Cloning, Characterization, and Expression of the Murine Cytomegalovirus Homologue of the Human Cytomegalovirus 28-kDa Matrix Phosphoprotein (UL99)

We have identified, characterized, and expressed in bacteria and recombinant vaccinia viruses a protein which likely represents the murine cytomegalovirus (MCMV) homologue of the human cytomegalovirus (HCMV) 28-kDa matrix phosphoprotein, the product of the HCMV UL99 open reading frame (ORF). This protein, referred to as the MCMV UL99, is encoded by a 336-nucleotide ORF within the HindIII G fragment of MCMV strain Smith (K181). Using a DNA probe that corresponded to the amino terminus of the ORF, we detected a transcript of 4.8 kb at 8 hr and additional transcripts of 0.88, 2.4, and 5.7 kb at 24-48 hr postinfection (p.i.) of NIH 3T3 cells with MCMV. The smallest transcript is unspliced, initiates 235 nucleotides upstream from the start of the ORF, and utilizes a polyadenylation site located 62 nucleotides downstream from the end of the ORF. The ORF encodes a protein of 112 amino acids, with a predicted mass of 11.8 kDa. Comparison of the derived amino acid sequence with that of the HCMV UL99 gene product reveals 34.8% identity in an overlap of 66 amino acids. Within the amino acid sequence are at least two potential protein kinase C and one potential casein kinase II target motifs for phosphorylation. The ORF was cloned into the pGEX-KG prokaryotic expression vector and bacterially expressed protein was used to generate a specific rabbit antiserum against the protein. Western blotting of MCMV-infected NIH 3T3 cells showed that the ORF was expressed as a doublet of 16.3 and 15.2 kDa at 48 hr p.i. only in the absence of phosphonoacetic acid, thus demonstrating that this protein is a member of the true late gene class. The immunoreactive protein in MCMVinfected cells comigrated with that produced in cells infected with recombinant vaccinia virus containing the ORF. The protein appears to be part of the MCMV virion, is phosphorylated in vivo, and generates a strong humoral immune response following MCMV infection of BALB/c mice.