Cloning and identification of regulatory gene UL76 of human cytomegalovirus

The major immediate-early promoter/enhancer (MIEP, -1139 to +52) of human c ytomegalovirus (HCMV) is regulated by cell type-specific transcriptional fa ctors, its own MIE proteins (IE2p40, IE1p55, IE1p72 and IE2p86) as well as viral proteins pUL69, pUL82 and pUL84. To investigate the hypothesis that t he regulation of HCMV MIEP is modulated by additional viral genes, HCMV (AD 169) genomic sublibraries were constructed and in vitro transient co-transf ection assays were performed to assess the ability of these sublibraries to modulate MIEP expression. In this study, enhancement of MIEP expression wa s exhibited by a number of sublibraries, from one of which a genomic clone was selected for;augmentation of expression. Subcloning the insert fragment led to the identification of the responsible locus, UL76. To generate a UL 76-specific antibody for immunodetection, the UL76 ORF was constructed as a histidine-tagged fusion protein that was produced in prokaryotic cells. A polyclonal antibody raised against the UL76 fusion protein immunoreacts wit h a protein of 38 kDa (pUL76) in UL76 ORF-transfected cells, Additionally, pUL76 is present in HCMV-infected cells at the immediate-early to late stag es of the reproductive cycle. Characterized by its highly basic composition (predicted pl 11.6), a free form of pUL76 tagged with green fluorescent pr otein was found to localize exclusively to the nucleus, In this report, pUL 76 is defined as a novel regulatory protein that modulates both activation and repression of gene expression, depending on the promoter context and th e ratio of transfected effector DNA.