Current Understanding of the Role of the Brd4 Protein in the Papillomavirus Lifecycle

The Brd4 protein is an epigenetic reader that is central to regulation of cellular transcription and mitotic bookmarking. The transcription and replication proteins of many viruses interact with Brd4. We describe the multiple roles of Brd4 in the papillomavirus lifecycle.


Introduction
Papillomaviruses (PVs) are an ancient group of viruses that have coevolved along with their hosts for millions of years. Each viral type infects only a particular host species and is trophic for a specific anatomical niche in the stratified epithelium of the skin or mucosa of the host. Papillomavirus infection is persistent and results in clinical outcomes, such as asymptomatic infection, verrucae, plantar and filiform warts and condylomata acuminata. A subset of oncogenic HPVs is associated with carcinomas of the oropharyngeal and anogenital tracts [1][2][3]. Despite the diversity of pathogenesis associated with papillomavirus infection, all papillomaviruses have similar small dsDNA genomes of approximately 8 kbp (see Figure 1), and each encodes only six to eight genes. Papillomaviruses rely on hijacking and manipulating host factors to maintain their lifestyle. Viral open reading frames are depicted as red arrows. The URR (upstream regulatory region) is expanded to show transcription and replication regulatory elements. Binding sites for the E2 protein (cyan circles) and the E1 binding site (blue rectangle) are shown. The early promoter and replication origin (ori) are indicated.
One key, cellular regulatory protein hijacked by all papillomaviruses (and other viral families) is a cellular chromatin binding protein, Brd4. Brd4 is an essential cellular protein that binds and marks chromatin to regulate several transcriptional processes. In this review, we will summarize the current knowledge pertaining to the role of Brd4 in papillomavirus infections.

The Papillomavirus Lifecycle
The stratified epithelium of the skin or mucosa consists of several layers of keratinocytes in various stages of differentiation, overlying the basal layer of proliferative cells. Papillomaviruses gain access to cells in the basal layer through a microabrasion. They infect these keratinocytes, induce their proliferation by expression of the viral E7 protein and establish a persistent infection therein. The viral genome is maintained as a low copy, extrachromosomal element in the nucleus of these cells, and only low levels of viral gene products are produced. When the infected cells divide, some of the daughter cells begin the process of stratification and differentiation and progress towards the surface of the epithelium. The papillomavirus lifecycle is finely tuned to this process of differentiation; viral transcription and replication switches from early to late modes as the cell differentiates. Vegetative viral DNA replication is initiated in the mid-layers of differentiated cells, and capsid synthesis is confined to the most differentiated layers of cells.

Viral Transcription
Papillomavirus genomes can be divided into three regions (see Figure 1). The upstream regulatory region (URR) contains transcriptional enhancers and promoters and the origin of replication. The early region encodes the E (early) proteins that are expressed at early and intermediate stages of infection. Last is the late region that contains the L (late) genes. In the most undifferentiated cells, viral RNAs are transcribed from the early promoter, terminate at the early polyadenylation site (pAE) and encode the E6, E7, E1, E2, E4 and E5 proteins (reviewed in [4]). As the cells progress through differentiation, the late promoter is activated, resulting in an intermediate class of transcripts that use the late promoter and the early polyadenylation signal. These transcripts encode high levels of the E4, E1 and E2, the latter two being required for high level vegetative viral DNA replication in the mid-layers of the epithelium. Exclusively late transcripts are expressed at an even later stage of differentiation; they use both the late promoter and late polyadenylation signal (pAL) and encode the L1 and L2 capsid proteins. Complex patterns of viral mRNA species are generated by the use of alternative splicing and viral gene expression is highly regulated by differential splicing and polyadenylation (reviewed in [4]).
The E2 protein is the primary regulator of early viral transcription [5]. E2 consists of a conserved amino terminal "transactivation" domain linked to a C-terminal DNA binding/dimerization domain by a poorly conserved linker (see Figure 2) [6]. E2 specifically binds to a consensus sequence of ACCGNNNNCGGT, several copies of which are located in the viral URR [7] (see Figure 1). E2 can activate transcription from certain viral promoters [5,[8][9][10], but the early viral promoter in the well-studied, oncogenic alpha-PVs is primarily repressed by E2 [11][12][13]. Early studies showed that E2 could repress the early promoter by sterically hindering the association of TBP and Sp1 with promoter elements adjacent to E2 binding sites 1 and 2 [14][15][16]. Subsequent studies have shown that much of this E2-mediated repression is mediated through interaction with factors that modulate cellular chromatin processes [17][18][19][20]. Disruption of E2-mediated repression (often due to HPV integration) results in increased expression of the E6 and E7 oncogenes [11,12,21]. In fact, reintroduction of the E2 protein into cervical cancer-derived lines (such as HeLa) leads to rapid senescence, as viability of these cells is completely dependent on E6 and E7 expression [22][23][24].
Shorter forms of the E2 protein that are lacking the transactivation domain also repress HPV transcription, in part by competing for binding to E2 binding motifs [25,26]. One of the best characterized is the E8^E2 protein (encoded from a spliced transcript), which also represses through the recruitment of chromatin-associated repressive factors [27]. As described in detail below, E2 interacts with the Brd4 protein, and this association is pivotal to transcriptional regulation of HPVs.

