Department of Molecular Genetics and Microbiology, The University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA1
Author for correspondence: Michelle Ozbun. Fax +505 272 9912. e-mail mozbun{at}salud.unm.edu
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Abstract |
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Introduction |
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According to current models, HPVs infect the mitotically active basal cell layer in vivo through a micro-abrasion or wound in the epithelium. The ability of HPVs to undergo a complete replication cycle resulting in the production of virions (i.e. infectious progeny) is tightly linked to the differentiation state of the infected cells (Laimins, 1996 ; Lowy & Howley, 2001
; Meyers et al., 1992
). Epidermal cells are not fully permissive for PVs at the onset of their cellular differentiation process, but become permissive with increasing differentiation. Viral genomes are replicated in three stages. In stage I, the autonomously replicating episomal viral DNA (vDNA) is established at low (10200) copy number per cell in the basal stem cells. This event is necessary for establishing virus persistence. Stage II occurs randomly during the cell cycle and provides daughter cells with an approximately equal copy number of the viral genome (Gilbert & Cohen, 1987
; Ravnan et al., 1992
). Stage III yields amplified copies of vDNA in differentiating cells (Bedell et al., 1991
; Ozbun & Meyers, 1998a
; Stoler et al., 1990
). Epithelial differentiation also results in the induction of late gene synthesis (Frattini et al., 1996
; Hummel et al., 1992
; Meyers et al., 1992
; Ozbun & Meyers, 1997
), leading to genome packaging and virion morphogenesis in the upper layers of the epithelium.
Major obstacles in the study of the initial stages of the HPV life-cycles, specifically those with high probability of inducing malignancies, have included the lack of high-titre infectious viral stocks and the lack of sensitive and quantitative in vitro assays for infection. Viral particles from most HPV types are produced in small amounts in vivo (Pfister, 1984 ). Although viral stocks of bovine PVs (BPVs) and cottontail rabbit PV (CRPV) can be readily purified from in vivo lesions (Crawford & Crawford, 1963
), the ability to obtain quantities of virions necessary for infectivity studies has been severely limited for HPVs. The number of virus particles in various human warts differs significantly, ranging from fewer than 103 particles/mg from laryngeal papillomas (probably HPV6 or 11) to 7x109 particles/mg from some plantar and common warts (probably HPV1 or 2) (Barrera-Oro et al., 1962
; Boyle et al., 1973
; Butel, 1972
). Purification of virus particles from the typically much smaller anogenital lesions has not been reported. In the last 10 years, the organotypic (raft) tissue culture system has greatly benefited HPV research by providing the level of epithelial differentiation required for a permissive HPV life-cycle and the biosynthesis of infectious virions (Meyers et al., 1992
, 1997
). We and others have demonstrated that HPV virions produced in the raft system have typical and well-formed morphology (Meyers et al., 1992
, 1997
, 2002
; Ozbun & Meyers, 1997
). Nevertheless, quantification of HPV infectivity remains elusive. Infectivity systems based on the final stages of the life-cycle (e.g. a plaque assay or an assay for viral particles) are not feasible because PVs are dependent on differentiation to complete their life-cycles, and the viruses are not lytic. Although BPV1 and BPV2 can be titrated in focus-forming assays, neither HPV from a plantar wart nor CRPV was able to induce cellular foci (Dvoretzky et al., 1980
). Data from the BPV1-induced focus assays suggest a particle-to-infectious-unit ratio of 104:1 (Roden et al., 1996
); however, no such information is available for HPVs. We and others have used RTPCR techniques for detection of viral RNAs following infection with HPV types 11, 16 and 18 (Meyers et al., 1997
; Smith et al., 1993
, 1995
; White et al., 1998
). Recently, Meyers et al. (2002)
demonstrated the production and infectivity of a chimeric HPV18/16 virus in the raft system.
