UL69 OF HUMAN CYTOMEGALOVIRUS, AN OPEN READING FRAME WITH HOMOLOGY TOICP27 OF HERPES-SIMPLEX VIRUS, ENCODES A TRANSACTIVATOR OF GENE-EXPRESSION

The UL69 open reading frame of human cytomegalovirus (HCMV) is homolog ous to the immediate-early protein ICP27 of herpes simplex virus, an e ssential viral regulatory protein involved in the transition from earl y to late gene expression. Genes with homology to ICP27 have been dete cted in all subclasses of herpesviruses so far. While the respective p roteins in alpha- and gammaherpesviruses have been defined as trans-re gulatory molecules, nothing is known about these genes in betaherpesvi ruses. This study was therefore undertaken in order to investigate exp ression from the UL69 gene locus of HCMV. Northern (RNA) blot experime nts revealed a complex pattern of transcripts that changed during the time course of the HCMV replicative cycle: two transcripts of 2.7 and 3.5 kb that were regulated differentially could be detected as early a s 7 h after infection. However, these transcripts could not be detecte d in the presence of cycloheximide. Additional, larger transcripts wer e present exclusively at late times after infection. To analyze protei n expression from the UL69 gene region, the UL69 open reading frame wa s expressed as a histidine-tagged protein in Escherichia coli. A speci fic antiserum was generated and used to detect the UL69 protein in HCM V-infected cells which revealed its localization within the intranucle ar inclusions that are characteristic for HCMV infection. In cotransfe ction experiments, an HCMV true late promoter could not be activated b y UL69, whereas an early promoter and several heterologous promoters w ere stimulated about 10-fold. Complementation studies showed that the UL69 protein cannot substitute for ICP27 in the contest of the HSV inf ection, suggesting functional differences between these two proteins. In summary, these experiments define a novel regulatory protein encode d by HCMV that is expressed as an early-late gene and appears to exert a broad stimulatory effect on gene expression,