Separation of quantitation of intracellular forms of poliovirus RNA by agarose gel electrophoresis

Hewlett, Martinez J.; Rozenblatt, Shmuel; Ambros, Victor R.; Baltimore, David

doi:10.1021/bi00631a027

Cited by 39 publications

(15 citation statements)

References 19 publications

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“…One picogram of poliovirus RNA corresponds to ϳ250,000 genomes. encapsidated (22). Nonetheless, the two sets of values clearly followed similar trends, as RNA yields and specific virus infectivities declined with increased numbers of replacement codons.…”

Section: Vol 80 2006 Deoptimization Of Synonymous Codon Usage In Pomentioning

confidence: 70%

Modulation of Poliovirus Replicative Fitness in HeLa Cells by Deoptimization of Synonymous Codon Usage in the Capsid Region

Burns

Shaw

Campagnoli

et al. 2006

J Virol

228

282

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We replaced degenerate codons for nine amino acids within the capsid region of the Sabin type 2 oral poliovirus vaccine strain with corresponding nonpreferred synonymous codons. Codon replacements were introduced into four contiguous intervals spanning 97% of the capsid region. In the capsid region of the most highly modified virus construct, the effective number of codons used (N C ) fell from 56.2 to 29.8, the number of CG dinucleotides rose from 97 to 302, and the G؉C content increased from 48.4% to 56.4%. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased in proportion to the number of replacement codons. Plaque areas decreased over an ϳ10-fold range, and virus yields decreased over an ϳ65-fold range. Perhaps unexpectedly, the synthesis and processing of viral proteins appeared to be largely unaltered by the restriction in codon usage. In contrast, total yields of viral RNA in infected cells were reduced ϳ3-fold and specific infectivities of purified virions (measured by particle/PFU ratios) decreased ϳ18-fold in the most highly modified virus. The replicative fitness of both codon replacement viruses and unmodified viruses increased with the passage number in HeLa cells. After 25 serial passages (ϳ50 replication cycles), most codon replacements were retained, and the relative fitness of the modified viruses remained well below that of the unmodified virus. The increased replicative fitness of high-passage modified virus was associated with the elimination of several CG dinucleotides. Potential applications for the systematic modulation of poliovirus replicative fitness by deoptimization of codon usage are discussed.The use of synonymous codons at unequal frequencies, the codon usage bias, is characteristic of all biological systems (26,27). The strength and direction of codon usage bias are related to the genomic GϩC content and the relative abundance of different isoaccepting tRNAs (reviewed in references 1, 16, and 53). Codon usage can affect the efficiency of gene expression. In bacteria (Escherichia coli) (26, 75), yeast (Saccharomyces cerevisiae) (5, 27), plants (Arabidopsis thaliana) (12), nematodes (Caenorhabditis elegans) (16), and insects (Drosophila melanogaster) (50), the most highly expressed genes use codons matched to the most abundant tRNAs (2). In contrast, in humans and other vertebrates, codon usage bias is much more strongly correlated with the GϩC content of the isochore where the gene is located (51, 71) than with the breadth or level of gene expression (16) or the number of corresponding tRNA genes (28, 30). Despite the weak correlation between codon usage and the levels of gene expression in mammalian cells (16,71), imbalances between codon usage and tRNA abundance can sharply reduce the levels of gene expression.Optimization of codon composition is frequently required for the efficient expression of genes in heterologous host systems (3,31,67,76). For example, the expression of human immunodeficiency virus type 1 ...

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Section: Vol 80 2006 Deoptimization Of Synonymous Codon Usage In Pomentioning

confidence: 70%

Modulation of Poliovirus Replicative Fitness in HeLa Cells by Deoptimization of Synonymous Codon Usage in the Capsid Region

Burns

Shaw

Campagnoli

et al. 2006

J Virol

228

282

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“…Fig. 4(a) shows that, as the infection proceeded, the ratio of Rfto 35S FMDV RNA increased to approximately 1 after 4 h as judged by ethidium bromide staining [Hewlett et al (1977) showed that poliovirus single-stranded RNA and double-stranded RNA gave similar yields of (Fig. 4b).…”

Section: The Replicative Form Of Fmdv Is Less Infectious Than 35s _rnmentioning

confidence: 94%

Studies on the Infectivity of Foot-and-Mouth Disease Virus RNA using Microinjection

Belsham

Bostock²

1988

Journal of General Virology

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“…Ribosomal RNA (2.0 A260 units) was incubated with 2 I~g of protein sample in 30 Ixl ofS0 mM-Tris-HC1 pH 7.6 containing 50 mM-KC1 and 5 mM-EDTA at 37 ~ for 15 minutes. After incubation, the RNA was analyzed by electrophoresis on a 1.0% agarose gel (14). The ribonuclease activity on homopolynucleotides was measured in 100 ~tl of 50 mM-Tris-HC1 pH 7.5 containing 50 mM-KC1, 50 ~tg of homopolynucleotide and 10 Ixg of protein as previously described (21).…”

Section: Analytical Proceduresmentioning

confidence: 99%

Isolation and characterization of inhibitors of animal cell-free protein synthesis from barley seeds

Asano

Svensson

Poulsen

1984

Carlsberg Res. Commun.

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Three inhibitors of animal cell-free protein synthesis were isolated from barley seeds by ammonium sulfate fractionation, CM-eellulose chromatography and gel filtration on Bio-Gel P-60. The three inhibitors were all single chain, basic proteins with molecular weights about 31,000 and with very similar amino acid compositions. In the rabbit reticulocyte lysate system, they inhibited in vitro translation to 50% at concentrations between 15 and 25 ng. ml", while in the wheat germ system, their effect was very weak. The three translation inhibitors showed no detectable ribonuclease activity neither on ribosomal RNA from rabbit liver nor on homopolynueleotides.

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Separation of quantitation of intracellular forms of poliovirus RNA by agarose gel electrophoresis

Cited by 39 publications

References 19 publications

Modulation of Poliovirus Replicative Fitness in HeLa Cells by Deoptimization of Synonymous Codon Usage in the Capsid Region

Modulation of Poliovirus Replicative Fitness in HeLa Cells by Deoptimization of Synonymous Codon Usage in the Capsid Region

Studies on the Infectivity of Foot-and-Mouth Disease Virus RNA using Microinjection

Isolation and characterization of inhibitors of animal cell-free protein synthesis from barley seeds

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