Variant Upstream Regulatory Region Sequences Diffe
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病菌学杂志 2005年第10期
Department of Dermatology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, Arkansas 72205
ABSTRACT
While the central role of the viral upstream regulatory region (URR) in the human papillomavirus (HPV) life cycle has been well established, its effects on viral replication factor expression and plasmid replication of HPV type 16 (HPV16) remain unclear. Some nonprototypic variants of HPV16 contain altered URR sequences and are considered to increase the oncogenic risk of infections. To determine the relationship between viral replication and variant URRs, hybrid viral genomes were constructed with the replication-competent HPV16 prototype W12 and analyzed in assays which recapitulate the different phases of normal viral replication. The establishment efficiencies of hybrid HPV16 genomes differed about 20-fold among European prototypes and variants from Africa and America. Generally, European and African genomes exhibited the lowest replication efficiencies. The high replication levels observed with American variants were primarily attributable to their efficient expression of the replication factors E1 and E2. The maintenance levels of these viral genomes varied about fivefold, which correlated with their respective establishment phenotypes and published P97 activities. Vegetative DNA amplification could also be observed with replicating HPV16 genomes. These results indicate that efficient E1/E2 expression and elevated plasmid replication levels during the persistent stage of infection may comprise a risk factor in HPV16-mediated oncogenesis.
Epidemiology.
Infection with human papillomaviruses (HPVs) leads to hyperproliferative lesions in humans at specific anatomical sites which are determined by the tropism of individual viral genotypes. Among the more than 100 types of HPVs identified to date, a subset of mucosotropic viruses, which infect the epithelial lining of the anogenital tract and oral cavity, are causally associated with the great majority of cervical cancers worldwide. This group of high-risk HPVs includes HPV type 16 (HPV16), HPV18, HPV31, HPV33, and HPV35, with HPV16 being the most prevalent. In malignant anogenital lesions, high-risk HPV DNA is present in infected cells and the viral oncogenes are expressed detectably. While type 16 is also found in over 80% of HPV-positive head and neck cancers (39, 40, 54), and less frequently in HPV-positive cutaneous squamous cell carcinomas (64), its etiology in these diseases is still unclear. Over the past decade, numerous HPV16 variants have been identified which exhibit one or more single nucleotide changes compared to the European prototype E (19, 48, 60). Typically, L1, E6, and also upstream regulatory region (URR) sequences of HPV16 DNA from cervical cancers were analyzed (69, 70, 73) and variant classes established for different continents (73). Recent epidemiologic studies indicate that non-European variants of HPV16 exhibit increased oncogenicity (71, 72), specifically members of the Asian-American class AA (6). The underlying causes for the pathogenic differences of HPV16 variants, particularly those with altered URR sequences, are not well understood.
Viral life cycle, DNA replication, and oncogenesis.
The life cycle of papillomaviruses is closely linked to the differentiation program of their host cells. HPVs infect dividing basal keratinocytes in the stratified epithelium and replicate as nuclear plasmids at low copy number. During this persistent stage, only a subset of viral genes is expressed to ensure stable viral plasmid replication in dividing cells without generating progeny virions (43). The productive stage of infection ensues as HPV-positive cells differentiate and migrate to the upper layers of the epithelium. At this time, viral genomes are amplified to a high copy number per cell, capsid genes are expressed, and progeny virions are assembled (30). Persistent infections with high-risk HPVs, such as types 16, 18, 31, and 33, may last for years, during which the continued expression of the viral oncogenes induces genetic instability in host cells and initiates malignant conversion (18, 75). High-risk HPV-mediated oncogenesis is commonly associated with integration of the viral DNA into the host genome (20). In tumor cells with integrated HPV DNA, high levels of the viral oncoproteins have been observed. As a consequence, the proliferative capacity of such cells is increased, which leads to a preferential expansion during cell culture (1, 21, 34, 35, 57). Viral DNA integration, however, is not required for HPV16-induced oncogenesis, as cervical cancers which harbor extrachromosomal HPV16 genomes have been identified. In such biopsies, analysis of the URR showed that YY1 factor binding sites were altered or deleted, which increased the transcriptional activity of the major viral promoter P97 (17). At present, the molecular parameters which abolish normal viral plasmid replication and lead to integration of HPV16 DNA into the host chromosome are not known. However, the behavior of YY1 mutants of HPV16 indicates that long-term viral plasmid stability may depend on higher transcriptional activities than those provided by the HPV16 prototype URR. More recently, elevated P97 activities have also been observed with biopsy-derived variant URRs which contain multiple single-nucleotide changes (36, 68).
