Brd4 is required for e2-mediated transcriptional activation but not genome partitioning of all papillomaviruses.

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.

Proteasome-mediated degradation of the papillomavirus E2-TA protein is regulated by phosphorylation and can modulate viral genome copy number.

The bovine papillomavirus E2 proteins regulate viral transcription, replication, and episomal genome maintenance. We have previously mapped the major phosphorylation sites of the E2 proteins to serine residues 298 and 301 and shown that mutation of serine residue 301 to alanine leads to a dramatic (10- to 20-fold) increase in viral DNA copy number. In this study we analyzed how phosphorylation regulates E2 protein function. S301 is located in a PEST sequence; these sequences are often found in proteins with a short half-life and can be regulated by phosphorylation. We show here that the E2 protein is ubiquitinated and degraded by the proteasome. Mutation of serine 301 to alanine increases the half-life of E2 from approximately 50 min to 160 min. Furthermore, the A301 E2 protein shows greatly reduced ubiquitination and degradation by the proteasome. These results suggest that the E2 protein level is regulated by phosphorylation, which in turn determines viral episomal copy number.

Production of infectious bovine papillomavirus from cloned viral DNA by using an organotypic raft/xenograft technique.

Bovine papillomavirus type 1 (BPV-1) induces fibropapillomas in its natural host and can transform fibroblasts in culture. The viral genome is maintained as an episome within fibroblasts, which has allowed extensive genetic analyses of the viral functions required for DNA replication, gene expression, and transformation. Much less is known about BPV-1 gene expression and replication in bovine epithelial cells because the study of the complete viral life cycle requires an experimental system capable of generating a fully differentiated stratified bovine epithelium. Using a combination of organotypic raft cultures and xenografts on nude mice, we have developed a system in which BPV-1 can replicate and produce infectious viral particles. Organotypic cultures were established with bovine keratinocytes plated on a collagen raft containing BPV-1-transformed fibroblasts. These keratinocytes were infected with virus particles isolated from a bovine wart or were transfected with cloned BPV-1 DNA. Several days after the rafts were lifted to the air interface, they were grafted on nude mice. After 6-8 weeks, large xenografts were produced that exhibited a hyperplastic and hyperkeratotic epithelium overlying a large dermal fibroma. These lesions were strikingly similar to a fibropapilloma caused by BPV-1 in the natural host. Amplified viral DNA and capsid antigens were detected in the suprabasal cells of the epithelium. Moreover, infectious virus particles could be isolated from these lesions and quantitated by a focus formation assay on mouse cells in culture. Interestingly, analysis of grafts produced with infected and uninfected fibroblasts indicated that the fibroma component was not required for productive infection or morphological changes characteristic of papillomavirus-infected epithelium. This system will be a powerful tool for the genetic analysis of the roles of the viral gene products in the complete viral life cycle.

Interaction of the papillomavirus E2 protein with mitotic chromosomes.

The bovine papillomavirus E2 transactivator protein is a multifunctional protein that activates viral transcription, cooperates in initiation of viral DNA replication, and is required for long-term episomal maintenance of viral genomes. We have shown previously that the E2 transactivator protein and bovine papillomavirus type 1 genomes are associated with mitotic chromosomes and have proposed that E2 links the genomes to cellular chromosomes to ensure segregation to daughter nuclei. In this study, we show that E2 is associated with cellular chromosomes at all stages of mitosis. We also further map the regions of E2 that are required for this association. The transactivation domain of E2 is necessary and sufficient to mediate the interaction with mitotic chromosomes; the DNA binding domain, and the flexible hinge region that separates the two domains, is not required. Furthermore, mutation of previously identified phosphorylation sites (serine residues 235, 298, and 301) has no effect on the ability of the E2 protein to bind mitotic chromosomes.

Papillomavirus DNA replication.

Papillomavirus genomes replicate and are maintained as stable extrachromosomal plasmid DNA (episomes) in many cell lines (reviewed in ref. 1). This process requires the viral E1 and E2 proteins and the origin of replication (2,3). The minimal origin of replication consists of an E1 binding site, an E2 binding site, and an AT rich region that probably facilitates origin unwinding (see Fig. 1). The E1 protein is an ATP-dependent helicase that specifically binds to and unwinds the origin (4-6). The E2 protein is the major transcriptional transactivator of the virus but it is also required for viral DNA replication. The E2 protein probably plays more of an auxiliary role in DNA replication; it has been shown to cooperatively bind to the origin with the E1 protein (7-11), to alleviate repression of replication by nucleosomes (12), and to interact with cellular replication proteins (RPA) (13). Fig. 1. Diagram of the BPV-1 replication origin.

