The long-term objectives of this research are to learn more about protein functions that underlie cytomegalovirus (CMV) replication, and to contribute to the development of new strategies for diagnosing and treating CMV-related diseases of man. In this application for renewed support, two sets of studies are proposed. The first is a continuation of ongoing work to learn more about specific proteins already under investigation. The second will exploit leads generated during the last grant period to learn more about several less well characterized proteins. Among the proteins being studied in more detail in ongoing work are two nonviron DNA-binding proteins that are expressed early during virus replication; a virion tegument phosphoprotein that elicits a strong response in man; an unusual protein that appears to be intimately associated with capsid assembly; and an envelope glycoprotein whose processing may be coupled with nucleocapsid envelopment. Proteins of more exploratory interest include three intrinsic capsid proteins whose structural role is unknown; two presumptive tegument proteins; and an acidic envelope glycoprotein that is highly O-glycosylated and may have been overlooked because of its poor antigenicity.
The specific aims are to (i) use existing and additional antisera to determine the location, intermolecular associations, and relatedness of these proteins; (ii) prepare antibodies to several less well studies proteins of interest for use in their further characterization; and (iii) use these antibodies in conjunction with lambda gt11 expression vector libraries to identify, map, and study CMV genes coding for proteins of interest. Procedures used will include protein and peptide purification; antibody production; antibody assays by immunofluorescence and immunoelectronmicroscopy, immunoprecipitation, and """"""""Towbin-type"""""""" gel replica immunoassay; infectivity analyses; and recombinant DNA methodologies for producing, propagating, and characterizing specific fragments of the CMV genome.
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