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Human papillomavirus (HPV) begins its life cycle by infecting the basal cells of the epithelium. Within these proliferating cells, the viral genomes are replicated, maintained, and passed on to the daughter cells. Using HPV episome-containing cell lines that were derived from naturally infected cervical tissues, we investigated the mode by which the viral DNAs replicate in these cells. We observed that, whereas HPV16 DNA replicated in an ordered once-per-S-phase manner in W12 cells, HPV31 DNA replicated via a random-choice mechanism in CIN612 cells. However, when HPV16 and HPV31 DNAs were separately introduced into an alternate keratinocyte cell line NIKS, they both replicated randomly. This indicates that HPV DNA is inherently capable of replicating by either random-choice or once-per-S-phase mechanisms and that the mode of HPV DNA replication is dependent on the cells that harbor the viral episome. High expression of the viral replication protein E1 in W12 cells converted HPV16 DNA replication to random-choice replication and, as such, it appears that the mode of HPV DNA replication in proliferating cells is dependent on the presence or the increased level of this protein in the host cell. The implications of these observations on maintenance, latency, and persistence are discussed.
The host tissue of human papillomaviruses is the stratified epithelium. This tissue is complex in that it is composed of layered sheets of nondividing cells in various stages of terminal differentiation, with the uppermost layer being the most differentiated. Only cells of the bottom-most layer of this tissue, the basal cells, proliferate. Although the HPV life cycle begins with the infection of a basal cell, it only comes to completion when the infected cell reaches the upper layers of the epithelium. As a consequence, the HPV DNA initially finds itself in the nucleus of a proliferating cell, but later in that of a differentiating (nonproliferating) one. This sequential mixture of cell states that the virus has to contend with has undoubtedly shaped the way by which HPV replicates its DNA throughout the varying milieux during its life cycle.
It is proposed that immediately after infection, the papillomavirus DNA copy number is amplified to a certain level (50 to 400) per cell (9). This first amplification replication is believed to be rapid and transient, after which the viral DNA is stably maintained at this level in subsequent divisions of the basal cell. This is thought to be achieved by maintenance replication, where the viral episomes approximately double their copy number during the S phase of the host cell cycle and segregate to the resulting two daughter cells. After differentiation of the host cell, the viral DNA undergoes another amplification step, the second amplification replication, which increases the HPV DNA copy number to several hundreds or thousands per cell. This is followed by packaging of the viral DNA into virus particles. Although this triphasic model has not been proven in its entirety, it is supported by various pieces of evidence. In situ hybridization studies of cervical epithelia show that the HPV DNA copy number does indeed increase concomitantly with terminal differentiation of the host cell (30). This increase correlates with the second amplification replication of the viral DNA. In contrast, the existence of a first amplification of the viral copy number is not concluded from such clear observations. Instead, it is deduced from the fact that HPV episome-containing cervical cell lines derived from clinical tissues harbor HPV DNA at about 100 to 1,000 copies per cell (15, 28). The likelihood that this number of viruses infected the precursor cell is low. It is more likely that the initial viral DNA introduced into the cell by the virus underwent amplification replication to the numbers stated above. The stable maintenance of the HPV DNA copy number per cell upon subsequent passaging constitutes the maintenance phase of HPV DNA, which is sustained by maintenance replication. These separate observations and deductions have established the hypothesized triphasic HPV DNA replication model.
Implicit in this model is the existence of two kinds of HPV DNA replication. The first is the amplification mode, which includes the first and second amplification during which HPV DNA copy number is increased by continuous replication either within a single S phase in the basal cell or in a prolonged or perpetual S phase that is induced by the virus in differentiating cells. The second kind of replication, which is the focus of this report, is the maintenance replication mode which maintains the HPV DNA at an approximately stable number in proliferating basal cells. There are two ways by which maintenance can be achieved. The first is by HPV DNA replicating only once per S phase, much like the cellular DNA. This form of replication is termed as ordered replication. The alternative is that HPV DNA replicates randomly, whereby some molecules replicate a few times per S phase, some replicate once, and some do not replicate at all. Both scenarios could in theory maintain the viral DNA at an approximately fixed number per cell on average throughout the proliferative phase of the host cell.
