Faculty Opinions recommendation of Interaction of the bovine papillomavirus E2 protein with Brd4 tethers the viral DNA to host mitotic chromosomes.

ABSTRACT Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal plasmids. It is therefore of vital importance for viruses to ensure nuclear retention and proper segregation of their viral DNA. The bovine papillomavirus (BPV) E2 enhancer protein plays a key role in these processes by tethering the viral DNA to the host cell chromosomes. Viral genomes that harbor phosphorylation mutations in the E2 gene are transformation defective, and for these mutant genomes, neither the viral DNA nor the E2 protein is detected on mitotic chromosomes, while other key functions of E2 in transcription and replication were wild type. Moreover, secondary mutations in both the E2 and E1 proteins lead to suppression of the phosphorylation mutant phenotype and resulted in reattachment of the viral DNA and the E2 protein onto mitotic chromosomes, suggesting that E1 also plays a role in viral genome partitioning. The E1 protein was cytologically always excluded from mitotic chromatin, either as a suppressor allele or as the wild type. In the absence of other viral proteins, an E2 protein containing alanine substitutions for phosphorylation substrates in the hinge region (E2-A4) was detected as wild-type on mitotic chromosomes. However, when wild-type E1 protein levels were increased in cells expressing either the A4 mutant E2 proteins or wild-type E2, the E2-A4 protein was much more sensitive to chromosomal dislocation than was the wild-type protein. In contrast, suppressor alleles of E1 were not capable of such abrogation of E2 binding (A4 or wild-type) to chromosomes. These results suggest that wild-type E1 can be a negative regulator of the chromosomal attachment of E2.

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Dynamic Localization of the Human Papillomavirus Type 11 Origin Binding Protein E2 through Mitosis While in Association with the Spindle Apparatus

Journal of Virology ◽

10.1128/jvi.80.10.4792-4800.2006 ◽

2006 ◽

Vol 80 (10) ◽

pp. 4792-4800 ◽

Cited By ~ 29

Author(s):

Luan D. Dao ◽

Aaron Duffy ◽

Brian A. Van Tine ◽

Shwu-Yuan Wu ◽

Cheng-Ming Chiang ◽

...

Keyword(s):

Human Papillomavirus ◽

Binding Protein ◽

Cellular Protein ◽

Bovine Papillomavirus ◽

Mitotic Spindles ◽

Mitotic Chromosomes ◽

Mitotic Apparatus ◽

Viral Origin ◽

E2 Protein ◽

Origin Binding Protein

ABSTRACT Papillomaviral DNA replicates as extrachromosomal plasmids in squamous epithelium. Viral DNA must segregate equitably into daughter cells to persist in dividing basal/parabasal cells. We have previously reported that the viral origin binding protein E2 of human papillomavirus types 11 (HPV-11), 16, and 18 colocalized with the mitotic spindles. In this study, we show the localization of the HPV-11 E2 protein to be dynamic. It colocalized with the mitotic spindles during prophase and metaphase. At anaphase, it began to migrate to the central spindle microtubules, where it remained through telophase and cytokinesis. It was additionally observed in the midbody at cytokinesis. A peptide spanning residues 285 to 308 in the carboxyl-terminal domain of HPV-11 E2 (E2C) is necessary and sufficient to confer localization on the mitotic spindles. This region is conserved in HPV-11, -16, and -18 and bovine papillomavirus type 4 (BPV-4) E2 and is also required for the respective E2C to colocalize with the mitotic spindles. The E2 protein of bovine papillomavirus type 1 is tethered to the mitotic chromosomes via the cellular protein Brd4. However, the HPV-11 E2 protein did not associate with Brd4 during mitosis. Lastly, a chimeric BPV-1 E2C containing the spindle localization domain from HPV-11 E2C gained the ability to localize to the mitotic spindles, whereas the reciprocal chimera lost the ability. We conclude that this region of HPV E2C is critical for localization with the mitotic apparatus, enabling the HPV DNA to sustain persistent infections.

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

Journal of Virology ◽

10.1128/jvi.00772-08 ◽

2008 ◽

Vol 82 (15) ◽

pp. 7298-7305 ◽

Cited By ~ 21

Author(s):

Juan Cardenas-Mora ◽

Jonathan E. Spindler ◽

Moon Kyoo Jang ◽

Alison A. McBride

Keyword(s):

Dna Binding ◽

Fluorescent Protein ◽

Binding Domain ◽

Bovine Papillomavirus ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Mitotic Chromosomes ◽

Dimerization Domain ◽

E2 Protein ◽

Binding Function

ABSTRACT 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.

