The broad and long term objectives of this application are to elucidate the molecular and cellular basis of growth and persistence of human cytomegalovirus (hCMV), an important human pathogen. Central to these objectives is determining what role viral encoded regulatory proteins, in particular the immediate -early (IE) class of genes, play in the life-cycle of hCMV. The first specific aim proposes to generate a synoptic library of site-directed mutants of the major IE proteins which will be analysed using a battery of assays. This high resolution mutational analysis will yield a functional map of side chains in the IE proteins important for determining their regulatory action. Moreover, this scanning mutant library provides a common resource for developing the remaining aims of the proposal and a powerful basis for future structural studies.
The second aim proposes to develop dominant and conditional interference assays for investigating immediate-early protein function based on selected mutants generated in aim 1. Specifically, defective mutants that are dominant or conditional (those that exhibit a temperature-sensitive phenotype)will be tested for their ability to inhibit viral gene expression and replication. These studies will provide insight in unravelling the role played by the IE proteins and will identify mutations which can be introduced into the virus using methods developed in aim 3. The third and final specific aim focuses on the development of genetically engineered CMV genomes, based on yeast artificial chromosomes, which can transfer infectious DNA to permissive cells. This system has potential for broad application in basic research and medicine. Here, it will be used to explore the function of the IE proteins in co-ordinating hCMV gene expression and replication. The results of these studies hold the promise for a better understanding of the function of key regulatory proteins in the growth control of hCMV and may provide a new way to identify novel anti-viral targets for drug development.
