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Synthesis of Infectious Human Papillomavirus Type 18 in Differentiating Epithelium Transfected with Viral DNA CRAIG MEYERS,* TIMOTHY J. MAYER,
AND
MICHELLE A. OZBUN
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Received 23 April 1997/Accepted 1 July 1997
The lack of a permissive system for the propagation of viral stocks containing abundant human papillomavirus (HPV) particles has hindered the study of infectivity and the early stages of HPV replication. The organotypic (raft) culture system has permitted the study of a number of the differentiation-specific aspects of HPV, including amplification of viral DNA, expression of late genes, and viral morphogenesis. However, these investigations have been limited to a single virus type, namely, HPV type 31 (HPV31). We have artificially introduced linearized HPV18 genomic DNA into primary keratinocytes by electroporation, followed by clonal expansion and induction of epithelial stratification and differentiation in organotypic culture. We report the synthesis of infectious HPV18 virions. Virus particles approximately 50 nm in diameter were observed by electron microscopy. HPV18 virions purified by isopycnic gradient were capable of infecting keratinocytes in vitro, as shown by the expression of multiple HPV18-specific, spliced transcripts. pression of HPV18-specific E6*I, E6*II, and E1∧E4 spliced transcripts in infected keratinocytes.
Cancer of the cervix is the most common cancer in developing countries and the second most common malignancy in women worldwide (26). Papillomaviruses are associated with greater than 90% of all cases of cervical cancer (29). Over 70 human papillomavirus (HPV) types have been identified, with subsets that are associated predominately with malignant or benign neoplasias of the anogenital area, particularly the cervix (29). Replication of HPVs is intimately connected to the differentiation program of the host tissue, squamous epithelium. The ability to synthesize HPV virions in vitro has been hindered by the lack of a reproducible tissue culture system proficient in viral morphogenesis. With the organotypic (raft) culture system, our laboratory and others have been able to achieve the differentiation-specific viral DNA amplification, late gene expression, and virion morphogenic stages of HPV type 31 (HPV31) (9, 19). However, culture systems continue to be deficient in the ability to propagate stocks of HPV competent for infectivity studies. Additionally, investigations of the differentiation-dependent life cycle of HPV in vitro are limited to a single viral type, HPV31 (9, 19). These limitations continue to hinder the ability to study the life cycle of the virus, in particular, the early stages of infection. A system whereby HPV DNA could be introduced into keratinocytes to yield virus stocks would be useful in the analysis of the early stages of infection of various HPV types. In addition, serologic and genetic analyses would also be made practical. We have investigated the probability that the artificial introduction of cloned HPV DNA into keratinocytes might yield sufficient quantities of infectious virus. Following epithelial stratification and differentiation in organotypic cultures, electron microscopy examination of raft culture tissue sections revealed virus particles approximately 50 nm in diameter. HPV18 virions were subsequently purified by isopycnic gradient and were able to infect keratinocytes. We report the ex-
MATERIALS AND METHODS Keratinocyte cultures, organotypic cultures, and electron microscopy. Primary human foreskin keratinocytes (HFK) and primary human ectocervical keratinocytes (HCK) were isolated from newborn circumcision and adult hysterectomy tissue specimens, respectively, as previously described (35). Keratinocytes were grown in monolayer culture by using E medium plus 5 ng of epidermal growth factor per ml in the presence of mitomycin-treated J2 3T3 feeder cells (18, 21, 22). Keratinocyte lines stably maintaining HPV18 DNA following electroporation were subcloned by limiting dilutions of cells and isolation of individual colonies on tissue culture plates with cloning cylinders. Organotypic (raft) epithelial culture tissues were grown as previously described (20–22, 24, 35). Briefly, keratinocytes transfected with HPV18 DNA were seeded onto rat tail type 1 collagen matrices containing J2 3T3 feeder cells not treated with mitomycin. After epithelial attachment and growth to confluence, collagen matrices were lifted onto stainless steel grids. Once lifted to the airliquid interface, epithelial raft cultures were fed by diffusion from underneath with