This question was previously addressed by analyzing replication of bovine papillomavirus 1 (BPV1) DNA in cultured mouse fibroblasts. These studies yielded varied results, with some reporting that BPV1 DNA replicated in an ordered once-per-S-phase fashion (3, 25) and others showing that BPV1 DNA replicated in a random fashion (11, 22, 23). Although the latter is generally regarded to be the actual case, it is still a question whether this conclusion can be extrapolated to the way by which HPV DNAs replicate in proliferating cells. After all, HPV DNAs replicate in the keratinocytes of the human cervix, whereas the BPV1 studies were carried out in murine fibroblasts. Although these studies may be relevant to BPV1, which infects and replicates in bovine fibroblasts (14), they may not necessarily be relevant for HPVs, which are strictly keratinocyte-specific viruses. We have analyzed the maintenance replication modes of HPV16 and HPV31 and found that HPV DNA can replicate randomly or in an ordered once-per-S-phase fashion depending on the cell in which it is located. In W12 cells, which are derived from a natural cervical lesion, the HPV16 DNA replicates once and only once per S phase. However, HPV16 DNA cloned from W12 cells will replicate randomly when introduced into another epithelial cell line, NIKS, which is HPV-free. Furthermore, when HPV replication protein E1 is ectopically expressed in W12 cells, HPV 16 DNA replication becomes random. These observations reveal the complexity and flexibility of HPV DNA replication, which appears to be dictated by the host cell.
Clones of W12 cells (originally isolated and described by Stanley et al. (28) were obtained from Paul Lambert (16). The results shown in the present study were obtained with the 20863 clone. Similar results were also obtained with the use of clone 20850 and the original polyclonal W12 cells. The growth of these cells can be irregular. As a result, there is no strict population doubling time. Notwithstanding this, 30 h is a close approximate. CIN 612 cells were obtained from Laimonis Laimins (15). These cells were cultured in modified Rheinwald medium (3 parts of F-12 Ham medium and 1 part Dulbecco modified Eagle medium supplemented with 5% fetal calf serum, adenine to 24 μg/ml, cholera toxin to 8.4 ng/ml, insulin to 5 μg/ml, hydrocortisone to 0.4 μg/ml, and epidermal growth factor to 10 ng/ml) and in the presence of lethally irradiated J2-3T3 cells. 5′ Bromouracil deoxyriboside (BUdR) was used at a final concentration of 30 μg/ml. Apart from experiments with CV-1 cells, 5-fluoro-2-deoxyuridine was not used. CV-1 cells were cultured in 5% fetal calf serum-containing Dulbecco modified Eagle medium.
Viral DNA was extracted from cells according to the method reported by Hirt (13), with some slight modifications. Cells were treated with trypsin, counted, and adjusted to a concentration of two million per ml of phosphate-buffered saline. Then, 500 μl of cells was added to 500 μl of 2× Hirt buffer (2% sodium dodecyl sulfate, 2 mM EDTA) in a vial. After 10 min, 250 μl of 5 M NaCl was added and mixed by inverting the tubes gently three times. The tubes were kept at 4°C overnight. After 15 min of centrifugation at top speed in a microfuge, supernatant containing viral DNA, small DNA fragments, RNA, and proteins was collected and subjected to two rounds of phenol-chloroform extraction, followed by ethanol precipitation. DNA precipitates collected after 15 min of centrifugation at top speed in a microfuge were resuspended in 500 μl of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA [pH 8]) and separated by centrifugation through a cesium chloride gradient. Cellular DNA recovered from the precipitate after centrifugation with the Hirt buffer was resuspended in 500 μl of Tris-EDTA buffer, and the DNA was fragmented by centrifugation through a QIAshredder (QIAGEN). After three rounds of phenol-chloroform extraction and ethanol precipitation, the cellular DNA was resuspended in 500 μl of Tris-EDTA buffer and separated in a cesium chloride gradient.
DNA was mixed with cesium chloride (CsCl), and the mixture was adjusted to 3 ml at the density of 1.753 g/ml (corresponding to a refractive index of 1.404). The DNA-CsCl solution was transferred to Beckman ultracentrifuge tubes and topped up with liquid paraffin. The samples were centrifuged at 30,000 rpm at 22°C for at least 55 h in either a SW55 rotor or an MLS50 Beckman rotor. After centrifugation, the tube was inserted into a gradient collector, a hole was punctured at the bottom of thetube, and fractions of 5 drops each were collected in Eppendorf tubes. Refractive indices of the fractions were measured with a refractometer, after which the fractions were slot blotted onto a positively charged nylon membrane. The wells of the slot blotter were washed twice with denaturation buffer (0.5 M NaOH, 0.5 M NaCl). The membrane was then air dried and UV cross-linked. The membranes were then blocked with prehybridization buffer for at least 30 min before hybridization with 32P-labeled DNA (HPV DNA or cellular DNA extracted from HPV-free NIKS cells) probes overnight. The washed membranes were exposed to either an X-ray film or to a phosphorimager screen, and the signals were measured by the phosphorimager reader.
