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Y V Svitkin

Publications and source records attributed to Y V Svitkin.

34 records · Page 2Linked to original sources
PubMed1992

Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA.

Initiation of translation on picornavirus RNAs is accomplished through internal binding of ribosomes to a complex cis-acting element. Here we show that efficient function of this element involves two appropriately spaced smaller elements: UUUCC and an AUG. This conclusion emerged from analysis of the genome structures of spontaneous revertants of mutant polioviruses with extended insertions between the UUUCC and AUG motifs. It was confirmed by the results obtained with specially designed constructs. A similarity to the prokaryotic translation initiation mechanism, which involves the Shine-Dalgarno sequence, is emphasized, but in the picornavirus system the position of the UUUCC must be strictly fixed relative to upstream cis-acting elements, and the AUG may not necessarily serve as an initiation codon.

Base Sequence↗
PubMed1991

Coupled mutations in the 5'-untranslated region of the Sabin poliovirus strains during in vivo passages: structural and functional implications.

All entero- and rhinovirus RNAs sequenced thus far possess A and U residues at positions corresponding to nucleotides 480 and 525, respectively, of poliovirus type 1. These two nucleotides have been proposed previously to form a base pair. The single exception to this rule appears to be the Sabin type 1 strain, which has a G480. Isolates of the Sabin 1 virus from healthy vaccinees were shown to have either a reversion to A480 or a second-site mutation U525----C, both restoring a potential for efficient base pairing. In vitro translation experiments demonstrated that poliovirus type 1 RNAs with either A480-U525 or G480-C525 are more efficient in promoting translation initiation as compared with the Sabin 1 RNA (G480-U525). The Sabin 2 strain has a U and an A at position 398 and 481, respectively, while its predecessor, strain P712, is shown to have C398 and G481. All the derivatives of the Sabin 2 isolated from vaccine-associated paralytic poliomyelitis cases shown reversion to G481, and most of them reverted also to C398. It is proposed that bases at positions 398 and 481 may be involved in a tertiary interaction. The in vitro template activity of the Sabin type 2 RNA (A481) is significantly lower than that of the isolate RNAs with G481, thus confirming the relation between attenuation and translation efficiency demonstrated previously for the type 1 and type 3 Sabin strains. The C----U change at position 398 exerted only a minor effect on the RNA template activity.

Base Sequence↗
PubMed1991

Use of protamine sulphate for elimination of substrate DNA in poliovaccines produced on continuous cell lines.

Cell substrate DNA was shown to be an abundant contaminant in the clarified preparations of the Sabin type 1, 2 and 3 poliovaccines produced on a continuous cell line (4647). The size of the DNA, as evaluated for the Sabin type 1 poliovaccine, was highly heterogeneous, ranging from 100 to 20,000 base pairs. In view of potential oncogenicity of this DNA a simple and efficient procedure for its elimination is proposed. The method is based on use of protamine sulphate which at the concentration of 2.0 mg ml-1 precipitated cell DNA almost completely without affecting the virus titres.

Animals↗
PubMed1990

Translation deficiency of the Sabin type 3 poliovirus genome: association with an attenuating mutation C472----U.

Previous studies have shown that the genome of Sabin type 3 poliovaccine strain (P3/Leon 12a1b) possesses a diminished translation efficiency as compared to genomes of closely related neurovirulent strains, the neurovirulent progenitor (P3/Leon/37), or a revertant (P3/119/70) of the vaccine (Y.V. Svitkin, S.V. Maslova, and V.I. Agol, 1985, Virology 147, 243-252). Here we attempted to evaluate the contribution of each mutation in the genome of the vaccine to this translation deficiency. Recombinants between P3/Leon 12a1b and P3/Leon/37 or P3/119/70 were constructed in vitro and their RNAs were translated in a cell-free system derived from Krebs-2 cells. The results show that of 10 nucleotide differences between the genomes of P3/Leon 12a1b and P3/Leon/37 9 have minor or no effect on translation and that the only mutation of significance is C472----U which is known to reduce the neurovirulence of the virus. Reversion from uridine to cytosine at position 472 in type 3 poliovaccine upon replication in the human gut resulted in an increase of both translation efficiency of polio RNAs and neurovirulence of corresponding strains. The data provide evidence for a common nucleotide sequence regulatory element for protein synthesis of the virus and its neurovirulence. In vitro translation assays may therefore prove to be useful for detection of attenuating mutations in the 5' noncoding region of poliovirus genome. The apparent involvement of the translation mechanism in the expression of neurovirulent or attenuated phenotype of poliovirus is briefly discussed.

Animals↗
PubMed1988

Point mutations modify the response of poliovirus RNA to a translation initiation factor: a comparison of neurovirulent and attenuated strains.