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Interaction of the Betapapillomavirus E2 Tethering Protein with Mitotic Chromosomes

Journal of Virology ◽

10.1128/jvi.01908-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 543-557 ◽

Cited By ~ 27

Author(s):

Vandana Sekhar ◽

Shawna C. Reed ◽

Alison A. McBride

Keyword(s):

Amino Acid ◽

Viral Genome ◽

Sequence Similarity ◽

Bovine Papillomavirus ◽

Mitotic Chromosomes ◽

E2 Protein ◽

Amino Acid Peptide ◽

Amino Acid Region ◽

Papillomavirus Infection ◽

Chromosome Binding

ABSTRACT During persistent papillomavirus infection, the viral E2 protein tethers the viral genome to the host cell chromosomes, ensuring maintenance and segregation of the viral genome during cell division. However, E2 proteins from different papillomaviruses interact with distinct chromosomal regions and targets. The tethering mechanism has been best characterized for bovine papillomavirus type 1 (BPV1), where the E2 protein tethers the viral genome to mitotic chromosomes in complex with the cellular bromodomain protein, Brd4. In contrast, the betapapillomavirus human papillomavirus type 8 (HPV8) E2 protein binds to the repeated ribosomal DNA genes that are found on the short arm of human acrocentric chromosomes. In this study, we show that a short 16-amino-acid peptide from the hinge region and the C-terminal DNA binding domain of HPV8 E2 are necessary and sufficient for interaction with mitotic chromosomes. This 16-amino-acid region contains an RXXS motif that is highly conserved among betapapillomaviruses, and both arginine 250 and serine 253 residues within this motif are required for mitotic chromosome binding. The HPV8 E2 proteins are highly phosphorylated, and serine 253 is a site of phosphorylation. The HPV8 E2 chromosome binding sequence also has sequence similarity with chromosome binding regions in the gammaherpesvirus EBNA and LANA tethering proteins.

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Conditional Mutations in the Mitotic Chromosome Binding Function of the Bovine Papillomavirus Type 1 E2 Protein

Journal of Virology ◽

10.1128/jvi.79.3.1500-1509.2005 ◽

2005 ◽

Vol 79 (3) ◽

pp. 1500-1509 ◽

Cited By ~ 18

Author(s):

Peng-Sheng Zheng ◽

Jane Brokaw ◽

Alison A. McBride

Keyword(s):

Amino Acid ◽

Bovine Papillomavirus ◽

Transactivation Domain ◽

Mitotic Chromosomes ◽

E2 Protein ◽

Binding Function ◽

Acid Domain ◽

Necessary And Sufficient ◽

Amino Acid Domain

ABSTRACT The papillomavirus E2 protein is required for viral transcriptional regulation, DNA replication and genome segregation. We have previously shown that the E2 transactivator protein and BPV1 genomes are associated with mitotic chromosomes; E2 links the genomes to cellular chromosomes to ensure efficient segregation to daughter nuclei. The transactivation domain of the E2 protein is necessary and sufficient for association of the E2 protein with mitotic chromosomes. To determine which residues of this 200-amino-acid domain are important for chromosomal interaction, E2 proteins with amino acid substitutions in each conserved residue of the transactivation domain were tested for their ability to associate with mitotic chromosomes. Chromatin binding was assessed by using immunofluorescence on both spread and directly fixed mitotic chromosomes. E2 proteins defective in the transactivation and replication functions were unable to associate with chromosomes, and those that were competent in these functions were attached to mitotic chromosomes. However, several mutated proteins that were defective for chromosomal interaction could associate with chromosomes after treatment with agents that promote protein folding or when cells were incubated at lower temperatures. These results indicate that precise folding of the E2 transactivation domain is crucial for its interaction with mitotic chromosomes and that this association can be modulated.

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The Human Papillomavirus Type 8 E2 Tethering Protein Targets the Ribosomal DNA Loci of Host Mitotic Chromosomes

Journal of Virology ◽

10.1128/jvi.01936-08 ◽

2008 ◽

Vol 83 (2) ◽

pp. 640-650 ◽

Cited By ~ 31

Author(s):

Atasi Poddar ◽

Shawna C. Reed ◽

Maria G. McPhillips ◽

Jonathan E. Spindler ◽

Alison A. McBride

Keyword(s):

