In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins

1993 ◽  
Vol 13 (2) ◽  
pp. 1238-1250
Author(s):  
K M Klucher ◽  
M Sommer ◽  
J T Kadonaga ◽  
D H Spector
Keyword(s):  
Human Cytomegalovirus ◽  
Hela Cells ◽  
Transcriptional Activation ◽  
Histone H1 ◽  
Early Gene ◽  
Immediate Early ◽  
Early Promoter ◽  
Drosophila Embryos

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The results of the in vitro experiments correlated well with those obtained in vivo. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein. Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter. However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold. The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Krüppel promoter in either the presence or absence of histone H1. These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.

scholarly journals In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins.

1993 ◽  
Vol 13 (2) ◽  
pp. 1238-1250 ◽  
Author(s):  
K M Klucher ◽  
M Sommer ◽  
J T Kadonaga ◽  
D H Spector
Keyword(s):  
Human Cytomegalovirus ◽  
Hela Cells ◽  
Transcriptional Activation ◽  
Histone H1 ◽  
Early Gene ◽  
Immediate Early ◽  
Early Promoter ◽  
Drosophila Embryos

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The results of the in vitro experiments correlated well with those obtained in vivo. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein. Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter. However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold. The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Krüppel promoter in either the presence or absence of histone H1. These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.

Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides

1994 ◽  
Vol 14 (5) ◽  
pp. 3484-3493
Author(s):  
T J Wu ◽  
G Monokian ◽  
D F Mark ◽  
C R Wobbe
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transcriptional Activation ◽  
Deletion Mutant ◽  
Full Length ◽  
Early Gene ◽  
Immediate Early ◽  
Simplex Virus

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

scholarly journals Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides.

1994 ◽  
Vol 14 (5) ◽  
pp. 3484-3493 ◽  
Author(s):  
T J Wu ◽  
G Monokian ◽  
D F Mark ◽  
C R Wobbe
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transcriptional Activation ◽  
Deletion Mutant ◽  
Full Length ◽  
Early Gene ◽  
Immediate Early ◽  
Simplex Virus

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

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

1989 ◽  
Vol 9 (3) ◽  
pp. 1342-1345
Author(s):  
H Lubon ◽  
P Ghazal ◽  
L Hennighausen ◽  
C Reynolds-Kohler ◽  
C Lockshin ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Specific Activity ◽  
Gene Promoter ◽  
Early Gene ◽  
Immediate Early ◽  
Upstream Region ◽  
Early Promoter ◽  
Major Immediate Early Promoter

In this paper we demonstrate that modulator sequences upstream of the enhancer of the major immediate-early 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 IE1 gene promoter-enhancer. 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.

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

1989 ◽  
Vol 9 (3) ◽  
pp. 1342-1345 ◽  
Author(s):  
H Lubon ◽  
P Ghazal ◽  
L Hennighausen ◽  
C Reynolds-Kohler ◽  
C Lockshin ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Specific Activity ◽  
Gene Promoter ◽  
Early Gene ◽  
Immediate Early ◽  
Upstream Region ◽  
Early Promoter ◽  
Major Immediate Early Promoter

In this paper we demonstrate that modulator sequences upstream of the enhancer of the major immediate-early 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 IE1 gene promoter-enhancer. 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.

scholarly journals Phosphorylation of the Human Cytomegalovirus 86-Kilodalton Immediate-Early Protein IE2

1998 ◽  
Vol 72 (7) ◽  
pp. 5481-5492 ◽  
Author(s):  
Noam Y. Harel ◽  
James C. Alwine
Keyword(s):  
Map Kinase ◽  
Human Cytomegalovirus ◽  
Transcriptional Activation ◽  
Immediate Early ◽  
Wild Type ◽  
Independent Manner ◽  
Early Protein ◽  
Mitogen Activated Protein

ABSTRACT We have investigated the phosphorylation state of the human cytomegalovirus 86-kDa immediate-early (IE) protein IEP86 from transfected and infected cells. We show that multiple domains of IEP86 are phosphorylated by cellular kinases, both in vitro and in vivo. Our data suggest that serum-inducible kinases play a significant role in cell-mediated IE protein phosphorylation and that a member of the mitogen-activated protein (MAP) kinase (MAPK) family, extracellular regulated kinase 2 (ERK2), phosphorylates several domains of IEP86 in vitro. Alanine substitution mutagenesis was performed on specific serines or threonines (T27, S144, T233/S234, and T555) found in consensus MAP kinase motifs. Analysis of these mutations showed that T27 and T233/S234 are the major sites for serum-inducible kinases and are the major ERK2 sites in vitro. S144 appeared to be phosphorylated in a serum-independent manner in vitro. All of the mutations except T555 eliminated specific phosphorylation in vivo. In transient transfection analyses, IEP86 isoforms containing mutations in S144 and, especially, T233/S234displayed increased transcriptional activation relative to the wild type, suggesting that phosphorylation at these sites in wild-type IEP86 may result in reduction of its transcriptional activation ability.

scholarly journals Human cytomegalovirus major immediate early transcripts arise predominantly from the canonical major immediate early promoter in reactivating progenitor-derived dendritic cells

2020 ◽  
Vol 101 (6) ◽  
pp. 635-644 ◽  
Author(s):  
Rebecca Mason ◽  
Ian J. Groves ◽  
Mark R. Wills ◽  
John H. Sinclair ◽  
Matthew B. Reeves
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Human Cytomegalovirus ◽  
Virus Production ◽  
Early Gene ◽  
Immediate Early ◽  
Genome Replication ◽  
Early Promoter ◽  
Major Immediate Early Promoter

Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) – the promoter that regulates immediate early (IE) gene expression – is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in in vitro generated dendritic cells or macrophages that support HCMV reactivation IE transcripts arise predominantly from the MIEP and not the intronic promoters. Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.

E1A-mediated activation of the adenovirus E4 promoter can occur independently of the cellular transcription factor E4F

1991 ◽  
Vol 11 (9) ◽  
pp. 4297-4305
Author(s):  
C Jones ◽  
K A Lee
Keyword(s):  
Transcription Factor ◽  
Hela Cells ◽  
Transcriptional Activation ◽  
Promoter Activity ◽  
Core Motif ◽  
Cellular Transcription Factor ◽  
The Core ◽  
Cellular Factors

The cellular factors E4F and ATF-2 (a member of the activating transcription factor [ATF] family) bind to common sites in the adenovirus E4 promoter and have both been suggested to mediate transcriptional activation by the viral E1A protein. To assess the role of E4F, we have introduced mutations into the E4F/ATF binding sites of the E4 promoter and monitored promoter activity in HeLa cells. We find that the core motif (TGACG) of the E4F/ATF binding site is important for E4 promoter activity. However, a point mutation adjacent to the core motif that reduces E4F binding (but has no effect on ATF binding) has no effect on E4 promoter activity. Together with previous results, these findings indicate that there are at least two cellular factors (a member of the ATF family and E4F) that can function with E1A to induce transcription of the E4 promoter. We also find that certain mutations strongly reduce E4 transcription in vivo but have no effect on ATF-2 binding in vitro. These results are therefore incompatible with the possibility that (with respect to members of the ATF family) ATF-2 alone can function with E1A to transactivate the E4 promoter in HeLa cells.

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