The long term objective of this research is to understand the molecular basis of cytomegalovirus (CMV) replication and use that information to develop new ways to diagnose and treat CMV-related diseases of man. This will be accomplished by identifying and studying the synthesis, structure, and function of specific viral proteins that are essential for virus replication. In this application for renewed support, two general sets of studies are proposed. One represents a continuation of ongoing work to learn more about proteins that are already under investigation. The other will pursue leads generated during the last grant period to better define two new sets of proteins. A major emphasis is placed on learning more about a """"""""scaffoldinglike"""""""" protein, referred to as the assembly protein and suspected to have a key role in capsid formation and possibly DNA-packaging. Experiments are outlined to identify, characterize, and genetically map the protease responsible for cleaving the precursor form of this protein. Chemical mutagenesis of the cloned assembly protein gene will be used in an attempt to generate temperature-sensitive mutants that will aid functional studies. Four proteins of the virion tegument will also be investigated. Two of these, referred to as the basic phosphoprotein and the lower matrix protein, appear to interact directly with proteins of the capsid and envelope. The nature of these associations will be explored by chemical, immunological, and genetic approaches. Two other tegument proteins, referred to as the high molecular weight protein and the 115K protein, cosediment as a possible heterodimer and are speculated to be related to the herpes simplex DNA cleavage/packaging complex. Experiments based on chemical dissociation, rate-velocity sedimentation, band-shift assays, and DNA-affinity chromatography are described to characterize the complex and determine whether it has properties of a cleavage/packaging enzyme. Studies of a more exploratory nature will try to identify the proteins encoded by three newly discovered HCMV genes that are homologous to cellular G protein-coupled receptors, and determine by recombinant DNA methods whether they are required for growth of the virus in cell culture.
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