Human Papillomavirus ◽

Ribosomal Dna ◽

Cellular Protein ◽

Bovine Papillomavirus ◽

Mitotic Chromosomes ◽

Protein Targets ◽

E2 Protein ◽

Viral Genomes ◽

Wide Range ◽

Polymerase I

ABSTRACT For many papillomaviruses, the viral protein E2 tethers the viral genome to the host mitotic chromosomes to ensure persistent, long-term maintenance of the genome during cell division. Our previous studies of E2 proteins from different genera of papillomaviruses have shown that they bind to different regions of the host chromosomes during mitosis. For example, bovine papillomavirus type 1 (BPV-1) E2 binds to all chromosomes as small speckles in complex with the cellular protein Brd4. In contrast, the human papillomavirus type 8 (HPV-8) E2 protein binds as large speckles at the pericentromeric regions of chromosomes. Here we show that these speckles do not contain Brd4, and unlike that of BPV-1, the N-terminal Brd4-interacting domain of HPV-8 E2 is not required for chromosome binding. In contrast to BPV-1 E2, the HPV-8 E2 protein targets the short arms of acrocentric mitotic chromosomes. Furthermore, the E2 protein interacts with the repeated ribosomal DNA genes found in this location and colocalizes with UBF, the RNA polymerase I transcription factor. Therefore, HPV-8 E2 genome tethering occurs by a Brd4-independent mechanism through a novel interaction with specific regions of mitotic chromosomes. Thus, a wide range of viruses have adopted the strategy of linking their genomes to host chromosomes, but individual viruses use different chromosomal targets. Characterization of these targets will enable the development of antiviral therapies to eliminate the viral genomes from infected cells.

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Inhibition of E2 Binding to Brd4 Enhances Viral Genome Loss and Phenotypic Reversion of Bovine Papillomavirus-Transformed Cells

Journal of Virology ◽

10.1128/jvi.79.23.14956-14961.2005 ◽

2005 ◽

Vol 79 (23) ◽

pp. 14956-14961 ◽

Cited By ~ 44

Author(s):

Jianxin You ◽

Michal-Ruth Schweiger ◽

Peter M. Howley

Keyword(s):

Viral Genome ◽

Mitotic Chromosome ◽

Transformed Cells ◽

Bovine Papillomavirus ◽

Complete Elimination ◽

Mitotic Chromosomes ◽

Viral Dna ◽

Viral Genomes ◽

Dividing Cells ◽

Phenotypic Reversion

ABSTRACT The bovine papillomavirus E2 protein tethers the viral genomes to mitotic chromosomes in dividing cells through binding to the C-terminal domain (CTD) of Brd4. Expression of the Brd4-CTD competes the binding of E2 to endogenous Brd4 in cells. Here we extend our previous study that identified Brd4 as the E2 mitotic chromosome receptor to show that Brd4-CTD expression released the viral DNA from mitotic chromosomes in BPV-1 transformed cells. Furthermore, stable expression of Brd4-CTD enhanced the frequency of morphological reversion of BPV-1 transformed C127 cells resulting in the complete elimination of the viral DNA in the resulting flat revertants.

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Interaction of Bovine Papillomavirus E2 Protein with Brd4 Stabilizes Its Association with Chromatin

Journal of Virology ◽

10.1128/jvi.79.14.8920-8932.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 8920-8932 ◽

Cited By ~ 73

Author(s):

Maria G. McPhillips ◽

Keiko Ozato ◽

Alison A. McBride

Keyword(s):

Cell Types ◽

Bovine Papillomavirus ◽

Chromosomal Protein ◽

Transactivation Domain ◽

Mitotic Chromosomes ◽

E2 Protein ◽

Viral Genomes ◽

Protein Protein Interaction ◽

Binding Function ◽

Interphase Cells

ABSTRACT The bovine papillomavirus E2 protein maintains and segregates the viral extrachromosomal genomes by tethering them to cellular mitotic chromosomes. E2 interacts with a cellular bromodomain protein, Brd4, to mediate the segregation of viral genomes into daughter cells. Brd4 binds acetylated histones and has been observed to diffusely coat mitotic chromosomes in several cell types. In this study, we show that in mitotic C127 cells, Brd4 diffusely coated the condensed chromosomes. However, in the presence of the E2 protein, E2 and Brd4 colocalized in punctate dots that were randomly distributed over the chromosomes. A similar pattern of E2 and Brd4 colocalization on mitotic chromosomes was observed in CV-1 cells, whereas only a faint chromosomal coating of Brd4 was detected in the absence of the E2 protein. Therefore, the viral E2 protein relocalizes and/or stabilizes the association of Brd4 with chromosomes in mitotic cells. The colocalization of E2 and Brd4 was also observed in interphase cells, indicating that this protein-protein interaction persists throughout the cell cycle. The interaction of E2 with Brd4 greatly stabilized the association of Brd4 with interphase chromatin. In both mitotic and interphase cells, this stabilization required a transcriptionally competent transactivation domain, but not the DNA binding function of the E2 protein. Thus, the E2 protein modulates the chromatin association of Brd4 during both interphase and mitosis. This study demonstrates that the segregation of papillomavirus genomes is not simply due to the passive hitchhiking of the E2/genome complex with a convenient cellular chromosomal protein.

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