Disruption of E2 Function by HPV Integration
In the majority of HPV associated cancers, the HPV genome is integrated into the host genome (reviewed in [28]). The viral genome is most often integrated in such a way as to disrupt the E2 gene and, thus, alleviate transcriptional repression of the early viral promoter [22][23][24]29]. In turn, this causes dysregulation of the E6 and E7 oncogenes and promotes malignant progression [30].

Viral Replication
Papillomavirus replication requires the E1 protein, the E2 protein and the origin of replication [31][32][33]. E1 is the primary replication protein, an ATP-dependent helicase that binds specifically to the origin and unwinds it in a bidirectional manner to permit access by the cellular replication machinery (reviewed in [34]). E2 functions to load the E1 helicase onto the origin [35]. As depicted in Figure 1, the minimal origin consists of the E1 binding site flanked by E2 binding sites and overlaps elements regulating the early promoter.
There are three modes of viral replication in the viral lifecycle. The first is a limited amplification that occurs when the virus particle infects a basal keratinocyte. The virus must undergo a few rounds of unlicensed DNA replication to establish the genome as a low copy, nuclear, extrachromosomal element. This phase requires E1, E2 and the minimal replication origin. The second phase of replication maintains the viral genome at a low copy number during division of the proliferating basal cells. This also requires E1, E2 and the minimal origin of replication, but there is an additional requirement for additional E2 binding sites in cis to the origin, thus implicating the E2 protein in maintenance replication [36]. Subsequent studies showed that the role of E2 was to tether the viral genomes to the host chromatin [37][38][39]. The interaction of E2 with Brd4 is crucial for the tethering function of many papillomaviruses and will be discussed in detail below. Further investigation showed that the E1 protein was not always necessary for maintenance replication and, presumably, in this situation viral replication, is initiated by cellular factors, while the genome is retained by the E2 protein [40,41].
The third phase of replication is vegetative amplification, when progeny virions are produced in large numbers in differentiated cells. This requires the E1 and E2 proteins, and their expression is upregulated in differentiated cells [42,43]. There is evidence that the mode of replication changes in differentiated cells [44], and incorporation of Rad51 into replication foci indicates that the virus may replicate using a recombination directed replication mechanism [45,46]. Furthermore, the cellular ataxia telangiectasia mutated (ATM) DNA damage response pathway is required for vegetative replication in differentiated cells [47]. Nuclear foci formed by either expression of the E1 and E2 proteins [48][49][50][51] or the replicating viral genome [46,47] recruit multiple cellular proteins required for the cellular DNA damage response and repair pathways.

