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E Ehrenfeld

Publications and source records attributed to E Ehrenfeld.

At least 91 records · Page 5Linked to original sources
PubMed1986

Synthesis of plus- and minus-strand RNA from poliovirion RNA template in vitro.

The poliovirus RNA polymerase, 3Dpol, was used to synthesize RNA in vitro in the presence of a host factor preparation from uninfected HeLa cells and poliovirion RNA as the template. The transcription products included molecules approximately twice the length of the template, apparently resulting from hairpin formation and template-directed elongation, as previously reported (D. C. Young, D. M. Tuschall, and J. B. Flanegan, J. Virol. 54:256-264, 1985). Other polyadenylated template RNAs also yielded products that were twice the length of the template. The polarity of the products synthesized from plus-strand poliovirus RNA template was analyzed by Southern blotting using labeled product RNA to probe single-stranded poliovirus DNAs cloned into M13 vectors. The results demonstrated that host factor-mediated polymerase products contain newly synthesized plus-strand sequences as well as the expected minus-strand sequences. Polymerase products primed with oligo(U) were all of minus-strand polarity.

Base Sequence↗
PubMed1985

Poliovirus protease does not mediate cleavage of the 220,000-Da component of the cap binding protein complex.

Poliovirus infection of HeLa cells results in a rapid shutoff of host protein synthesis but does not inhibit the translation of poliovirus mRNA. It has been suggested that this virus-induced translational control is mediated by the inactivation of a cap binding protein (CBP) complex, and it has been shown that the 220,000-Da component(s) (p220) of the CBP complex is cleaved in infected HeLa cells to form antigenically related peptides of 100,000-130,000 Da. To determine whether the known viral protease (peptide 3C) was the mediator of the cleavage of p220, we used immunoblot techniques to analyze partially purified infected HeLa cell extracts for cleavage activity. We report here that p220 cleavage activity does not copurify with viral peptide 3C or with any precursors containing 3C sequences. We also show that cleavage of p220 can be demonstrated in vitro in HeLa cell extracts under conditions where the functional activity of the poliovirus protease is inhibited by specific antibody.

Carrier Proteins↗
PubMed1984

Two forms of poliovirus VPg result from amino acid modification of a single viral protein.

The protein (VPg) covalently attached to the 5' terminus of poliovirus RNA has been reported to resolve into two forms, separable by electrofocusing, yet the viral gene sequence predicts only one apparent gene for VPg. The two VPg species were separated and analyzed for structural differences. The protein contains no phosphorylated amino acid residues other than the junction tyrosine linked to the nucleic acid moiety. After isolation, the acidic form of VPg remains stable, but the more basic form again generates a distribution of both species. This suggests some lability or modification of at least one amino acid residue postsynthesis. The position of the alteration in the protein was localized to the amino-terminal tryptic peptide, containing nine amino acids.

Amino Acid Sequence↗
PubMed1984

Structure of poliovirus replicative intermediate RNA. Electron microscope analysis of RNA cross-linked in vivo with psoralen derivative.

The structure of the poliovirus replicative intermediate RNA was examined by electron microscopy after cross-linking in vivo with 4'-aminomethyl-4,5',8-trimethylpsoralen. After purification from infected cells, undenatured RI appeared as a double-stranded backbone of genome length, with an average of three (and occasionally up to eight) nascent, single-stranded tails. After denaturation, however, only single strands of heterogeneous length were visualized, indicating that the RI in the cell contains little or no duplex structure, and thus nascent chains are only transiently hydrogen-bonded to their template over short regions. The double-stranded backbone of undenatured RI, observed previously by others and in these experiments, is due to collapse of complementary chains during the deproteinization and purification procedures. The effectiveness of the in vivo cross-linking procedure was demonstrated by the complete inhibition of viral RNA synthesis in treated cells and by direct binding of [3H]AMT to RI molecules in vivo. Mature polio virions are impermeable to AMT; however, growth of virus in cells incubated with AMT in the dark resulted in normal yields of virus particles containing RNA genomes, whose infectivity could be subsequently photo-inactivated. The frequency of AMT-induced cross-linking was determined by analyses of double-stranded poliovirus RNA (RF). Cross-linking in vitro followed by spreading for electron microscopy under denaturing conditions yielded bubbled duplex structures with a minimum of one interstrand cross-link per 80 base-pairs. RF cross-linked in vivo also showed extensive cross-linking, decreased about fivefold from the in vitro cross-linked value. Thus, the failure to detect cross-linked RI under these conditions indicates that extensive base-pairing does not exist in vivo.

Base Composition↗
PubMed1984

Demonstration in vitro that eucaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus-infected HeLa cells.

