Bromodomain protein 4 (Brd4) has been identified as the cellular binding target through which the E2 protein of bovine papillomavirus type 1 links the viral genome to mitotic chromosomes. This tethering ensures retention and efficient partitioning of genomes to daughter cells following cell division. E2 is also a regulator of viral gene expression and a replication factor, in association with the viral E1 protein. In this study, we show that E2 proteins from a wide range of papillomaviruses interact with Brd4, albeit with variations in efficiency. Moreover, disruption of the E2-Brd4 interaction abrogates the transactivation function of E2, indicating that Brd4 is required for E2-mediated transactivation of all papillomaviruses. However, the interaction of E2 and Brd4 is not required for genome partitioning of all papillomaviruses since a number of papillomavirus E2 proteins associate with mitotic chromosomes independently of Brd4 binding. Furthermore, mutations in E2 that disrupt the interaction with Brd4 do not affect the ability of these E2s to associate with chromosomes. Thus, while all papillomaviruses attach their genomes to cellular chromosomes to facilitate genome segregation, they target different cellular binding partners. In summary, the E2 proteins from many papillomaviruses, including the clinically important alpha genus human papillomaviruses, interact with Brd4 to mediate transcriptional activation function but not all depend on this interaction to efficiently associate with mitotic chromosomes.To date, more than 100 different types of papillomaviruses, representing 17 distinct genera, have been identified in a wide range of mammals as well as in birds and reptiles (10, 13). Members of the Papillomaviridae family have a specific tropism for squamous epithelium. For example, mucosal human papillomavirus (HPV) types, such as HPV6, HPV11, HPV16, and HPV31, infect genital and sometimes oral epithelia. Cutaneous HPV types, such as HPV1, -2, -3, and -4, cause plantar and palmar warts, whereas HPV5 and HPV8 are associated with epidermodysplasia verruciformis. Certain fibropapillomaviruses, such as bovine papillomavirus type 1 (BPV1), can also infect fibroblasts. Despite this diversity, all papillomaviruses are capable of establishing persistent infections by maintaining their viral genomes as low-copy-number extrachromosomal elements in mitotically active cutaneous and mucosal epithelial cells.Papillomavirus genomes do not contain centromeric sequences and hence require an alternative and efficient means of retaining extrachromosomal viral genomes within the nucleus and distributing them to daughter cells. The mechanism of papillomavirus genome segregation and chromosomal attachment in BPV1 has been extensively studied (25,31,47). BPV1-transformed mouse cells maintain extrachromosomal viral genomes indefinitely and therefore provide a useful system in which to study genome maintenance (30). Faithful segregation of BPV1 genomes is achieved through the actions of the multifunctional viral protein, E2 (5,25,...

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.Papillomaviruses are small DNA viruses that infect the squamous epithelium of the skin and mucosa, causing benign tumors or papillomas (22). A key step in the viral life cycle occurs upon infection of basal epithelial cells, when the viral genome must be maintained as an extrachromosomal element to ensure persistence of the infection in dividing cells. Transient viral DNA replication requires the E2 transcriptional transactivator, the E1 helicase protein, and the origin of replication (54,55,57,61). Long-term, persistent replication requires, in addition, numerous high-affinity E2 binding sites in cis with the replication origin (47). The E2 transcriptional transactivator protein maintains the viral genomes as multicopy extrachromosomal elements by tethering them to mitotic chromosomes to ensure their nuclear retention and segregation to daughter cells (24,32,51).The E2 transactivator protein can be divided into three domains. The amino-terminal ϳ200 amino acids comprise the transactivation domain of E2, and the carboxy-terminal ϳ100 amino acids mediate DNA binding and dimerization. These domains are linked by a flexible region called the hinge. The transactivation domain and DNA binding domain are conserved among E2 proteins of different papillomaviruses, whereas the length and amino acid sequence of the hinge region vary greatly (reviewed in reference 42). The transactivation domain is critical for the segregation, DNA replication, and transcriptional regulatory functions of the E2 protein (5,41,51,58).Several groups have mapped residues important for the transactivati...

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.Persistent papillomavirus infections are established in the mitotically active basal cells of the squamous epithelium. Certain papillomaviruses, including bovine papillomavirus type 1 (BPV-1), can also infect fibroblast cells. Within these dividing cells, the viral genome is amplified to a low copy number and stably maintained as a persistent extrachromosomal element in the cell nucleus. As infected cells undergo division, papillomaviruses, like other extrachromosomal viruses, must position their genomes in such a way as to ensure an equal distribution of viral DNA to daughter cells and to avoid loss following nuclear membrane disassembly. BPV-1 has served as a model with which to study this process in papillomaviruses due to its ability to efficiently replicate and maintain the viral genome extrachromosomally through many cell divisions (26).BPV-1 viral genomes are tethered to cellular mitotic chromosomes via a protein-protein interaction mediated by the viral protein E2 (20,29,50). By this action, viral genomes are efficiently segregated into daughter cells, thus ensuring the longevity of the viral infection. The central role of the viral E2 protein in mitotic tethering and genome segregation has been well documented (2,20,29,45,50). Pirsoo et al. first demonstrated that both the E2 protein and E2 DNA binding sites are required for long-term extrachromosomal genome maintenance (45). S...

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.Human papillomaviruses (HPVs) are a family of epitheliotropic viruses that infect both cutaneous and mucosal epithelia. HPV infection most commonly results in benign papillomas or warts; however, on rare occasions, malignant lesions can develop following infection with a high-risk HPV type and integration of the virus genome into the host genome (18). HPV-16 is the most significant member of this high-risk subgroup, being associated with approximately 60% of cervical carcinoma cases worldwide (52).Transcription of the 8.0-kb virus genome generates a number of transcripts as a result of a complex program of alternative splicing and polyadenylation (44). Viral mRNAs are translated to yield six early proteins, expressed throughout the virus life cycle (primarily involved in episomal maintenance of the genome, transcriptional regulation, and cell transformation) (52) and two late proteins, the capsid proteins L1 and L2. Expression of the capsid proteins is restricted to cells undergoing terminal differentiation in the uppermost layers of the stratified epithelium (31) but because late transcripts are expressed in less-differentiated epithelial cells (43), control of late-gene expression is largely attributed to posttranscriptional mechanisms. cis-acting inhibitory elements identified within the late region of several papillomaviruses (40) appear to regulate gene expression via distinct RNA-based mechanisms. However, the overall effect is the same in that each element probably acts in a position-and orientation-dependent manner through interactions with cellular factors (40) to reduce the levels of polyadenylated viral late mRNAs in the cytoplasm of undifferentiated cells. The best-understood example is bovine papillomavirus type 1 (BPV-1), in which regulation of lategene expression by a short negative...