A Novel Silencer Element in the Bovine Papillomavirus Type 4 Promoter Represses the Transcriptional Response to Papillomavirus E2 Protein

Vance, Keith W.; Campo, M. Saveria; Morgan, Iain M.

doi:10.1128/jvi.75.6.2829-2838.2001

Cited by 12 publications

(16 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…WT and E2 TopBP1 were titrated into 293T cells, along with an E2 reporter containing six E2 DNA binding sites upstream from a tk promoter driving luciferase (62). Cells were harvested, and luciferase and protein assays were carried out.…”

Section: Fig 4 E2 Topbp1 Retains Transcription Function (A) E2mentioning

confidence: 99%

An Interaction between Human Papillomavirus 16 E2 and TopBP1 Is Required for Optimum Viral DNA Replication and Episomal Genome Establishment

Donaldson

Mackintosh

Bodily

et al. 2012

J Virol

Self Cite

View full text Add to dashboard Cite

bIn human papillomavirus DNA replication, the viral protein E2 forms homodimers and binds to 12-bp palindromic DNA sequences surrounding the origin of DNA replication. Via a protein-protein interaction, it then recruits the viral helicase E1 to an A/T-rich origin of replication, whereupon a dihexamer forms, resulting in DNA replication initiation. In order to carry out DNA replication, the viral proteins must interact with host factors that are currently not all known. An attractive cellular candidate for regulating viral replication is TopBP1, a known interactor of the E2 protein. In mammalian DNA replication, TopBP1 loads DNA polymerases onto the replicative helicase after the G 1 -to-S transition, and this process is tightly cell cycle controlled. The direct interaction between E2 and TopBP1 would allow E2 to bypass this cell cycle control, resulting in DNA replication more than once per cell cycle, which is a requirement for the viral life cycle. We report here the generation of an HPV16 E2 mutant compromised in TopBP1 interaction in vivo and demonstrate that this mutant retains transcriptional activation and repression functions but has suboptimal DNA replication potential. Introduction of this mutant into a viral life cycle model results in the failure to establish viral episomes. The results present a potential new antiviral target, the E2-TopBP1 interaction, and increase our understanding of the viral life cycle, suggesting that the E2-TopBP1 interaction is essential. There are more than 100 types of human papillomavirus (HPV) involved in a host of epithelial lesions, ranging from hand warts and genital warts to cervical cancer (69). So-called high-risk HPVs are those associated with cancer, and type 16 is the most commonly detected, being present in ca. 50% of cervical carcinomas and increasingly detected in head and neck cancers (30). All HPV encode two proteins, E1 and E2, required for replication of their double-stranded DNA genome in association with cellular partner proteins. The E2 protein forms homodimers and binds to 12-bp palindromic sequences surrounding the origin of replication and via a protein-protein interaction recruits the E1 protein to the A/T-rich origin (9,40,61). E1 then forms a dihexameric helicase that interacts with the cellular DNA polymerase machinery, resulting in DNA replication initiation (36,38,46,55). The origin of replication is located in the long control region (LCR), a noncoding part of the genome that controls the initial transcription from the viral genome by cellular factors (50). The E2 protein can also regulate viral genome transcription; it can act as either an activator or a repressor of viral oncogene expression depending upon E2 levels and the cell type under study (10,15,60). The carboxyl terminus domain of E2 is required for homodimerization and DNA binding, while the amino terminus interacts with E1 and a number of cellular transcription factors (16,47,54,56,63). E2 can also associate with mitotic chromatin and is proposed as a viral genome segregation fact...

show abstract

Section: Fig 4 E2 Topbp1 Retains Transcription Function (A) E2mentioning

confidence: 99%

An Interaction between Human Papillomavirus 16 E2 and TopBP1 Is Required for Optimum Viral DNA Replication and Episomal Genome Establishment

Donaldson

Mackintosh

Bodily

et al. 2012

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…293T cells transiently transfected with E2 and TopBP1 depletion plasmids were subjected to cytoplasmic/nuclear fractionation as depicted in Fig. 1C (17). Figure 1D shows that depletion of TopBP1 results in E2 proportionally redistributing into the chromatin pellet.…”