Differences in Transcription and Replication among Papillomaviruses
To date, there are over 240 named papillomavirus genomes that have been classified into 37 different genera [52]. The best studied are human viruses from the alpha and beta genera that infect primarily the mucosa and skin, respectively. The human mu virus, HPV1, and the ungulate delta virus, BPV1, are also well characterized. While each of these viruses has a similar organization of genes to that of HPV18 (an alpha-PV) shown in Figure 1, the number and position of the E2 binding sites can vary considerably. BPV1 has 11 E2 binding sites in the URR and six elsewhere in the genome [53], while most human alpha viruses have only the four E2 sites shown in Figure 1. In BPV1, E2 is primarily an activator of transcription [5], while E2 predominantly represses the major early promoter of alpha-PVs.
All viruses require the minimal replication origin and the E1 and E2 proteins to initiate replication, but the requirements for maintenance replication are more complex. E2 binding sites are essential for initiation of replication and for transcriptional regulation making it very difficult to separate and elucidate the role of individual sites in maintenance replication of the viral genome. Maintenance replication is best understood for BPV1, where it has been shown that at least eight E2 binding sites are required for persistent replication [36]. In the alpha-PV HPV31, only three of the four E2 binding sites are required for maintenance replication of the viral genome [54]. In agreement with this finding, using a novel complementation assay, we find that a region encompassing the 3' half of the URR of HPV18 (containing E2 binding sites 1-3) is sufficient for long-term maintenance in the presence of the E1 and E2 proteins [55].

Brd4 Structure and Function
Brd4 was first described as an unusual chromatin binding factor that remained bound to chromosomes throughout mitosis [56]. It is a member the BET (bromodomain and extra-terminal domain) family of chromatin binding proteins and, therefore, its name was changed from MCAP (mitotic chromosome-associated protein) to bromodomain containing protein 4 (Brd4) [57]. Brd4 is an essential protein [58] that is ubiquitous in proliferating cells. The tandem bromodomains of Brd4 interact with acetylated tails of H3 and H4 histones [59], and Brd4 has been shown to be a mitotic bookmark that marks genes, which are expressed shortly after mitotic exit [60,61]. Brd4 decompacts chromatin and recruits transcriptional initiation and elongation factors to rapidly activate early G1 genes post-mitosis, as well as later in interphase [62]. Brd4 recruits the transcriptional elongation factor, p-TEFb, to promoters to enhance phosphorylation of the C-terminal tail (CTD) of RNA polymerase II promoters to stimulate transcription [63,64]. Brd4 further promotes transcription by directly phosphorylating the RNA polymerase II CTD [65]. This fundamental role of Brd4 in transcriptional regulation places it at the center of many diverse biological activities.
The Brd4 gene encodes two proteins; the short form of Brd4 contains the two bromodomains and the extra-terminal (ET) region (important for many protein-protein interactions), while the longer form of Brd4 has an additional, long unique C-terminal region (see Figure 3). The structures of both bromodomains and the ET domains have been solved [66,67]. The bromodomains bind to specific acetylated lysines on H3 and H4, but BD2 (bromodomain 2) can also interact with acetylated residues in other proteins, such as cyclin T1 (the p-TEFb subunit) and the relA subunit of NFκB [67][68][69].
The extra-terminal domain consists of three alpha-helices, and it seems to be a site of protein-protein interaction [70]. It binds proteins involved in transcriptional regulation, such as NSD3 (also known as WHSC1L1), a histone methyl transferase, JMJD6, a histone demethylase, and CHD4, a component of the NuRD (nuclear remodeling and deacetylase) repressor complex. Thus, Brd4 has the potential to assemble multifaceted positive and negative regulatory complexes on promoters [71].
The function of the C-terminal region is not well-characterized, except for the last 100 amino acids, which is important for interacting with the papillomavirus E2 protein [72] and with p-TEFb [73] (see Figure 3). Thus, p-TEFb interacts with two independent regions of the Brd4 protein [68]. p53 has also recently been shown to be a binding partner of Brd4, and analysis of this interaction revealed a conformational switch in Brd4 structure and protein-protein interactions modulated by CK2 phosphorylation [74]. A region between the second bromodomain and the ET domain contains two interaction regions, BID (basic residue enriched interaction domain) and NPS (N-terminal cluster of phosphorylation sites), that modulate this switch. When unphosphorylated, the NPS region interacts with the PDID (phosphorylation dependent interaction domain) that encompasses bromodomain 2, thus preventing Brd4 from binding to acetylated histones (see Figure 3). p53 binds to the BID region when Brd4 is unphosphorylated. Upon phosphorylation of NPS, p53 is released from binding to BID, and both p53 and BID now associate with phosphorylated NPS, thus exposing the bromodomain 2 in PDID and activating the complex [74].

