Pseudorabies virus infection and RNA metabolism in hamster kidney cells

Abrahams, Judith C (1972) Pseudorabies virus infection and RNA metabolism in hamster kidney cells. PhD thesis, University of Glasgow.

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In this thesis, some effects of pseudorabies virus infection on RNA metabolism in BHK-21/C15 cells were examined; in particular the production of 4S RNA and 5S RNA was studied. 1. The concentration of uridine in the medium that allows maximum incorporation of isotopically-labelled uridine into RNA in CI5 cells was determined. 2. A 10% polyacrylamide gel electrophoresis system was calibrated and used to fractionate low molecular weight cytoplasmic RNA, excellent separation between 5S RNA and 4S RNA being obtained. 5. Infection of exponentially-growing cells with pseudorabies virus caused a steady decrease (to 15% of the uninfected level) in incorporation of 3H uridine into RNA by 5 hours after infection. The production of cytoplasmic 28S and 18S RNAs was found to be most strongly inhibited while the production of 4S and 5S RNAs was much less affected; the decrease in synthesis of 4S RNA from either total cytoplasm, cell sap or ribosomes was found to be virtually identical. 4. In some experiments, serum-depleted "resting" cells were used; in these cells DNA synthesis was shown to be <1% of the exponential, level, and the rates of synthesis of 28S, 18S, 4S and 5S RKAs were all found to be decreased. 5. In the uninfected, exponentially-growing CI5 cell, a pulse-label of 3H-uridine for a period of about 30-60 min produces cytoplasmic low molecular weight RNA in which almost all of the radioactivity is in 5S RNA and 4S RNA. It was found that in pseudorabies virus-infected cells, such a pulse-label produced radioactive low molecul8.r weight RM mainly of a size between 5S and 4S. This material eluted as a single heterodisperse peak from a Sephadex G100 column, but resolved on 10% polyacrylamide gel electrophoresis into two peaks; the peaks; nearer RNA was designated species I and the other species II. 6c Severa.1 lines of evidence suggested that species I and II in the pseudorabies virus-infected cell are precursors to tRNA and that they remain so clearly labelled because their maturation to 4S RNA is retarded as a result of virus-infection. Such evidence included experiments involving time-courses of labellings in vivo "chase" experiments using actinomycin D, and in vitro incubation experiments. It was also shown that pre-tRNA from uninfected CI5 cells (pulse-labelled for 5-10 min) electrophoresed on 10% polyacrylamide gels as two peaks, analogous to peaks I and XI from the virus-infected cell, 7. In vitro incubation experiments failed to demonstrate any difference in the ability of cell extracts from virus-infected or uninfected cells to convert precursor RNA to 4S RNA; both extracts successfully matured unpurified or partially-purified species I and II to 4S RNA, These experiments supported evidence from the kinetic experiments that species I my become species II en route to 4S RNA. 8. After infection of "resting" cells with pseudorabies virus, 4S RNA synthesis v/as found not to be decreased and possibly even increased, until late in infection; this may be correlated with appearance of species I Eind II indicating retardation of the maturing process. Alterations in level of methylation and nucleotide composition of 4S RNA after infection of "resting" cells were shown to be negligible. 9. The increase in 5S RNA synthesis, relative to other cytoplasmic RNA species, which occurs after pseudorabies virus-infection of exponentially- growing cells, was found to be due to two factors; (a) the synthesis of ribosome-associated 5S RNA is not strongly decreased after infection (much less than that of the larger rRNAs) and (b) some 5S RKA appears to be a precursor to tRNA; a similar situation may occur in the uninfected cell. 10. After infection of "resting" cells, it was found that ribosome-associated RNA synthesis did not seem to be further decreased. Significant levels of cell sap 5S RNA probably pre-tRNA, appeared late in infection. After infection of "resting" cells, there were no significant alterations in nucleotide composition of 5S RNA or in the sequence as determined by electrophoretic separation of oligonucleotides ("fingerprinting").

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J Hay
Keywords: Virology
Date of Award: 1972
Depositing User: Enlighten Team
Unique ID: glathesis:1972-72741
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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