The experiments above were carried out to study the way in which HPVs replicate their DNA in proliferating cells, that is, maintenance replication. This question has been addressed previously with the BPV1 system, but the results were conflicting. Initially, BPV1 DNA was reported to replicate in an ordered once-per-S-phase fashion (3
). However, more rigorous experiments with nocodazole showed that BPV1 DNA replicated in a random fashion (11
). Although the mode of BPV1 DNA replication in proliferating cells is now clear, it is questionable whether this applies to HPV DNA as well, not least because HPVs are strictly keratinocyte-specific viruses, whereas the BPV1 experiments were carried out in murine fibroblasts. To address this question directly for HPVs, we studied the maintenance replication of HPV16 and HPV31 DNAs in W12 (28
) and CIN612 (15
) cells, respectively. These are the only naturally derived cell lines harboring replicating HPV DNA episomes. Since these lines were derived from naturally infected cervical epithelium, they are undoubtedly most suitable to be used to answer the question posed above.
Our initial results with W12 cells indicated that HPV16 DNA replicated in a manner similar to that of cellular DNA. Although suggestive, these findings were not a proof because the apparent similarity of replication profiles between viral and cellular DNA can also be obtained even if different modes of replication were used by these two DNAs. Hence, a more surgical approach was used. This was to see whether HH HPV16 DNA appears in W12 cells that have not replicated more than once (and whose DNA is only LL or HL). Interestingly, the results after a shorter time of BUdR feeding, where W12 cells replicated at most only once, showed no HH HPV16 DNA. Instead, like cellular DNA, only HL and LL viral DNAs were detected. This lends a more legitimate support to the notion that HPV16 DNA replicates in the same way as the cellular DNA. Confirmation of this was obtained when HPV16 DNA in nocodazole-treated W12 cells, even after 31 h, did not rereplicate. Together, this evidence leads to the conclusion that in proliferating W12 cells, HPV16 DNA replication is controlled in a manner that is similar to that of the cellular DNA, which is once and only once per S phase. As such it was therefore surprising to observe that HPV31 DNA replicated randomly in CIN612 cells. To ascertain whether the difference was due to the different cell lines used (W12 and CIN612), we tested the replication of HPV16 and HPV31 DNA in NIKS cells and saw that HPV16 and HPV31 DNAs both replicated randomly. It is clear that the mode by which HPV DNA replicates in proliferating cells is dictated by the host cell.
The random replication mode of HPV DNA is in accordance with the prevailing notion of HPV DNA replication based on conclusions drawn from BPV1. However, the ordered replication of HPV16 DNA is not. It is on the one hand unexpected but on the other hand not entirely surprising since there are at least two other viruses whose DNA replication is controlled by the cell. The most studied is Epstein-Barr virus (EBV), whose DNA is replicated once and only once per S phase in latently infected cells (33
). Consistent with this, cellular proteins that regulate similar ordered timing of cellular DNA replication, MCMs, and ORC (2
) were observed to be associated with the oriP
of EBV (4
). Likewise, MCM and ORC proteins also assemble on the origin of replication of latent Kaposi's sarcoma-associated herpesvirus (KSHV) DNA (29
). It is particularly noteworthy that MCM and ORC proteins associate with latent EBV and latent KSHV DNA. It may well be that by yielding the control of their genome replication to the cell, these viruses minimize expression of their proteins in the host cell. This is a trait that is particularly advantageous and may even be a prerequisite for a successful latent phase in the virus life cycle. The latency of HPV has been a subject of thought for a long time since it is not clear whether it occurs and, if it does, what mechanism is used. Based on the observations presented above, HPV, by conceding control of its genome replication to the cell, may actually favor the establishment of latency.
It is not clear what the differences are between W12, CIN612, and NIKS cells, but it is thought that in the basal cells of the epithelium, two types of keratinocytes exist. The first are stem cells, which replicate infrequently and serve as a supply source of transit-amplifying cells, which are the other cell type that constitute the basal cell population. Unlike stem cells, which have the capacity to proliferate perhaps indefinitely, transit amplifying cells proliferate only a limited number of times before they cease and leave the basal layer to begin the process of terminal differentiation. It is conceivable that HPV DNA introduced into these two different cell types by infection is replicated differently. For example, W12 may have originated from an HPV-infected cervical epithelial stem cell, as suggested by Kim et al. (18
), whereas CIN612 cells may have originated from an HPV-infected cervical epithelial transit-amplifying cell, or perhaps vice versa. Whatever the case may be, it is a notion worth considering since it is possible that infection of epithelial stem cells may be a prerequisite for the latency and persistence of HPVs. As such, it would be important to understand the replication of HPV DNA in such cells.