Bovine papillomavirus type 1 genomes and the E2 transactivator protein are closely associated with mitotic chromatin.

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.

Transient viral DNA replication and repression of viral transcription are supported by the C-terminal domain of the bovine papillomavirus type 1 E1 protein.

The bovine papillomavirus type 1 E1 protein is important for viral DNA replication and transcriptional repression. It has been proposed that the full-length E1 protein consists of a small N-terminal and a larger C-terminal domain. In this study, it is shown that an E1 polypeptide containing residues 132 to 605 (which represents the C-terminal domain) is able to support transient viral DNA replication, although at a level lower than that supported by the wild-type protein. This domain can also repress E2-mediated transactivation from the P89 promoter as well as the wild-type E1 protein can.

The transactivation and DNA binding domains of the BPV-1 E2 protein have different roles in cooperative origin binding with the E1 protein.

The bovine papillomavirus E2 transactivator protein enhances the ability of the E1 protein to bind to the viral origin of replication which contains an E1 binding site flanked by two E2 binding sites. To determine which regions and functions of the E2 protein are important for this cooperative interaction, a series of mutated E2 proteins were assayed for their ability to enhance E1 origin-specific binding. Cooperative origin binding required at least one E2 DNA binding site, an intact functional E2 DNA binding domain, and an intact transactivation domain. The hinge region of the E2 proteins was dispensable for this activity. To further examine the role of the E2 C-terminal domain, a series of chimeric proteins were generated that substituted the yeast GAL4 DNA binding domain for the E2 DNA binding domain. These chimeric proteins were able to cooperatively bind to a hybrid origin that contained GAL4 binding sites in place of the E2 binding sites. These studies indicate that the E2 transactivation domain is sufficient for interaction with the E1 protein and that the E2 DNA binding domain is required for interaction with origin DNA sequences.

The bovine papillomavirus type 1 E2 transactivator and repressor proteins use different nuclear localization signals.

The E2 gene of bovine papillomavirus type 1 encodes at least three nuclear phosphoproteins that regulate viral transcription and DNA replication. All three proteins have a common C-terminal domain that has DNA-binding and dimerization activities. A basic region in this domain forms an alpha helix which makes direct contact with the DNA target. In this study, it is shown that in addition to its role in DNA binding, this basic region functions as a nuclear localization signal both in the E2 DNA-binding domain and in a heterologous protein. Deletion of this signal sequence resulted in increased accumulation of the E2 transactivator and repressor proteins in the cytoplasm, but nuclear localization was not eliminated. In the full-length transactivator protein, another signal, present in the N-terminal transactivation domain, is used for transport to the nucleus, and the C-terminal nuclear localization signal(s) are masked. The use of different nuclear localization signals could potentially allow differential regulation of the subcellular localization of the E2 transactivator and repressor proteins at some stage in the viral life cycle.

Amino acids critical for the functions of the bovine papillomavirus type 1 E2 transactivator.

The N-terminal domain of the bovine papillomavirus type 1 E2 protein is important for viral DNA replication, for transcriptional transactivation, and for interaction with the E1 protein. To determine which residues of this 200-amino-acid domain are important for these activities, single conservative amino acid substitutions have been generated in 17 residues that are invariant among all papillomavirus E2 proteins. The resulting mutated E2 proteins were tested for the ability to support viral DNA replication, activate transcription, and cooperatively bind to the origin of replication with the E1 protein. We identified five mutated proteins that were completely defective for transcriptional activation and either were defective or could support viral DNA replication at only low levels. However, several of these proteins could still interact efficiently with the E1 protein. In addition, we identified several mutated proteins that were unable to efficiently cooperatively bind to the origin with the E1 protein. Although a number of the mutated proteins demonstrated wild-type activity in all of the functions tested, only 3 out of 17 mutated viral genomes were able to induce foci in a C127 focus formation assay when the mutations were generated in the background of the entire bovine papillomavirus type 1 genome. This finding suggests that the E2 protein may have additional activities that are important for the viral life cycle.

Suppression of cellular proliferation by the papillomavirus E2 protein.