HPV DNAs were excised from their backbone vectors (pSP64 for HPV16 and pBR322 for HPV31), and 5 μg was self-ligated in 2 ml of 1× DNA ligase buffer (New England Biolabs) with 2,000 U of T4 DNA ligase. Ligation reactions were carried out overnight at 16°C. The DNA was isolated using miniprep kit from QIAGEN. The eluted DNA, together with equal amounts of pCDNA6 were used to transfect NIKS cells using Effectene according to the manufacturer's protocol (QIAGEN). The next day, blasticidin was added to the cells at a concentration of 7 μg/ml. After 3 days, when all of the control cells were dead, the surviving transfectants were passed into fresh plates and fed with BUdR (30 μg/ml) for the times stated in Results.
To determine the mode of HPV DNA replication, we sought to determine how many times HPV DNAs replicate in a single S phase. This could be ascertained by feeding cells with BUdR, which is an analogue of thymidine, but with a greater density. Viral DNAs that have replicated once would incorporate BUdR on a single DNA strand. This would increase the density of one strand of the DNA, and hence this species is called “heavy-light” (HL). Those that replicated more than once will incorporate BUdR into both strands and are termed “heavy-heavy” (HH), and those that failed to replicate will have no BUdR in their DNA and are termed “light-light” (LL). These three species, with sufficiently different densities should lend themselves to separation in a cesium chloride gradient. To set up the optimal conditions, in terms of labeling and separation resolution, for this experiment, we used the simian virus 40 (SV40) DNA as control marker for the three species of DNA. It is known that, like polyomavirus (12), SV40 replicates randomly in the host cell, and as a consequence SV40-infected cells would be expected to contain HH, HL, and LL DNA. According to the previously published protocol for such experiments (12), we infected CV-1 cells that were grown for 5 h in the presence of 1 μCi of [3H]thymidine/ml, with SV40. At 27 h after infection, the cells were fed with medium containing 1 μCi of 3HTdR/ml, 5 μg of BUdR/ml, and 15 μg of 5-fluoro-2-deoxyuridine/ml. After 24 h, DNA was extracted according to the Hirt method (13), which separates small DNA, such as viral DNAs, from cellular DNA. After centrifugation of the DNA through a CsCl gradient, fractions of 5 drops each were collected from the bottom of the centrifuge tube. Radiolabeled DNA in each fraction was quantitated in a scintillation counter. As seen in Fig. Fig.1,1, SV40 DNA from CV-1 cells was successfully separated in the CsCl gradient into the HH peak at 1.795 g/ml (fraction 9), the HL peak at 1.753 g/ml (fraction 17), and the HH peak at 1.709 g/ml (fraction 25). As expected, only LL and HL CV-1 DNAs were detected. This is because although most of the CV-1 cells replicated only once (HL DNA) during the 24-h period in the presence of BUdR, SV40 DNA replicated randomly, with some copies of its genome replicating multiple times, some once, and others not at all. This result shows that BUdR labeling of cells was efficient and that the cesium chloride gradient centrifugation method used provided sufficient resolution to separate the three forms of DNA based on their BUdR-altered densities.
Having established that the method above can separate and distinguish the three types (HH, HL, and LL) of DNA by CsCl density centrifugation, we proceeded to use it on HPV16 DNA in W12 cells, which are cervical keratinocytes that harbor several hundred copies of HPV16 DNA as episomes (28) (Fig. (Fig.2).2). Because the HPV16 DNA in W12 cells is not as abundant as the replicating SV40 DNA in infected CV-1 cells, it was decided that it would be more accurate and sensitive if viral DNA and cellular DNA in the CsCl fractions were detected by slot blotting using DNA probes specific for the respective DNAs, followed by quantitation with a phosphorimager. As such the counts on the y axis of the following figures are values quantitated using the phosphorimager. In the first experiment, W12 cells were fed BUdR for 36 h prior to harvesting and analysis of their DNA. As shown in Fig. Fig.3,3, the HH, HL, and LL peaks of HPV16 bear very strong resemblance to those of the cellular DNA. Although this may indicate that the viral and cellular DNA replicated in step, it is not by any means a proof of this. It is possible that the similarity of HH, HL, and LL patterns was merely coincidental. Knowing that cellular DNA replication occurs only once per cell division, we sought to determine whether viral DNA would also do that. This was done by analyzing the viral and cellular DNA at time points shorter than the doubling time of W12 cells, which is approximately 30 h. After 16 h (Fig. (Fig.4a),4a), only HL and LL DNA peaks of W12 DNA were present. This is as it should be since this time point is well short of the doubling time of W12. Interestingly, the HPV16 DNA peaks were also of HL or LL species with no HH type detected, indicating that HPV16 DNA, like cellular DNA, did not replicate more than once within one cell cycle. After 24 h, most of the W12 DNA was HL, and the same was true for HPV16 DNA (Fig. (Fig.4b).4b). The similarity of the patterns between cellular DNA and viral DNA is very clear, and the most noteworthy point is that no HH viral DNA was detected. These results point to HPV16 DNA being replicated once and only once per S phase. Suggestive as they were, by themselves, these results still do not prove that replication of HPV16 DNA is controlled by the same mechanism that controls replication of W12 DNA. It is possible that HPV16 DNA replicates extremely slowly and, if mitosis were to be delayed, rereplication of the viral DNA within one cell division would be seen, while cellular DNA cannot reinitiate another round of DNA replication before an intervening mitosis. To test this, we treated W12 cells with BUdR and nocodazole. The latter prevents the division of cells by inhibiting the process of mitosis. After 31 h, HPV16 DNA was extracted and analyzed, and Fig. Fig.55 shows that virtually all of the viral DNA was HL. Once again, no rereplicated (HH) viral DNA was detected, indicating that HPV16 DNA does indeed replicate in an ordered once-per-S-phase manner, just like cellular DNA.
