Intracellular Sites of Synthesis of Encephalomyocarditis Virus Components in Krebs-2 Ascites Tumour Cells

Bellett, A. J. D.; Burness, A. T. H.

doi:10.1099/00221287-30-1-131

Cited by 27 publications

(4 citation statements)

References 13 publications

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“…With these nucleus isolation techniques, it has been shown, in confirmation of earlier work, that NDV, like other RNA viruses (2,13), matures in the cytoplasm and PYV in the nuclei.…”

supporting

confidence: 76%

Application of Cell Fractionation Techniques in the Study of Cells Infected with Polyoma Virus and Newcastle Disease Virus

1966

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Kingston), HIDEMI MATSU-MIYA, AND MASANOBU AZUMA. Application of cell fractionation techniques in the study of cells infected with polyoma virus and Newcastle disease virus. J. Bacteriol. 91:1645-1651. 1966.-Techniques which permitted rigorous separation of nuclei and cytoplasm were applied to the study of the formation of Newcastle disease virus (NDV) in an established line of Chinese hamster cells and of polyoma virus (PYV) in mouse embryo fibroblasts. The results obtained for hemagglutinin and plaque-forming titers during virus growth were in agreement with those obtained by others, using different techniques. These indicated that NDV matures in the cytoplasm, and PYV in the nucleus, of host cells.

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“…With these nucleus isolation techniques, it has been shown, in confirmation of earlier work, that NDV, like other RNA viruses (2,13), matures in the cytoplasm and PYV in the nuclei.…”

supporting

confidence: 76%

Application of Cell Fractionation Techniques in the Study of Cells Infected with Polyoma Virus and Newcastle Disease Virus

1966

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“…There was, however, a late increase in the infectivity of cytoplasmic SDS-extracted RNA. Hours after infection Ir&acellular location of phenol-emtracted RNA There was no long period of accumulation of infective phenol-extracted RNA in the nuclear fraction of L-cells infected by EMC virus, in contrast to our previous results with Krebs-2 ascites cells (Bellett & Burness, 1963); nevertheless, when the results of several experiments were compared it was found that the increase in infective RNA extracted from the nuclear fraction by phenol occurred consistently 30-60 min. before the increase in cytoplasmic infective RNA (Fig.…”

Section: Iintracellular Location Of Sds-extracted Rnacontrasting

confidence: 53%

Sites of Synthesis of Encephalomyocarditis Virus Components in Infected L-Cells

Bellett

Harris

Sanders

1965

Journal of General Microbiology

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SUMMARYL-cells were fractionated at various times after infection with EMC virus and assayed for haemagglutinin, virus and phenol-extractable infective RNA. A second fraction of infective RNA was obtained by sodium dodecyl sulphate (SDS) extraction of the samples after phenol extraction. The infectivity of SDS-extracted RNA increased earlier than that of phenol-extracted RNA, but the maximum titre was less than 10 yo of the maximum titre extracted by phenol. The early rise in SDS-extracted RNA was largely confined to the nuclear fraction, and the infectivity of phenol-extracted RNA appeared to increase in the nuclear fraction before the cytoplasmic fraction. There was a simultaneous increase in the titres of both fractions of RNA, haemagglutinin and virus in the cytoplasmic fraction from 3 to 5 hr after infection, and the maximum titres of infective RNA were found in the cytoplasm 4-54hr after infection. It was concluded that synthesis of viral RNA begins near the nuclear membrane and then spreads outwards through the cytoplasm, but that there is no conclusive evidence as to whether this process begins in the nuclei or the perinuclear cytoplasm.

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“…Strain K2 of EMC virus (10) was grown in Krebs II ascites cells (11) and purified either by the procedure of Burness (12) or, more recently, by that of Kerr and Martin (13). RNA was extracted from purified virus by the method of Kerr et al (14), precipitated with ethanol, dialyzed against oocyte injection medium (2), and stored at -70°.…”

Section: Methodsmentioning

confidence: 99%

Translation of Encephalomyocarditis Viral RNA in Oocytes of Xenopus laevis

Laskey

Gurdon²,

Crawford

1972

Proc. Natl. Acad. Sci. U.S.A.

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RNA from encephalomyocarditis virus was injected into oocytes of Xenopus laevis. After incubation of the oocytes in [35S]methionine, polyacrylamide gel electrophoresis showed that a new series of polypeptides had been synthesized. They were identical in size to the polypeptides that appeared in ascites cells after infection with this virus. Electrophoretic and chromatographic analysis of the methionine-containing tryptic peptides from three of the induced polypeptides confirmed that they were virus-specific. All of the bands that appeared in ascites cells after infection also appeared in oocytes after injection of RNA from the virus. We conclude that Xenopus oocytes translate a mammalian viral mRNA faithfully and extensively, and perform normal post-translational modifications.Cell organelles or solutions of macromolecules can be injected into living oocytes of Xenopus laevis (1, 2). When messenger RNA for globin or calf-lens crystallin is injected into such oocytes, it is translated (2, 3). Gurdon et al. (2) estimated that each molecule of injected globin mRNA is translated once every 5-10 min for 25 hr. Since the purest sources of natural mRNA currently available are RNA viruses, this paper investigates the ability of oocytes to translate a viral mRNA. Encephalomyocarditis (EMC), a picornavirus, was chosen for this study because the virus-specific proteins synthesized in infected cells have been identified (4), and the activity of the virion RNA as a messenger has been studied in vitro (5-9). EMC RNA has only been partially translated in vitro (7,8), and its translation products in vivo undergo an extensive series of post-translational modifications. MATERIALS AND METHODSAscites Cells and EMC Virus. Strain K2 of EMC virus (10) was grown in Krebs II ascites cells (11) and purified either by the procedure of Burness (12) Lyophilized precipitated proteins were oxidized with performic acid (200,1/50-,ug sample) for 1 hr at 0°, then lyophilized twice. Oxidized proteins were digested with 10 yg of TPCK-treated trypsin (Worthington) in 100jul of triethylamine carbonate (pH 9.0) for 1 hr at 370, followed by addition of a further 10 ,ug of trypsin and 3 hr of incubation. Digests were lyophilized twice to remove triethylamine carbonate, suspended in electrophoresis buffer, and applied to 20 X 40 cm Polygram CEL 300 cellulose-coated thin-layer sheets for electrophoresis at pH 3.5 (5% acetic acid-0.5% pyridine) for 2 hr at 1 kV (25 V/cm). After location of peptides by autoradiography, the cellulose layer was divided into zones, scraped off, and chromatographed on cellulose thin layers in butanol-pyridine-acetic acid-water 5:1:4:4 (18).

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Intracellular Sites of Synthesis of Encephalomyocarditis Virus Components in Krebs-2 Ascites Tumour Cells

Cited by 27 publications

References 13 publications

Application of Cell Fractionation Techniques in the Study of Cells Infected with Polyoma Virus and Newcastle Disease Virus

Application of Cell Fractionation Techniques in the Study of Cells Infected with Polyoma Virus and Newcastle Disease Virus

Sites of Synthesis of Encephalomyocarditis Virus Components in Infected L-Cells

Translation of Encephalomyocarditis Viral RNA in Oocytes of Xenopus laevis

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