E medium lacking epidermal growth factor. As viral gene expression has been shown to peak at 12 days in the raft system (25), raft cultures were allowed to stratify and differentiate for 12 days. Electron microscopy was performed on raft tissue cross sections and purified virus preparations (19). Electroporation of primary keratinocytes. HPV18 DNA, a generous gift from Harold zur Hausen, was linearized at nucleotide (nt) 2440 by restriction digestion with EcoRI, interrupting the E1 open reading frame. Viral DNA and a plasmid encoding a hygromycin B-selectable marker were electroporated into HFK or HCK (21). Cells were selected with 25 mg of hygromycin B per ml beginning 72 h after electroporation. Selection lasted 3 to 4 days, when the cells began to detach from the plate. Stocks of the electroporated cell lines were prepared and stored in liquid nitrogen until further use. HPV particle isolation and Southern (DNA) blot hybridization. Virions were isolated by first scraping the raft culture epithelium off the collagen with a scalpel and washing the tissue with 0.15 M NaCl. Tissue was ground in a mortar with sea sand, resuspended in buffer 1 (1 M NaCl, 0.05 M Na2HPO4 [pH 8.0]), and centrifuged for 10 min at 8,000 3 g. This supernatant was removed and saved on ice; the pellet was reextracted with buffer 1 and centrifuged for 10 min at 8,000 3 g. The pellet was discarded, and supernatants from both centrifugations were pooled and centrifuged for 1 h at 130,000 3 g. Following centrifugation at 130,000 3 g, the supernatants were discarded and the pellet was resuspended in buffer 2 (0.05 M NaCl, 0.1 M EDTA, 0.05 M Na2HPO4 [pH 7.4]) and centrifuged for 10 min at 8,000 3 g. This supernatant was removed and saved on ice; the pellet was reextracted with buffer 2 and centrifuged for 10 min at 8,000 3 g. Supernatants from these last two centrifugations were pooled, CsCl was added to a final concentration of 1.3 g/ml, and the samples were centrifuged for 24 h at 135,000 3 g. Fractions of 0.5 ml each were collected from the gradient and dialyzed against Tris-EDTA (TE) buffer (pH 8.0). Total cellular DNA and viral DNA were isolated as previously described (19, 21), and 5 mg was electrophoresed per lane in a 0.7% agarose gel. Southern blot hybridization was performed as previously described (19).
* Corresponding author. Mailing address: Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA 17033. Phone: (717) 531-6240. Fax: (717) 5314600. E-mail:
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FIG. 1. Examination of HPV18 DNA-electroporated HFK raft cultures for the presence of virus particles. Raft culture tissue cross sections were fixed with glutaraldehyde and stained with uranyl acetate. (A) Representative nucleus with numerous virions scattered throughout the intranuclear space (arrows point to representative viral particles). (B) Enlargement of a second representative nucleus containing virions (arrows point to representative viral particles). Bars, 100 nm.
HPV infectivity analyses. Gradient fractions shown by Southern blot hybridization analysis and electron microscopy to contain HPV18 virions were pooled. Virions isolated from the parental cell line HCK 18:1B and two subclones derived from this parental line, HCK 18:1Bj and HCK 18:1Bs, were used. Growth media were removed from subconfluent plates of normal HFK cultures, and an inoculum of 50, 100, or 200 ml of a viral stock added to 500 ml of growth medium was applied. Mock-infected plates were also prepared. Plates were rocked every 15 min for 2 h, and then growth media were added. The cells were incubated for 24 or 72 h or maintained by continuous passage postinfection. Total RNA was extracted from HPV18-infected and mock-infected keratinocytes at 24 and 72 h and after the fourth passage in culture. RNA was DNase I treated and subjected to reverse transcription (RT) as previously described (24, 25). HPV18 cDNAs were PCR amplified by using primers to E6 sequence nt 116 to 136 (59-TGAGGATCCAACACGGCGACC-39) and to E4/E2 sequence nt 3609 to 3631 (59-GGTGTAGCTGCACCGAGAAGTGG-39). PCR primers were synthesized by Operon Technologies (San Diego, Calif.) and used at 0.5 mM. The thermocycling profile was as follows: 4-min time delay at 94°C; 35 cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 2 min; and 15 min of extension at 72°C. The PCR products were subjected to electrophoresis in a 1.2% agarose gel, followed by Southern blot hybridization. PCR-amplified HPV18 cDNAs were cloned by using the TA Cloning Kit (Invitrogen, San Diego, Calif.). Double-stranded dideoxy sequencing was performed by using the Sequenase version 2.0 kit (U.S. Biochemical, Cleveland, Ohio).
