Characterization of a human cytomegalovirus 1.6-kilobase late mRNA and identification of its putative protein product.

In a previous study (J. Martinez, R. S. Lahijani, and S. C. St. Jeor, J. Virol. 63:233-241, 1989), we identified a late, unspliced 1.6-kb mRNA that maps to the HindIII R fragment of human cytomegalovirus (HCMV) AD169. In the present study, the direction of transcription of this mRNA was determined by Northern (RNA) analysis with strand-specific probes. Primer extension was used to precisely map the 5' end of the mRNA. An open reading frame (ORF) designated ORF 2-1, located 176 nucleotides downstream from the cap site of the 1.6-kb mRNA, was identified. A synthetic peptide was made representing a hydrophilic region in the amino terminus of ORF 2-1. Immunoprecipitation and Western immunoblot analysis of infected HEL cell lysates, using affinity-purified antibody to the peptide (anti-P2-1), detected a viral protein with an apparent molecular mass of 58 kDa late in infection. Further support for the presence of this protein in infected-cell lysates was obtained by an enzyme-linked immunosorbent assay. Expression of viral antigens in intact infected HEL cells was assessed by immunofluorescence. General cytoplasmic staining was observed at 62 h postinfection, in contrast to a localized staining observed in the nuclear and perinuclear region at 96 h postinfection.