Upon translation of poliovirus RNA in reticulocyte lysates, initiation occurs largely "incorrectly," that is, at sites in the middle of the viral genome rather than at the beginning of the polyprotein reading frame; the anomaly appears to be due to an initiation factor deficiency. Here, a fraction which stimulated initiation at the correct site, provisionally called "initiation correcting factor" (ICF), was partially purified from Krebs-2 cells. The ICF activity appeared to copurify with a complex of initiation factors eIF-2 and eIF-2B. The ability of ICF to stimulate, in reticulocyte lysates, the correct initiation of translation on the RNAs from neurovirulent and attenuated type 1 and type 3 poliovirus strains was investigated. Like crude initiation factor preparations, ICF appeared to be relatively less active with the RNAs from attenuated strains, the difference being especially pronounced for the type 3 strains. For the latter strains, the data suggested an important role of the nucleotide at, and perhaps around, position 472 in determining a response to the addition of ICF. It is proposed that interaction of a specific segment of the viral RNA with one or more of initiation factors plays an important part in the mechanism of translation of the picornavirus genomes, poliovirus attenuation, and, possibly, pathogenesis of poliomyelitis.

Cell Line↗
PubMed1985

The genomes of attenuated and virulent poliovirus strains differ in their in vitro translation efficiencies.

In mRNA-dependent extracts of Krebs-2 cells, RNAs from attenuated strains of poliovirus type 1 and type 3 exhibited diminished template activity as compared to RNAs from the respective virulent counterparts. This defect appeared to be due to the impaired initiation of viral polyprotein synthesis as evidenced by a relatively low level of accumulation of polypeptide 1a (which corresponds to an NH2-terminal region of the polyprotein) in samples programmed with RNAs from attenuated strains. In reticulocyte lysates, where poliovirus RNA is translated predominantly from abnormal (internal) sites [Dorner et al. (1984) J. Virol. 50, 507-514], this difference in the overall template activity of the attenuated and virulent poliovirus genomes was less pronounced, but the correct initiation (as judged by polypeptide 1a accumulation) was again more efficient on RNAs from virulent strains. It is suggested that template deficiency is a factor contributing to the attenuated phenotype of poliovirus strains studied. A possible involvement of nucleotide sequences located far upstream from the initiator codon in the control of translation of poliovirus genome is briefly discussed.

Cell Line↗
PubMed1984

Differences between translation products of tick-borne encephalitis virus RNA in cell-free systems from Krebs-2 cells and rabbit reticulocytes: involvement of membranes in the processing of nascent precursors of flavivirus structural proteins.

Tick-borne encephalitis virus (TBEV) RNA was translated in extracts from Krebs-2 cells and in rabbit reticulocyte lysates. In the former system, two polypeptides, p53 and p13, corresponding to envelope (E) and core (C) proteins of the virion, respectively, were synthesized preferentially. In contrast, the major product in reticulocyte lysates was represented by a heterogeneous set of high-molecular-weight polypeptides which did not appear to include p53 or p13. The reticulocyte lysates, however, acquired the ability to produce structural proteins (p53 at least) after addition of purified membranes isolated from the rough endoplasmic reticulum of Krebs-2 cells. On the other hand, the ability of Krebs-2 extracts to generate identifiable viral structural proteins was lost after degradation of membranes by the nonionic detergent Triton X-100. These findings strongly suggest that membrane-dependent processing of protein precursors is involved in the formation of TBEV structural proteins. Evidence has been obtained that only nascent precursor polypeptides can be processed efficiently into structural proteins in the membrane-dependent reaction.

Animals↗
PubMed1983

Translational barrier in central region of encephalomyocarditis virus genome. Modulation by elongation factor 2 (eEF-2).

A fractionated cell-free system from Krebs-2 cells was prepared which contained ribosomes and a high-speed supernatant. When this system was programmed with encephalomyocarditis virus RNA, the synthesis of a precursor of capsid proteins, polypeptide preA, proceeded at a rate not very different from that observed in unfractionated extracts, whereas the synthesis of more distally encoded proteins, in particular polypeptide F, was greatly retarded, if not abolished. A protein was purified from the cytoplasmic extracts of Krebs-2 cells which greatly enhanced production of polypeptide F as well as other noncapsid proteins in the fractionated system. By several criteria, this protein was identified as eukaryotic elongation factor 2 (eEF-2). By using the ADP-ribosylation assay, it was found that the fractionated system contained about 15% of the amount of eEF-2 present in the unfractionated extracts. The results suggest that changes in the eEF-2 content may affect the elongation rate differently at different regions of the RNA template.

Cell-Free System↗
PubMed1974

Efficiency of translation of viral and cellular mRNA's in extracts from cells infected with encephalomyocarditis virus.

The relative efficiency of translation of host cell mRNA and encephalomyocarditis (emc) virus RNA has been investigated in cell-free preparations from uninfected and EMC virus-infected Drebs-II cells. The ability of cell-free extracts from infected cells to translate exogenously-added EMC virus RNA and total Krebs-II cell mRNA was markedly diminished, but no evidence for the selective inhibition of translation of host cell mRNA in these systems was obtained.

Animals↗