UBC Theses and Dissertations
Genome properties and non-productive infections of herpesviruses Mosmann, Timothy Richard
Herpesvirus infections have been studied with the purpose of establishing conditions under which control of latent infection could be investigated. Several relevant properties of the viruses were also studied. Murine cytomegalovirus (MCV) preparations grown in vitro in mouse embryo cells contained large numbers of multiple capsid virions. These multiples were apparently infectious, but were not the main cause of the unusual enhancement of infectivity of MCV caused by application of centrifugal force during adsorption. The DNA of three herpesviruses was studied. Herpes simplex (HSV) DNA did not contain a significant number of cross-links. By comparison with bacteriophage T4-DNA (molecular weight 110 x 10⁶) after neutral sucrose gradient sedimentation, the molecular weights of MCV and HSV-DNA's were calculated as 132 x 10⁶ and 85 x 10⁶ respectively. Both these viral DNA's contained alkali sensitive single strand interruptions, and in the case of MCV-DNA it was found that the number of interruptions was dependent on the purification procedure, and preparations were obtained in which some molecules were free, of alkali sensitive regions in both strands. Human cytomegalovirus preparations contained a DNA component with an approximate molecular weight of 3 x 10⁸. MCV-DNA was analyzed by centrifugation to equilibrium in cesium chloride solution, and evidence obtained for a heterogeneous distribution of density along the molecule. Intact molecules banded at a single density corresponding to a G + C content of 59%, whereas fragmented molecules of 18 x 10⁶ molecular weight banded as two components, at densities corresponding to 57.5 and 61.5% G + C. This heterogeneity was confirmed by analysis of UV-absorbance/ temperature profiles of MCV-DNA, which provided evidence for two or possibly three components present in unequal amounts. Evidence was obtained from DNA-RNA hybridization studies to suggest that there was differential transcription or degradation of the viral RNA synthesized during infection, from the two DNA components separated on cesium chloride gradients. After infection of a continuous line of human cells with MCV, the titre of infectious virus rapidly declined to undetectable levels. The growth rate of the infected cells decreased, and the morphology of the cells was altered. Both these changes lasted for a few weeks, after which the cultures reverted to the normal growth rate and morphology. DNA-DNA annealing was used in an attempt to detect the presence and synthesis of viral DNA in these cells. In most cases, neither the presence nor absence of MCV-DNA at the level of one genome per cell genome, could be established, but in one experiment, synthesis of viral DNA apparently occurred in MCV infected cells lU days after infection. Viral RNA synthesis in these cells was undetectable, i.e. less than two parts in 10⁵ of the total RNA synthesis.
Item Citations and Data