Date of Award
Doctor of Philosophy (PhD)
Richard M. Stenberg
Timothy J. Bos
Ann E. Campbell
An Escherichia coli lac repressor-based system was developed to study the roles of human cytomegalovirus (HCMV) genes during viral replication. To this end, a recombinant HCMV expressing the lac repressor was generated (RVlac), and an HCMV-specific promoter was targeted for conditional expression by inserting the lac operator sequence. The promoter of a nonessential gene was chosen in order to be able to assess parameters of repression and derepression of the operator-containing promoter in the endogenous locus, without having virus growth dependent on the specific inducer isopropylthiogalactoside (IPTG). The feasibility of this approach to conditionally express an HCMV promoter was demonstrated by analyzing lac operator-containing, HCMV US9 promoters in CAT reporter constructs, using RVlac and IPTG in transient assays. This study demonstrates that efficient repression mediated by the lac repressor can be achieved, and this repression is efficiently reversed by IPTG. In addition, the operator-containing US9 promoters were inserted into the endogenous locus to investigate the impact of the operator insertion on basal promoter expression in the context of the virus, before these constructs are used to study conditional expression in the viral genome. It was observed that US9 endogenous promoter expression was not affected significantly by the operator insertions. Because attempts to isolate a recombinant virus containing the operator-containing US9 promoter and expressing the lac repressor were unsuccessful, future studies should target the insertion of the operator-containing US9 promoter and the lac repressor gene into two separate recombinant viruses. These viruses could be used in coinfection experiments to address conditional US9 gene expression. Furthermore, alternative sites for insertion of the operator sequence within the US9 promoter should also be evaluated. After demonstrating the feasibility of this approach in the context of the viral genome, the system can then be adapted to target putative essential HCMV genes.
Cageao-Luchetti, Laura F..
"Construction of an Escherichia coli lac Repressor-Based System to Study Human Cytomegalovirus Gene Expression"
(1999). Doctor of Philosophy (PhD), Dissertation, , Old Dominion University, DOI: 10.25777/5rtn-3n63