Association of Brd4 with Disease
The first association of Brd4 with human disease was the discovery that the aggressive NUT midline carcinoma (NMC) was due to a translocation (t(15;19)(q13;p13)), resulting in the fusion of Brd4 with NUT (nuclear protein in testes) [75]. The NUT protein recruits the histone acetyl transferase, p300, resulting in a feed-forward loop of histone acetylation and Brd4-NUT recruitment, which gives rise to hyperacetylated, but inactive, chromosomal foci [76]. Sequestration of Brd4 and p-TEFb to these foci promotes proliferation of Brd4-NUT cells. This can be reversed and differentiation restored, by siRNA to Brd4, by histone deacetylase inhibitors that promote global acetylation and by BET protein-specific histone mimics [77][78][79].
Brd4 is also a modifier of breast cancer metastasis [80] and a promising target for several cancers, because of its fundamental role in transcriptional processes. Targeted inhibition of BET bromodomain binding is potentially therapeutic for glioblastoma, lung adenocarcinoma, ALL (acute lymphoblastic leukemia) and MLL (mixed-lineage leukemia) [81][82][83][84]85].

Brd4 and Papillomaviruses
Brd4 was discovered to be a major interactor of the papillomavirus E2 protein by proteomic analyses [20,72], yeast two hybrid screening [86]. It was also investigated as an E2 target, because the analogous tethering protein in KSHV (LANA) interacts with the BET family member, Brd2 [87][88][89]. The E2 protein had previously been shown to bind and tether viral genomes to host mitotic chromosomes [37][38][39]90], and the Brd4 protein colocalized completely with these chromatin bound speckles of E2 [72,87,89,91]. The E2 protein is a multifunctional protein involved in papillomavirus transcription, maintenance and partitioning of extrachromosomal viral genomes and initiation of viral DNA replication. Many subsequent studies have dissected the role of Brd4 in each of these processes.

Interaction between Papillomavirus E2 and Brd4 Proteins
The Brd4 protein binds primarily to the transactivation domain of the E2 proteins (see Figure 2) [72]. The transactivation domain contacts a peptide at the extreme C-terminus of the Brd4 protein [91], and a C-terminal domain of Brd4 (CTD; residues 1047-1362) has proven useful as a dominant-negative inhibitor of the E2-Brd4 interaction [72]. The region of the E2 transactivation domain that makes contact with the Brd4 CTD is highly conserved; yet, there is a wide range of binding affinities between Brd4 and E2 proteins from different papillomaviruses [92]. The C-terminal DNA binding domain of E2 does not seem necessary for Brd4 binding, but the dimerization function of this domain greatly increases E2-Brd4 binding both in vivo and in vitro. There are hints that there may be additional contacts between E2 and Brd4: HPV11 E2 binds to the Brd4 CTD, but also to a region encompassing bromodomain 2 (BD2). The DNA binding domain of the KSHV LANA protein (a tethering protein analogous to E2 and with a DNA binding domain of similar structure) interacts with the ET domain of Brd4 [93,94]. It is likely that the interaction between Brd4 and the E2 protein will be multifactorial.

Brd4 Modulates the Stability of the E2 Proteins
Several groups have noted that the E2 protein is stabilized by interaction with Brd4 [95,96] or with the Brd4 CTD [97]. This interaction prevents proteasomal degradation of E2 by the E3 ligase cullin-3 [97] and may enhance many E2 functions, such as transcriptional regulation and stable tethering of genomes on host chromatin.