To ascertain whether the above observation with HPV16 DNA is general for other papillomavirus types, we analyzed HPV31 DNA replication in CIN612 cells, which possess approximately 150 copies of HPV31 DNA as episomes (15). As is seen in Fig. Fig.6a,6a, the HPV31 DNA replicated randomly, with HH, HL, and LL DNA species present, while cellular DNA was only of LL and HL density (Fig. (Fig.6b).6b). This shows that some HPV31 DNA replicated more than once per S phase. The difference between HPV16 and HPV31 DNA replication was repeatedly observed in separate experiments. Thus, it appears that, although HPV16 and HPV31 are the closest HPV types based on DNA sequence, they have profoundly different modes of maintenance replication of their genomes.
The difference between HPV16 DNA replication in W12 cells and that of HPV31 DNA in CIN612 cells can be accounted for by two possibilities. The first is that HPV16 and HPV31 are indeed different in the way they replicate their DNA in proliferating cells. The second is that the nonidentical cellular background of W12 and CIN612 accounts for the differences seen. To test this, we prepared recircularized HPV16 and HPV31 DNA from their respective bacterial vectors. These DNAs, together with pcDNA6, which expresses the blasticidin resistance protein, were transfected into NIKS, which are non-HPV-containing keratinocytes that can support the replication and maintenance of transfected HPV genomes (10). Two weeks after blasticidin selection and expansion of the cultures, the cells were fed BUdR and nocodazole for 24 h, and the DNA was isolated and analyzed as described above. Interestingly, both HPV16 and HPV31 DNA were seen to replicate randomly in NIKS cells, with HH, HL, and LL viral DNAs present, whereas only HL and LL cellular DNA was detected (Fig. (Fig.7).7). Similar results were obtained when individual clones of NIKS with HPV16 DNA or HPV31 DNA were used in the experiments. Together, these results show that to maintain a stable copy number of HPV DNA per cell, the viral DNA can either replicate in an ordered manner or randomly. The choice of maintenance replication mode appears to be dependent on the host cell that harbors the viral DNA.
It has long been established that replication of papillomavirus DNA is carried out by the concerted action of two viral proteins, E1 and E2 (5, 6, 21, 31), and the host's DNA replication machinery. Since HPV16 DNA replication is restricted to once per S phase in W12 cells, it is likely to be restricted by the cellular DNA replication control mechanism. If so, we sought to determine whether the control of HPV16 DNA replication in W12 cells can be broken by expressing the HPV DNA replication proteins E1 and E2. Since the E2 protein is already present in W12 cells at readily detectable levels (1) (Fig. (Fig.8a),8a), we tested the effect of introducing a plasmid capable of expressing codon-optimized E1 (32) (Fig. (Fig.8a)8a) into W12 cells, followed by BUdR feeding as described above. As is shown in Fig. 8b and c, whereas cellular DNA was of only HL and LL types after 16 h in presence of BUdR, HPV16 DNA in these E1-expressing W12 cells was of the HH, HL, and LL types, indicating that the HPV16 E1 protein had indeed broken the rigid once-per-S-phase restriction that was imposed on HPV16 DNA replication. Experiments where E2 expression plasmids were transfected into W12 cells to increase the already substantial amounts of endogenous E2 protein did not demonstrate any difference from control transfected cells (data not shown). Hence, it appears that only the E1 protein is able to subvert the once-per-S-phase replication control of the HPV16 DNA.
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, 25). However, more rigorous experiments with nocodazole showed that BPV1 DNA replicated in a random fashion (11, 22, 23). 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, 8, 24, 27). 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. Fig.8.8. 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.
Funding for this study was provided by the United Kingdom Medical Research Council and the Swiss Cancer League.
We are grateful to the Virology Division of the National Institute for Medical Research, Mill Hill, for their support. We thank Margaret Stanley for the W12 cells and Laimonis Laimins for the CIN612 cells. NIKS cells and W12 clones were generously provided by Paul Lambert. Codon-optimized E1 was a generous gift from W. L. McClements.