HPV31b is the best-characterized high-risk HPV resulting from the study of the differentiation of latently infected CIN-612 9E cells in the methylcellulose suspension or raft tissue culture systems (reviewed in Ozbun & Meyers, 1999a ; Stubenrauch & Laimins, 1999
). These systems have permitted the analysis of various aspects of the latter parts of the differentiation-dependent HPV life-cycle, including the expression of viral proteins (Frattini et al., 1996
; Mayer & Meyers, 1998
; Meyers et al., 1992
; Pray & Laimins, 1995
), regulation and structural characterization of viral transcripts (Hummel et al., 1992
, 1995
; Klumpp & Laimins, 1999
; Ozbun & Meyers, 1997
, 1998a
, b
, 1999b
; Stubenrauch et al., 2000
; Terhune et al., 1999
, 2001
) and the replication of vDNA (Bedell et al., 1991
; Ozbun & Meyers, 1998a
). Yet, the early events in HPV infection of host cells remain to be characterized. In addition, a detailed analysis of infection of keratinocytes in vitro by virions synthesized in organotypic cultures has not been described thus far.
In the present study, we have purified high-risk HPV31b virions from raft tissue cultures grown from the latently infected CIN-612 9E cell line and used these virions to begin to characterize the process of HPV infection. Methods for the safe isolation of large quantities of HPV virus were identified as well as techniques to quantify virus stocks. Furthermore, we used infection of the HaCaT cell line to characterize the HPV31b transcripts expressed following infection.
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Methods |
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Virion purification and quantification.
CIN-612 9E raft tissues were extracted using a modified protocol of Favre et al. (Favre et al., 1975 ; Meyers et al., 1997
). Briefly, 5075 raft tissues were ground with sea sand (Fisher Scientific) in a mortar using 25 ml of buffer A (1 M NaCl, 0·05 M sodium phosphate buffer, pH 8·0). The debris was pelleted at 8000 g for 10 min at 4 °C. The supernatants were kept on ice while the pellet was re-extracted with 25 ml of buffer A and pelleted again under the same conditions. The efficacy of virus extraction was also tested by grinding the tissues in buffer A using a sealed BeadBeater (BioSpec Products) homogenizer and 1·0 mm glass beads. Both extractions proceeded as described below. Supernatants from the first set of extractions were pooled and the vDNA-containing particles were pelleted based on a sedimentation coefficient of 296S300S for full' vDNA-containing particles (Crawford & Crawford, 1963
) for 1 h at 4 °C in a swinging bucket rotor at 130000 g. The supernatants were discarded and the virus pellets were suspended in 2 ml of buffer B (0·05 M NaCl, 0·01 M EDTA, 0·05 M sodium phosphate buffer, pH 7·4) using a Dounce homogenizer. The debris was pelleted at 8000 g for 10 min at 4 °C. The supernatants were kept on ice while the pellet was re-extracted with 2 ml of buffer B and then pelleted again under the same conditions. Caesium chloride was added to the pooled supernatants and the refractive index was used to verify the density at 1·3 g/ml. A gradient was formed by centrifugation at 135000 g for 24 h at 4 °C. The tubes were punctured and 0·5 ml fractions were collected from the bottom of the gradient. Refractive indices of the fractions were measured. Each fraction was placed in SpectraPor tubing (1200014000 Mr cut-off) and the fractions were dialysed against four to five changes of PBS. The vDNA was extracted from 50 µl of each fraction by incubating for 2 h at 55 °C with 25 mM EDTA, 0·5% SDS, 100 µg/ml proteinase K and 50 µg/ml sheared salmon sperm DNA in a total volume of 400 µl. The solution was extracted twice with phenolchloroformisoamyl alcohol (25:24:1) and then ethanol-precipitated. The DNA preparations were restricted with EcoRI to linearize the vDNA and analysed alongside cloned vDNA copy number controls by 0·8% agarose gel electrophoresis and Southern blot hybridization using a 32P-labelled HPV31 probe, as previously described (Ozbun & Meyers, 1998a
). As an alternative to vDNA extraction, 10 µl of each fraction containing CsCl or purified cloned vDNA copy number controls in CsCl were incubated with 0·25 M NaOH, 0·5 M NaCl for 10 min to release and/or denature the vDNA. The DNA solutions were applied to a dot-blot apparatus containing a GeneScreen Plus membrane (NEN Life Science) and allowed to attach to the membrane for 30 min at room temperature. Following vacuum application, the membrane was hybridized using a 32P-labelled HPV31 probe, as described (Ozbun & Meyers, 1998a
). Blots were analysed by Phosphor Image analysis. A standard curve was plotted using copy number controls and samples were quantified accordingly. Fractions found to contain vDNA were subjected to dialysis in PBS as described above. Virion stocks were stored at -80 °C. Homogenizations and extractions of infectious HPV were performed in a tissue culture hood, under strict BSL2 conditions, and with the investigator wearing a lab. coat, gloves, eye protection and HEPA filter mask. Surfaces were decontaminated with 70% ethanol (Roden et al., 1997
).
