Aspects of protein synthesis in pseudorabies virus infected cells

Chowdhury, K. B.M. Mamun R (1980) Aspects of protein synthesis in pseudorabies virus infected cells. PhD thesis, University of Glasgow.

Abstract

Inhibition of protein synthesis has been studied in cells infected with PRV. Infection of HeLa, C13 and BS-C-1 cells with PRV showed that in the case of HeLa and C13 cells inhibition was rapid and substantial but for B-SC-1 cells it was gradual and apparently limited. Moreover, C13 and BS-C-1 cells showed some stimulation of protein synthesis at 1 hr post-infaction with subsequent inhibition. Because of the simple Inhibition kinetics, HeLa cells were mostly used for further studies. Cell-free translation studies were carried out using mRNA from PRV infected cells. Immediate-early mRNA's (mRNA made in the infected cells before any viral protein synthesis) and late mRNA's were Isolated from Hela cells infected with PRV for 5 hrs in the presence and in the absence of Cycloheximide respectively. Hybridisation data for the poly(A) containing RNA showed the presence of, at least, 5% of viral transcripts in the Immediate-early mRNA preparation. Cell-free translation of this preparation in the Messenger Dependent reticulocyte lysate system showed the presence of some viral proteins but a large number of host proteins were also synthesized. Simultaneous addition of both viral and host mRNA's did not result in the preferential translation of viral mRNA in the cell free system and both cytoplasmic and polysomal late poly(A) RNA's allowed the synthesis of the same spectrum of proteins. K+ dependence studies in the MDL system did not reveal any difference in the optima for the translation of host, IE and late mRNA's though polyacrylamide gel electrophoresis of the translation products showed that at least one host and one viral protein were slightly resistant to high K+ concentrations in the system. Studies in the wheat germ extract showed an identical K+ optima for all these mRNA's. Again, protein synthesis vivo under hypertonic conditions showed that synthesis of both host and IE proteins were susceptible to elevated salt concentrations to the same extent. When protein synthesis was studied in the presence of inhibitor of elongation rate, viral mRNA's were not apparently more efficient than host mRNA's in initiating protein synthesis. So there appears no selectivity in the inhibition of host protein synthesis. Investigations of the inhibition of protein synthesis revealed that both PRV and HSV-1 caused marked polysome disaggregation in the absence of viral protein synthesis. There was an Increase in the extent of polysome disaggregation by PRV when the number of infecting virus particles was increased. Moreover, heat and u.v. inactivated PRV caused substantial disaggregation of host polysomes. In similar studies, both normal, infective and inactivated PRV caused an almost equal extent of inhibition of protein synthesis at 8 hrs post infection. By 24 hrs post infection, infective virus produced cytopathic effects whereas inactivated viruses did not. Again, infective viruses caused about 80% inhibition of RNA synthesis by 8 hrs post infection whereas by that time inactivated-viruses caused only 25% inhibition. In the cell-free translation system, exogenous mRNA translation was inhibited by both normal and Inactivated PRV and this inhibition was not due merely to addition of exogenous proteins. The extent of inhibition increased with the addition of increasing amounts of both normal and inactivated PRV. So inhibition of protein synthesis is caused by the virus particles which causes polysome disdggregation and might act in some other steps of protein synthesis. By 6 hr post infection there was induction of an RNase activity which under cell-free conditions selectively degraded host mRNA. No mRNase activity was associated with either normal or inactivated PRV. These experiments revealed that in PRV infected HeLa cells, translatable host mRNA's are present when IE proteins are being made. There is no evidence of any selective translation of viral mRNA either in the Intact cell or in cell-free systems. The only evidence for selectivity resides in the mRNase which degrades host mRNA but not viral mRNA. Again in the Intact cell or in cell-free systems, inhibition of protein synthesis is caused by the virus particles themselves and does not involve infected cell protein synthesis. This inhibition also involves polyribosome disaggregation by the virus particles.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Virology.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Stevely, Dr. W.S.
• Date of Award: 1980
• Depositing User: Enlighten Team
• Unique ID: glathesis:1980-72051
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 13:14
• Last Modified: 03 Aug 2022 13:13
• Thesis DOI: 10.5525/gla.thesis.72051
• URI: https://theses.gla.ac.uk/id/eprint/72051