Bovine Papillomavirus Type 1 Genomes and the E2 Transactivator Protein Are Closely Associated with Mitotic Chromatin

1998 ◽  
Vol 72 (3) ◽  
pp. 2079-2088 ◽  
Author(s):  
Mario H. Skiadopoulos ◽  
Alison A. McBride
Keyword(s):  
Bovine Papillomavirus ◽  
Transactivation Domain ◽  
Mitotic Chromosomes ◽  
Viral Genomes ◽  
Binding Function ◽  
Dividing Cells ◽  
Transactivator Protein ◽  
Episomal Maintenance ◽  
Mitotic Chromatin

ABSTRACT The bovine papillomavirus type 1 E2 transactivator protein is required for viral transcriptional regulation and DNA replication and may be important for long-term episomal maintenance of viral genomes within replicating cells (M. Piirsoo, E. Ustav, T. Mandel, A. Stenlund, and M. Ustav, EMBO J. 15:1–11, 1996). We have evidence that, in contrast to most other transcriptional transactivators, the E2 transactivator protein is associated with mitotic chromosomes in dividing cells. The shorter E2-TR and E8/E2 repressor proteins do not bind to mitotic chromatin, and the N-terminal transactivation domain of the E2 protein is necessary for the association. However, the DNA binding function of E2 is not required. We have found that bovine papillomavirus type 1 genomes are also associated with mitotic chromosomes, and we propose a model in which E2-bound viral genomes are transiently associated with cellular chromosomes during mitosis to ensure that viral genomes are segregated to daughter cells in approximately equal numbers.

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

2005 ◽  
Vol 79 (3) ◽  
pp. 1500-1509 ◽  
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.

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

2005 ◽  
Vol 79 (14) ◽  
pp. 8920-8932 ◽  
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.

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

2008 ◽  
Vol 82 (15) ◽  
pp. 7298-7305 ◽  
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.

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

2005 ◽  
Vol 79 (23) ◽  
pp. 14956-14961 ◽  
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.

scholarly journals Hitchhiking of Viral Genomes on Cellular Chromosomes

2019 ◽  
Vol 6 (1) ◽  
pp. 275-296 ◽  
Author(s):  
Tami L. Coursey ◽  
Alison A. McBride
Keyword(s):  
Viral Genome ◽  
Viral Infections ◽  
Genome Replication ◽  
Mitotic Chromosomes ◽  
Dna Viruses ◽  
Viral Genomes ◽  
Barr Virus ◽  
Dividing Cells ◽  
Viral Genome Replication ◽  
Epstein Barr

Persistent viral infections require a host cell reservoir that maintains functional copies of the viral genome. To this end, several DNA viruses maintain their genomes as extrachromosomal DNA minichromosomes in actively dividing cells. These viruses typically encode a viral protein that binds specifically to viral DNA genomes and tethers them to host mitotic chromosomes, thus enabling the viral genomes to hitchhike or piggyback into daughter cells. Viruses that use this tethering mechanism include papillomaviruses and the gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This review describes the advantages and consequences of persistent extrachromosomal viral genome replication.

Detection and characterization of viral genomes and search for tumoral antigens in two hamster cell lines derived from tumors induced by bovine papillomavirus type 1

1981 ◽  
Vol 27 (5) ◽  
pp. 693-702 ◽  
Author(s):  
FrançOise Breitburd ◽  
Michel Favre ◽  
Rima Zoorob ◽  
Dominique Fortin ◽  
Gérard Orth
Keyword(s):  
Cell Lines ◽  
Bovine Papillomavirus ◽  
Viral Genomes

scholarly journals E1 Protein of Bovine Papillomavirus Type 1 Interferes with E2 Protein-Mediated Tethering of the Viral DNA to Mitotic Chromosomes

2002 ◽  
Vol 76 (7) ◽  
pp. 3440-3451 ◽  
Author(s):  
Christian Voitenleitner ◽  
Michael Botchan
Keyword(s):  
Negative Regulator ◽  
Bovine Papillomavirus ◽  
Wild Type ◽  
Mitotic Chromosomes ◽  
Viral Dna ◽  
Nuclear Retention ◽  
E2 Protein ◽  
Protein Levels ◽  
E1 Protein ◽  
Dividing Cells

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.

scholarly journals Brd4 Is Involved in Multiple Processes of the Bovine Papillomavirus Type 1 Life Cycle

2006 ◽  
Vol 80 (7) ◽  
pp. 3660-3665 ◽  
Author(s):  
Ivar Ilves ◽  
Kristina Mäemets ◽  
Toomas Silla ◽  
Kadri Janikson ◽  
Mart Ustav
Keyword(s):  
Life Cycle ◽  
Transcription Activation ◽  
Bovine Papillomavirus ◽  
Viral Genomes ◽  
Therapeutic Value ◽  
Multiple Processes ◽  
Chromosome Attachment ◽  
Dependent Mechanism

ABSTRACT Brd4 protein has been proposed to act as a cellular receptor for the bovine papillomavirus type 1 (BPV1) E2 protein in the E2-mediated chromosome attachment and mitotic segregation of viral genomes. Here, we provide data that show the involvement of Brd4 in multiple early functions of the BPV1 life cycle, suggest a Brd4-dependent mechanism for E2-dependent transcription activation, and indicate the role of Brd4 in papillomavirus and polyomavirus replication as well as cell-specific utilization of Brd4-linked features in BPV1 DNA replication. Our data also show the potential therapeutic value of the disruption of the E2-Brd4 interaction for the development of antiviral drugs.

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

Virology ◽  
2013 ◽  
Vol 439 (2) ◽  
pp. 132-139 ◽  
Author(s):  
David Gagnon ◽  
Hélène Sénéchal ◽  
Claudia M. D’Abramo ◽  
Jennifer Alvarez ◽  
Alison A. McBride ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Bovine Papillomavirus ◽  
Transactivation Domain ◽  
Dimerization Interface

scholarly journals Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division

2010 ◽  
Vol 84 (21) ◽  
pp. 11175-11188 ◽  
Author(s):  
Toomas Silla ◽  
Andres Männik ◽  
Mart Ustav
Keyword(s):  
Transcriptional Activation ◽  
Human Herpesvirus ◽  
Bovine Papillomavirus ◽  
Transactivation Domain ◽  
Mitotic Chromosomes ◽  
Herpesvirus Type ◽  
Barr Virus ◽  
Human Herpesvirus Type ◽  
Multiple Copies ◽  
Epstein Barr

ABSTRACT Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV) genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV, and KSHV segregation are E2, LANA1, and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral genome segregation. Because E2 transcriptional activation and chromatin attachment functions are not mutually exclusive, we aimed to determine the requirement of these activities during segregation by analyzing chimeric E2 proteins. This approach allowed us to separate the two activities. Our data showed that attachment of the segregation protein to chromatin is not sufficient for proper segregation. Rather, formation of a segregation-competent complex which carries multiple copies of the segregation protein is required. Complementation studies of E2 functional domains indicated that chromatin attachment and transactivation functions must act in concert to ensure proper plasmid segregation. These data indicate that there are specific interactions between linker molecules and transcription factors/complexes that greatly increase segregation-competent complex formation. We also showed, using hybrid E2 molecules, that restored segregation function does not involve interactions with Brd4.

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