HPV31b infections.
Cells were seeded at 3x105 cells per well in 4 cm2 wells or at 5x105 cells per well in 9 cm2 wells and allowed to attach overnight. The cells were 6080% confluent at the time of infection. Virion stocks were thawed from -80 °C at room temperature and were sonicated for 20 s at 0 °C. Virus dilutions were added to each well in 0·25 ml (for 4 cm2 wells) or 0·5 ml (for 9 cm2 wells) of normal HaCaT medium (see above), and the plates were rocked for 1 h at 4 °C. Additional medium was added and the cells were transferred to 37 °C. In most cases, the virus inoculum was removed and the cells were washed with an excess of normal medium. The cells were re-fed with normal medium and moved to 37 °C. The medium was changed every other day, and cells were expanded when they reached confluence.
Virus neutralization assays and DNase I treatment.
Virion stocks were incubated with dilutions of anti-PV monoclonal antibodies (a generous gift of Neil Christensen, Penn State College of Medicine) at 37 °C for 1 h. Virion stocks were treated with 0·4 units of RQ DNase I (Promega) in 10 mM MgCl2 and Tris buffer for 15 min at 37 °C. Normal medium was added and cells were exposed to the suspensions for 1 h at 4 °C with rocking. The inoculum was removed, and the cells were washed and re-fed as described above.
Nucleic acid extraction and RTPCR analysis.
Total RNAs were extracted from cells using TRIzol reagent (Invitrogen Life Technologies). RNA samples were treated with RNase-free DNase I (Promega) to remove co-purifying viral and cellular DNA (Ozbun & Meyers, 1997 , 1998a
). RNA concentrations were determined by optical density measurement; RNA concentrations and qualities were verified by electrophoresis through agarose gels containing ethidium bromide. RNAs were reverse-transcribed using random hexamer primers and PCR was performed using a GeneAmp RNA PCR kit and AmpliTaq Gold DNA polymerase as instructed by the manufacturer (Applied Biosystems). Oligonucleotide primers (Table 1
) were synthesized by Sigma Genosys. The PCR thermocycling profile was as follows: 10 min at 95 °C; 3050 cycles of 95 °C for 4560 s, 60 °C for 3060 s and 72 °C for 30120 s; 7 min extension at 72 °C. PCR products were analysed by electrophoresis through 2% agarose gels containing ethidium bromide. Primer pairs were optimized for annealing temperatures and MgCl2 concentration using serial tenfold dilutions of template cDNA copy number controls from 105 to 100 copies per PCR reaction. Serial tenfold dilutions of template cDNA copy number controls were also used to determine the relative sensitivities of primer pairs for a cDNA template. For the nomenclature of the amplicons, the open reading frames (ORFs) included are given with asterisks (*) or carets (
) signifying splicing; subscripts indicate the HPV31 nucleotides at the junctions. A Roman numeral denotes that more than one splice variant exists for a given larger ORF. For example, the structures of the major early spliced transcripts of HPV31b are described by the following: E6*I210
413 indicates a splice in the E6 ORF contains a junction from nt 210 to 413; in transcript E1*I,E2877
2646, E1*I begins in the E1 ORF and contains a splice joining nt 877 to 2646; E1
E4877
3295 indicates the E1
E4 fusion protein using the 877
3295 splice junction; and E8
E2C1296
3295 represents the fusion of the E8 ORF with the C terminus of the E2 ORF using the splice joined at nt 1296 and 3295.
