Monoclonal antibody 1B4, previously shown to be protective in vivo and to cross-react with both virally encoded and normal host cell proteins, was used to screen a lambda gt11 cDNA library derived from mRNA harvested from mouse embryo fibroblasts 24 hr after infection with murine cytomegalovirus (MCMV). A 700-bp cDNA was identified representing the 5' terminus of a 2460-bp open reading frame (ORF) with significant homology to the human cytomegalovirus UL25 ORF. The UL25 ORF of MCMV potentially encodes an 820 amino acid viral tegument protein with an estimated molecular weight of approximately 90 kDa. Amino acid homology with eukaryotic nucleo[ins was identified in the acidic N-terminal third of the MCMV UL25 protein, suggesting that the protein may be involved in transcriptional activation or interactions with chromatin. Northern analysis and S1 nuclease data indicated that the gene is expressed late in infection as an approximately 3-kb transcript and that expression is dependent on viral DNA replication. An epitope recognized by MAb 1B4 was identified using recombinant pGEX plasmids expressing fusion proteins representing the N-terminal region of the MCMV UL25 protein. The identification of the MCMV UL25 ORF as a member of the CMV-specific UL25/UL35 gene family provides an opportunity for the investigation of the role these genes and their products in CMV pathogenesis in an animal model. (C) 1994 Academic Press, Inc.