Biomedical subjects

Alison A McBride

Publications and source records attributed to Alison A McBride.

14 recordsLinked to original sources

PubMed2026

Differentiation-independent activation of HPV genome replication by the lncRNA DINO.

Human papillomaviruses (HPVs) rely on multiple host cell factors to replicate the viral genome, yet the contribution of host long non-coding RNAs (lncRNAs) to viral genome maintenance and amplification in the productive life cycle remains poorly understood. In this study, we show that the lncRNA damage-induced long non-coding RNA (DINO) is a driver of HPV DNA replication. DINO levels increase during keratinocyte differentiation, and ectopic expression of DINO promotes both HPV genome replication and the formation of replication foci, and this is independent of keratinocyte differentiation signals. Ectopic DINO expression increases select early viral transcript levels, including E1^E4, E1, and E2. Notably, DINO's subcellular localization is also context-dependent: during DNA damage, DINO is predominantly cytoplasmic, but during keratinocyte differentiation, nuclear retention is observed. This differential localization suggests that DINO has distinct functional roles in keratinocyte differentiation and HPV biology. Our findings highlight DINO as a lncRNA that promotes HPV genome replication and suggest that lncRNAs may play underappreciated roles in host-virus interactions. This work provides a foundation for further exploration of lncRNAs as potential therapeutic targets in HPV-associated diseases.IMPORTANCEHuman papillomaviruses (HPVs) are the causative agents of many anogenital tract and oral cancers, yet the host factors that trigger and support viral genome replication during the productive life cycle are incompletely understood. This study identifies the long non-coding RNA DINO as a host regulator that promotes HPV DNA replication, replication focus formation, and early viral gene expression independently of keratinocyte differentiation. We further show that DINO exhibits context-dependent subcellular localization, suggesting distinct functional roles in cellular stress responses and HPV biology. These findings reveal an underappreciated role for host lncRNAs in virus-host interactions and provide new insight into cellular pathways that support HPV genome replication.

Virus Replication↗

PubMed2026

Differentiation-independent Activation of HPV Genome Replication by the lncRNA DINO.

Human papillomaviruses (HPV) rely on multiple host cell factors to replicate the viral genome, yet the contribution of host long noncoding RNAs (lncRNAs) to viral genome maintenance and amplification in the productive life cycle remains poorly understood. In this study, we show that the lncRNA DINO is a driver of HPV DNA replication. DINO levels increase during keratinocyte differentiation and ectopic expression of DINO promotes both HPV genome replication and the formation of replication foci, and this is independent of keratinocyte differentiation signals. Ectopic DINO expression increases select early viral transcript levels including E1^4, E1, and E2. Notably, DINO's subcellular localization is also context-dependent: during DNA damage DINO is predominantly cytoplasmic, but during keratinocyte differentiation nuclear retention is observed. This differential localization suggests that DINO has distinct functional roles in keratinocyte differentiation and HPV biology. Our findings highlight DINO as a lncRNA that promotes HPV genome replication and suggest that lncRNAs may play underappreciated roles in host-virus interactions. This work provides a foundation for further exploration of lncRNAs as potential therapeutic targets in HPV-associated diseases.

Journal Article↗

PubMed2025

Chromatin assembly by the histone chaperone HIRA facilitates Human Papillomavirus replication.

The circular, double-stranded DNA genomes of Human papillomaviruses (HPV) exist in a nucleosomal state throughout the infectious cycle and rely on host histone epigenetic modifications and chromatin assembly processes to promote various phases of the viral life cycle. Here, we show that the histone H3.3 chaperone HIRA and its associated complex members are recruited to HPV replication factories during the late phase of the HPV life cycle. HIRA is also recruited to HPV replication factories generated by amplification of a replicon with a minimal origin and expression of the viral replication proteins E1 and E2, demonstrating that the E1 and E2 proteins are sufficient for HIRA recruitment. Downregulation of HIRA expression reduces HPV31 DNA amplification and viral transcription in differentiated keratinocytes. Histone H3.3 that is highly phosphorylated on serine residue 31 is also enriched at sites of HPV replication and this modification links the DNA damage response to chromatin that supports rapid gene activation. We propose that deposition of histone H3.3 generates viral minichromosomes that are highly primed to support the late stages of the HPV life cycle.