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Results |
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To investigate the expression of the four major spliced HPV31b transcripts following infection, single RT reactions for each RNA sample were divided into PCR amplifications targeting E6*I210413 (Fig. 3d
), E1*I,E2877
2646 (Fig. 3e
), E1
E4877
3295 (Fig. 3f
) and E8
E2C1296
3295 (Fig. 3g
), with the spliced RNA of cellular
-actin as a control (Fig. 3c
). HPV31b spliced RNAs from both E6*I and E1
E4 were detected using a single round of RTPCR from HPV31b-infected cells inoculated with a dose as low as 1·0 vDNA-containing particle per cell (Fig. 3d
, f
, lanes 35, 810). Amplimers derived from spliced E1*I,E2 RNAs were detected at a viral dose as low as 10 vDNA-containing particles per cell (Fig. 3e
, lanes 45, 910), whereas new E8
E2C transcripts were not detected following HPV31b infection of HaCaT cells under the described conditions (Fig. 3g
). Targeting the E1
E4 RNAs by RTPCR was the most sensitive means of detecting HPV31b infection at 4 days p.i. The use of serial tenfold dilutions of cloned cDNA template copy number controls demonstrated that the primer pairs used for detecting E6*I and E1*I,E2 were each able to detect targets present at
102 copies per PCR reaction, whereas the primer pairs for E1
E4 could detect
101 copies per PCR reaction (Table 2
). Testing the E6*I,E7,E1*I,E2 and E6*I,E7,E1
E4 templates with primers E6A+742B yielded equal sensitivities of 102 copies indicating that the template controls were equivalently quantified and diluted. The primer pairs for E8
E2C were able to detect control templates at
104 copies per PCR reaction. Thus, it is possible that E8
E2C transcripts are present early in infection, but are at levels below the sensitivity of this assay. Infections and RTPCR experiments were repeated to verify the results.
Antibody-mediated neutralization assays and DNase I treatment of virions were performed to confirm the specificity of infection. Monoclonal antibodies raised to HPV31, HPV16 and CRPV VLPs (Christensen & Kreider, 1991 ; Christensen et al., 1996
) were tested for neutralization activity against HPV31b virion preparations as described in Methods. As expected, antibodies against CRPV and HPV16 had no effect on HVP31b infectivity (Fig. 4a
, lanes 16 and 1318, respectively). The monoclonal antibody H31.A6 raised to HPV31 VLPs completely neutralized HPV31b infection of HaCaT cells (Fig. 4a
, lanes 712). Additionally, pretreatment of virion stocks with DNase I did not affect their infectivity (Fig. 4a
, lanes 1921), verifying that unpackaged vDNA was not contributing to our observations. These data indicate that detection of spliced viral transcripts in infected cells is a result of bona fide HPV31b infection.
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Discussion |
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As there is currently no way to quantitatively titre HPVs based on infectivity, we have defined the dose of viral infection based on the number of vDNA-containing particles. Newly synthesized, spliced viral RNAs were targeted by RTPCR as a qualitative indication of infection. We have found the detection of HPV infection to be inconsistent among various isolates of low passage human foreskin keratinocytes (Meyers et al., 1997 ; Ozbun, 2002
). Comparing HPV31b infections among a number of human keratinocyte cell lines, we found infection of the HaCaT cell line to be the most efficient and reproducible (Ozbun, 2002
). Spliced viral RNAs were detected by RT and a single round of PCR in a population of HaCaT cells infected with a dose as low as 1·0 viral genome per cell. Furthermore, we showed that HPV31b-infected HaCaT cells synthesize late gene transcripts on epithelial raft tissue differentiation (Ozbun, 2002
). This suggests that early infection events in HaCaT cells reflect an accurate view of HPV31b infection biology.