RESULTS Intranuclear synthesis of virions following electroporation of HPV18 DNA in keratinocytes. To investigate the possibility of producing infectious virus of a preselected HPV type, HCK and HFK were electroporated with linearized HPV18 DNA. Following electroporation, cells were selected with hygromycin B and individual plates were pooled. Electroporations were performed multiple times, and the recircularization and maintenance of episomal HPV18 viral genomes were confirmed in each experiment by Southern blot hybridization (data not shown). HCK and HFK lines stably maintaining episomal copies of HPV18 DNA were subsequently allowed to grow as stratified and differentiated epithelial tissues in the organotypic culture system. Electron microscopy of thin sections of organotypic culture tissue demonstrated nuclei containing virus particles approximately 50 nm in diameter (Fig. 1). The presence of virus particles was limited to upper suprabasal
layers of the epithelium, and the particles observed were similar to those detected in clinical biopsy material from low-grade lesions (8, 15, 23, 27, 34). The observations of HPV18 virion synthesis were reproducible by using several HPV18 DNAtransfected HCK and HFK lines. Preparation of HPV18 viral stocks for infectivity assays. We sought to use HPV18-infected keratinocyte lines that were shown by electron microscopy to synthesize virus particles in differentiated raft tissue to propagate and purify virions for use in infectivity assays. However, the yield of viral particles from these parental cell lines was consistently low. We reasoned that the small amounts of virus obtained would be insufficient to pursue studies concerning infectivity and early stages of replication. We considered the hypothesis that the parental cell populations might lack homogeneity with respect to copy numbers of episomal HPV18 DNA. To address this, we subcloned the parental cell lines. Southern blot hybridization analysis was used to determine the copy number and maintenance of episomal HPV18 DNA in the subcloned lines. Results obtained with representative subcloned lines of the HCK 18:1B parental line are shown in Fig. 2. Nearly half of the subcloned lines contained no detectable amounts of HPV18 DNA (Fig. 2, lanes t and k, and data not shown). Each subcloned cell line with detectable levels of HPV18 DNA maintained the viral DNA episomally. The subcloned cell lines maintaining HPV18 varied in viral genomic DNA copy number (Fig. 2). This illustrated that parental cell lines consisted of a heterogeneous population of cells with respect to HPV18 DNA content. The parental line HCK 18:1B and the subcloned lines HCK 18:1Bj and HCK 18:1Bs (Fig. 2) were allowed to stratify and differentiate in the raft culture system. Viral particles were extracted from raft tissues and purified by isopycnic gradient centrifugation as described in Materials and Methods. With the parental HCK 18:1B cell line, it was difficult to visualize a band on an isopycnic gradient corresponding to the density expected for virus particles. However, the subcloned lines
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FIG. 2. Southern (DNA) blot hybridization of HPV18 DNA-electroporated, subcloned HCK cell lines. Lanes: 1000, 100, and 10, controls of 1,000, 100, and 10 viral genomic DNA copies per 106 cells, respectively; t, s, k, i, and j, total DNAs isolated from representative subcloned cell lines. The lowest band indicates form I (supercoiled) viral DNA. The upper bands represent form II (nicked) and concatemerized viral DNA (3, 37). The arrows on left indicate the positions of HindIII-digested l DNA molecular size markers (from the top, 23,130, 9,416, 6,557, 4,361, 2,322, and 2,027 bp).
