Study of the relationship between virus and host cell

The author began work in the virus field at the University of Edinburgh in 1946 in collaboration with Dr. C. E. van Rooyen. Interest was first centred in the pro - blem of obtaining fowl pox virus in a highly purified state and subjecting it to chemical and physical analysis. After some months of work it became apparent that the most interesting and fundamental problem in virology was the process of viral multiplication, that this was a study in biochemical dynamics and that the collection of analytical data for another purified virus was not likely o contribute much to its solution. At that time the view that virus multiplication was similar to that of bacteria was still widely held, but it seemed to the author that this hypothesis was open to doubt.While most bacteria have the complete metabolic apparatus which will enable them to grow in a liquid medium often remarkably simple in composition, and can adapt themselves to grow under a variety of conditions, no virus has yet been found to grow in the absence of its host cell. Since virus particles apparently have none of the metabolic functions required for growth, it followed that during their intracellular growth the host must play more than a purely passive role. The idea was then conceived that the virus attached itself to the host and from that time on directed the course of intracellular reactions. Under this influence the cell was supposed to cease its synthesis of cell constituents and instead synthesize new virus. The onus for virus production was thereby placed entirely on the cell and the invading particle took little active part after initiating the infection. While this was a considerable departure from prevailing views, there were already indications in the literature that such a mechanism had been considered by others (Green, Science, 82, 443, 1935; Laidlaw, Virus Diseases and Viruses, Cambridge Univ. Press, 1938). It is now known that a number of research groups in Europe and North America were working with a similar hypothesis in mind at the end of 1946 andlas will be shown later, this hypothesis in a more elaborate form #has now gained wide acceptance.Work was begun by the author in 1947 at the University of Toronto to determine whether it might be possible to label an animal or bacterial virus with P32. If this proved successful, it was proposed firstly to study the behaviour of the labelled virus particle on infection of its host and secondly to investigate intracellular virus synthesis utilizing isotope techniques.The main technical requirements in such a study were (a) a virus host system which was relatively simple and subject to experimental control; (b) a virus that could be grown readily in high yield, was easily purified from host constituents and whose chemical and physical properties had already been worked out in some detail; and (c) a virus that contained one per cent or more of phosphorus so that it would take up sufficient P32 during growth to make the labelled virus a useful experimental tool. Two systems satisfied these requirements, (1) influenza virus adapted to growth in the embryonated egg and (2) the seven T bacteriophages active on Escherichia coli. Work was therefore initiated on both systems.It was found that influenza virus could be labelled with P³² by growing it in the allantoic membrane of the embryonated egg with inorganic p³² placed in the allantoic fluid. Control experiments and chemical analysis indicated that the label was incorporated into the virus during its growth, and there was no direct exchange of P³² between virus and inorganic phosphate. Chemical analyses of the phosphorus constituents in allantoic membrane were also carried out in preparation for experiments on membrane infected with labelled virus.The remainder of this thesis is concerned with research on the set of seven bacteriophages, active on the host E. coli, commonly referred to as the T group. When the present work was begun more exact knowledge had been accumulated about the biological properties of bacteriophages than any other type of virus. Bacteriophages are relatively easy to handle and had attracted the attention of a number of outstanding workers among whom d'Herelle, Burnet, Craigie and Delbruck were pre-eminent.Each of the T phages can mutate during growth in a number of known ways and probably a variety of unknown ways. The host bacteria also mutate and it is relatively easy to obtain variants of the original strain resistant to one or more of the seven phages. Some of the mutants of both phage and cells are extraordinarily useful as experimental tools in biochemical experiments and the problems involved in the mutation of the phages provide the main approach to the genetic side of the problem.As described in the present work, it was found that T2 bacteriophage could readily be labelled with P²³ and that at least 95% of the isotope was contained in the desoxyribonucleic acid moiety of the virus. when the cell was infected with the labelled virus there was a rapid and extensive breakdown of the particle although up to 350 of the P²³ appeared in the viral progeny. The extent of breakdown and the contribution of parental P²³ to progeny varied considerably with experimental conditions and were studied in some detail. In a later section the significance of these findings is discussed in the light of recent knowledge on virus multiplication