On comparing the efficiency of infection between cells where the virus inoculum remained on the cells and cells in which the unbound virions were washed away after a 1 h binding at 4 °C, we found the minimal detectible doses were similar. This suggests that viral particles capable of binding to the cells were able to bind in 1 h at 4 °C, and is in agreement with the findings of Volpers et al. (1995) , showing that approximately 70% of HPV33 VLPs were bound to cells under these conditions. However, Christensen et al. (1995)
found that maximum foci production in C127 cells by BPV1 was not obtained unless the virus inoculum remained on the cells for 8 h, suggesting that BPV1 binding leading to infection was relatively slow. The focus assay is a quantitative assessment of infection, whereas our RTPCR detection is a qualitative assay. Therefore, we are probably unable to detect a quantitative change in infectivity by basic RTPCR. Nevertheless, the sensitivity of RTPCR permitted us to qualitatively assay viral transcripts resulting from infection following the binding of virions to the cells for 1 h at 4 °C.
Our RTPCR amplification from spliced viral RNAs is a sensitive assay for HPV31b infection. Using optimized PCR primer pairs targeting the known early spliced HPV31b RNAs, we found that the primers specific to spliced E1E4 transcripts were able to reproducibly detect targets with the greatest sensitivity at 4 days p.i. Targeting spliced E6*I and E1
E4 transcripts, we detected infection in HaCaT cells inoculated with a dose as low as 1·0 vDNA-containing particle per cell. We have used the primer pairs listed in Table 2
to characterize the temporal initiation of viral transcripts following HPV31b infection (Ozbun, 2002
). Others found that detection of newly spliced viral transcripts in HPV11- and HPV16-infected HaCaT cells required the use of RT and nested PCR (Smith et al., 1995
; White et al., 1998
, 1999
). Our ability to detect PV infection of cells at an apparently lower viral dose and using RT with a single round of PCR could be due to a number of experimental variables. RTPCR for PV transcripts is not a standardized technique. Differences in primer sensitivities, the amount of input RNA, the efficiency of reverse transcription, reaction conditions like magnesium concentration and primer annealing temperatures, type of polymerase and the number of PCR cycles are each expected to affect the sensitivity of the assay. Infectivity also could vary among HPV and animal PV types. We are in the process of devising standardized techniques to quantitatively compare the infectivities of various PV types. Although we were unable to assess the number of infectious units for HPVs, if we assume the particle-to-infectious-unit ratio is
104:1, as reported for BPV1 (Roden et al., 1996
), then we calculate that our RTPCR system is capable of detecting
50 infectious events in a background of 5x105 cells.
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Acknowledgments |
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References |
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Barrera-Oro, J. G., Smith, K. O. & Melnick, J. L. (1962). Quantitation of papova virus particles in human warts. Journal of the National Cancer Institute 29, 583-595.
Bedell, M. A., Hudson, J. B., Golub, T. R., Turyk, M. E., Hosken, M., Wilbanks, G. D. & Laimins, L. A. (1991). Amplification of human papillomavirus genomes in vitro is dependent on epithelial differentiation. Journal of Virology 65, 2254-2260.[Medline]
Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A. & Fusenig, N. E. (1988). Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. Journal of Cell Biology 106, 761-771.[Abstract]
Boyle, W. F., Riggs, J. L., Oshiro, J. S. & Lennette, E. H. (1973). Electron microscopic identification of papovavirus in laryngeal papilloma. Laryngoscope 83, 1102-1108.[Medline]
Butel, J. S. (1972). Studies with human papilloma virus modeled after known papovavirus systems. Journal of the National Cancer Institute 48, 285-299.[Medline]
Christensen, N. D. & Kreider, J. W. (1991). Neutralization of CRPV infectivity by monoclonal antibodies that identify conformational epitopes on intact virions. Virus Research 21, 169-179.[Medline]
Christensen, N. D., Cladel, N. M. & Reed, C. A. (1995). Postattachment neutralization of papillomaviruses by monoclonal and polyclonal antibodies. Virology 207, 136-142.[Medline]
Christensen, N. D., Dillner, J., Eklund, C., Carter, J. J., Wipf, G. C., Reed, C. A., Cladel, N. M. & Galloway, D. A. (1996). Surface conformational and linear epitopes on HPV-16 and HPV-18 L1 virus-like particles as defined by monoclonal antibodies. Virology 223, 174-184.[Medline]
Crawford, L. V. & Crawford, E. M. (1963). A comparative study of polyoma and papilloma viruses. Virology 21, 258-263.[Medline]
de Villiers, E.-M. (1989). Heterogeneity of the human papillomavirus group. Journal of Virology 63, 4898-4903.[Medline]
de Villiers, E.-M. (1999). Introduction. Seminars in Cancer Biology 9, 337.
