were consistent with the results of in vivo SELEX experiment, which demonstrated that infectious virus could contain Ser or Thr at position 219 and hydrophobic Val, Leu, Ile, as well as Arg (whose side chain has a nonpolar part) at position 216. A similar thumb-palm domain interaction may be a general feature of several RdRPs and its possible functional significance is discussed. (+info)
Cellular protein synthesis shutoff by mengovirus: translation of nonviral and viral mRNA's in extracts from uninfected and infected Ehrlich ascites tumor cells.
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The mechanism whereby picornaviruses inhibit host protein synthesis while their own synthetic processes proceed unabated has remained elusive. One of our approaches to this problem was to study the ability of cell-free extracts derived from uninfected and mengovirus-infected Ehrlich ascites tumor cells to translate viral and nonviral mRNA's under various conditions of incubation. Our results indicate that viral messengers (from mengovirus and encephalomyocarditis virus) and cellular messengers [L cell and Ehrlich ascites tumor poly(A)-containing mRNA's, rabbit globin mRNA, and chicken embryo lens crystallin mRNA] are translated equally well in both extracts. We also examined the simultaneous translation of viral and nonviral mRNA's in extracts from uninfected Ehrlich ascites tumor cells. Our results indicate that under certain conditions mengovirus RNA can suppress completely the translation of globin mRNA. The significance of these results in terms of the shutoff of host protein synthesis is discussed. (+info)
Considerations on structure-activity relationships with mengovirus of substituted 5-amino-4-cyanopyrazoles.
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In a screening program using the agar diffusion-plaque inhibition test, 22 out of 27 substituted aminopyrazoles tested showed marked antiviral activity against mengovirus in FL cell cultures. The efficacy of the different derivatives was compared by determining their effect on the yield of infectious virus after a one-step growth cycle. The antiviral activity against mengovirus correlated with the chain length of the substituents and increased or decreased according to the position of the side chain. (+info)
Mode of action in vitro against mengovirus of substituted 5-amino-4-cyanopyrazoles.
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Substituted amino pyrazoles were found to exhibit plaque reduction and inhibition of the cytopathic effect of mengovirus on FL cells. Their antiviral activity was not caused by a virucidal effect or by inhibition of viral adsoprtion or penetration but by suppression of the virus multiplication. During a one-step growth cycle maximum suppression of virus yield occurred after compound addition from 0 to 2 h after infection. Progressively less viral inhibition appeared when compound was applied during the later part of virus replication. The antiviral effect was reversible by removal of the compound, and no inhibitor-resistant period occurred. (+info)
Alanosine toxicity in Novikoff rat hepatoma cells due to inhibition of the conversion of inosine monophosphate to adenosine monophosphate.
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2-Amino-3-(hydroxynitrosoamino)propionic acid (alanosine), at a concentration as low as 2.7 muM, completely inhibits the incorporation of hypoxanthine into adenosine triphosphate by cultured Novikoff rat hepatoma cells. Alanosine inhibits the first step in the conversion of inosine monophosphate to adenosine monophosphate because inosine monophosphate, but not adenylosuccinate, accumulates in treated cells. However, the alanosine inhibition is not prevented by aspartic acid, even at a concentration of 1 mM. Alanosine treatment results in the inhibition of cell division, DNA synthesis, RNA and protein synthesis (in this order), and a depletion of the cells of adenosine triphosphate. Some of the cells accumulate in late G2 or M, but the remainder become arrested in other stages of the cell cycle. All effects are due to the inhibition of adenosine monophosphate synthesis and the consequent depletion of the adenosine triphosphate pool since they are completely prevented or reversed by addition of adenine, but not hypoxanthine, to the medium. Pyrimidine nucleotide synthesis is not significantly inhibited by alanosine, since the uridine triphosphate pool is not affected and uridine fails to reverse the cytotoxicity of alanosine. Alanosine also inhibits the transport of aspartic acid, but has a much lower affinity for this transport system than aspartic acid. (+info)
Processing of mengovirus precursor polypeptides in the presence of zinc ions and sulfhydryl compounds.
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The effect of zinc ions on the post-translational cleavage of mengovirus polypeptides has been examined. The cleavage of the "A" precursor, which gives rise to the capsid proteins, was the most sensitive at concentrations of zinc chloride from 0.1 to 1.0 mM. Beta-mercaptoethanol and dithiothreitol antagonized the zinc-promoted inhibtion of clevage. Our results indicate that zinc ions interfere with the proper folding of the nascent polypeptide precursor rather than inhibit the proteases responsible for the cleavages. Thus, proper folding of mengovirus polypeptide "A" appears to be necessary for subsequent processing by proteases. (+info)
Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II.
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Noroviruses, an important cause of gastroenteritis, are excreted by infected individuals and are therefore present in wastewater. We quantified norovirus genogroup I (GI) and GII in wastewater at different locations in France and evaluated removal by a range of treatment types, including basic (waste stabilization pond), current industry standard (activated sludge), and state-of-the-art (submerged membrane bioreactor) treatments. Noroviruses were quantified using real-time reverse transcription-PCR (rRT-PCR). Mengovirus was used as a virus extraction control, and internal controls were used to verify the level of GI and GII rRT-PCR inhibition. A total of 161 (81 influent and 79 effluent) samples were examined; GI and GII were detected in 43 and 88% of the influent samples, respectively, and in 24 and 14% of the effluent samples, respectively. Physicians in France report far more cases of GII than GI during outbreaks; thus, the frequent presence of GI was unexpected. The GI influent concentrations were more variable, the peak GI influent concentrations were higher than the peak GII influent concentrations at all four sites (up to 1 x 10(9) and 6 x 10(7) genome copies/liter, respectively), and the average positive influent concentrations of GI were higher than the average positive influent concentrations of GII. The maximum effluent breakthrough concentrations were 6 x 10(6) and 3 x 10(6) genome copies/liter for GI and GII, respectively, indicating that the four treatment systems studied decreased the norovirus contamination load in receiving waters. (+info)
Human fibroblast and leukocyte interferons show different dose-response curves in assay of cell protection.
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Preparations of human fibroblast and leukocyte interferons of similar potency show markedly different dose-response curves in an assay which measures the degree of protection of tissue cultures against virus c.p.e. The effect is observed with both vesicular stomatitis virus (VSV) and Mengovirus, and is not altered by purification of the interferons. (+info)