URR-dependent replication of HPV16.
Earlier efforts to genetically analyze the regulation of HPV16 DNA replication were hampered by the lack of a cell culture system which could support normal viral plasmid replication. Following the recent isolation of the HPV16 prototype W12 (22), however, stable replication of these viral genomes was demonstrated by different laboratories (22, 62). Using this replication-competent HPV16 prototype, the hypothesis was tested in this study that variant URR sequences, which alter P97 activity, also modulate viral DNA replication. To determine the viral establishment, maintenance, and vegetative replication phenotypes of HPV16, URR sequences from the European prototype (E) and five African (Af1aB2, Af1aD1, Af2aE1, Af2aF1, and Af2aF3) and two American (AAc and NA1) variants (36) were analyzed in the background of an HPV16W12 genome, adapting methodologies initially developed for the related HPV types 18 and 31 (23-25, 47). Comparison of the replication behavior of the HPV16 genomes with their published transcriptional phenotypes (36) indicates that the low replication levels observed with European and African hybrids correlate with inefficient expression of the viral replication factors E1 and E2. In contrast, American URR variants were found to replicate at much higher levels, which may be related to their elevated oncogenicity (8).
MATERIALS AND METHODS
Plasmids. The parental HPV16 genome pBRmin3-HPV16W12 (542.203) was derived from pEFHPV16W12E (provided by P. Lambert, Madison, WI) (22), which had been isolated from a clonal population (34, 35) of the W12 biopsy cell line (63). The viral genome was excised from pEFHPV16W12E by BamHI digestion and cloned into pBRmin3, a minimal vector with bacterial origin, ?-lactamase, and rop genes, which was obtained from pBRmin-HPV31 (31) and adapted by inserting a BamHI linker at its unique EcoRI site. Nucleotide numbering for the W12 genome is based on its published sequence (22). Numbering for the non-W12 URRs is based on the revised HPV16R sequence (48) of the European prototype E (19, 60). Hybrid HPV16W12 genomes were prepared by transferring URR fragments (PmlI at nucleotide [nt] 7268 to PpuMI at nt 111) from the European prototype E (provided by L. Turek, Iowa City, IA) and variants Af1aB2, Af1aD1, Af2aE1, Af2aF1, Af2aF3, AAc, and NA1 (36) (pALuc reporter constructs provided by C. Wheeler, Albuquerque, NM) into pBRmin3-HPV16W12, which was opened at PmlI (nt 7266) and PpuMI (nt 111). Two control plasmids were also created in pBRmin3-HPV16W12. (i) E1TTL (543.301) contains a six-frame translational termination linker (52), 5'-d(gcgcCTAACCTAGGTTAG), with KasI-compatible ends at KasI (nt 1310), which terminates translation of E1 after codon 150. (ii) E2FS (808.103) was prepared by partial digestion of pBRmin3-HPV16W12 with BstXI, blunt ending with T4 DNA polymerase, and religation. The deletion of nt 2896 to 2899 results in a frameshift mutation in E2 which terminates translation after codon 75. pSK-E116 and pSK-E216 (58), provided by P. Howley, Boston, MA, are expression vectors which contain a human cytomegalovirus promoter and intact E1 or E2 coding sequences, respectively, derived from the European HPV16 prototype plasmid p1203 (55). Both vectors have been shown to support replication of HPV16 origin plasmids (58).
