Cell-specific activity of the modulator region in the human cytomegalovirus major immediate-early gene.

Abstract

In this paper we demonstrate that modulator sequences upstream of the enhancer of the major immediateearly promoter of human cytomegalovirus exert a differential effect on the level of transcription in a variety of cells and that this region has the capacity to interact with specific nuclear protein. Depending on the cell type, these modulator sequences increased or decreased transcriptional activation from the IEl gene promoterenhancer. The cell lines identified in this report should be useful to study the molecular mechanism of cell-specific transcriptional repression and activation exerted by the major immediate-early promoter upstream region.Although human cytomegalovirus (HCMV) nonproductively infects cells of many types from humans (8,16,17) and other species (2, 11), the virus replicates in vitro only in fibroblasts of human origin. Transcription of the first viral genes expressed, the immediate-early (IE) genes, are mainly dependent on host cell factors (1, 5-7, 20, 21), suggesting that this may constitute the primary event at which expression of the viral genome is regulated. Regulatory sequences of the HCMV major IE gene, the IEl gene, encompass a polymerase II promoter between +1 and -65 (6, 19); a strong and complex enhancer between -66 and -524 (1, 5, 7), which is active in many different cell types; a cluster of nuclear factor 1-binding sites between -600 and -720 (9, 10); and modulator sequences between -750 and -1145 (14, 15). These modulator sequences negatively regulate expression in nonpermissive undifferentiated teratocarcinoma (Tera-2) cells but positively influence expression in permissive differentiated cells (14,15). In the present study we have addressed the question of whether the modulator region is functional in cell types other than the embryonal carcinoma cell system and whether indeed it may contribute to cellspecific expression.Recombinant plasmids containing the modulator (A1145) or with the modulator sequences truncated (A525), which were previously used to define the modulator region (15) A1145 and A525 in cells of various lineages were measured by scintillation counting of conversion rates from chloramphenicol to its acetylated forms. All cells were cotransfected with pRSV-luciferase as a transfection control (3), and the results with A1145 and A525 were adjusted according to the relative luciferase activity in the cell extracts. The effect of the modulator sequences on CAT activity differed depending on cell type (Fig. 1). A twofold negative effect was observed in H9, U937, and SW480 cells, but expression in Jurkat, 293, Raji, and Namalwa cells was barely affected. In B cells the expression was augmented about three-to fourfold by the upstream sequences. These results clearly indicate that the HCMV modulator sequences can influence IE promoterenhancer activity in vivo in cell types other than teratocarcinoma cells. In particular, the cis-acting sequences negatively modulate expression in some T cells and rectal epithelial cells, in which HCMV is found during natural infection...

Human Cytomegalovirus: Recent Aspects from Molecular Biology

“…In transient expression assays this element was able to modulate gene expression in non-permissive cells in a negative manner, whereas it positively influences expression in permissive cells (Nelson et al, 1987). This was confirmed by in vitro transcription studies in which nuclear extracts of various cell lines were tested for their ability to mediate transcription in the presence or absence of the IE1 upstream region between positions -1145 and -524 (Lubon et al, 1989). This region, also referred to as the modulator region, participates in certain cell lines in transcriptional repression, whereas it augments transcription in other cells.…”

“…This region, also referred to as the modulator region, participates in certain cell lines in transcriptional repression, whereas it augments transcription in other cells. This suggests a cell-type specific regulatory mechanism of IE1 gene expression (Lubon et al, 1989).…”

Ets-2 Repressor Factor (ERF) mediates repression of the human cytomegalovirus major immediate-early promoter in undifferentiated non-permissive cells

“…For example, there are numerous nuclear factor-1 (NF-1) sites within the MIEP and the 18 and 19 bp repeat sequences contain NF-kB and cyclic AMPresponsive element binding protein (CREB) binding sites respectively (Meier & Stinski, 1996). Conversely, several studies using non-permissive cells have shown that the modulator and the 21 bp repeats are responsible for the inhibition of MIEP activity in such cells (Nelson et al, 1987;Lubon et al, 1989;Kothari et al, 1991).…”

“…For example, there are numerous nuclear factor-1 (NF-1) sites within the MIEP and the 18 and 19 bp repeat sequences contain NF-kB and cyclic AMPresponsive element binding protein (CREB) binding sites respectively (Meier & Stinski, 1996). Conversely, several studies using non-permissive cells have shown that the modulator and the 21 bp repeats are responsible for the inhibition of MIEP activity in such cells (Nelson et al, 1987;Lubon et al, 1989;Kothari et al, 1991).In vivo, monocytes have been identified as an important site of HCMV latency (Taylor-Wiedeman et al, 1991); these cells carry the viral genome in the absence of lytic (productive) gene expression. Experiments in our laboratory and those of others have shown that the differentiation of peripheral blood monocytes to macrophages (monocytederived macrophages; MDMs) results in the induction of endogenous HCMV IE gene expression and reactivation of virus (Taylor-Wiedeman et al, 1994;Soderberg-Naucler et al, 1997).…”

Tumour necrosis factor stimulates the activity of the human cytomegalovirus major immediate early enhancer/promoter in immature monocytic cells

“…The HCMV promoter is strongly influenced by an upstream enhancer region with binding sites for various cellular transcription factors (Boshart et al, 1985;Niller & Henninghausen, 1991;Stinski & Roehr, 1985;Thomsen et al, 1984). Upstream of the enhancer region there is a modulator region which regulates the cell-type-specific expression of HCMV by binding further cell-specific transcription factors (Lubon et al, 1989).…”