The Role of Brd4 in Viral Transcription
The E2 protein can both activate or repress viral transcription, depending on whether it binds to sites that are distal or proximal to promoter elements [5,[8][9][10]. Binding of E2 to promoter proximal sites represses the early viral promoter in the oncogenic alpha-PVs [11][12][13]. A number of mutational analyses identified residues in the transactivation domain of E2 that were important for E2-mediated transcriptional activation or replication [98][99][100][101][102][103]. Prominent were two highly conserved residues (R37 and I73) that when mutated, abrogated E2-mediated transactivation, but not replication. Subsequently, it was shown that these residues were located on two adjacent alpha helices on the same face of the E2 transactivation domain [104], and later, these residues were shown to make direct contact with a C-terminal peptide (residues 1343-1362) of Brd4 [91]. In addition to binding to the Brd4 C-terminus, HPV11 E2 also interacts with Brd4 residues 280-580, which encompasses the BD2 bromodomain.
It became clear that Brd4 was essential for the transcriptional activation function of E2. A dominant negative C-terminal peptide encompassing the Brd4 CTD interfered with the E2-Brd4 interaction and inhibited transactivation by many papillomavirus E2 proteins [92,105,106]. Initially, the role of Brd4 in transcriptional repression was controversial, but it has now been proven that Brd4 is also involved in E2-mediated transcriptional repression [18][19][20]. However, in some cases, the dominant negative Brd4 CTD interferes with E2-mediated transactivation, but not repression, implying that there are additional or alternative modes of interaction between the Brd4 and E2 proteins [18]. Another factor that contributes to repression of the HPV early promoter is the histone acetyl transferase complex, NuA4/TIP60 [18,107]. TIP60 preferentially acetylates K14 of histone H3 and K5, K8, K12 and K16 of histone H4 [108], which are all targets of the Brd4 bromodomains [66]. Notably, the HPV E6 protein destabilizes TIP60, thereby alleviating repression of its own promoter [107].
One of the key functions of Brd4 is to recruit p-TEFb to promoters to stimulate elongation of RNA polymerase II transcription. The C-terminal region of Brd4 interacts with both E2 and p-TEFb, suggesting that these complexes are mutually exclusive. Brd4 recruitment of P-TEFb to the early promoter is required for viral transcription, and E2 disrupts this interaction [109]. In this study (unlike [18]), the dominant negative BRD4 CTD was able to inhibit E2-mediated repression.
Most HPV repression studies have analyzed viral transcription in cervical carcinoma-derived cell lines that harbor integrated HPV genomes. However, E2 may preferentially repress integrated genomes compared to episomally replicating genomes, and so, further studies are needed to define the role of Brd4 in HPV transcription [110].

The Role of Brd4 in Viral Genome Replication
Most studies have indicated that the E2-Brd4 interaction is important for transcriptional regulation and tethering of viral genomes to host chromatin. Mutated E2 proteins that are unable to bind Brd4 are able to efficiently support transient replication of an origin containing plasmid [87,98,99,105,106,[111][112][113]. Ilves et al. demonstrated that the dominant negative Brd4 CTD could inhibit the replication of BPV1 genomes or origins in rodent cells, but not in human C-33A cells [113]. Furthermore, this inhibition was not dependent on the interaction of Brd4 with E2 (at least through the R37 and I73 residues) and was not specific for papillomavirus replication [113].
Wang et al. find that Brd4 is recruited to foci formed by HPV16 E1 and E2 in a replication origin-dependent fashion in C-33A cells [114]. An E2 protein mutated in both Brd4 interacting residues (R37 and I73) is defective in replication, thus leading the authors to propose that Brd4 is required for replication. However, as previously found by others, E2 proteins with a single substitution in I73 are not defective in replication, despite an inability to bind to Brd4 [87,99,105,106,[111][112][113]. Because downregulation of Brd4 has detrimental effects on cell growth and proliferation (making it difficult to interpret HPV replication experiments), Wang et al. demonstrated that Brd4 could stimulate HPV replication in vitro [114].
Somewhat similarly, we find that Brd4 is recruited to replication foci formed by the E1-E2 proteins in keratinocytes [49], in a process that is completely dependent on E1, E2 and Brd4. However, we find that Brd4 is displaced to the periphery of these foci in the presence of an actively replicating origin or genome, and Brd4 is no longer required for their formation [115]. Brd4 is also found in a satellite pattern around late replication foci that contain amplified genomes in differentiated keratinocytes [115], but it does not seem to be essential for viral DNA amplification [116]. The HPV replication foci induce a cellular DNA damage response and recruit repair proteins [46][47][48][49][50], and it is tempting to speculate that Brd4 is involved in these processes. The replication factor, RF/C, is found in Brd4 [117] and E2-Brd4 protein complexes [20,118]. Furthermore, the alternative RFC1 subunit, ATAD5, which is involved in the DNA damage response, interacts with the ET domain of Brd4 [71], suggesting that it might play a role in HPV replication. Clearly, more studies are required to elucidate the exact role of Brd4 in the papillomavirus replication process, but there are several hints that Brd4 might be involved in viral and cellular DNA replication and repair processes.