Protein synthesis initiation factor preparations from poliovirus-infected HeLa cells have reduced ability to initiate translation on capped mRNA. The defect in initiation factors has been variously attributed to inactivation of eucaryotic initiation factor 3 (eIF3), eIF4B, or a cap-binding protein (CBP) complex. We have developed a series of in vitro protein synthesis assays to show that eIF3 is active but a CBP complex activity is inactivated after poliovirus infection. eIF3 activity, when determined in the presence of purified CBP complex, is present in sucrose gradients of factors from both infected and uninfected cells. CBP complex activity, determined in the presence of eIF3 from poliovirus-infected cells, is present in uninfected cells only and comigrates on sucrose gradient with an activity which restores the ability of crude initiation factors from infected cells to translate capped globin mRNA. This is the first demonstration by a fractionated translation system that an activity which is attributable to CBP complex is inactivated in poliovirus-infected cells. The results also indicate that eIF3 is undetectable or has greatly reduced activity in the absence of CBP complex.

Carrier Proteins↗
PubMed1983

The effect of poliovirus infection on the translation in vitro of VSV messenger ribonucleoprotein particles.

Messenger ribonucleoprotein (mRNP) particles were isolated and examined for the presence of factors involved in the inhibition of protein synthesis induced by poliovirus infection. Vesicular stomatitis virus (VSV) mRNPs were used as a model for cellular mRNPs. These mRNPs were translated in HeLa cell extracts with a similar efficiency and optimal conditions to that of purified mRNA, but they were not translated in extracts prepared from poliovirus-infected HeLa cells, which have been shown to be defective in cap-binding protein activity. We conclude that mRNP proteins do not include cap-binding protein activity, since the mRNPs were not able to bypass the restriction on translation of capped mRNAs in polio-infected cell extracts. In addition, VSV mRNPs were isolated from polio-superinfected cells, in which their translation was inhibited. These mRNPs were translated in vitro as well as normal VSV mRNPs. No evidence of a modification or a blocking factor on the mRNPs which prevented their translation following polio infection was observed. Thus, within the limits of the in vitro translation assays used, no factors involved in the discrimination between polioviral and cellular or VSV mRNA could be detected in the mRNP particle.

Carrier Proteins↗
PubMed1983

Analysis of Theiler's virus isolates from persistently infected mouse nervous tissue.

The DA strain of Theiler's virus causes a chronic progressive demyelination in mice following intracerebral inoculation. Virus was isolated from chronically infected mice, and then grown in cell culture, and the isolates were compared with the parent virus used for inoculation. No defective interfering particles or temperature-sensitive virus were recovered, and capsid proteins appeared identical by SDS-PAGE. One of three isolates had evidence of genomic mutation by Tl ribonuclease oligonucleotide fingerprinting. The significance of these findings with regard to the generation and maintenance of persistence and to adaptation to cell culture is discussed. Also of interest was the marked difference between the DA fingerprint and that of GD VII, a serologically related strain with different biological activity.

Animals↗
PubMed1982

Genetic and physiological analysis of conjugation in Streptococcus faecalis.

In an effort to elucidate the mechanisms of conjugal plasmid transfer in Streptococcus faecalis, a genetic analysis of the sex pheromone-dependent tetracycline resistance plasmid pCF-10 was initiated. Rare transconjugants obtained from short matings with wild-type donors not exposed to sex pheromones were screened for increased donor potential in a subsequent mating. From this screening, a mutant plasmid, designated pCF-11, whose transfer functions are expressed in the absence of pheromone induction was isolated. Cells carrying pCF-11 spontaneously clump when grown in broth culture but do not excrete sex pheromones active against wild-type donors. In the course of initial experiments, it was observed that physiological conditions could affect plasmid transfer frequency. Therefore, a set of standardized optimal mating conditions was defined. The experiments carried out to determine these conditions revealed that a transient increase in transfer frequency of about 2 order of magnitude occurred in early-exponential-phase donor cells. This peak of activity is independent of sex pheromone response, since it was observed with induced or uninduced donor cells carrying either pCF-11 or pCF-10.

Conjugation, Genetic↗
PubMed1982

Association of cap-binding protein with eucaryotic initiation factor 3 in initiation factor preparations from uninfected and poliovirus-infected HeLa cells.

Extracts from poliovirus-infected HeLa cells are unable to translate vesicular stomatitis virus or cellular mRNAs in vitro, probably reflecting the poliovirus-induced inhibition of host cell protein synthesis which occurs in vivo. Crude initiation factors from uninfected HeLa cells are able to restore translation of vesicular stomatitis virus mRNA in infected cell lysates. This restoring activity separates into the 0 to 40% ammonium sulfate fractional precipitate of ribosomal salt wash. Restoring activity is completely lacking in the analogous fractions prepared from poliovirus-infected cells. The 0 to 40% ammonium sulfate precipitates from both uninfected and infected cells contain eucaryotic initiation factor 3 (eIF-3), eIf-4B, and the cap-binding protein (CBP), which is detected by means of a cross-linking assay, as well as other proteins. The association of eIF-3 and cap binding protein was examined. The 0 to 40% ammonium sulfate precipitate of ribosomal salt wash from uninfected and infected cells was sedimented in sucrose gradients. Each fraction was examined for the presence of eIF-3 antigens by an antibody blot technique and for the presence of the CBP by cross-linking to cap-labeled mRNAs. From uninfected cells, a major proportion of the CBP cosedimented with eIF-3; however, none of the CBP from infected cells sedimented with eIF-3. The results suggest that the association of the CBP with eIF-3 into a functional complex may have been disrupted during the course of poliovirus infection.

