SUMMARYHuman cytomegalovirus (HCMV) purified from cell culture contains two dominant structural phosphoproteins with apparent molecular weights of 65000 and 150000, designated as pp65 and pp 150 respectively. The humoral immune response of infected individuals against pp65 is relatively weak and is not always detectable by Western blot analyses. This report shows that recent clinical isolates of HCMV do not necessarily have pp65 as a prominent constituent, suggesting that the low immune reaction is due to variable expression of the pp65 in natural infections. However, the HCMV strains tested in this study produced the large structural phosphoprotein (pp 150) in about equal amounts. The pp150 is remarkably immunogenic, if compared with all other virion constituents; serum pools and individual sera from HCMV-infected patients recognized this particular protein intensively in immunoblot assays. Thus, phosphoprotein pp 150 seems to be the primary polypeptide candidate for expression cloning in order to develop reagents for novel ways of HCMV diagnosis.

Human cytomegalovirus particles contain a phosphoprotein of 150,000 (ppl50) apparent molecular weight in their matrix; the protein appears particularly reactive in Western blot analyses with human antisera. The gene for ppl50 was mapped by screening a bacteriophage lambda gtll cDNA expression library with monospecific rabbit antisera. Subsequent hybridization of cDNA with cosmid and plasmid clones containing the human cytomegalovirus strain AD169 genome mapped the gene to HindIII fragments J and N. The gene is transcribed into a late 6.2-kilobase RNA. The nucleotide sequence of this region was determined, and a transcription initiation site and two polyadenylation sites of an abundant transcript were located by primer extension and nuclease protection experiments. The reading frame for ppl50, deduced from computer analyses, gives rise to a polypeptide of 1,048 amino acids in length; protein secondary structure analysis revealed multiple n-pleated sheets in hydrophilic clusters, providing a possible explanation for the immunogenic properties of the polypeptide.

SUMMARYThe large phosphorylated matrix protein pp 150 of human cytomegalovirus (HCMV) is the polypeptide most frequently reactive in immunoblotting analyses with human antisera when compared with other viral proteins. Several defined regions of ppl50 were expressed as ]~-galactosidase fusion proteins and these were tested for their immunoreactivity with human sera and their immunogenicity. One antigenic region could be expressed in large amounts and was found to carry immunodominant epitopes, as shown by immunoblotting and ELISA. A rabbit antiserum raised against recombinant ppl50 antigens produced in bacteria proved to be useful for immunofluorescence and immunohistochemistry studies of HCMV-infected cells and tissues. The results suggest that this anti-pp 150 serum will help to elucidate the process of virus assembly and antigen detection in infected cells.

Parts of the large phosphorylated tegument protein, pp150, of human cytomegalovirus (HCMV) were expressed in bacteria. The resulting fusion proteins were tested in a Western blot (immunoblot) assay for reactivity with a monoclonal antibody against pp150, with a polyspecific rabbit antiserum, and with human reconvalescent-phase sera. Those fusion proteins that performed well in the Western blot assay were used as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against HCMV. Five different recombinant beta-galactosidase fusion proteins were evaluated by ELISA using 62 seropositive and 38 seronegative human serum samples. Of all the proteins tested, one peptide representing 162 amino acids of pp150 was superior to the others with regard to sensitivity and specificity. All sera known to be positive for antibodies against HCMV were identified by combining the results of the ELISAs with the different pp150 fusion proteins. Therefore, it appears that peptides from a single protein of HCMV might be sufficient to identify HCMV-seropositive individuals by recombinant ELISA.

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