The observations described above also bring to the fore the question of replication mechanisms that are involved in the two modes of replication. To further this line of investigation, we will be looking to see whether the cellular DNA replication licensing proteins such as ORCs and MCMs are associated with HPV16 DNA in W12 cells. It will also be important to ascertain the role of the E1 protein in both forms of HPV DNA replication. It is interesting that the E1 protein, which is an ATPase, helicase, and origin-binding protein of HPV, bears many similarities to the MCM proteins of the cell. Just like SV40 large T antigen (20
), E1 can be seen to function as the viral replication license. However, whereas MCMs can license cellular DNA for replication only once per S phase (2
), the E1 protein is able to trigger HPV16 DNA replication continuously, as seen in the experiment described in Fig. . Although the molecular mechanism of how HPV16 DNA replication is limited to once per S phase in W12 cells is not known, two hypotheses can be proposed. The first posits that HPV16 DNA replication is carried out in W12 cells by cellular proteins independently of E1. The MCM proteins, by substituting for E1, replicate the viral DNA only once per S phase in W12 cells. This strict regulation is abrogated when E1 is present, allowing random replication of HPV16 DNA. Although the suggestion that HPV DNA can replicate without E1 protein is rather troubling at first sight and contrary to the prevailing model of HPV DNA replication, there is increasing evidence to support this view. Kim et al. (19
) reported that whereas E1 protein was required for the establishment of BPV1 DNA as episomes in cells, it was not necessary for the maintenance of these episomes upon subsequent cell divisions. Furthermore, in a more recent separate report, Kim et al. showed that HPV16 DNAs are replicated and maintained as episomes in Saccharomyces cerevisiae
in the absence of any viral gene expression (17
). Indirectly relevant to this point is the report that a plasmid without any known human origin of replication replicated in a once-per-S-phase mode in CHO and HeLa cells and that ORC and MCM proteins were attached to this plasmid (26
). In sum, these observations support the possibility that after establishment as stable episomes in W12 cells, the HPV16 DNA copy number is stably maintained via cell-controlled replication. If so, HPV DNA's presence in cells may be maintained at minimal expense to the virus, in a stealthy way with regard to immune surveillance. This may be why EBV and KSHV have also evolved to use this strategy to support their latent phase in the host cell.
An alternative suggestion would be that HPV DNA maintenance replication in W12 is indeed E1-driven but that, in these cells, E1 is only able to license HPV DNA for replication once per S phase. Although there is no direct evidence to support the idea that E1's activity is limited to only once per S phase, the report by Deng et al. (7
) demonstrating that E1's localization to the nucleus is regulated by the cyclin E-cdk2 phosphorylation opens an avenue to this hypothesis. It is possible that the cell-cycle-regulated entry of E1 into the nucleus may confer once-per-S-phase activity to E1. This regulation may be overcome by excess amounts of E1 protein, as occurs when W12 cells were transfected with codon-optimized E1. This is also the case when E1 is expressed at high levels in transient DNA replication assays. Whatever the mechanism may be, it is clear that random replication of HPV DNA can be attained in the presence of sufficient E1 protein, and this may be the case in CIN612 and NIKS cells. Since it is not possible to detect and compare endogenous levels of E1 protein in W12, CIN612, and NIKS cells (the E1 proteins are undetectable in all cell lines), this line of investigation cannot be easily pursued directly and will have to be addressed with more complex methods.
Since no other naturally occurring cell lines bearing HPV episomes are available for more testing, it will be necessary to resort to other experimental systems to answer the questions that have arisen from this work. Until then, we draw attention to the conclusion and purpose of this study, which is that HPV DNAs can be maintained as replicating episomes in dividing cells either by replicating once per S phase or by random replication. Either of these two mechanisms can sustain the maintenance of HPV DNA in the infected tissue. Whether these two modes of viral DNA replication impinge on pathogenesis, latency, and persistence are intriguing questions worth exploring.