Characterisation of in vitro translation conditions for messenger RNA prepared from pseudorabies virus infected cell

McGrath, Bernadette M (1978) Characterisation of in vitro translation conditions for messenger RNA prepared from pseudorabies virus infected cell. PhD thesis, University of Glasgow.

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Poly-(A) containing RNA was isolated by affinity chromatography from HeLa cells 5h after infection with Pig Herpesvirus I (Pseudorabies virus PrV) or mock-infection. 54% of the RNA from infected cells was shown by molecular hybridisation to be complementary to virus specific sequences. Both species of poly-(A) containing RNA stimulated incorporation of radioactive amino acids into TCA precipitable material in a wheat germ cell free translation system. It was concluded, therefore, that these RNAs contained significant amounts of mRNA. The In vitro translation products were examined by polyacrylamide gel electrophoresis and fluorography. Preliminary classification of the in vitro products of infected cell mRNA as viral or cellular coded was carried out by comparison of their electrophoretic mobilities with those of the in vitro products of mock-infected cell mRNA. The mobilities of the vitro products were also compared with those of the polypeptides present in infected and mock-infected cell lysates and, with a few exceptions, vivo labelled polypeptides which comigrated with those synthesised in vitro could be identified. Further evidence for the viral origin of eight infected cell mRNA products was obtained by immune precipitation of vitro products with antisera to the major capsid protein of prV and by examining the products synthesised in vitro when infected cell mRNA had been hybridised to PrV DNA prior to addition to the translation system. The mRNAs were also translated in a mRNA dependent reticulocyte lysate and a similar spectrum of products was obtained. This cell free system, however, was more efficient in the synthesis of high molecular weight polypeptides. Translation of the mRNAs in a Krebs II ascites cell free system was also investigated. The in vitro translation systems were used to examine the applicability of the model for virus-induced shut-off of host cell protein synthesis proposed by Carrasco (1977). This hypothesis attributes the virus-induced shut-off to an increase in monovalent cation concentration in infected cells. In the wheat germ system the optimum conditions for translation of infected and mock-infected cell mRNA were similar whereas reticulocyte mRNA and encephalomyocarditis virus RNA showed slightly different requirements. Investigation of the optimum {K+} for translation in the Krebs II ascites system also suggested that translation conditions for infected and mock-infected cell mRNA were similar. Hence it was concluded that changes in the intracellular monovalent cation concentration are unlikely to have a role in the PrV-induced shut-off of host cell protein synthesis. Possible differences in the initiation rates for viral and cellular mRNA were also investigated in vivo. Protein synthesis in cells grown in hypertonic medium was compared with synthesis under isotonic conditions both early (2h) and late (6h) after PrV-infection or mock-infection. After infection with a number of other viruses protein synthesis has been found to be less susceptible to a hypertonic initiation block than synthesis in uninfected cells and the resistant polypeptides have been shown to be virus coded (Nuss et al , 1975). PrV infection did not result in any resistance to this initiation block and it was concluded that PrV mRNA is not more readily translated than HeLa cell mRNA under limiting conditions. Hence it did not seem likely that the PrV-induced inhibition of cellular protein synthesis could be due to an overall inhibition of protein synthesis because under such conditions PrV mRNA translation would be inhibited also. The increase in average polysome size in PrV-infected cells which has been reported by Ben-Porat et al., (1971) and which has also been observed in this laboratory could be due to an increase in the average length of the translated region of infected cell mRNAs. This was investigated by allowing completion in vitro of the nascent polypeptide chains on different size classes of polysomes isolated from cells late after infection or mock-infection. THe results showed that such a model could not account for the increased loading phenomenon. Thus no differences between PrV mrNA and HeLa cell mRNA which might account for the changes in protein synthesis which occur in PrV-infected HeLa cells could be detected.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: W S Stevely
Keywords: Biochemistry
Date of Award: 1978
Depositing User: Enlighten Team
Unique ID: glathesis:1978-72091
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 13:03
Last Modified: 17 May 2019 13:03

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