H3.3 phosphorylation↗

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↗

PubMed2006

Repression of HPV16 early region transcription by the E2 protein.

HPV16 DNA is often integrated in cancers, disrupting the E1 or E2 genes. E2 can repress the E6/E7 promoter, but other models have been proposed to explain why integration promotes malignant progression. E1 and E2 are required for viral replication, and so genetic analysis of their role in transcriptional regulation is complex. Therefore, we developed an extrachromosomal vector containing HPV16 to undertake a genetic analysis of the E1 and E2 genes. We demonstrate that the E2 protein is primarily a transcriptional repressor when expressed from the virus. Furthermore, repression requires both the transactivation function of E2 and specific binding of E2 to the LCR. We find no evidence that the E1 protein directly modulates HPV16 gene expression. However, certain E1 mutations modulated transcription indirectly by altering splicing of E2 mRNA species. These data provide important insight into which E1 and E2 functions are optimal targets for anti-viral therapies.

Base Sequence↗

PubMed2006

Variations in the association of papillomavirus E2 proteins with mitotic chromosomes.

The E2 protein segregates episomal bovine papillomavirus (BPV) genomes to daughter cells by tethering them to mitotic chromosomes, thus ensuring equal distribution and retention of viral DNA. To date, only the BPV1 E2 protein has been shown to bind to mitotic chromosomes. We assessed the localization of 13 different animal and human E2 proteins from seven papillomavirus genera, and we show that most of them are stably bound to chromosomes throughout mitosis. Furthermore, in contrast to the random association of BPV1 E2 with mitotic chromosomes, several of these proteins appear to bind to more specific regions of mitotic chromosomes. Using human papillomavirus (HPV) type 8 E2, we show that this region is adjacent to centromeres/kinetochores. Therefore, E2 proteins from both HPV and animal papillomavirus bind to mitotic chromosomes, and there are variations in the specificity of this binding. Only the alpha-papillomavirus E2 proteins do not stably associate with mitotic chromatin throughout mitosis. These proteins closely associate with prophase chromosomes and bind to chromosomes in telophase but not in metaphase. However, extraction of mitotic cells before fixation results in alpha-E2 proteins binding to the pericentromeric region of metaphase chromosomes, as observed for HPV8 E2. We postulate that this is the authentic target of these E2 proteins but that additional factors or a specialized cellular environment is required to stabilize this association. Thus, E2-mediated tethering of viral genomes to mitotic chromosomes is a common strategy of papillomaviruses, but different viruses have evolved different variations of this theme.

Bovine papillomavirus 1↗

PubMed2006

Molecular basis for phosphorylation-dependent, PEST-mediated protein turnover.

Proteasomal-mediated rapid turnover of proteins is often modulated by phosphorylation of PEST sequences. The E2 protein from papillomavirus participates in gene transcription, DNA replication, and episomal genome maintenance. Phosphorylation of a PEST sequence located in a flexible region accelerates its degradation. NMR analysis of a 29 amino acid peptide fragment derived from this region shows pH-dependent polyproline II and alpha helix structures, connected by a turn. Phosphorylation, in particular that at serine 301, disrupts the overall structure, and point mutations have either stabilizing or destabilizing effects. There is an excellent correlation between the thermodynamic stability of different peptides and the half-life of E2 proteins containing the same mutations in vivo. The structure around the PEST region appears to have evolved a marginal stability that is finely tunable by phosphorylation. Thus, conformational stability, rather than recognition of a phosphate modification, modulates the degradation of this PEST sequence by the proteasome machinery.