The Role of Brd4 in Viral Genome Maintenance and Partitioning
The E1 and E2 proteins support transient replication, but long-term persistence of viral-derived DNA requires additional E2 binding sites in cis to the replication origin [36]. The first clue to the role of E2 in viral DNA persistence was the observation that both viral DNA and the BPV1 E2 protein are localized in punctate foci on the host mitotic chromosomes [37]. This led to the model (as shown in Figure 4) that E2 associates with host chromosomes through the transactivation domain [39], while the DNA binding domain binds to E2 sites in the viral genomes and tethers them to the host chromosomes to promote retention and partitioning [119]. This tethering mechanism ensures that the low copy viral genome is retained in the nucleus and is partitioned to daughter cells. At least for BPV1, the cellular target that mediates mitotic tethering is the Brd4 protein [72,87,89]. Brd4 is usually observed as a diffuse cloud (if at all) around the condensed mitotic chromosomes [59], but in the presence of E2, both proteins colocalize in punctate foci [89]. Brd4 has a high on-off rate and can be easily extracted from chromatin [59], but E2 dramatically stabilizes the interaction of Brd4 with chromatin forming a stable anchor in interphase and mitosis [89]. As described above, two highly conserved residues in the transactivation domain of E2 (R37 and I73) mediate the interaction with Brd4, and mutations in these residues abrogate the interaction of E2 with Brd4 and mitotic chromatin [87]. The DNA binding domain of E2 is not required for the interaction with Brd4, but it is required to link the viral genomes to chromatin [119]. The dimerization function of the E2 C-terminal domain greatly increases the affinity of Brd4 for chromatin both in vivo and in vitro, most likely by promoting the assembly of higher order E2-Brd4 complexes [120]. Expression of the dominant negative Brd4 CTD resulted in inhibition of E2 mitotic chromosome binding and loss of BPV1 genomes from BPV1 transformed cells [89,113,121]. Moreover, BPV1 E2-mediated plasmid maintenance could be reconstituted in Saccharomyces cerevisiae by exogenously expressed Brd4 [122].
Although the E2 proteins from all papillomaviruses interact with the C-terminal region of Brd4 (through the R37 and I73 residues in E2), there are differences in the strength of binding [92,118].
Furthermore, not all E2 proteins are as readily observed bound to mitotic chromosomes as BPV1 E2 [92,123]. A careful analysis of the E2 proteins from numerous different papillomaviruses showed that there were three different phenotypes of mitotic chromosome binding that segregated perfectly according to the phylogeny of papillomaviruses [123]. These groups were comprised of the alpha-papillomaviruses, a large genus that encompasses mainly human viruses that infect the oral and genital mucosa; the beta and gamma papillomaviruses, another large group containing mainly human viruses that infect the cutaneous epithelium in an asymptomatic manner and a diverse group of viruses from the delta (BPV1), mu (HPV1), kappa (OcPV1 and SfPV1) and other genera. The E2 proteins from this latter group bind tightly to Brd4, stabilize its association with interphase chromatin and colocalize with Brd4 on the arms of mitotic chromosomes in punctate dots [92,123].
