JOURNAL OF VIROLOGY, May 1992, p. 3220-3224 0022-538X/92/053220-05$02.00/0 Copyright ©D 1992, American Society for Microbiology

Vol. 66, No. 5

Replication of Plasmid-Derived Human Papillomavirus Type 11 DNA in Cultured Keratinocytes SALVATORE MUNGAL,1 BETTIE M. STEINBERG,2AND LORNE B. TAICHMANl* Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8702, 1 and Department of Otolaryngology, Long Island Jewish Medical Center, New Hyde Park New York 110422 Received 8 January 1992/Accepted 19 February 1992

Plasmid-derived human papillomavirus type 11 (HPV11) DNA has been shown to replicate episomally and semiconservatively following transfection of primary human foreskin keratinocytes. HPV11 DNA was excised from the bacterial plasmid, religated to form circular molecules, and cotransfected along with pSV2Neo. Transfectants were selected and shown to contain replicated episomal HPV DNA. Once selected, HPV1I DNA persists in cells through at least two additional passages. The natural host of human papillomaviruses (HPVs) is the keratinocyte of stratified squamous epithelia (11). In benign warts and condylomas induced by these viruses, HPV DNA replicates as a multicopy episome in a highly regulated fashion. In the basal layer of the lesion, papillomavirus DNA undergoes a stable but low-copy maintenance replication, while in the suprabasal differentiating cells, virus DNA accumulates in vegetative replication. The most commonly used model for the study of papillomavirus DNA replication has been bovine papillomavirus type 1 (BPV1) in established lines of rodent cells. BPV1 morphologically transforms and replicates episomally in these cells but does not express late gene functions or undergo vegetative replication (10). What has made this model so useful is the fact that plasmid-derived BPV1 DNA replicates episomally following transfection by various physical means (14, 23). The ability to work with cloned BPV1 DNA has allowed identification of viral gene products and cis-acting sequences necessary for episomal viral replication (23). For the HPVs, development of a similar model for the study of episomal replication has been particularly difficult. Keratinocytes transfected with plasmid-derived HPV DNA and selected on the basis of immortalization contain integrated viral DNA (5, 8, 19, 24), thereby rendering the model useless for the study of episomal replication. The single published report of an unsuccessful attempt to detect transient episomal replication of HPV DNA in primary keratinocytes (6) is likely to be an underrepresentation of the actual number of failed attempts. Several considerations led us to formulate a new approach to the question of how to induce and detect episomal replication of plasmid-derived HPV DNA in primary keratinocytes. First, the efficiency of transfection is low in primary keratinocytes (12), and detection of events in the transfected population is therefore likely to require cotransfection and selection with a dominant selectable marker. Second, it was recently shown that HPV type 11 (HPV11)-positive cells cultured from laryngeal papillomas plate with lower efficiency than HPV11-negative cells and are thus selected against (4). Utilization of transfected cells without passaging is therefore likely to avoid this complication. Third, plasmid *

sequences are known to reduce BPV1 transformation (18) and simian virus 40 replication (13). These sequences should therefore be removed. And fourth, when keratinocytes are transfected with a 3-galactosidase reporter gene that has been linearized in the enzyme-coding region, there is no recovery of transient activity. We interpret this to mean that religation of linearized DNA does not take place to any appreciable degree in keratinocytes. It seems prudent, therefore, to religate the excised HPV11 DNA prior to transfection. These considerations were incorporated in the following way. Human newborn foreskin keratinocytes were cultured (16) in serum-containing medium (25) on feeder layers of lethally irradiated Neor-3T3 cells. Stocks of Neor-3T3 were developed by transfection with pLJ DNA (9) and selection with G418. HPV11 DNA was excised from pHPV11 (3) by restriction with BamHI followed by electrophoresis in lowmelting-point agarose. The 7.9-kb HPV11 fragment was removed and purified by Gene Clean (Bio 101, La Jolla, Calif.). Purified HPV11 DNA at 5 to 10 ng/ul was incubated with T4 DNA ligase (1 U/50 RI of reaction mixture) for 16 h at 15°C. Ligation under these conditions resulted in the formation of predominantly monomeric HPV11 molecules (Fig. 1, lane 11). The keratinocyte cultures were cotransfected with 5 ,ug of excised ligated HPV11 and 5 ,ug of pSV2Neo DNA (21) by the modified calcium phosphate coprecipitation method (17). Twenty-four hours after transfection, selection with G418 (active, 400 ,ug/ml; GIBCO) was begun and continued for 4 to 7 days until the mock-transfected controls had died. From an initial inoculum of 3 x 105 keratinocytes in a 60-mm-diameter culture dish, we usually observed 3 to 12 colonies. It was important to begin G418 selection early after seeding, before the attached keratinocytes had multiplied beyond the one- to four-cell stage. Selection at later times, when larger colonies were present, was less effective. Keratinocytes are interconnected through desmosomal junctions. A single transfected Neor cell linked to nontransfected Neos cells is likely to be lost when the sensitive cells become necrotic and detach from the dish. Following G418 selection, cultivation was continued for 14 to 21 days in nonselective medium without passage until confluence was attained. Any adherent 3T3 cells were removed with EDTA prior to harvesting. To determine the state of HPV11 DNA, extracted cellular DNA was electrophoresed and analyzed by Southern hy-

