Induction of enzymes in mammalian cells infected with herpes simplex virus

Perera, Ponnamperuma A. J (1970) Induction of enzymes in mammalian cells infected with herpes simplex virus. PhD thesis, University of Glasgow.

Abstract

Herpes simplex virus and BHK cells provided an excellent system for the study of virus-host cell interactions. The main emphasis of this work was laid on the induction of new enzymes after virus infection. During this investigation, evidence for the induction of a new deoxycytidine kinase was obtained, while there were indications of an isoenzyme. This is the first instance of a virus-induced deocycytidine kinase that has been reported. The principal evidence which led to the above conclusion came from studies on inhibition and heat stability of host and virus-induced deoxycytidine kinases. Deoxycytidine triphosphate at 2 x 10-5 M strongly inhibited host cell deoxycytidine kinase but had very little effect on the virus-induced dCyd kinase. At a ten-fold higher concentration deoxycytidine triphosphate inhibited both host and virus-induced kinases. In contrast, deoxycytidine triphosphate at this concentration activated virus-induced deoxythymidine kinase whilst inhibiting the host deoxythymidine kinase. Deoxythymidine triphosphate was a potent inhibitor of both dCyd and deoxythymidine kinases. At 2 x 10-4M, deoxythymidine triphosphate inhibited host deoxythymidine kinase and deoxycytidine kinases of control arid infected cells but had only a small effect on the virus-induced dThd kinase. This suggested the induction of a new deoxycytidine kinase in BHK cells following herpes infection, which was distinct from host and virus-indiiced deoxythymidine kinases and host deoxycytidine kinase. Additional evidence for the induction of a new deoxycytidine kinase came from the heat sensitivity studies of the pyrimidine deoxyribonucleoside kinases. The substrate-stabilised profiles of these enzymes against heat inactivation were clearly different. Furthermore, mixing studies with control and virus-infected cell extracts proved the absence of a possible activator or an inhibitor in this system which increased the activity of host deoxycytidine kinase following herpes infection. The requirement for the synthesis of protein for the occurrence of this increase in activity came from studies with inhibitors of macromolecular synthesis. The virus-induced deoxycytidine kinase was resolved into two components during fractionation on DEAE-cellulose. In order to understand the functions of the virus-induced pyrimidine deoxyribonucleoside kinases an investigation was carried out into the variation of acid-soluble pool size within the host cell following virus-infection. It was found that the uptake of pyrimidine deoxyribonucleoside precursors into the acid-soluble pool increased in a manner which paralleled the increase in activity of pyrimidine deoxyribonucleoside kinases. The discovery of an increased pool size within the infected cell suggested an alteration of host cell's mechanism of controlling nucleotide synthesis and may explain the requirement for new virus-coded enzymes which are less susceptible to feedback inhibition. The high levels of activity of purine deoxyribonucleoside kinases in the uninfected cell relative to the activity of the pyrimidine kinases and the strong inhibitory effect of deoxyadenosine triphosphate on host DNA synthesis perhaps provide satisfactory reasons for the absence of induction of purine deoxyribonucleoside kinases after herpes infection. The discovery of two possibly virus-coded DNA exonucleases and the high levels of deoxyribonucleoside kinases within the infected cell strongly suggested the control of host DNA synthesis and the regulation of the production of viral progeny DNA through the coordination of virus-induced enzymes. There was a progressive incorporation of labelled host deoxyribonucleotides into four buoyant density bands of DNA in addition to double-stranded herpes DNA, following infection of BHK cells by herpes virus. These appeared simultaneou.sly with the production of progeny virus. One has been tentatively identified as being single- stranded viral DNA. The others have very high buoyant densities and may represent DNA-RNA hybrids. These putative hybrids may represent transcription of late functions of herpes virus. It is likely that cut-off of early virus-induced enzymes which is recognised to be one of the late viral functions may be mediated by the synthesis of such late viral RNA.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J M Morrison
• Keywords: Virology
• Date of Award: 1970
• Depositing User: Enlighten Team
• Unique ID: glathesis:1970-73199
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73199