Binding of cellular repressor protein or the IE2 protein to a cis-acting negative regulatory element upstream of a human cytomegalovirus early promoter

Huang, Lili; Stinski, Mark F.

doi:10.1128/jvi.69.12.7612-7621.1995

Cited by 31 publications

(7 citation statements)

References 59 publications

(92 reference statements)

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“…The selection may result from IE2 binding directly to sites in the viral genome or to host proteins (or another viral protein) that bind to specific sites in the viral promoter. A crs-like sequence to which IE2 is known to bind is not located in or nearby many of the IE2-activated promoters, but non-crs-like elements can also be bound by IE2 as has been described for HCMV early promoters [55,56]. Alternatively, IE2 might bring about transcription from a set of weakly active viral promoters as a consequence of the onset of viral DNA replication that rendered the genome more accessible to host GTF that interact with IE2.…”

Section: Discussionmentioning

confidence: 99%

Human cytomegalovirus IE2 drives transcription initiation from a select subset of late infection viral promoters by host RNA polymerase II

Ball

Collins

et al. 2020

PLoS Pathog

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Herpesvirus late promoters activate gene expression after viral DNA synthesis has begun. Alphaherpesviruses utilize a viral immediate-early protein to do this, whereas beta-and gammaherpesviruses primarily use a 6-member set of viral late-acting transcription factors (LTF) that are drawn to a TATT sequence in the late promoter. The betaherpesvirus, human cytomegalovirus (HCMV), produces three immediate-early 2 protein isoforms, IE2-86, IE2-60, IE2-40, late in infection, but whether they activate late viral promoters is unknown. Here, we quickly degrade the IE2 proteins in late infection using dTag methodology and analyze effects on transcription using customized PRO-Seq and computational methods combined with multiple validation methods. We discover that the IE2 proteins selectively drive RNA Pol II transcription initiation at a subset of viral early-late and late promoters common to different HCMV strains, but do not substantially affect Pol II transcription of the 9,942 expressed host genes. Most of the IE2-activated viral late infection promoters lack the TATT sequence bound by the HCMV UL87-encoded LTF. The HCMV TATT-binding protein is not mechanistically involved in late RNA expression from the IE2-activated TATT-less UL83 (pp65) promoter, as it is for the TATT-containing UL82 (pp71) promoter. While antecedent viral DNA synthesis is necessary for transcription from the late infection viral promoters, continued viral DNA synthesis is unnecessary. We conclude that in late infection the IE2 proteins target a distinct subset of HCMV early-late and late promoters for transcription initiation by RNA Pol II. Commencement of viral DNA replication renders the HCMV genome late promoters susceptible to late-acting viral transcription factors. PLOS PATHOGENSPLOS Pathogens | https://doi.org/10.1371/journal.ppat.

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Section: Discussionmentioning

confidence: 99%

Human cytomegalovirus IE2 drives transcription initiation from a select subset of late infection viral promoters by host RNA polymerase II

Ball

Collins

et al. 2020

PLoS Pathog

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“…IE2 86 can interact with itself and the viral 75 kDa early protein (UL84), as well as with the components of the basal transcription complex (TBP and TFIIB), several TBP-associated factors, and specific transcription factors and products of the tumor suppressor genes p53 and Rb (Caswell et al, 1993;Chiou et al, 1993;Choi et al, 1995;Furnari et al, 1993;Hagemeier et al, 1992;Jupp et al, 1993;Lang et al, 1995;Lukac et al, 1994Lukac et al, , 1997Schwartz et al, 1996;Scully et al, 1995;Sommer et al, 1994;Spector and Tevethia, 1994;Speir et al, 1994;Yoo et al, 1996). IE2 86 can also bind to DNA in a site-specific but sequence-tolerant manner (Arlt et al, 1994;Cherrington et al, 1991;Huang and Stinski, 1995;Liu et al, 1991;Pizzorno and Hayward, 1990;Rodems et al, 1998;Schwartz et al, 1994;Scully et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

