The papillomavirus E2 proteins are pivotal to the viral life cycle. The E2 open reading frames of most papillomaviruses encode both a full-length E2 protein containing the transactivation domain, the hinge region, and the DNA binding and dimerization domain and shorter “repressor” proteins that contain only the C-terminal domain and hinge sequences. The full-length protein can both activate and repress transcription and initiates DNA replication in complex with the E1 protein. The shorter E2 proteins can antagonize these functions. For several papillomaviruses, the full-length E2 proteins link the viral genome to the host chromosomes to support maintenance, stability, and partitioning of the viral genome.
For BPV1, both the N-terminal transactivation domain and C-terminal DNA binding domain of E2 are required for efficient mitotic chromosome interaction and genome partitioning (3
). The N-terminal domain is required for interaction with cellular factors bound to mitotic chromosomes, such as the Brd4 protein (4
), and the C-terminal domain is required for binding to DNA sites in the viral genome (19
). Specific residues within the transactivation domain of E2 are required for interaction with Brd4 (4
), and the C-terminal dimerization function of E2 augments the E2-Brd4-mitotic chromosome interaction (5
). We have previously shown that the domains required for chromosomal association of HPV8 E2 protein are different from those required for BPV1 E2 (44
). Unlike BPV1 E2, the N-terminal domain of HPV8 E2 is not required for chromosomal association; instead, the C-terminal DNA binding and dimerization domain and the hinge region are essential. To date, no truncated E2 “repressor” proteins have been identified in the beta-HPVs, but there is a splice acceptor in the N-terminal region of the HPV5 E2 hinge that could generate such a protein (17
). Should such proteins exist, we predict that they would be able to support maintenance and partitioning of the viral genome.
The betapapillomavirus E2 proteins have a unique intracellular localization in both interphase and mitotic cells. In interphase, the betapapillomavirus E2 proteins are observed in a nucleolus-excluded, fine granular pattern, throughout the nucleus, similar to the E2 proteins from other genera. However, the betapapillomavirus E2 proteins are additionally localized to nuclear speckles, which are enriched in splicing factors and are adjacent to sites of active transcription (25
; this study). In mitosis, the beta-HPV E2 proteins associate with pericentromeric and rDNA loci on mitotic chromosomes. Figure shows a model of cell cycle-specific localization of betapapillomavirus E2 proteins. In interphase cells, HPV8 E2 localizes to splicing speckles and transcription and replication foci but is excluded from the nucleoli. Because the rDNA genes are sequestered in the nucleoli in interphase, the E2 protein is free to participate in viral transcription and replication. When the nucleolar envelope breaks down in mitosis, E2 is able to interact with specific regions on the mitotic chromosomes. The number of tandemly repeated rDNA units (400 per cell) will greatly increase the local concentration of the E2 binding target. RNA polymerase I and associated transcription factors remain bound to the rDNA loci in mitosis (36
), and these regions also have specialized mechanisms for replication, cohesion, condensation, and segregation (8
). The resulting, unique chromatin structure could provide unique targets for the betapapillomavirus E2 proteins.
FIG. 9. Model of HPV8 E2 interactions at different stages of the cell cycle. In human interphase cells, HPV8 E2 localizes to splicing speckles and transcription and replication foci but is excluded from the nucleoli. When the nucleoli break down in mitosis, E2 (more ...)
