Our long-term objective is to aid in the development of new diagnostic and therapeutic approaches to herpesvirus related diseases of man. We plan to accomplish this through a more complete understanding of the synthesis, structure and function of specific viral proteins that are essential for herpesvirus replication. The studies proposed here will be done with human and simian strains of cytomegalovirus (CMV) and will focus on two sets of virus proteins. The first is comprised of proteins associated with virus particles, and includes (i) the B-capsid assembly protein, which has been shown to have a slowly processed precursor and appears to be involved in capsid assembly, (ii) the enveloped glycoproteins, which are of interest in terms of their biogenesis and function, and of importance as subunit vaccine candidates, (iii) the basic phosphoprotein, which is a major tegument constituent, the principal phosphate acceptor in vitro for the particle-associated kinase, and is unusually reactive in """"""""Western"""""""" immunoassays of AIDS sera, and (iv) the virion-associated protein kinase, which can be resolved into two activities based on differences in size and behavior during ion-exchange chromatography. The second set of proteins includes two early, nuclear DNA-binding species, DB51/53 and DB129/140.
The specific aims are to (i) produce antibodies and use them to study these proteins and others, (ii) study the synthesis, processing and function of the assembly protein and envelope glycoproteins, (iii) determine whether and how DB51 and DB129 interact with DNA, (iv) establish whether transition B-capsids are DNA packaging intermediates, and (v) obtain oligonucleotide probes for specific CMV genes by sequencing peptides prepared from the proteins and screening expression-vector libraries of CMV DNA. Procedures used will include virus and protein purification; antibody production; colloidal gold immunoelectronmicroscopy; active-site radiolabeling with enzyme-specific reagents; protein analyses by SDS-PAGE, HPLC and """"""""Western' immunoassays; and recombinant DNA methodologies for producing, propagating and characterizing specific fragments of CMV DNA.
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