Dickens, P., Srivastava, G., Loke, S. L. & Larkin, S. (1991). Human papillomavirus 6, 11, and 16 in laryngeal papillomas. Journal of Pathology 165, 243-246.[Medline]
Dvoretzky, I., Shober, R., Chattopadhyay, S. K. & Lowy, D. R. (1980). A quantitative in vitro focus assay for bovine papilloma virus. Virology 103, 369-375.[Medline]
Evander, M., Frazer, I. H., Payne, E., Qi, Y. M., Hengst, K. & McMillan, N. A. J. (1997). Identification of the 6 integrin as a candidate receptor for papillomaviruses. Journal of Virology 71, 2449-2456.[Abstract]
Favre, M., Breitburd, F., Croissant, O. & Orth, G. (1975). Structural polypeptides of rabbit, bovine, and human papillomaviruses. Journal of Virology 15, 1239-1247.[Medline]
Frattini, M. G., Lim, H. B. & Laimins, L. A. (1996). In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression. Proceedings of the National Academy of Sciences, USA 93, 3062-3067.
Gilbert, D. M. & Cohen, S. N. (1987). Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle. Cell 50, 59-68.[Medline]
Goldsborough, M. D., DiSilvestre, D., Temple, G. F. & Lorincz, A. T. (1989). Nucleotide sequence of human papillomavirus type 31: a cervical neoplasia-associated virus. Virology 171, 306-311.[Medline]
Hummel, M., Hudson, J. B. & Laimins, L. A. (1992). Differentiation-induced and constitutive transcription of human papillomavirus type 31b in cell lines containing viral episomes. Journal of Virology 66, 6070-6080.[Abstract]
Hummel, M., Lim, H. B. & Laimins, L. A. (1995). Human papillomavirus type 31b late gene expression is regulated through protein kinase C-mediated changes in RNA processing. Journal of Virology 69, 3381-3388.[Abstract]
Joyce, J. G., Tung, J.-S., Przysiecki, C. T., Cook, J. C., Lehman, E. D., Sands, J. A., Jansen, K. U. & Keller, P. M. (1999). The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes. Journal of Biological Chemistry 274, 5810-5822.
Klein, C. E., Steinmayer, T., Mattes, J. M., Kaufmann, R. & Weber, L. (1990). Integrins of normal human epidermis: differential expression, synthesis and molecular structure. British Journal of Dermatology 123, 171-178.[Medline]
Klumpp, D. J. & Laimins, L. A. (1999). Differentiation-induced changes in promoter usage for transcripts encoding the human papillomavirus type 31 replication protein E1. Virology 257, 239-246.[Medline]
Laimins, L. A. (1996). Human papillomaviruses target differentiating epithelia for virion production and malignant conversion. Seminars in Virology 7, 305-313.
Lancaster, W. D. & Olson, C. (1982). Animal papillomaviruses. Microbiological Reviews 46, 191-207.
Lowy, D. R. & Howley, P. M. (2001). Papillomaviruses. In Fields Virology , pp. 2231-2264. Edited by D. M. Knipe & P. M. Howley. Philadelphia:Lippincott Williams & Wilkins.