In this study, we have mapped the regions of the HPV8 E2 protein required for localization to interphase splicing speckles. Lai et al. have shown that the HPV5 E2 protein is also associated with splicing factors and can augment splicing (25
). In HPV8 E2, the hinge region is essential for nuclear speckle localization, but it is not sufficient; the C-terminal domain of E2 is also required for efficient nuclear speckle association. Progressive removal of SR dipeptide motifs in the hinge region results in loss of nuclear speckle localization (Fig. ). However, expression of the hinge region alone results in nucleolar localization. In addition, proteins containing truncated regions of the hinge region fused to the C-terminal domain, which no longer associates with nuclear speckles, also localize to the nucleolus. Thus, in the presence of the DNA binding domain, nuclear speckle localization is dominant, but in the absence of this domain, E2 proteins derived from hinge and CTD are often observed in the nucleolus. It is not clear whether the nucleolar localization is of physiological significance. The hinge region contains many GR-rich regions that may function as RNA binding regions that could associate with the large amounts of RNA found in the nucleolus. We have shown that the hinge region is important for the colocalization of HPV8 E2 with UBF and the ribosomal DNA loci on mitotic chromosomes (44
), so localization to the nucleoli in interphase might not be unexpected. However, the mitotic chromosome binding region of the hinge (residues 240 to 255) does not localize E2 to the nucleolus. Furthermore, E2 proteins that do localize to the nucleoli are usually found in a diffuse pattern throughout the nucleolus and are not located specifically in the fibrillar centers or dense fibrillar components that contain the rDNA genes and UBF factor.
In this study, we have identified a 16-amino-acid chromosome binding region in the hinge that, when fused to CTD, is sufficient for E2 chromosomal association. Further analyses identified two specific residues within the mapped region, arginine 250 and serine 253, which are critical for the E2 mitotic chromosome binding function. Notably, the identified R250XXS253 motif, along with the arginine 250 and the serine 253 residues, is completely conserved among the 29 betapapillomaviruses that have been sequenced thus far. We have also demonstrated that the analogous chromosomal binding region in the HPV5 E2 protein confers an identical chromosomal binding phenotype. Thus, evolutionary conservation of this region in an otherwise divergent part of the E2 protein highlights its functional significance in the viral life cycle.
Arginine 250 and serine 253 lie within a consensus kinase motif, R250
, and we have demonstrated that serine 253 is phosphorylated and that mutation of arginine 250 greatly decreases this modification. An overlapping R251
motif is not required for mitotic chromosomal foci, and serine 254 is not a major phosphorylation site. However, mutation of arginine 251 also results in greatly reduced phosphorylation, although the resulting protein is not defective for chromosome binding. Therefore, an RRXS motif is required for phosphorylation, but only RXXS is required for localization to mitotic chromosomal foci. Thus, phosphorylation of serine 253 does not seem to be required for chromosomal binding of the 240-255-C E2 protein, unless this modification is cell cycle regulated and the residual phosphorylation observed is found primarily in mitotic cells. A second possibility is that the RXXS motif is directly required for mitotic chromosomal association but this interaction is blocked by phosphorylation. The model in Fig. shows how E2 phosphorylation might directly regulate chromosome binding in mitosis. Another possibility is that phosphorylation might regulate another property of the E2 protein that is related to but not directly required for mitotic chromosome association. For example, the BPV1 E2 proteins are phosphorylated in the hinge region by CK2 and this modification triggers a conformational switch that targets the E2 protein for proteolysis through the ubiquitin-proteosome pathway. Thus, phosphorylation regulates the overall levels of the BPV1 E2 protein, which ultimately determines the number of genomes attached to mitotic chromosomes (32
The RXXS motif is a consensus sequence for many protein kinases, including protein kinases A and C, calcium-calmodulin-dependent kinase, Aurora kinase B, and the ROCK kinases. Notably, some of these, such as Aurora kinase B, play an important role during mitosis in regulating host chromosomal segregation and cytokinesis. Phosphorylation of the E2 chromosome binding region requires more than the minimal R250XXS253 motif, since R251A results in decreased phosphorylation and the S254 in the overlapping R251XXS254 motif is not phosphorylated. The requirements for S253 phosphorylation indicate that the kinase recognition motif might be RRXS, a protein kinase A site.