Carcinogenic progression of a human papillomavirus (HPV)-infected cell is often associated with integration of the viral genome in a manner which results in the loss of expression of the viral regulatory protein E2. One function of E2 is the regulation of expression of the viral oncogenes, E6 and E7. Introduction of the bovine papillomavirus type 1 (BPV-1) E2 transactivator (E2-TA) in HeLa cells, an HPV type 18 (HPV-18)-positive cervical carcinoma cell line results in growth arrest. In this study, we have found that the HPV-16 and HPV-18 E2 proteins share with BPV-1 E2-TA the ability to suppress HeLa cell growth. This property was not observed for the BPV-1 E2 transcriptional repressor (E2-TR). Analysis of various mutant E2 proteins for growth suppression revealed a requirement for the intact transactivation and DNA binding domains. A HeLa cell line (HeLa-tsE2) which expressed a conditional mutant E2 protein that was functional only at the permissive temperature (32 degrees C) was established, permitting an analysis of the molecular and cellular consequences of E2 expression. Our data indicate that one mechanism by which E2 suppresses cellular growth is through repression of E6 and E7 expression, thereby enabling the cellular targets of E6 and E7 to resume regulation of the cell cycle.

Domains of the BPV-1 E1 replication protein required for origin-specific DNA binding and interaction with the E2 transactivator.

The viral E1 and E2 proteins are required for replication of bovine papillomavirus type 1 DNA. Both proteins bind as a complex to the replication origin, which consists of an E1 binding site flanked on either side by E2 binding sites. The E1 protein has properties common to replication initiator proteins such as sequence-specific origin binding and DNA helicase activities. The E2 protein is a transcriptional transactivator that forms a complex with the E1 protein and enhances binding of E1 to the replication origin. We have mapped the regions of the E1 protein required for sequence-specific DNA binding, for cooperative binding with the E2 protein to the origin region, and for interaction with the E2 protein. These studies demonstrate that a region between amino acids 162 and 378 of the E1 protein is important for origin-specific DNA binding. The C-terminal half of the E1 protein is required in addition to the DNA binding domain (residues 162 to 605) for cooperative binding to the origin with the E2 protein. Binding studies confirmed that this region is also required for efficient complex formation with the E2 protein.

Binding of bovine papillomavirus E1 to the origin is not sufficient for DNA replication.

Replication of the bovine papillomavirus (BPV-1) DNA requires both the viral E1 and E2 gene products. The minimal origin of replication, which resides in a 60-basepair fragment centered on the unique HpaI site of the BPV-1 genome, can be bound by the E1 protein and is flanked by E2 binding sites. The integrity of the region surrounding the unique HpaI restriction enzyme site is important for E1 binding and DNA replication, but the E2 binding sites are not required. The ability of E1 to complex with E2 and the requirement for both factors in DNA replication have led to the hypothesis that the E1/E2 complex may have a direct role in replication. Therefore we have studied mutations in the BPV-1 origin region for E1, E2, and E1/E2 complex binding and for their effect on transformation and replication in the context of the complete viral genome. Transformation and DNA replication were observed with some mutated viral genomes dramatically reduced for E1 binding. These mutated origins could bind significant amounts of E1 in the presence of E2, suggesting that binding of the E1/E2 complex could compensate for the loss of E1 binding in DNA replication. One mutation, with an eight-nucleotide insertion at the HpaI site, was still bound by E1 and the E1/E2 complex, yet did not replicate or transform efficiently. Thus although the binding of E1 or the E1/E2 complex to the origin appears to be necessary for replication, it is not sufficient.

Separation of the transcriptional activation and replication functions of the bovine papillomavirus-1 E2 protein.

Replication of bovine papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these domains are highly conserved among different papillomaviruses. An internal hinge region separates the two functional domains. The region varies in amino acid sequence and length among the E2 proteins of different papillomaviruses. A series of mutations were constructed within the E2 open reading frame which delete various regions of the conserved DNA binding and transactivation domains as well as the internal hinge region. Two mutated E2 proteins that lack portions of the conserved DNA-binding domain but which support DNA replication were identified using transient replication assays. These mutated E2 proteins were unable to function as transcriptional activators. Conversely, two E2 proteins containing large deletions of the hinge region were able to activate transcription, but were defective for replication. Thus, the replication and transactivation functions of the E2 protein are separable.

Conserved cysteine residue in the DNA-binding domain of the bovine papillomavirus type 1 E2 protein confers redox regulation of the DNA-binding activity in vitro.

The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfhydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfhydryl group(s) may be protected by DNA binding. Sensitivity to sulfhydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

Bovine papillomavirus with a mutation in the E2 serine 301 phosphorylation site replicates at a high copy number.