HCK 18:1Bj and HCK 18:1Bs formed readily observable bands at the expected density following isopycnic gradient centrifugation (Fig. 3). Gradient fractions were collected and analyzed by Southern blot hybridization, confirming the presence of HPV18 DNA (Fig. 4). Gradient fractions 7, 8, and 9 were found to contain HPV18 DNA at high levels (Fig. 4). Fractions positive for HPV18 DNA correlated with fractions where the banded virus was observed in the gradient (Fig. 3 and 4) and had the buoyant density reported for papillomaviruses (29). Gradient fractions positive for HPV18 DNA were investigated for the presence of viral particles. As demonstrated by electron microscopy, both the parental HCK 18:1B cell line (Fig. 5D) and the subcloned HCK 18:1Bj and HCK 18:1Bs cell lines (Fig. 5A to C, E, and F) were able to support the production of HPV18 virions, albeit in vastly different quantities. Data from Southern blot hybridization analysis indicated
FIG. 3. HPV18 virions banded on isopycnic gradients. Subcloned HPV18 DNA-electroporated line HCK 18:1Bj (J) and HCK 18:1Bs (S) raft cultures were chosen as representative examples. Bands were visualized under normal lighting. The arrowheads indicate bands of HPV18 virions (corresponding to 1.3 to 1.4 g/cm3).
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FIG. 4. Southern blot hybridization of fractions collected from isopycnic gradients of virus preparations from subcloned HPV18 DNA-electroporated line HCK 18:1Bs raft culture tissues. Lanes: 1 to 9, series of fractions with lane 1 representing the bottom of the gradient and viral DNA appearing in lanes 7, 8, and 9; 10, 100, and 1000, controls of 10, 100, and 1,000 viral genomic DNA copies per 106 cells, respectively. The arrows on right indicate the positions of DNA molecular size markers as in Fig. 2.
that viral inocula contained between 1 3 109 and 2 3 109 particles per ml for the subclonal cell lines HCK 18:1Bj and HCK 18:1Bs and greater than 1 order of magnitude fewer particles for the parental line HCK 18:1B. The levels of virions produced in the subcloned lines resulted in clumping of the particles (Fig. 5A and B), as has been seen with higher concentrations of cottontail rabbit papillomavirus and bovine papillomavirus purified from infected tissues (4). To obtain electron micrographs of individual HPV18 virions, virus preparations from subcloned cell lines were diluted (Fig. 5C and E to F). The cosedimentation of both HPV18 DNA and viral particles within the same fractions suggested that these were complete HPV18 virions. HPV infectivity analyses. We next investigated whether our HPV18 viral stocks were capable of infection, the final step in the viral life cycle. Aliquots of HPV18 stocks were allowed to attach to and infect monolayer cultures of HFK. Due to the lack of an infectivity assay for HPV, we assayed for the expression of spliced viral transcripts in infected cells. Following 24 or 72 h or four passages in culture, total RNA was isolated from HPV18-infected and mock-infected HFK monolayer cultures (Fig. 6). Little is known concerning the structures of HPV18-specific mRNAs (2). By using as a model the various spliced E6 transcripts (designated E6*I, II, III, and IV) identified for HPV16 (2, 32), we assayed for similarly spliced transcripts in our HPV18-infected cells. HPV18-specific E6*spliced transcripts were demonstrated