Biomedical subjects

Maria G McPhillips

Publications and source records attributed to Maria G McPhillips.

6 recordsLinked to original sources

PubMed2006

Partitioning viral genomes in mitosis: same idea, different targets.

Papillomavirus infections are long-lived and persistent. The circular DNA of the viral genome is maintained in dividing epithelial cells as an extrachromosomal element. The E2 protein of the virus binds to the viral genome and tethers it to mitotic chromosomes to ensure that the genome is retained and faithfully partitioned in dividing cells. This mechanism has been best studied for bovine papillomavirus type 1. Recent evidence indicates that while this is a common strategy among papillomaviruses, different viruses have selected different chromosomal targets.

Animals↗

PubMed2005

Interaction of bovine papillomavirus E2 protein with Brd4 stabilizes its association with chromatin.

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.

Animals↗

PubMed2005

The mitotic chromosome binding activity of the papillomavirus E2 protein correlates with interaction with the cellular chromosomal protein, Brd4.

The papillomavirus transcriptional activator, E2, is involved in key functions of the viral life cycle. These include transcriptional regulation, viral DNA replication, and viral genome segregation. The transactivation domain of E2 is required for each of these functions. To identify the regions of the domain that mediate binding to mitotic chromosomes, a panel of mutations has been generated and their effect on various E2 functions has been analyzed. A structural model of the bovine papillomavirus type 1 (BPV1) E2 transactivation domain was generated based on its homology with the solved structure of the human papillomavirus type 16 (HPV16) domain. This model was used to identify distinct surfaces of the domain to be targeted by point mutation to further delineate the functional region of the transactivation domain responsible for mitotic chromosome association. The mutated E2 proteins were assessed for mitotic chromosome binding and, in addition, transcriptional activation and transcriptional repression activities. Mutation of amino acids R37 and I73, which are located on a surface of the domain that in HPV16 E2 is reported to mediate self-interaction, completely eliminated mitotic chromosome binding. Mitotic chromosome binding activity was found to correlate well with the ability to interact with the cellular chromosomal associated factor Brd4, which has recently been proposed to mediate the association between BPV1 E2 and mitotic chromosomes.

Animals↗

PubMed2004

Brd4: tethering, segregation and beyond.

Papillomaviruses segregate their genomes in dividing cells by tethering them to mitotic chromosomes via the viral E2 protein. A recent report has shown that this interaction is mediated by the cellular bromodomain protein Brd4. This discovery provides new insight into the mechanism of viral genome segregation and raises many exciting questions about the regulation and nature of the interaction of this complex with mitotic chromosomes.

Animals↗

PubMed2004

SF2/ASF binds the human papillomavirus type 16 late RNA control element and is regulated during differentiation of virus-infected epithelial cells.

Pre-mRNA splicing occurs in the spliceosome, which is composed of small ribonucleoprotein particles (snRNPs) and many non-snRNP components. SR proteins, so called because of their C-terminal arginine- and serine-rich domains (RS domains), are essential members of this class. Recruitment of snRNPs to 5' and 3' splice sites is mediated and promoted by SR proteins. SR proteins also bridge splicing factors across exons to help to define these units and have a central role in alternative and enhancer-dependent splicing. Here, we show that the SR protein SF2/ASF is part of a complex that forms upon the 79-nucleotide negative regulatory element (NRE) that is thought to be pivotal in posttranscriptional regulation of late gene expression in human papillomavirus type 16 (HPV-16). However, the NRE does not contain any active splice sites, is located in the viral late 3' untranslated region, and regulates RNA-processing events other than splicing. The level of expression and extent of phosphorylation of SF2/ASF are upregulated with epithelial differentiation, as is subcellular distribution, specifically in HPV-16-infected epithelial cells, and expression levels are controlled, at least in part, by the virus transcription regulator E2.

3' Untranslated Regions↗

PubMed2003

Activity of the human papillomavirus type 16 late negative regulatory element is partly due to four weak consensus 5' splice sites that bind a U1 snRNP-like complex.

The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the squamous epithelia that it infects. Capsid proteins, and hence mature virions, are produced in the outermost layer of differentiated cells. As late gene transcripts are produced in the lower layers, posttranscriptional mechanisms likely prevent capsid protein production in less differentiated cells. For HPV type 16 (HPV-16), a 79-nucleotide (nt) negative regulatory element (NRE) inhibits gene expression in basal epithelial cells. To identify key NRE sequences, we carried out transient transfection in basal epithelial cells with reporter constructs containing the HPV-16 late 3' untranslated region with deletions and mutations of the NRE. Reporter gene expression was increased over 40-fold by deletion of the entire element, 10-fold by deletion of the 5' portion of the NRE that contains four weak consensus 5' splice sites, and only 3-fold by deletion of the 3' GU-rich region. Both portions of the element appear to be necessary for full repression. Inactivating mutations in the 5' splice sites in the 5' NRE partially alleviated repression in the context of the 79-nt NRE but caused full derepression when assayed in a construct with the 3' NRE deleted. All four contribute to the inhibitory effect, though the second splice site is most inhibitory. Sm proteins, U1A and U1 snRNA, but not U1 70K, could be affinity purified with the wild-type NRE but not with the NRE containing mutations in the 5' splice sites, indicating that a U1 snRNP-like complex forms upon the element.

5' Untranslated Regions↗