BPV-1 establishes its genome in transformed cells as a multicopy nuclear plasmid with the ability to amplify its genome, and as such it seems likely that the key segregation function mediated by E2 is nuclear retention. Upon nuclear membrane breakdown, viral plasmids untethered to mitotic chromosomes may be lost after reassembly of the new nuclear membranes in late telophase. This model would be consistent with the very rapid and catastrophic loss of plasmids, as detected as sectored colonies by fluorescent in situ hybridization in earlier studies of the A4 mutant genome. It seems possible that the BPV-1 minichromosomes are too large to efficiently reenter the nucleus after cytosolic dispersion.
These issues clearly bring into focus the problem of how effectively and by what mechanisms these viruses deliver the initial minichromosomes to the nucleus in the first place. These questions aside, as E2 is not known to be associated with the virion, the A4 mutations in E2 do not diminish any direct replication or transcriptional activity of E2. Thus, the genetics clearly separated the tethering pathway from other known activities of the proteins. The central purpose of the present study was to understand more directly the nature of the mutations and the role played by the suppressor alleles of E1.
The key data and conclusions presented here are that phosphorylation of E2 must only indirectly affect the tethering activity of the regulatory protein. When expressed from recombinant PAVA viruses, no differences in chromosomal binding by the proteins were found even at limiting E2 levels. The other important finding is that E1 serves, when its levels increase significantly above those of E2, as a negative regulator of such tethering activities. It seems rather obvious but perhaps inescapable to posit that the E1-E2 complex forms in the cell and that this complex blocks the E2 activation domain from finding a chromosomally bound factor to interact with. What role would phosphorylation play in such a regulatory pathway? We suspect that there is no simple model as yet that can explain all of our data, though several points indicate that the tethering process may require a pathway rather then a simple binding step.
The disruption of the major phosphorylation sites in E2 at residues 298 and 301 by themselves lead to an increase in E2 trans
-acting activity in transient assays (12
) and increased E2 protein accumulation and a longer half-life for mutant E2 (16
). A simple model might then include an overexpression of E2 in the A4 mutant genomic context, leading subsequently to an overexpression of E1. Such an imbalance of E1 could then in turn “squelch” E2 from its tethering functions. In previous studies, we found that the E2-A3 genomes stably transformed cells with a higher copy number than the wild type, perhaps because of increased E2 activity. However, no further increase in accumulated E2 levels could ever be detected in comparing the E2-A3 to the E2-A4 alleles in transient assays (12
), yet the A4 genomes are severely transformation defective. Furthermore, the simplest model does not at all account for our finding that the E2-A4 protein is much more sensitive to E1 squelching than is the wild-type allele when the factors are expressed from recombinant vectors and chromosomal binding is measured (Fig. ).
Another idea that must be considered for an understanding of the mutant phenotypes is that phosphorylation of E2 may disrupt E1-E2 complexes and that the A4 alleles of E2 allow a more stable complex and accumulate throughout interphase. Data from a previous study (14
) indicated that E1 would only associate with E2 that was not modified by phosphorylation. Our data are consistent with a somewhat increased affinity (two- to threefold) between the unmodified and modified forms. This small difference may indeed be of significance when one considers the abundance and affinity that E2 might have for the unknown cellular chromosomal factor that E2 must interact with. Perhaps this factor is not as abundant as E1 and has a comparable binding affinity. In such a scenario, very small effects on E1-E2 interactions may be relevant. In the absence of any data relevant to receptor concentrations and affinities, such speculation is of course premature.
We must, however, point out that this concept—phosphorylation by itself destabilizes inactive complexes—does not simply explain why the suppressor alleles of E1 are not capable of squelching either the wild-type E2 or the A4 mutant form. In our experiments, both forms of E1 accumulated in mitotic cells to the same level, as judged from fluorescent signals, and no indication of differences in levels of proteins was measured by Western blotting (data not shown). Thus, the E1 suppressors have lost some activity that does not manifest in standard E1 experiments. For example, its ability to form a complex with the mutant and wild-type E2 forms of the protein is identical, a point that by itself would be somewhat problematic with a simple requirement for phosphorylation of E2 in the tethering pathway. In a final scenario, some combination of E1 levels, E2 modification, and perhaps other factors may be playing a role in this pathway.
The squelching effects of excess E1 on the mitotic tethering functions of E2 may also be relevant to the requirements for the E2C or E8:E2C repressor protein in the wild-type genome. In repressor-minus BPV-1 plasmids, transient replication is higher than wild type, yet stable transformation is not measurable. Moreover, in the E2-A3 background, the repressor forms are not required for stable transformation. A speculation that would explain these findings might be that for the wild-type case, the repressors are needed to keep E1 levels below a threshold to keep E1 from blocking E2's late step in the cell cycle for plasmid maintenance. In the case of the A3 allele, an increased level of E2 due to increased stability may in this case throw the balance between E1 and E2 back towards a manageable ratio.
We would posit that a single phosphorylation site in the E2-A3 allele at residue 235 in the hinge region can, when modified, serve to weaken the E1-E2 complex just enough to allow tethering. In the E2-A4 genome, this site for modification is not available, and E1 levels may be intolerable for stable maintenance. Continuing along this line of thought, the E1 suppressor mutations, through some unknown effect, alleviate the squelching behavior.
Figure summarizes our current data and ideas pertaining to the roles of E2 and E1 in plasmid replication and tethering. To understand the E2-A4 allele phenotypes, an excess of E1 may squelch E2 chromosomal binding, but, as argued above, E2 modification by itself does not reasonably account for all these data. Clearly E1 may be modified (or unmodified) in post-S-phase periods, and such modifications as the sumoylation of BPV-1 E1 (18
) do indeed affect E1 localization in the cell. Such loss (or gain) of modification or the association with other factors may release E2 from inactive E1-E2 complexes in the nucleus.
FIG. 8. Model for the function of the E1 and E2 proteins in viral replication and nuclear retention of the viral genome during mitosis. E1 (green circles) and E2 (red circle/square) interaction facilitates the origin-specific binding of E1 to the viral DNA. It (more ...)
A major goal is to uncover the nature of the chromosomal protein to which E2 binds for tethering. Perhaps insight into the behavior of this activity will provide further clues to the pathway by which stable BPV-1 plasmids are segregated.