by RT-PCR in the subcloned HCK18:1Bj producer line and in HPV18-infected HFK lines but not in mock-infected cells (Fig. 6). The splice donor and splice acceptor sites for HPV18 E6* and E1∧E4 were previously reported (30), and our results are in agreement (Fig. 6). Additionally, we detected E6* spliced transcripts corresponding in size to E6*I and E6*II that were previously observed in HPV18-associated cervical cancer-derived cell lines but were not sequenced or otherwise characterized (13). Interestingly, RT-PCR products with sizes corresponding to four spliced transcripts (E6,E7,E1∧E4, E6*I,E7,E1∧E4, E6*II,E7,E1∧E4, and E6*IV,E2) were observable in the HPV18:1Bj producer line (Fig. 6, lanes 1 and 2) but only E6*I,E7,E1∧E4 and E6*II,E7,E1∧E4 were detected in HPV18infected lines (Fig. 6, lanes 5, 9, and 13). This implies a difference between the splice acceptors used early in infection and those used in more established HPV18-associated lines. Furthermore, sequencing of cDNAs from the HCK18:1Bj producer line verified transcripts corresponding to HPV18 E6*I,E7,E1∧E4 and E6*III. The E2 splice acceptor (illustrated in Fig. 6, transcript E6*IV,E2) was also identified by sequenc-
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FIG. 5. Electron micrographs of HPV18 virions approximately 50 nm in diameter. Isopycnic gradient fractions determined to be positive for HPV18 DNA by Southern blot hybridization were stained with phosphotungstic acid and examined by electron microscopy. (A and B) Concentration of virions produced in subclone line HCK 18:1Bj resulted in clumping of the virions, as seen here (the arrows point to representative viral particles). Bars in A and B, 50 nm. Purified virions from subcloned cell lines were diluted 20-fold in TE buffer [pH 8.0] to prevent clumping; individual particles were observed (C, E, and F). (C) Subclone line HCK 18:1Bj. (D) Parental line HCK 18:1B (undiluted preparation). (E and F) Subclone line HCK 18:1Bs. The magnification in F is approximately twice that of C to E. All virus particles are approximately 50 nm in diameter.
ing a cDNA corresponding to E6*,E7,E1∧E2 (data not shown). In addition, we detected an amplification product of approximately 430 bp in one of the infected cell lines 24 h postinfection (Fig. 6, lane 7) and in the HPV18:1Bj cell line (Fig. 6, lanes 1 and 2). The structure of the transcript corresponding to this product is currently under investigation. As the introduction of HPV18 early genes into keratinocytes has been shown to increase their life span (10, 12), we assessed the life span of our HPV18-infected cell lines. Mock-infected keratinocytes grew in culture for 6 to 8 weeks and then senesced. By using separate viral stocks from the parental HCK 18:1B line and the subcloned lines HCK 18:1Bj and HCK 18:1Bs, we found that six of six infected lines were able to grow a month longer in culture than the mock-infected and normal keratinocyte cell lines. Infected cell lines with extended life spans also expressed HPV18 spliced transcripts (Fig. 6, lanes 9 to 11). DISCUSSION The ability to study the complete life cycle of HPVs has been limited by the lack of an efficient system yielding infectious viral stocks. We have used DNA electroporation in combination with the organotypic (raft) culture system to propagate sizable quantities of virions of a preselected HPV type in vitro.