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FIG. 1. Replication of HPV11 DNA in primary foreskin keratinocytes. Second-passage keratinocytes (3.0 x 105) were seeded in 60-mm-diameter culture dishes containing 5 x 105 gamma-irradiated 3T3 cells. Twenty-four hours later, cultures were transfected with 5 ,ug of ligated HPV11 DNA and 5 ,ug of pSV2Neo and selected with G418. Southern hybridization was performed with 1.7 ,ug of DNA in each lane. Lane 1, mock culture transfected with only pSV2Neo. Lanes 2 to 7 contain DNA from cultures cotransfected with ligated HPV11 DNA and pSV2Neo and selected with G418. Lane 2, uncut DNA; lane 3, MboI treated; lane 4, DpnI treated; lane 5, BclI treated; lane 6, MluI treated; lane 7, NcoI treated. BclI, MluI, and NcoI do not cut HPV11 DNA. Lanes 8 to 10 contain DpnI-treated pHPV11 DNA at a concentration equivalent to 1, 5, and 25 copies per cell, respectively. Lane 11 contains 22.5 pg of ligated HPV1l DNA used for transfection. All of the material in this figure was run on a single gel. Rearrangement of lane 11 was done for illustrative purposes. The positions of HindIII fragments of phage lambda are indicated in kilobases at the far right. The asterisk indicates the position of excised linear HPV1l DNA prior to ligation.

bridization (20), using a 1.5-kb AosI-EcoRV fragment from the URR region of HPV11 labeled with 32p with a randomprime DNA labeling kit (Boehringer Mannheim). In cellular DNA run without prior enzymatic restriction, the HPV11 DNA formed two major bands with apparent sizes close to 8 kb (Fig. 1, lane 2). Additional high-molecular-weight bands were present, reminiscent of the catenated forms of HPV16 DNA seen in keratinocytes cultured from a cervical lesion

(2). To assess HPV11 replication, cellular DNA was cut with DpnI and MboI. Both enzymes recognize the palindrome GATC, but DpnI cleaves only if the adenine is methylated and MboI cleaves only if the adenine is not methylated. Since bacteria methylate this nucleotide and eukaryotes do not, plasmid-derived HPV11 DNA that has replicated in keratinocytes will be resistant to DpnI but sensitive to MboI. With the probe we have employed, we expect to see a 2.3-kb band following MboI treatment as evidence of HPV11 DNA replication. In Fig. 1, such a band is present in MboIdigested DNA (lane 3). A smaller fraction of the viral DNA is resistant to MboI digestion, indicating its failure to replicate. Furthermore, with DpnI digestion (lane 4), most of the material is resistant to digestion, indicative of its replication in the keratinocytes. On the basis of the relative intensities of resistant and sensitive DNA, it would appear that about 75% of the viral DNA present was replicated. On the basis of

0O Fraction FIG. 2. Episomal and semiconservative replication of transfected HPV11 DNA. Keratinocytes were cotransfected with ligated HPV1l DNA and pSV2Neo and selected with G418. Cultures were fed medium containing 100 Fig of bromodeoxyuridine per ml daily for 4 days prior to harvesting. A Hirt lysate was prepared, and the supernatant was centrifuged to equilibrium in a CsCl density gradient (4). The positions of half-substituted (HL) and fully substituted (HH) HPV1l DNA are indicated.