The Use of Recombinant Baculoviruses for Sustained Expression of Human Cytomegalovirus Immediate Early Proteins in Fibroblasts

et al. 2001

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The isolation of viruses with mutations in essential genes requires that they be propagated in cells expressing the wild-type proteins. This has been a particularly challenging problem for studying mutations in the human cytomegalovirus (HCMV) immediate early (IE) gene, IE2 86. In the past, we tried a number of approaches to derive human fibroblasts expressing wild-type IE2 86, but were unable to maintain expression of a fully functional protein. To overcome this obstacle, we developed a strategy whereby recombinant baculoviruses were used as vectors for the expression of HCMV IE proteins in primary human fibroblasts (FFs). The IE2 86 and IE1 72 cDNAs, as well as the genomic fragment of the UL122-123 region under the control of a chicken actin promoter, were introduced into the baculovirus genome by site-specific transposition in Escherichia coli. Recombinant "bacmid" DNAs were then transfected into Sf9 cells to generate recombinant baculoviruses. FFs infected at high m.o.i. with these baculoviruses expressed high levels of the HCMV protein for at least 1 week, as determined by immunofluorescence assays and Western blots. Moreover, the IE2 86 protein was found to be fully functional with respect to its ability to activate the HCMV UL112-113 early promoter. Recombinant baculoviruses expressing IE1 72 were also able to efficiently complement HCMV ie1 mutants. These data demonstrate the potential of using recombinant baculoviruses as vectors for the expression of toxic viral genes in human cells and for subsequent isolation of mutant HCMV lacking these essential genes.

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“…It has been demonstrated that IE86 alone strongly activates the UL4 and UL112-113 early genes in transiently transfected cells (5,27,42) while the IE72 protein can act synergistically with IE86 to activate these genes (5,25,45,47,62). The IE86 protein has been shown to bind to the cis-acting negative regulatory element on the UL4 promoter and negate its repressive effect (18). Transactivation of the UL112 gene is mediated through an interaction between IE86 and the cellular transcription factor CREB (1,29,30).…”

mentioning

confidence: 99%

Transcription Factor Sp1 Mediates Cell-Specific trans -Activation of the Human Cytomegalovirus DNA Polymerase Gene Promoter by Immediate-Early Protein IE86 in Glioblastoma U373MG Cells

Jun

O'Neill

Barbosa

1998

J Virol

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Human cytomegalovirus (HCMV) gene expression is highly cell and tissue specific. Cell factor-mediated regulatory interactions are involved in regulating the restricted expression of the HCMV major immediate-early (IE) gene (J. F. Baskar, P. P. Smith, G. Nilaver, R. A. Jupp, S. Hoffmann, N. J. Peffer, D. J. Tenney, A. M. Colberg-Poley, P. Ghazal, and J. A. Nelson, 70:3207–3213, 1996). To gain an understanding of HCMV early gene activation, we studied the effect of each of the three major IE proteins, IE72, IE86, and IE55, on the HCMV DNA polymerase gene (pol; UL54) promoter. In transient-expression assays, the IE86 protein alone was able to transactivate the polpromoter, but IE72 and IE55 were not, in permissive U373MG cells. However, we were unable to detect IE86-mediated transactivation in nonpermissive HeLa or C33-A cells. Using electrophoretic mobility shift assays (EMSAs), we found that expression of the IE86 protein in U373MG cells resulted in specific binding of a DNA complex to an inverted-repeat element, IR1, of the pol promoter. Antibody supershifting and EMSA-Western blotting experiments further showed that IE86 and the cellular transcription factor Sp1 were components of the IR1 DNA-binding complex. Furthermore, we found that binding of DNA by Sp1 was dramatically increased in the presence of IE86. Interestingly, this IE86-induced DNA-binding activity of Sp1 was inhibited by a repressor activity presented in HeLa cells. In summary, our study suggests that a viral regulatory protein can modulate the DNA binding activity of a cellular transcription factor, resulting in cell-specific transactivation of viral genes.

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Binding of cellular repressor protein or the IE2 protein to a cis-acting negative regulatory element upstream of a human cytomegalovirus early promoter

Cited by 31 publications

References 59 publications

Human cytomegalovirus IE2 drives transcription initiation from a select subset of late infection viral promoters by host RNA polymerase II

Human cytomegalovirus IE2 drives transcription initiation from a select subset of late infection viral promoters by host RNA polymerase II

The Use of Recombinant Baculoviruses for Sustained Expression of Human Cytomegalovirus Immediate Early Proteins in Fibroblasts

Transcription Factor Sp1 Mediates Cell-Specific trans -Activation of the Human Cytomegalovirus DNA Polymerase Gene Promoter by Immediate-Early Protein IE86 in Glioblastoma U373MG Cells

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