Messenger RNA synthesis in HeLa cells infected with pseudorabies virus

Vass, John Keith (1975) Messenger RNA synthesis in HeLa cells infected with pseudorabies virus. PhD thesis, University of Glasgow.

Abstract

Polysomes isolated from HeLa cells infected with pig herpesvirus 1 (pseudorabies virus or PrV) were found to sediment more rapidly than those isolated from mock-infected cells. This result has been previously reported to occur in rabbit kidney cells, infected with PrV (Ben-Porat et al., 1971). These authors also reported that polysomes disaggregate after infection with PrV, and this was also noted in this study and the effect was quantitated, for the first time. The effects of the metabolic inhibitors actinomycin D and cordycepin were also investigated and both were found to increase the rate of polysome disaggregation in infected cells. These inhibitor studies could be interpreted in terms of the theory of Leibowitz & Penman (1971), who postulated that the disaggregation of cellular polysomes in HeLa cells infected with poliovirus was at least partly due to the inhibition of the synthesis of a small molecular weight cellular ENA species, with a translational control function. Polyadenylated RNA was isolated from whole polysomal RNA by poly=(U) sepharose chromatography and shown to contain mRNA by its ability to stimulate the incorporation of labelled amino acids into protein in a cell free system. The polyadenylated ENA was characterised by polyacrylamide gel electrophoresis and found to be heterogeneous in size in both infected and mock-infected cells. Polysome size did not mirror the average size of mRNA - large mRNA species were found on small polysomes and small mRNA species were found in large polysomes. The most likely explanation of this was thought to be that the frequency of initiation of polypeptide synthesis was the rate limiting process in protein synthesis, and that mRNAs with a high frequency of initiation would he associated with a larger number of ribosomes than those with a low initiation frequency. The PAGE profiles showed that mRNA in infected and mock-infected situations were of similar size; this together with the larger polysome size in infected cells indicates that more ribosomes may be found per unit length of viral mRNA than on the same length of cellular mRNA. It is possible that one mechanism whereby the switch from cellular to viral protein synthesis takes place is that viral mRNA promotes more efficient initiation than cellular mRNA. Polysomal RNA synthesis was monitored in cells infected in the presence or absence of cycloheximide. This inhibitor of eukaryote protein synthesis was included to find if any species of viral RNA, detectable by PAGE, were only present if viral proteins had first been synthesised. Two species of polysomal RNA which normally appear around 3 - 4 hours post-infection, were still not synthesised by 5 hours postinfection in the presence of cycloheximide. Using molecular hybridisation techniques Rakusanova et al. (1971) found that only a subset of the viral RNA sequences normally present late in infection were synthesised in cells infected in the presence of cycloheximide. The specific effect of cycloheximide, reported in this thesis, confirms the results of Rakusanova et al. (1971) by another technique. This result shows that the production of viral mRNA is a controlled process and that some mRNA species do not appear in the cytoplasm until viral protein synthesis has begun. The first group of viral proteins probably affect the transcription or post-transcriptional processing of later classes of mRNA.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Bill Stevely
• Keywords: Biochemistry, Virology
• Date of Award: 1975
• Depositing User: Enlighten Team
• Unique ID: glathesis:1975-72841
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 11 Jun 2019 11:06
• URI: https://theses.gla.ac.uk/id/eprint/72841