copy number reconstructions (lanes 8 to 10), there appear to be about 25 copies of replicated HPV11 per cell, on average. To verify that the viral DNA was episomal, cellular DNA was treated with three restriction enzymes that do not cut HPV11 (Fig. 1, lanes 5 to 7). The position of the HPV11 DNA following treatment with each of these enzymes was unchanged and identical to the position seen following no enzyme treatment. These results indicate that all of the HPV DNA detected was episomal. To ensure that replication was semiconservative, transfected keratinocytes were exposed to bromodeoxyuridine 4 days prior to harvesting. The Hirt supernatant (7) from these cells was centrifuged to equilibrium in gradients of CsCl, and fractions were spotted onto cellulose nitrate and probed for HPV11 DNA (Fig. 2). The bound radioactive HPV11 DNA probe was quantified by liquid scintillation spectroscopy, and the position of nonreplicated light DNA was identified by the location of phage lambda DNA which was added to the Hirt supematant prior to centrifugation. Cellular DNA was measured in each gradient fraction by Hoesch 33258-induced fluorescence (4). Hybrid and fully substituted HPV11 DNAs were present, indicative of single and multiple cycles of semiconservative replication (Fig. 2). In repeat experiments, all of the viral DNA detected in the selected cells was in the replicated form

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FIG. 3. Factors altering HPV1l DNA replication. (A) To determine whether selection with G418 was required, keratinocytes were cotransfected with ligated HPVll DNA and pSV2Neo, but selection with G418 was not performed. Lane 1, uncut DNA. Lane 2, 100 pg of input ligated HPV1l DNA. In this and all other panels of this figure, the positions of HindlIl bands of phage lambda are indicated by Roman numerals as follows: I, 2.0 kb; II, 2.3 kb; III, 4.4 kb; IV, 6.6 kb; V, 9.4 kb; VI, 23.1 kb. The asterisk indicates the position of excised linear HPV11 DNA. (B) To determine whether linear HPVll DNA would also replicate, 5 p.g of excised linear HPV11 DNA was cotransfected into keratinocytes along with pSV2Neo, and transfectants were selected with G418. Lane 1, uncut DNA; lane 2, DpnI treated; lane 3, MboI treated; lane 4, BclI treated; lane 5, NcoI treated; lane 6, MluI treated. (C) To rule out spurious plasmid replication, keratinocytes were transfected with 5 pug of Bluescript DNA and 5 ,ug of pSV2Neo DNA. Cells were selected with G418. The 448-bp PvuII fragment of the LacZ gene of Bluescript was gel purified, labeled with 32P by random priming, and used as a probe. Lane 1 was mock transfected with pSV2Neo and selected. Lanes 2 to 7 contain DNA from cells cotransfected with Bluescript and pSV2Neo and selected. Lane 2, uncut DNA; lane 3, BclI treated; lane 4, NcoI treated; lane 5, MluI treated; lane 6, DpnI treated (inadvertently underloaded); lane 7, MboI treated. The arrow indicates the position of Bluescript DNA used in the transfection.

(data not shown). These results prove that HPV11 DNA does replicate as an episome in a semiconservative fashion when introduced into primary human keratinocytes. Several parameters in the protocol were examined further. First, to determine whether selection of transfectants was vital, transfection and growth were repeated, but without G418 selection. No HPV11 DNA was detected (Fig. 3A, lane 1). Second, to determine whether the use of recircularized HPV11 DNA was essential, linear HPV11 DNA was cotransfected along with pSV2Neo and the cells were selected. Analysis of cellular DNA with DpnI and MboI revealed that all of the HPV11 DNA in the cells was replicated (Fig. 3B, lanes 2 and 3). However, after restriction with three enzymes that do not cut HPV11, dissimilar bands of viral DNA were evident in the three lanes (Fig. 3B, lanes 4 to 6), indicating that some or all of the HPV11 DNA was integrated. HPV11 replication was therefore a consequence of chromosomal replication. Third, preliminary experiments with HPV11 DNA linked to the plasmid sequence indicate a reduced level of replication (data not shown). To rule out spurious replication of HPV11, we repeated the transfection with pSV2Neo and Bluescript. Bluescript is a commercially available plasmid (Stratagene) that contains no sequences that could sustain replication in a eukaryotic cell. Transfectants were selected with G418, and cellular DNA was probed for P-galactosidase DNA (Fig. 3C). When cellular DNA was treated with restriction enzymes that do

not cut Bluescript, there was no alteration in the banding patterns (Fig. 3C, lanes 3 to 5), indicating that the Bluescript