Carrier Proteins↗
PubMed1982

Characterization of a cell culture persistently infected with the DA strain of Theiler's murine encephalomyelitis virus.

We established a persistent infection in L 929 cells with the DA strain of Theiler's murine encephalomyelitis virus. Our studies showed that only a small number of cells in the cultures contained infectious virus or viral antigen. A role for interferon in the maintenance of persistence was suggested. Viral isolates from the cultures were not temperature sensitive, nor did they contain viral capsid polypeptide mutations or defective interfering particles. T1 oligonucleotide maps showed evidence of mutation in two of three isolates.

Animals↗
PubMed1982

Localization of cap-binding protein in subcellular fractions of HeLa cells.

The 26,000-M(r) cap-binding protein was analyzed by a cross-linking assay in cell fractions from uninfected and poliovirus-infected HeLa cells. Cap-binding protein was found in the postribosomal supernatant (S-200) and in the ribosomal salt wash. The cap-binding protein in the S-200 had a sedimentation coefficient of 5 to 7S and lacked the ability to restore translation in extracts of poliovirus-infected cells.

Cell Fractionation↗
PubMed1981

Stability of poliovirus RNA in cell-free translation systems utilizing two initiation sites.

The stability of purified poliovirus RNA in cell-free translation systems prepared from HeLa cells or rabbit reticulocytes has been examined. Degradation of the RNA occurs with a t1/2 of approximately 35 min at 30 degrees C under conditions used for in vitro translation. Degradation is due in part to activity in the cell lysate, and in part to contaminants in the commercial preparations of creatine phosphokinase used in the energy-regenerating system. Addition of crude preparations of initiation factors significantly slows degradation, presumably as a result of protein-RNA interactions which confer resistance to nuclease action. Prior treatment of RNA with methylmercury hydroxide has no effect on degradation rates. On the other hand, endogenous mRNA, present as a messenger ribonucleoprotein particle in extracts from poliovirus-infected HeLa cells, remains completely intact during in vitro translation. These infected cell extracts synthesize the normal complement of viral proteins and utilize two different initiation sites for translation. Treatment of the infected cell extract with micrococcal nuclease destroys the endogenous mRNA. Subsequent addition of exogenous RNA to the same extract results in the formation of a protein-associated RNA particle with sedimentation properties slightly different from the endogenous messenger ribonucleoprotein, and the added RNA is unstable. We conclude that two initiation sites can be utilized on intact poliovirus mRNA, and fragmentation of the RNA is not prerequisite for generation of a second site in this RNA.

Kinetics↗
PubMed1981

Presence of the cap-binding protein in initiation factor preparations from poliovirus-infected HeLa cells.

Crude preparations of initiation factors from mock-infected and poliovirus-infected HeLa cells were analyzed for the presence of proteins which could be cross-linked to the 5' cap group of mRNA. A protein having an apparent molecular weight of 26,000, similar to the cap-binding protein in rabbit reticulocytes described by Sonenberg and Shatkin (Proc. Natl. Acad. Sci. U.S.A. 75:4843-4847, 1978), was found in the ribosomal salt wash from both uninfected and infected cells. Cross-linking of this polypeptide was inhibited by the cap analog m7GMP. In addition, cross-linking of a protein having an approximate molecular weight of 60,000 was similarly inhibited by cap analog. The smaller cap-binding protein fractionated in a 0 to 40% ammonium sulfate precipitate of ribosomal salt wash; the larger protein was found in the 40 to 70% ammonium sulfate fraction. Although the cap-binding proteins were present in both mock-infected and poliovirus-infected ribosomal salt wash, only preparations from uninfected HeLa cells were able to restore translation of capped vesicular stomatitis virus mRNA by extracts prepared from poliovirus-infected cells.

Carrier Proteins↗
PubMed1981

Two forms of VPg on poliovirus RNAs.

The protein (VPg) linked to the 5' termini of poliovirus RNAs resolved into two species when subjected to non-equilibrium electrofocusing. The differently charged forms of VPg were not due to protein phosphorylation nor to variability of the number of phosphate residues associated with the nucleotide moiety remaining after RNase digestion of the nucleoprotein. Single-stranded viral RNA isolated from mature virions contained predominantly the more basic form of VPg, whereas unpackaged single-stranded RNa remaining in cells at the end of the virus replication cycle contained predominantly the more acidic form of VPg. Replicative-form (RF) molecules also contained both species of VPg, with the more acidic form representing the major species. Both plus and minus RNA strands in RF had similar VPg compositions; however, there appeared to be a strongly selective loss of VPg from only the minus strand in RF, particularly at late times postinfection.

HeLa Cells↗