Mapping and expression of a human cytomegalovirus major viral protein

Abstract

We constructed a DNA fragment map of low-passage Towne strain cytomegalovirus by analyzing cross-blot hybridization and hybridizations of isolated recombinant clones. The abundant late transcripts were located on this map by hybridization of labeled total RNA of virus-infected cells to blotted DNA fragments. The most abundant late transcript, carried by the 11.7-kilobase EcoRI fragment (EcoRI-G), was precisely mapped. The EcoRI fragment was fragmented and subcloned in a plasmid carrying simian virus 40 sequences (pSV-OH, constructed by Chi-Bom Chae, Department of Biochemistry, University of North Carolina, Chapel Hill). One resulting recombinant plasmid, pHD713SV2, was transferred to simian virus 40-transformed monkey kidney cells (COS-1) by DNA transfection. Synthesis of a cytomegalovirus-specific 67-kilodalton protein was detected in these cells by reaction of blotted proteins with virus-specific monoclonal antibody. The 67-kilodalton protein is a major phosphorylated protein found in virions; it is not glycosylated. The location of the gene for this 67-kilodalton protein is therefore assigned to the center of the L-unique region of human cytomegalovirus, at 0.37 to 0.39 map units.