The E2 open reading frame of bovine papillomavirus type 1 (BPV-1) encodes at least three proteins with transcriptional regulatory properties. The full-length E2 open reading frame encodes a transcriptional transactivator, and the 3' region encodes two smaller polypeptides that repress E2-mediated transactivation. The full-length gene product is also required for viral DNA replication. We have demonstrated that the BPV-1 E2 polypeptides are phosphorylated primarily on two serine residues at a site adjacent to the carboxy-terminal DNA binding domain, which is common to all three E2 proteins (A. A. McBride, J. B. Bolen, and P. M. Howley, J. Virol. 63:5076-5085, 1989). These serine residues, at amino acid positions 298 and 301, were substituted with alanine residues in the context of the entire BPV-1 genome. The mutated BPV-1 genomes were introduced into rodent cell lines and assayed for focus formation, viral gene expression, and extrachromosomal viral DNA replication. Viral DNAs containing the E2 serine-to-alanine substitution mutants transformed C127 cells with efficiencies comparable to that of wild-type BPV-1. However, the viral genome containing the serine-to-alanine substitution at position 301 of the E2 polypeptide replicated to a copy number 20-fold higher than that of wild-type DNA.

Phosphorylation sites of the E2 transcriptional regulatory proteins of bovine papillomavirus type 1.

The E2 open reading frame of bovine papillomavirus type 1 (BPV-1) encodes three transcriptional regulatory proteins. The full-length open reading frame encodes a protein of 410 amino acids which functions as a transcriptional transactivator. Two transcriptional repressor proteins, E2-TR and E8/E2, contain the C-terminal 249 and 204 amino acids, respectively. We have expressed both the full-length E2 protein and the E2-TR repressor protein in insect cells, by using recombinant baculoviruses, and in mammalian COS-1 cells, by using a chimeric simian virus 40/BPV-1 virus. Analysis of the E2 proteins revealed that both the transactivator and repressor forms are phosphorylated predominately on serine residues at similar sites in both expression systems. By a combination of peptide mapping and site-directed mutagenesis techniques, the serine residues at positions 298 and 301 were determined to be the major phosphorylation sites of the BPV-1 E2 proteins.

E2 polypeptides encoded by bovine papillomavirus type 1 form dimers through the common carboxyl-terminal domain: transactivation is mediated by the conserved amino-terminal domain.

The E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) encodes positive- and negative-acting factors that regulate viral gene expression. The full-length ORF encodes a transactivator, and two transcriptional repressors are expressed from the 3' half of the ORF. Previous analysis has shown that a conserved C-terminal region of 101 amino acids, which is shared by E2 transactivator and repressor proteins, contains the specific DNA binding activity. Further analysis of the E2 transactivator shows that a conserved N-terminal domain of approximately 220 amino acids is crucial for the transcriptional activation function, whereas the variable internal region is not required. The E2 proteins bind to a sequence, ACCGN4CGGT, several copies of which are sufficient to constitute an E2-dependent enhancer. By using a gel retardation assay and proteins derived by in vitro transcription and translation, we were able to show that the E2 polypeptides bind as dimers to a single DNA binding site. The dimeric E2 proteins are stable in the absence of DNA and dimerization is mediated through the DNA binding domain. This may reveal an additional mechanism of repression that could potentially result from the formation of inactive heterodimers between transactivator and repressor species.

Functional analysis of the papilloma virus E2 trans-activator in Saccharomyces cerevisiae.

The papilloma virus E2 transcriptional trans-activator is representative of a class of transcriptional modulators that activate transcription through direct binding to cis-acting DNA sequences. In this study we measured the capacity for this mammalian virus factor to function in Saccharomyces cerevisiae. When expressed in the yeast, the bovine papilloma virus E2 trans-activator could stimulate transcription from a yeast promoter having E2 DNA-binding sites present in cis. Whereas a single E2 DNA-binding site was sufficient for trans-activation, a strong cooperative effect was observed with two E2 DNA-binding sites. The level of trans-activation was dependent on the position of the E2 DNA-binding sites in relation to the yeast promoter, with the maximal effect demonstrated when the binding sites were positioned upstream. Deleted E2 proteins, lacking part of the trans-activation or DNA-binding domains, failed to activate transcription in yeast, similar to their behavior in mammalian cells. Replacement of the amino-terminal region of the E2 trans-activation domain with a synthetic amphipathic helix partially restored the trans-activation function; however, it did not result in a molecule that exhibited cooperativity between neighboring E2 DNA-binding sites.