A Strong Negative Transcriptional Regulatory Region between the Human Cytomegalovirus UL127 Gene and the Major Immediate-Early Enhancer

Abstract

The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18–25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

“…The modulator's role is largely unknown, although a cell-type specific regulatory function has been suggested [87][88][89]. "Rightward" transcription from the MIEP is suppressed by the unique region which binds cellular homeobox proteins and appears to function as an insulator between the enhancer and UL127 [90][91][92][93][94][95] ( Figure 1). The core promoter is sufficient, yet not required, for low-level transcription to the "leftward" direction of the major IE gene [76,96].…”

Neuropathogenesis in cytomegalovirus infection: indication of the mechanisms using mouse models

“…The third gene found in the predominant IE gene, referred to as MCMV ie2 (M128), is transcribed in the opposite direction from the ie1 and ie3 tran-scripts in this region ( Figure 5A) [65]. The MCMV ie2 gene is not related to a HCMV gene, but it is the positional homologue of HCMV UL 127 gene [66].…”

Differentiation-Coupled Induction of Human Cytomegalovirus Replication by Union of the Major Enhancer Retinoic Acid, Cyclic AMP, and NF-κB Response Elements

“…HCMV-GFP has a green fluorescent protein (GFP) gene driven by the viral native UL127 promoter that is expressed with early/late kinetics ( 30 ). HCMV recombinants rCRE − (rC − ), rkB − (rK − ), rCRE − .kB − (rCK − ), and rWT CK− have been reported previously ( 22 , 23 ) and were derived from HCMV strain Towne in a bacterial artificial chromosome (BAC) ( 31 ).…”