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 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.

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 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 Long-Term Episomal Maintenance of Bovine Papillomavirus Type 1 Plasmids Is Determined by Attachment to Host Chromosomes, Which Is Mediated by the Viral E2 Protein and Its Binding Sites

1999 ◽  
Vol 73 (5) ◽  
pp. 4404-4412 ◽  
Author(s):  
Ivar Ilves ◽  
Sirje Kivi ◽  
Mart Ustav
Keyword(s):  
Host Cell ◽  
Binding Sites ◽  
Regulatory Region ◽  
Host Cells ◽  
Bovine Papillomavirus ◽  
E2 Protein ◽  
Multiple Binding Sites ◽  
Stable Maintenance ◽  
Necessary And Sufficient

ABSTRACT Papillomavirus genomes are stably maintained as extrachromosomal nuclear plasmids in dividing host cells. To address the mechanisms responsible for stable maintenance of virus, we examined nuclear compartmentalization of plasmids containing the full-length upstream regulatory region (URR) from the bovine papillomavirus type 1 (BPV1) genome. We found that these plasmids are tightly associated with the nuclear chromatin both in the stable cell lines that maintain episomal copies of the plasmids and in transiently transfected cells expressing the viral E1 and E2 proteins. Further analysis of viral factors revealed that the E2 protein in trans and its multiple binding sites in cis are both necessary and sufficient for the chromatin attachment of the plasmids. On the other hand, the BPV1 URR-dependent plasmid replication and chromatin attachment processes are clearly independent of each other. The ability of the plasmids to stably maintain episomes correlates clearly with their chromatin association function. These data suggest that viral E2 protein-mediated attachment of BPV1 genomes to the host cell chromatin could provide a mechanism for the coupling of viral genome multiplication and partitioning to the host cell cycle during viral latent infection.

scholarly journals Interaction of the Betapapillomavirus E2 Tethering Protein with Mitotic Chromosomes

2009 ◽  
Vol 84 (1) ◽  
pp. 543-557 ◽  
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.

scholarly journals An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export

2003 ◽  
Vol 160 (7) ◽  
pp. 1029-1040 ◽  
Author(s):  
Frederic Kendirgi ◽  
Dianne M. Barry ◽  
Eric R. Griffis ◽  
Maureen A. Powers ◽  
Susan R. Wente
Keyword(s):  
Amino Acid ◽  
Hela Cells ◽  
Mrna Export ◽  
Nucleocytoplasmic Transport ◽  
Nucleocytoplasmic Shuttling ◽  
Cell Permeable Peptide ◽  
A Cell ◽  
Acid Domain ◽  
Necessary And Sufficient ◽  
Amino Acid Domain

Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four–amino acid segment, whereas hGle1B has a COOH-terminal 43–amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP–hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39–amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide–treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.

scholarly journals Characterization of the Functional Activities of the Bovine Papillomavirus Type 1 E2 Protein Single-Chain Heterodimers

2006 ◽  
Vol 80 (22) ◽  
pp. 11218-11225 ◽  
Author(s):  
Reet Kurg ◽  
Helena Tekkel ◽  
Aare Abroi ◽  
Mart Ustav
Keyword(s):  
Binding Site ◽  
Full Length ◽  
Viral Gene ◽  
Epithelial Tissue ◽  
Bovine Papillomavirus ◽  
Transactivation Domain ◽  
Single Chain ◽  
E2 Protein

ABSTRACT Papillomaviruses are small DNA viruses which establish persistent infection in the epithelial tissue of various animal species. Three papillomavirus proteins encoded by the bovine papillomavirus type 1 E2 open reading frame have a common C-terminal DNA binding and dimerization domain and function as dimeric proteins in the regulation of viral gene expression, genome replication, and maintenance. The full-length E2 protein, expressed usually at the lowest level of the three, is an activator, while shorter forms of E2, lacking the transactivation domain, serve as repressors of replication and transcription. In virally infected cells, the full-length E2 protein forms heterodimers with repressor forms of the E2 protein and the biological activities of such heterodimers are poorly known. In order to study the functionality of E2 heterodimers, we joined the full-length E2 protein and E2 repressor by a flexible polypeptide hinge so that they formed a single-chain intramolecular dimer. The single-chain E2 heterodimers folded correctly to form genuine pseudodimers capable of binding to the specific E2 protein binding site with high affinity. Characterization of the activities of this protein in transcription showed that it functions as an effective transcriptional activator, which is comparable to what was found for the full-length E2 protein. The single-chain heterodimer is dependent to some extent on Brd4 protein and is able to support papillomavirus origin replication; however, it does not support the partitioning of the multimeric E2 binding site containing plasmids in dividing cells. Our results suggest that E2 heterodimers serve as activators of transcription and replication during the viral life cycle.

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