Amino Acid Motifs↗

PubMed2005

Reconstitution of papillomavirus E2-mediated plasmid maintenance in Saccharomyces cerevisiae by the Brd4 bromodomain protein.

The papillomavirus E2 protein functions in viral transcriptional regulation, DNA replication, and episomal genome maintenance. Viral genomes are maintained in dividing cells by attachment to mitotic chromosomes by means of the E2 protein. To investigate the chromosomal tethering function of E2, plasmid stability assays were developed in Saccharomyces cerevisiae to determine whether the E2 protein could maintain plasmids containing the yeast autonomous replication sequence replication element but with the centromeric element replaced by E2-binding sites. E2 expression was not sufficient to maintain such plasmids, but plasmid stability could be rescued by expression of the mammalian protein Brd4. In the presence of both Brd4 and E2 proteins, plasmids with multiple E2-binding sites were stable without selection. S. cerevisiae encodes a homolog of Brd4 named Bdf1 that does not contain the C-terminal domain that interacts with the E2 protein. A fusion protein of Bdf1 and the Brd4 C-terminal "tail" could support E2-mediated plasmid maintenance in yeast. Using a panel of mutated E2 proteins, we determined that plasmid stability required the ability of E2 to bind DNA and to interact with Brd4 and mammalian mitotic chromosomes but did not require its replication initiation and transactivation functions. The S. cerevisiae-based plasmid maintenance assays described here are invaluable tools for dissecting mechanisms of episomal viral genome replication and screening for additional host protein factors involved in plasmid maintenance.

Binding Sites↗

PubMed2005

An acidic amphipathic helix in the Bovine Papillomavirus E2 protein is critical for DNA replication and interaction with the E1 protein.

The papillomavirus E2 proteins function in viral transcriptional regulation, and genome replication and episomal maintenance. The transactivation domain is essential for these activities. To identify functional regions, a structural model of the BPV1 E2 transactivation domain was used to target surface residues for mutation. Mutation of several previously uncharacterized regions yielded proteins specifically disrupted in the replication activity of E2. Mutations in an amino-terminal acidic amphipathic helix disrupted the interaction of the E1 and E2 proteins and a peptide derived from this helix blocked cooperative origin binding of E1 and E2. Mutation of clusters of charged residues, R47, K48, K49, R58, and H61 or R172, D175, E176, and R179, or residue R68 in the previously described putative E1 interaction region, specifically disrupted replication while retaining the ability to bind to the E1 protein. Thus, this approach has identified novel regions that are required for the replication function of E2.

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

Conditional mutations in the mitotic chromosome binding function of the bovine papillomavirus type 1 E2 protein.

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.

Amino Acid Sequence↗

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

Casein Kinase II phosphorylation-induced conformational switch triggers degradation of the papillomavirus E2 protein.

The major phosphorylation sites of the bovine papillomavirus E2 transactivator protein are two serine residues, 298 and 301, that are located in a flexible hinge region between the DNA binding and transactivation domains. Phosphorylation of serine residue 301 promotes ubiquitination and rapid degradation of the E2 protein by the proteasome pathway. To understand the mechanism through which phosphorylation regulates the intracellular levels of this unique papillomavirus regulatory protein, we have carried out an extensive mutational analysis of the region surrounding the phosphorylation sites of the E2 protein. Our results indicate that casein kinase II phosphorylates serine 301. However, phosphorylation of serine 301 is not a sufficient recognition motif for proteasomal degradation; other residues that directly surround the phosphorylation sites are crucial for E2 degradation. The phenotypes of E2 proteins mutated in this region indicate that phosphorylation of serine 301 induces a conformational change that leads to degradation of the E2 protein. In support of this model, circular dichroism studies of the conformational tendencies of peptides from this region indicate that phosphorylation at position 301 decreases the local thermodynamic stability of this region. Thus, this region appears to have evolved to display a marginal local thermodynamic stability that can be regulated by phosphorylation, leading to targeted degradation of the E2 protein.

Amino Acid Motifs↗

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↗