By using this system, we produced HPV18 virions, as demonstrated by isopycnic gradient sedimentation, Southern blot analysis, electron microscopy, and infectivity. Our data show that cell populations homogeneous in the maintenance of episomal viral DNA were efficient at in vitro synthesis of infectious HPV. Interestingly, the two subcloned cell lines described in this report differed 10-fold in HPV18 DNA content (Fig. 2) but produced equivalent amounts of viral particles. To overcome the lack of an in vitro infectivity assay for HPVs, we investigated the expression of HPV18-specific, spliced transcripts. The presence of these spliced transcripts in HPV18-infected cells confirms entry of the virus into the cell and the initiation of early viral transcription. Furthermore, our data suggest changes in viral transcript expression during the first 3 days postinfection (Fig. 6, 24 h postinfection versus 72 h postinfection). An in vitro raft culture system capable of complete vegetative replication of HPV18 will permit the description of HPV18 transcripts and their temporal expression pattern, similar to what we have done with HPV31 (25). The present work also supports the concept that epithelial differentiation is required for HPV virion production. The requirement of host tissue differentiation for efficient virus replication is not unusual and has been described for other viral systems, such as cytomegalovirus (36), Epstein-Barr virus (5, 7, 17), Friend virus (11), human immunodeficiency virus (6,
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FIG. 6. Identification of HPV18-specific, spliced transcripts in HPV18-infected keratinocytes. (A) Southern blot of RT-PCR products. Lanes: 1, 3 ml of the PCR product of HCK 18:1Bj cell line cDNA; 2, 0.3 ml of the PCR product of HCK 18:1Bj cell line cDNA; 3 to 12, 30 ml of each indicated PCR sample; 3 to 5, keratinocytes 72 h postinfection; 6 to 8, keratinocytes 24 h postinfection; 9 to 11, keratinocytes four passages postinfection; 12, control with no RNA added to RT-PCR mixture. j and P represent viruses isolated from subclone HCK 18:1Bj and parental HCK 18:1B raft culture tissues, respectively. At the left, DNA size markers of (from the top) 2,322, 2,027, 1,353, 1,078, 872, 603, and 310 bp are indicated. (B) Splicing patterns of HPV18 transcripts shown schematically, along with the corresponding HPV18 nucleotide numbers of the splice sites and the predicted sizes of the RT-PCR products. HPV18-specific, spliced transcripts identified in the HCK 18:1Bj cell line and in the infected keratinocytes are represented by corresponding symbols in A and B. P105 is the major early promoter of HPV18. The placement and orientation of PCR primers are illustrated by open arrows.
14), measles virus (31), polyomavirus (1), Pichinde virus (28), Rift Valley fever virus (16), and visna virus (33). We have described an in vitro tissue culture system capable of the synthesis of infectious HPV of a preselected type. We expect that any naturally occurring HPV type or mutant for which cloned DNA is available can be propagated in this system. This will allow comparisons of the replication cycles of different HPVs, genetic analysis of the HPV life cycle, and investigation of the early stages of HPV infection. This system also would be particularly useful in the preparation of viral stocks for viral serology. Studies of these kinds have not been possible before and will be valuable not only for the understanding of the biology of papillomaviruses but also for the development and evaluation of antiviral therapies.
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ACKNOWLEDGMENTS We thank H. zur Hausen for the HPV18 DNA clone, D. Huber and R. Myers for excellent technical assistance, and N. Christensen and R. Courtney for critical reading of the manuscript. This work was supported by NIH training grant CA-60395 (T.J.M.), Public Health Service fellowship CA-66316 from the National Cancer Institute (M.A.O.), American Cancer Society grant IRG-196 (C.M.), and National Cancer Institute grant CA-64624 (C.M.). REFERENCES 1. Atencio, I. A., and L. P. Villarreal. 1994. Polyomavirus replicates in differentiating but not in proliferating tubules of adult mouse polycystic kidneys. Virology 201:26–35. 2. Baker, C., and C. Calef. 1996. A compilation and analysis of nucleic acid and amino acid sequences, p. III-3-III-13. In G. Myers, C. Baker, C. Wheeler, A. Halpern, A. McBride, and J. Doorbar (ed.), Human papillomaviruses 1996. Los Alamos National Laboratory, Los Alamos, N.Mex. 3. Choo, K. B., W. F. Cheung, L. N. Liew, H. H. Lee, and S. H. Han. 1989. Presence of catenated human papillomavirus type 16 episomes in a cervical carcinoma cell line. J. Virol. 63:782–789. 4. Christensen, N. Personal communication. 5. Crawford, D. H., and I. Ando. 1986. EB virus induction is associated with B-cell maturation. Immunology 59:405–409. 6. Cullen, B. R., and W. C. Greene. 1989. Regulatory pathways governing HIV-1 replication. Cell 58:423–426. 7. Davies, A. H., R. J. A. Grand, F. J. Evans, and A. B. Rickinson. 1991.
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