plasmid has remained episomal in keratinocytes. When treated with DpnI, all of the Bluescript DNA was detected as a 1.0-kb band as expected (lane 6; another 192-bp band was expected but ran off the gel), and when treated with MboI, all of the Bluescript DNA was resistant (lane 7). These results indicate a lack of replication. Bluescript DNA that is being detected in these selected cells is therefore input plasmid DNA that has not replicated. These results show that replication of HPVii DNA is specific for sequences that have known eukaryotic replicons. Passage of laryngeal papilloma cells with low-copy-number HPV DNA and passage of keratinocytes infected with HPV1 result in loss of viral DNA (4, 15). This phenomenon appears to result from a selective plating advantage of HPV-negative cells in the culture. To determine whether HPVl1 DNA would persist in the G418-selected transfectants when the cells were passaged, the selected cells were put through two additional passages in the absence of G418 and the DNA was examined. As seen in Fig. 4, HPV DNA was present in the first and second passages and seemed to increase with passaging (lanes 2 and 3). The patterns of bands further suggested that the viral DNA had remained episomal. In transfected cells that were not selected with G418, HPV11 DNA could not be detected (Fig. 4, lane 4). Continued growth of these cells through two passages did

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We thank Teresa DiLorenzo for technical assistance with the density gradient analysis.

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REFERENCES 1. Bedell, M. A., J. B. Hudson, T. R. Golub, M. E. Turyk, M. 0

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4. 5. -2.3 6. FIG. 4. Persistence of HPV11 DNA following cell passage. Keratinocytes cotransfected with ligated HPV11 and pSV2Neo DNA were selected with G418 for 7 days. Cultivation was continued, and just prior to confluence, the cells were carried through two cell passages. Lane 1, uncut DNA from cells after transfection but prior to passaging. Lanes 2 and 3, DNA from transfected and selected cells after one and two passages, respectively. Lanes 4 to 6, DNA from cells that were transfected but not selected with G418. DNA in lane 4 is from transfected cells prior to passaging, and DNA in lanes 5 and 6 is from cells after the first and second passage, respectively. The positions of HindIIl fragments of phage lambda are indicated in kilobases at the right.

7.

8. 9.

10. 11.

not select for cells with HPV11 DNA (lanes

5 and 6). It that when all of the cells contain HPV11 DNA, as they would following selection with G418, there is no population with a selective advantage. A similar conclusion was drawn when laryngeal papilloma cells with a high viral copy number were passaged and no drop in viral DNA copy number was noted (4). It was concluded that in this situation, all of the laryngeal cells were likely to contain HPV11 DNA and thus were not selected against during passage. The reason for the selective disadvantage to HPV-positive cells remains unknown. In summary, transfection of plasmid-derived HPV11 does result in episomal replication of the newly introduced DNA. Factors which improved replication are removal of bacterial plasmid sequences, recircularization of the excised HPV11 DNA, and avoidance of cell passage prior to selection with G418. However, the key variable appears to be selection of the transfectants by cotransfection with a dominant selectable gene. It is unlikely that there is either amplification of HPV11 DNA or late viral gene expression under the culture conditions used in this study. Amplification of episomal HPV31b is seen only when the cells are cultured on collagen rafts at the air-liquid interface (1), and late gene expression of HPV16 is seen only when the cells are inoculated into athymic mice (22). The ability to employ cloned HPV11 DNA for replication studies affords a new opportunity to define viral and cellular components involved in the papillomavirus life cycle.

appears

This research was supported by Institutes of Health (DC00203).

a grant

from the National

12. 13. 14. 15. 16. 17. 18.

19.

20. 21.

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22. Sterling, J., M. Stanley, G. Gatward, and T. Minson. 1990. Production of human papillomavirus type 16 virions in a keratinocyte cell line. J. Virol. 64:6305-6307. 23. Ustav, M., and A. Stenlund. 1991. Transient replication of BPV-1 requires two viral polypeptides encoded by the El and E2 open reading frames. EMBO J. 10:449-457. 24. Woodworth, C. D., J. Doniger, and J. A. DiPaolo. 1989. Immortalization of human foreskin keratinocytes by various human

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papillomavirus DNAs corresponds to their association with cervical carcinoma. J. Virol. 63:159-164. 25. Wu, Y.-J., L. M. Parker, N. E. Binder, M. A. Beckett, J. H. Sinand, C. T. Griffiths, and J. G. Rheinwald. 1982. The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia. Cell 31:693-703.