Notably, the chromosome binding regions of LANA and EBNA have been mapped to short peptide sequences that have sequence similarities to the chromosome binding region of HPV8 E2 that we have mapped in this study. As shown in Fig. , the identified RXXS kinase motif is also present in the chromosome binding region of the LANA and EBNA1 tethering proteins. In EBNA1, several of the RXXS motifs overlap with AT hook regions that are required for chromosome binding and partitioning (47
). In LANA, substitution of residues overlapping the RXXS motif also disrupts episomal persistence, chromosome binding, and interaction with histones H2A and H2B (2
). In addition, the chromosome binding regions all contain GR motifs. In EBNA1, these arginine residues are methylated (51
), the serine residues are phosphorylated, and substitution of the serines disrupts the EBNA partitioning function (10
). EBNA1 is thought to associate with mitotic chromosomes by either interacting directly with chromosomal DNA through AT-hook DNA binding regions (47
) and/or or by interacting with a host nucleolar protein human EBP2 (7
). These observations suggest that, although these proteins may have different chromosomal targets, phosphorylation by certain cell cycle-dependent kinases could be a common mechanism of regulation of chromosome binding function.
FIG. 10. Similarities among the chromosome binding regions (CBSs) of HPV8 E2, EBNA1, and LANA. AT hook regions are underlined, and RXXS motifs are shown in red. The E2 serine 253 residue, shown to be phosphorylated in this study, is indicated by a P above the (more ...)
The C-terminal domain of HPV8 E2 is required for mitotic chromosome association and localization to nuclear speckles. This domain mediates dimer formation (31
) and specifically binds to a DNA motif that is found at multiple positions in the viral genome (1
). Either the DNA binding or dimerization function could be required for interaction with mitotic chromosomes. In fact, we have found that the C-terminal dimerization function is required for efficient association of BPV1 E2 with Brd4 and with mitotic chromosomes (5
). Another possibility is that the C-terminal domain is required for direct association with cellular proteins on mitotic chromosomes and that this interaction cooperates with the binding or regulatory role of the RXXS motif. There is precedence for this association in studies of the LANA C-terminal domain; mutations that do not disrupt DNA binding or dimerization of LANA can abrogate mitotic chromosome binding (23
). Notably, the LANA C-terminal region also localizes to pericentromeric regions of mitotic chromosomes (22
), suggesting that LANA and E2 might have a common target.
Future studies will address the molecular interactions of the chromosome binding region of the betapapillomavirus E2 proteins. Several functions and protein interactions have already been assigned to the PV E2 hinge region. For example, the HPV8 E2 hinge region can activate transcription by recruitment of Sp1 to cellular promoters (53
). The studies presented here show that the full-length HPV8 E2 protein interacts with several different complexes that direct its intracellular localization. One of the strongest associations is with nuclear speckles. As mentioned above, the hinge of HPV5 E2 interacts with splicing factors and enhances splicing. The N-terminal domain of HPV8 E2 binds the Brd4 protein, and this interaction strengthens the association of Brd4 with cellular chromatin (35
). However, Brd4 is not required for the prominent foci observed bound to the rDNA loci on mitotic chromosomes (44
). In the absence of the N-terminal domain and the RS regions of the hinge, the E2 protein was observed in the nucleolus. As discussed above, this localization might be due to putative RNA binding regions of the hinge region or might be due to interaction with nucleolar proteins.
The betapapillomavirus E2 proteins have properties distinct from those of E2 proteins from other papillomavirus genera. To date, interphase localization to nuclear speckles and mitotic association with rDNA loci has been characterized only for the beta-HPV E2 proteins. HPV8 E2 can give rise to tumors in transgenic mice (43
), and it will be of interest to see whether any of these unique properties are connected and relate to the unique pathogenesis of beta-HPV infection. The experiments described here have characterized sequences required for the interaction of the HPV8 E2 protein with mitotic chromosomes. By analogy with other papillomaviruses, we assume that this interaction is crucial for the virus to tether its genome to host chromosomes during persistent infection. Understanding the mechanism by which the E2 protein tethers the viral genome to host chromosomes during persistent infection will assist in the development of antiviral therapies to inhibit E2 interaction with mitotic chromosomes and thus eliminate viral genomes from infected cells.