In contrast, the beta-PV E2 proteins bind strongly to pericentromeric regions of mitotic chromosomes that overlap the loci for the ribosomal RNA genes [123,124]. The determinants of the E2 protein required for this perichromosomal binding are quite different from those required for the E2-Brd4 chromosomal foci. The primary requirements are the E2 DNA binding domain and a short peptide from the hinge region that facilitates interaction with chromatin [125]. Phosphorylation of this peptide by PKA (protein kinase A) stabilizes the E2 protein and promotes chromosomal binding [126] in a manner analogous to that of the KSHV LANA tethering protein [127]. However, the beta E2 proteins do have high affinity for Brd4, and if the chromosomal binding peptide in the hinge is mutated, E2-directed foci of Brd4 can be observed on mitotic chromosomes [128]. The significance of these two binding modes has yet to be determined, as beta-PV genomes do not readily replicate in cell culture.
For alpha-PVs, the mechanism of E2-mediated viral genome tethering and E2-Brd4 chromosomal binding is still elusive. Alpha-PV E2 proteins bind to Brd4 relatively weakly, they do not stabilize the association of Brd4 with host chromatin and cannot be easily detected on mitotic chromosomes, except in late telophase [123,129]. When cells are pre-extracted before fixation, the alpha-PV E2 proteins bind to the peri-centromeric regions of host chromosomes in a Brd4-independent manner similar to that of the beta-PVs [123]. Difficulties in detecting alpha-PV E2-Brd4 mitotic foci have led to the proposal of other targets, such as the mitotic spindle [130], a mitotic kinesin-like protein, MKlp2 [131], ChlR1 (an ATP-dependent DNA helicase important for sister chromatid cohesion) [132] and TopBP1 [129]. Furthermore, HPV31 genomes that encode a Brd4 binding defective E2 protein (an I73L mutation) can still maintain extrachromosomal viral genomes and undergo amplification in differentiated keratinocytes [54,105]. Recent findings indicate that Brd4 colocalizes with nuclear foci formed by the alpha-PV E1 and E2 proteins [114,115], and we find that the alpha-PV E1-E2 protein complex binds to the same regions of host chromatin as the stable HPV1 E2-Brd4 complex in C-33A cells [133].
Therefore, many questions remain, and it seems that the interaction of E2 and Brd4 with host chromatin is complex. The tethering mechanism is likely to be coupled with transcriptional and replication processes. Silla et al. have shown that simple attachment of genomes to chromatin is not sufficient [134]; chromatin attachment and transactivation functions must cooperate to ensure proper plasmid segregation. A genome-wide ChIP-on-chip analysis showed that BPV1 E2 and Brd4 were bound to transcriptionally active regions of chromatin, perhaps to ensure that the viral genome localized to transcriptionally active regions of the nucleus [135]. Brd4 is recruited to HPV replication centers containing alpha-PV proteins that do not tightly associate with Brd4 [114,115]. Thus, transcription, replication and genome partitioning are most likely intertwined processes.

Association of Brd4 with Other Viruses
Papillomaviruses are not the only viruses that have discovered the versatility and usefulness of BET proteins [136]. The EBNA and LANA tethering proteins of the gamma herpes viruses, EBV and KSHV, interact with Brd2 and Brd4 for transcriptional regulation [88,93,137,138]. Polyoma viruses also recruit Brd4 to viral replication centers [139]. Brd4 represses HIV expression by competing with the HIV TAT transactivator for recruitment of p-TEFb to the HIV promoter [73]. Inhibition of Brd4 with BET inhibitors reactivates latent HIV with great therapeutic potential [140][141][142][143].

Conclusions
Viruses have always alerted us to the key players in cellular processes. Papillomaviruses, in particular, have small genomes with limited coding capacity and rely almost completely on using and manipulating cellular factors for viral processes. Brd4 is clearly a central player in HPV biology, and a complete understanding of its role in essential viral processes will provide deeper insight into its role in host biology.