Dimerization of the Human Papillomavirus Type 16 E2 N Terminus Results in DNA Looping within the Upstream Regulatory Region

Hernandez-Ramon, Elena E.; Burns, Julie E.; Zhang, Wenke; Walker, Hannah F.; Allen, Stephanie; Antson, A.A.; Maitland, Norman J.

doi:10.1128/jvi.02388-07

Cited by 17 publications

(14 citation statements)

References 43 publications

(48 reference statements)

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“…For E2 from human papillomavirus type 16 (HPV16), dimerization of the TAD was found to involve two of the three α-helices that form part of the Brd4-binding surface (Antson et al, 2000). This TAD–TAD interface was shown to occur between different E2 dimers as it can promote DNA-looping between E2 dimers bound at distant sites in vitro (Antson et al, 2000; Hernandez-Ramon et al, 2008). A very different TAD dimerization interface was identified for BPV1 E2.…”

Section: Introductionmentioning

confidence: 99%

Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1

et al. 2013

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The bovine papillomavirus type 1 (BPV1) E2 protein binds as a dimer to the viral genome to promote its transcription, replication and maintenance in keratinocytes. Although BPV1 E2 dimerizes primarily through its DNA-binding domain, it was shown previously that its transactivation domain (TAD) can also dimerize in vitro through formation of a disulfide bond between cysteine 57 (C57) of adjacent monomers and of an ion pair between arginine 172 (R172) and aspartic acid 175 (D175). The function of this TAD dimerization interface in vivo remains unknown. Here, we report the effects of substituting C57, R172 and D175 by alanine on the transactivation activity of BPV E2 as well as on its ability to support viral DNA replication using a novel luciferase-based assay. Results for this mutational analysis suggest that the TAD dimerization interface is not essential for either process but may contribute to the DNA replication activity of BPV1 E2.

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Section: Introductionmentioning

confidence: 99%

Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1

et al. 2013

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“…The E2 transactivation domain has also been implicated in self-interaction, and the looping of DNA containing E2 binding sites has been observed previously by electron microscopy for BPV-1, human papillomavirus type 16 (HPV-16), and HPV-11 E2 proteins (3,11,15,29). For HPV-16 E2, selfinteraction involves residues R37 and I73, which are also crucial for Brd4 binding (1,5,20), and thus, self-interaction may modulate Brd4 binding.…”

Section: Discussionmentioning

confidence: 99%

Dimerization of the Papillomavirus E2 Protein Is Required for Efficient Mitotic Chromosome Association and Brd4 Binding

Cardenas-Mora

Spindler

McBride

2008

J Virol

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The E2 proteins of several papillomaviruses link the viral genome to mitotic chromosomes to ensure retention and the efficient partitioning of genomes into daughter cells following cell division. Bovine papillomavirus type 1 E2 binds to chromosomes in a complex with Brd4, a cellular bromodomain protein. Interaction with Brd4 is also important for E2-mediated transcriptional regulation. The transactivation domain of E2 is crucial for interaction with the Brd4 protein; proteins lacking or mutated in this domain do not interact with Brd4. However, we found that the C-terminal DNA binding/dimerization domain of E2 is also required for efficient binding to Brd4. Mutations that eliminated the DNA binding function of E2 had no effect on the ability of E2 to interact with Brd4, but an E2 protein with a mutation that disrupted C-terminal dimerization bound Brd4 with greatly reduced efficiency. Furthermore, E2 proteins in which the C-terminal domains were replaced with the dimeric DNA binding domain of EBNA-1 or Gal4 bound efficiently to the Brd4 protein, but the replacement of the E2 C-terminal domain with a monomeric red fluorescent protein did not rescue efficient Brd4 binding. Thus, E2 bound to Brd4 most efficiently as a dimer. To prove this finding further, the E2 DNA binding domain was replaced with an FKBP12-derived domain in which dimerization was regulated by a bivalent ligand. This fusion protein bound Brd4 efficiently only in the presence of the ligand, confirming that a dimer of E2 was required. Correspondingly, E2 proteins that could dimerize were able to bind to mitotic chromosomes much more efficiently than monomeric E2 polypeptides.The papillomavirus E2 protein has several functions in the viral life cycle. It is the major viral transcriptional regulatory protein and, along with the E1 helicase, binds to the origin of replication to initiate DNA replication. The E2 protein also ensures that the infection is transmitted to daughter cells through many cell divisions by attaching the viral genomes to cellular mitotic chromosomes. For all papillomaviruses studied to date, the E2 transcriptional regulatory function involves interaction with the cellular protein Brd4 (12,20,27,28,34). Brd4 binds to acetylated chromatin via two bromodomains and regulates transcriptional elongation (13,33). An E2-Brd4 complex binds to viral promoters and is involved in both the activation and the repression of viral transcription (26,33,34).For a subset of papillomaviruses, the E2-Brd4 complex is also involved in genome tethering to mitotic chromosomes (5,20,35). The interaction of the bovine papillomavirus type 1 (BPV-1) E2 protein with both Brd4 and mitotic chromosomes has been well studied; BPV-1 E2 and Brd4 colocalize on both interphase chromatin and mitotic chromosomes in punctate speckles (21, 35). Furthermore, E2 greatly increases the affinity of interphase Brd4 for chromatin and relocalizes mitotic Brd4 from a diffuse cloud to punctate speckles on condensed chromosomes (21).The interaction of BPV-1 E2 and Brd4 has been wel...

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“…Such structure will result in the tissue-specific enhancers shifting closer to the core transcription complex for transcriptional activation [20,21]. Meanwhile, some studies have indicated that binding of the intact E2 to the LCR sequences may spatially prevent the transcriptional machine to active the promoter, which are the main molecular mechanism for E2 transcriptional repression [22,23].…”

Section: Discussionmentioning

confidence: 99%

A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

et al. 2012

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BackgroundThe human papillomavirus (HPV) E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa) substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells.ResultsCAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA) showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2.ConclusionsThese results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

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Dimerization of the Human Papillomavirus Type 16 E2 N Terminus Results in DNA Looping within the Upstream Regulatory Region

Cited by 17 publications

References 43 publications

Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1

Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1

Dimerization of the Papillomavirus E2 Protein Is Required for Efficient Mitotic Chromosome Association and Brd4 Binding

A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

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