mentioning

confidence: 99%

TopBP1 Regulates Human Papillomavirus Type 16 E2 Interaction with Chromatin

Donaldson

Boner

Morgan

2007

J Virol

Self Cite

View full text Add to dashboard Cite

Human papillomavirus type 16 (HPV16) E2 regulates transcription from and replication of the viral genome, in association with viral and cellular factors. HPV16 E2 interacts functionally with TopBP1, a cellular protein essential for the initiation of cellular, and potentially viral, DNA replication. This report demonstrates that the absence of TopBP1 results in the redistribution of HPV16 E2 into an alternative cellular protein complex, resulting in enhanced affinity for chromatin. This redistribution does not significantly alter the ability of HPV16 E2 to either activate or repress transcription. We also show colocalization of both proteins on chromatin at late stages of mitosis, suggesting that TopBP1 could be the mitotic chromatin receptor for HPV16 E2. The possible significance of the results for the regulation of the viral life cycle is discussed.All human papillomaviruses (HPVs) encode an E2 protein which regulates the replication of and transcription from the viral genome (5). We have previously identified TopBP1 as a functional cellular interacting partner for HPV type 16 (HPV16) E2 (hereafter called E2) which increases its transcriptional and DNA replication activity (2, 3). TopBP1 is a BRCT repeat-containing protein that can regulate many aspects of nucleic acid metabolism, including transcription, replication, and DNA damage and repair processes (6,10,12,14,(19)(20)(21)(22). As both E2 and TopBP1 are chromatin-associated proteins, we investigated whether TopBP1 may be a chromatin receptor for E2 and a regulator of E2 function.TopBP1 is not essential for E2 transcriptional activity. 293T cells (chosen due to their high transfectability and ease of TopBP1 knockdown as demonstrated below) were cultured and used in transcription assays as previously described (18). TopBP1 was depleted or mock depleted using pSUPERTopBP1 or pSUPER plasmids (8). From the results shown in Fig. 1A, it is clear that removal of TopBP1 has little effect on the ability of E2 to activate transcription from the thymidine kinase promoter or to repress transcription from the HPV18 promoter. Thirty micrograms of protein from each lysate from the assays whose results are shown in Fig. 1A was Western blotted and probed, as described previously (3), for TopBP1 and E2 (Fig. 1B). Good depletion of TopBP1 can be observed in samples transfected with the pSUPER-TopBP1 plasmid as opposed to the control plasmid. In samples where TopBP1 is depleted, the levels of E2 are markedly elevated. Using the same protein preparation technique, we demonstrated that this increased level of E2 was not due to enhanced stability of the E2 protein (not shown). We therefore investigated the ability of TopBP1 to alter the subcellular localization, and therefore potentially the solubilization, of E2.Depletion of TopBP1 alters the subcellular localization of E2. 293T cells transiently transfected with E2 and TopBP1 depletion plasmids were subjected to cytoplasmic/nuclear fractionation as depicted in Fig. 1C (17). Figure 1D shows that depletion of TopBP1 results in E...

show abstract

“…This plasmid was kindly prepared by Dr. Roni Wright (Gene Regulation Program, Center for Genomic Regulation, Barcelona, Spain) by cloning five Gal4 binding sites, which were amplified by PCR from the pG5Luc vector, into the pTK6E2Luc vector using the KpnI and NheI restriction sites. The p6xE2-3bp-Luc vector has been described previously (23).…”

Section: Plasmidsmentioning

confidence: 99%

“…In telomerase-negative cells, the TR promoter is more active than the TERT promoter, and in telomerase-positive cells, there is not much difference between the two promoters, suggesting more specificity by using the TERT promoter for this application. The optimization of this new TSTA system by incorporating E2-responsive promoters more active in epithelial cells into the system along with the TERT promoter is discussed (22,23). Finally, we describe efficient and selective cell killing of telomerase-positive cells with incorporation of the full-length tumor necrosis factor-related apoptosisinducing ligand (TRAIL) gene into this new TSTA system.…”

Section: Introductionmentioning

confidence: 99%

A novel two-step transcriptional activation system for gene therapy directed toward epithelial cells

Arendt

Nasir

Morgan

2009

Molecular Cancer Therapeutics

Self Cite

View full text Add to dashboard Cite

The two-step transcriptional activation (TSTA) mechanism in gene therapy amplifies cell type-specific promoter activity, allowing for increased levels of gene expression in target tissues. In this system, the specific promoter drives expression of a strong transcriptional activator that binds to DNA target sequences located upstream from a second promoter controlling the expression of the therapeutic gene. The majority of previous studies have exploited a fusion between the DNA binding domain of the yeast transcriptional activator Gal4 fused to the VP16 activation domain of herpes simplex virus 1 as the transcriptional activator. In this report, an alternative to this system is described based on a fusion protein containing the DNA binding domain of the bovine papillomavirus 1 transcriptional activator E2 fused to VP16 that induces target gene expression following binding to a minimal bovine papillomavirus 4 promoter containing upstream E2 binding sites and only 3 bp of promoter sequence upstream from the TATA box. VP16-E2 is superior to Gal4-VP16 as the transcriptional activator in a TSTA system driven by either of the two potentially cancer-specific promoters telomerase RNA and telomerase reverse transcriptase in several cell lines. Results also suggest that this new system has an advantage in epithelial cells and is therefore ideal for potential targeting of carcinomas. By incorporating the TRAIL gene as a transgene in the VP16-E2 TSTA system, selective killing of telomerase-positive cells occurs. We propose that our new system should be considered in future TSTA, particularly when targeting epithelial-derived cells.

show abstract

A Novel Silencer Element in the Bovine Papillomavirus Type 4 Promoter Represses the Transcriptional Response to Papillomavirus E2 Protein

Cited by 12 publications

References 40 publications

An Interaction between Human Papillomavirus 16 E2 and TopBP1 Is Required for Optimum Viral DNA Replication and Episomal Genome Establishment

An Interaction between Human Papillomavirus 16 E2 and TopBP1 Is Required for Optimum Viral DNA Replication and Episomal Genome Establishment

TopBP1 Regulates Human Papillomavirus Type 16 E2 Interaction with Chromatin

A novel two-step transcriptional activation system for gene therapy directed toward epithelial cells

Contact Info

Product

Resources

About