The DNA Binding Domain of a Papillomavirus E2 Protein Programs a Chimeric Nuclease To Cleave Integrated Human Papillomavirus DNA in HeLa Cervical Carcinoma Cells

Horner, Stacy M.; DiMaio, Daniel

doi:10.1128/jvi.00232-07

Cited by 12 publications

(14 citation statements)

References 62 publications

(47 reference statements)

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“…Thus, competition for adjacent binding sites and steric hindrance most presumably accounted for the inhibition of JunB/Fra-1 binding. It is tempting to speculate that similar mechanisms may occur at additional E2 binding sites which have been recently identified in cellular DNA (23).…”

Section: Discussionmentioning

confidence: 99%

Human Papillomavirus Type 8 E2 Protein Unravels JunB/Fra-1 as an Activator of the β4-Integrin Gene in Human Keratinocytes

Ołdak

Maksym

Sperling

et al. 2010

J Virol

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The papillomavirus life cycle parallels keratinocyte differentiation in stratifying epithelia. We have previously shown that the human papillomavirus type 8 (HPV8) E2 protein downregulates ␤4-integrin expression in normal human keratinocytes, which may trigger subsequent differentiation steps. Here, we demonstrate that the DNA binding domain of HPV8 E2 is sufficient to displace a cellular factor from the ␤4-integrin promoter. We identified the E2-displaceable factor as activator protein 1 (AP-1), a heteromeric transcription factor with differentiation-specific expression in the epithelium. ␤4-Integrin-positive epithelial cells displayed strong AP-1 binding activity. Both AP-1 binding activity and ␤4-integrin expression were coregulated during keratinocyte differentiation suggesting the involvement of AP-1 in ␤4-integrin expression. In normal human keratinocytes the AP-1 complex was composed of JunB and Fra-1 subunits. Chromatin immunoprecipitation assays confirmed that JunB/Fra-1 proteins interact in vivo with the ␤4-integrin promoter and that JunB/Fra-1 promoter occupancy is reduced during keratinocyte differentiation as well as in HPV8 E2 positive keratinocytes. Ectopic expression of the tethered JunB/Fra-1 heterodimer in normal human keratinocytes activated the ␤4-integrin promoter, while coexpression of HPV8 E2 reverted the JunB/Fra-1 effect. In summary, we identified a novel mechanism of human ␤4-integrin regulation that is specifically targeted by the HPV8 E2 protein mimicking transcriptional conditions of differentiation. This may explain the early steps of how HPV8 commits its host cells to the differentiation process required for the viral life cycle.

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Section: Discussionmentioning

confidence: 99%

Human Papillomavirus Type 8 E2 Protein Unravels JunB/Fra-1 as an Activator of the β4-Integrin Gene in Human Keratinocytes

Ołdak

Maksym

Sperling

et al. 2010

J Virol

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“…It has recently been demonstrated, using an E1-defective mutant of HPV16, that E1 protein is dispensable for maintenance replication and essential for initial and productive replication of HPV16 [9]. The E2 protein is a transcription factor that acts as an activator or repressor of the transcriptional activity of all HPV genes, and it has been observed that this protein is also involved in viral DNA replication via an interaction with protein E1 and episome maintenance [10, 11]. Therefore, E2 is a regulatory protein relevant for the establishment of infection and vital for the virus to complete the life cycle.…”

Section: The Natural History Of Cervical Lesionsmentioning

confidence: 99%

HPV-Based Screening, Triage, Treatment, and Followup Strategies in the Management of Cervical Intraepithelial Neoplasia

Peralta‐Zaragoza

Deas

Gómez-Cerón

et al. 2013

Obstetrics and Gynecology International

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Cervical cancer is the second most common cause of death from cancer in women worldwide, and the development of new diagnostic, prognostic, and treatment strategies merits special attention. Many efforts have been made to design new drugs and develop immunotherapy and gene therapy strategies to treat cervical cancer. HPV genotyping has potentially valuable applications in triage of low-grade abnormal cervical cytology, assessment of prognosis and followup of cervical intraepithelial neoplasia, and in treatment strategies for invasive cervical cancer. It is known that during the development of cervical cancer associated with HPV infection, a cascade of abnormal events is induced, including disruption of cellular cycle control, alteration of gene expression, and deregulation of microRNA expression. Thus, the identification and subsequent functional evaluation of host proteins associated with HPV E6 and E7 oncoproteins may provide useful information in understanding cervical carcinogenesis, identifying cervical cancer molecular markers, and developing specific targeting strategies against tumor cells. Therefore, in this paper, we discuss the main diagnostic methods, management strategies, and followup of HPV-associated cervical lesions and review clinical trials applying gene therapy strategies against the development of cervical cancer.

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“…One potential advantage of this approach is that the genome sequence information on DNA viruses of interest should be adequate for the application. Prior to our study, Horner and DiMaio reported the first application of an artificial endonuclease to cleave integrated HPV DNA in HeLa cervical carcinoma cells [16]. This group generated a chimeric nuclease (designated BEF in reference [16]) comprising the DNA binding domain of bovine papillomavirus type 1 (BPV1) E2 protein and the DNA-cleavage domain of a FokI endonuclease such as zinc-finger nucleases (ZFNs; [17]) that harbor an engineered zinc-finger protein as their DNA-binding domain and the FokI cleavage domain, and demonstrated clearly that the BEF nuclease effectively cleaved its target DNA in vitro and in HeLa cells.…”

Section: Introductionmentioning

confidence: 99%

“…Prior to our study, Horner and DiMaio reported the first application of an artificial endonuclease to cleave integrated HPV DNA in HeLa cervical carcinoma cells [16]. This group generated a chimeric nuclease (designated BEF in reference [16]) comprising the DNA binding domain of bovine papillomavirus type 1 (BPV1) E2 protein and the DNA-cleavage domain of a FokI endonuclease such as zinc-finger nucleases (ZFNs; [17]) that harbor an engineered zinc-finger protein as their DNA-binding domain and the FokI cleavage domain, and demonstrated clearly that the BEF nuclease effectively cleaved its target DNA in vitro and in HeLa cells. Although the adenovirus-delivered BEF nuclease induced cellular senescence in HeLa cells, molecular analysis revealed that the DNA-binding domain of the BPV1 E2 protein, but not the FokI domain of this nuclease, was responsible for senescence.…”

Section: Introductionmentioning

confidence: 99%

Gene- and Protein-Delivered Zinc Finger–Staphylococcal Nuclease Hybrid for Inhibition of DNA Replication of Human Papillomavirus

et al. 2013

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Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP–SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP–SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP–SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP–SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

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The DNA Binding Domain of a Papillomavirus E2 Protein Programs a Chimeric Nuclease To Cleave Integrated Human Papillomavirus DNA in HeLa Cervical Carcinoma Cells

Cited by 12 publications

References 62 publications

Human Papillomavirus Type 8 E2 Protein Unravels JunB/Fra-1 as an Activator of the β4-Integrin Gene in Human Keratinocytes

Human Papillomavirus Type 8 E2 Protein Unravels JunB/Fra-1 as an Activator of the β4-Integrin Gene in Human Keratinocytes

HPV-Based Screening, Triage, Treatment, and Followup Strategies in the Management of Cervical Intraepithelial Neoplasia

Gene- and Protein-Delivered Zinc Finger–Staphylococcal Nuclease Hybrid for Inhibition of DNA Replication of Human Papillomavirus

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