Lowy, D. R., Kirnbauer, R. & Schiller, J. T. (1994). Genital human papillomavirus infection. Proceedings of the National Academy of Sciences, USA 91, 2436-2440.[Abstract]
Mayer, T. J. & Meyers, C. (1998). Temporal and spatial expression of the E5a protein during the differentiation-dependent life cycle of human papillomavirus type 31b. Virology 248, 208-217.[Medline]
McCance, D. J., Kopan, R., Fuchs, E. & Laimins, L. A. (1988). Human papillomavirus type 16 alters human epithelial cell differentiation in vitro. Proceedings of the National Academy of Sciences, USA 85, 7169-7173.[Abstract]
Meyers, C. (1996). Organotypic (raft) epithelial tissues culture system for the differentiation-dependent replication of papillomavirus. Methods in Cell Science 18, 201-210.
Meyers, C., Frattini, M. G., Hudson, J. B. & Laimins, L. A. (1992). Biosynthesis of human papillomavirus from a continuous cell line on epithelial differentiation. Science 257, 971-973.[Medline]
Meyers, C., Mayer, T. J. & Ozbun, M. A. (1997). Synthesis of infectious human papillomavirus type 18 in differentiating epithelium transfected with viral DNA. Journal of Virology 71, 7381-7386.[Abstract]
Meyers, C., Bromberg-White, J. L., Zhang, J., Kaupas, M. E., Bryan, J. T., Lowe, R. S. & Jansen, K. U. (2002). Infectious virions produced from a human papillomavirus type 18/16 genomic DNA chimera. Journal of Virology 76, 4723-4733.
Moore, C. E., Wiatrak, B. J., McClatchey, K. D., Koopmann, C. F., Thomas, G. R., Bradford, C. R. & Carey, T. E. (1999). High-risk human papillomavirus types and squamous cell carcinoma in patients with respiratory papillomas. Otolaryngology-Head Neck Surgery 120, 698-705.
Ozbun, M. A. (2002). Human papillomavirus type 31b infection of human keratinocytes and the onset of early transcription. Journal of Virology (in press).
Ozbun, M. A. & Meyers, C. (1997). Characterization of late gene transcripts expressed during vegetative replication of human papillomavirus type 31b. Journal of Virology 71, 5161-5172.[Abstract]
Ozbun, M. A. & Meyers, C. (1998a). Human papillomavirus type 31b E1 and E2 transcript expression correlates with vegetative viral genome amplification. Virology 248, 218-230.[Medline]
Ozbun, M. A. & Meyers, C. (1998b). Temporal usage of multiple promoters during the life cycle of human papillomavirus type 31b. Journal of Virology 72, 2715-2722.
Ozbun, M. A. & Meyers, C. (1999a). Human papillomavirus type 31b transcription during the differentiation-dependent viral life cycle. Current Topics in Virology 1, 203-217.
Ozbun, M. A. & Meyers, C. (1999b). Two novel promoters in the upstream regulatory region of human papillomavirus type 31b are negatively regulated by epithelial differentiation. Journal of Virology 73, 3505-3510.
Pfister, H. (1984). Biology and biochemistry of papillomaviruses. Reviews in Physiology, Biochemistry and Pharmacology 99, 111-181.
Pray, T. R. & Laimins, L. A. (1995). Differentiation-dependent expression of E1E4 proteins in cell lines maintaining episomes of human papillomavirus type 31b. Virology 206, 679-685.[Medline]
Ravnan, J.-B., Gilbert, D. M., Ten Hagen, K. G. & Cohen, S. N. (1992). Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines. Journal of Virology 66, 6946-6952.[Abstract]
Roden, R. B. S., Greenstone, H. L., Kirnbauer, R., Booy, F. P., Jessie, J., Lowy, D. R. & Schiller, J. T. (1996). In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype. Journal of Virology 70, 5875-5883.[Abstract]
Roden, R. B. S., Lowy, D. R. & Schiller, J. T. (1997). Papillomavirus is resistant to desiccation. Journal of Infectious Diseases 176, 1076-1079.[Medline]
Rowson, K. E. K. & Mahy, B. W. J. (1967). Human papova (wart) virus. Bacteriological Reviews 31, 110-131.[Medline]
Sakakura, A., Yamamoto, Y., Takasaki, T., Makimoto, K., Nakamura, M. & Takahashi, H. (1996). Recurrent laryngeal papillomatosis developing into laryngeal carcinoma with human papilloma virus (HPV) type 18: a case report. Journal of Laryngology Otology 110, 75-77.
Schoop, V. M., Mirancea, N. & Fusenig, N. E. (1999). Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts. Journal of Investigative Dermatology 112, 343-353.
Smith, L. H., Foster, C., Hitchcock, M. E. & Isseroff, R. (1993). In vitro HPV-11 infection of human foreskin. Journal of Investigative Dermatology 101, 292-295.[Abstract]
Smith, L. H., Foster, C., Hitchcock, M. E., Leiserowitz, G. S., Hall, K., Isseroff, R., Christensen, N. D. & Kreider, J. W. (1995). Titration of HPV-11 infectivity and antibody neutralization can be measured in vitro. Journal of Investigative Dermatology 105, 438-444.[Abstract]
Stoler, M. H., Whitbeck, A., Wolinsky, S. M., Broker, T. R., Chow, L. T., Howett, M. K. & Kreider, J. W. (1990). Infectious cycle of human papillomavirus type 11 in human foreskin xenografts in nude mice. Journal of Virology 64, 3310-3318.[Medline]
Stubenrauch, F. & Laimins, L. A. (1999). Human papillomavirus life cycle: active and latent phases. Cancer Biology 9, 379-386.
Stubenrauch, F., Hummel, M., Iftner, T. & Laimins, L. A. (2000). The E8E2C protein, a negative regulator of viral transcription and replication, is required for extrachromosomal maintenance of human papillomavirus type 31 in keratinocytes. Journal of Virology 74, 1178-1186.
Terhune, S. S., Milcarek, C. & Laimins, L. A. (1999). Regulation of human papillomavirus type 31 polyadenylation during the differentiation-dependent life cycle. Journal of Virology 73, 7185-7192.
Terhune, S. S., Hubert, W. G., Thomas, J. T. & Laimins, L. A. (2001). Early polyadenylation signals of human papillomavirus type 31 negatively regulate capsid gene expression. Journal of Virology 75, 8147-8157.
Volpers, C., Unckell, F., Schirmacher, P., Streeck, R. E. & Sapp, M. (1995). Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells. Journal of Virology 69, 3258-3264.[Abstract]
Walboomers, J. M. M., Jacobs, M. V., Manos, M. M., Bosch, F. X., Kummer, J. A., Shah, K. V., Snijders, P. J. F., Peto, J., Meijer, C. J. L. M. & Muñoz, N. (1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. Journal of Pathology 189, 12-19.[Medline]
White, W. I., Wilson, S. D., Bonnez, W., Rose, R. C., Koenig, S. & Suzich, J. A. (1998). In vitro infection and type-restricted antibody-mediated neutralization of authentic human papillomavirus type 16. Journal of Virology 72, 959-964.
White, W. I., Wilson, S. D., Palmer-Hill, F. J., Woods, R. M., Ghim, S. J., Hewitt, L. A., Goldman, D. M., Burke, S. J., Jenson, A. B., Koenig, S. & Suzich, J. A. (1999). Characterization of a major neutralizing epitope on human papillomavirus type 16 L1. Journal of Virology 73, 4882-4889.
Yoon, C.-S., Kim, K.-D., Park, S.-N. & Cheong, S.-W. (2001). 6 integrin is the main receptor of human papillomavirus type 16 VLP. Biochemical and Biophysical Research Communications 283, 668-673.[Medline]
Zhou, J., Gissmann, L., Zentgraf, H., Müller, H., Picken, M. & Müller, M. (1995). Early phase in the infection of cultured cells with papillomavirus virions. Virology 214, 167-176.[Medline]
zur Hausen, H. (1996). Papillomavirus infectionsa major cause of human cancers. Biochimica et Biophysica Acta 1288, F55-F78.[Medline]
Received 1 April 2002;
accepted 10 July 2002.