Short-term replication. Viral establishment efficiency was analyzed in transient replication assays essentially as previously described for HPV31 (31). Briefly, viral genomes were released from bacterial vector sequences and unimolecularly religated at a low DNA concentration. HPV-negative human squamous cell carcinoma 13 (SCC13) (53) cells (5 x 106) were transfected by electroporation either with ligated HPV genomes (5 μg of viral DNA) by themselves or together with equimolar amounts of pSK-E116 and pSK-E216 each. After expanding the transfected SCC13 cells on J2-3T3 fibroblast feeder layers for 5 days, low-molecular-weight DNA was extracted by a modified Hirt DNA procedure. Prior to gel electrophoresis, sample DNAs were digested to completion with NcoI to linearize HPV16 genomes and DpnI to remove bacterially methylated input DNA.
Long-term replication. Viral maintenance efficiency was analyzed in stable replication assays as published for HPV31 (31). Briefly, ligated viral genomes were prepared as described for the transient assay. Subconfluent, low-passage, primary human foreskin keratinocytes (HFKs; isolated from anonymous neonatal donors) were transfected with HPV16 genomes (5 μg of viral DNA) and a 0.5 molar equivalent of the marker plasmid pSV2neo using FuGENE 6 (Roche) or Lipofectamine (Invitrogen). Transfected HFKs were transferred to J2-3T3 fibroblast feeder layers and selected briefly with G418. Drug-resistant colonies from each transfection were pooled within 3 weeks and expanded as mass cultures. After removal of fibroblast feeders, total cellular DNA was isolated from HFKs 4 to 6 weeks posttransfection using proteinase K digestion and differential salt precipitation to minimize shearing (31). Gel electrophoresis was performed with DpnI-digested total cellular DNA samples (5 μg) which were either sheared or digested with the single-cut enzyme NcoI.
Vegetative replication. Viral DNA amplification efficiency was analyzed by in vitro differentiation of stably transfected cells as previously described for HPV31 (31, 56). Briefly, fibroblast feeders were removed from the expanded, HPV16-positive mass cultures (see above), and HFKs trypsinized. After collection, cell pellets were divided equally into two samples and suspended separately in methylcellulose (1.6%, wt/vol)-containing E medium. Cells were rinsed out from methylcellulose cultures immediately or after 24 h of incubation at 37°C. Total cellular DNA was isolated for the two time points, and sheared samples analyzed as in the stable replication assay (see above).
Southern hybridization and analysis. DNA samples from all replication assays were resolved on agarose gels (0.8%, wt/vol) and transferred to positively charged nylon membranes (Magna Probe; Osmonics) under alkali conditions as previously described for HPV31 (31). HPV16W12-derived DNA standards of 500, 25, 2.5, and 0.5 pg of genome size viral DNA were included on each blot. Prehybridization and hybridization were performed with dextran sulfate- and formamide-containing solutions at 42°C (31). HPV16-specific probe was prepared by labeling genome length viral DNA (W12 prototype) with [-32P]deoxycytosine triphosphate by random priming (Prime-It-RmT; Stratagene). After stringent washing, viral DNA was visualized by storage phosphor-mediated autoradiography (Molecular Dynamics). Autoradiograms were quantified with ImageQuant software (Molecular Dynamics) or IPLab gel (Signal Analytics), and figures assembled with Photoshop (Adobe) and Freehand (Macromedia), performing only linear adjustments.
Sequence analysis. The published HPV16R (48) sequence was used as a base for variant URRs Af1aB2, Af1aD1, Af2aE1, Af2aF1, Af2aF3, AAc, and NA1 (36). The approximate positions of variant nucleotides within the URR are shown in Fig. 1B. The search for potential factor binding sites in the URR was performed with DNA Strider (Christian Marck, CEA, France) as described in the legend to Fig. 1.
Research compliance. All experiments with human cells and human papillomavirus DNAs were in compliance with federal, state, and institutional regulations and were approved by the chairman of the Institutional Review Board at the University of Arkansas for Medical Sciences.
RESULTS
Establishment efficiencies of URR variants are strongly modulated by E1 and E2 expression. The viral establishment phase is the first phase of the HPV life cycle during which a stable low plasmid copy number is attained within an infected cell population. In this phase, viral plasmids replicate more frequently than the host genome for multiple cell division cycles. For a transient replication assay which mimics the viral establishment phase, permissive human epithelial cells are transfected with HPV genomes and their replication levels are quantified after 5 days. In this assay, the expression efficiency and functionality of the viral replication factors E1 and E2 can be analyzed, as well as the function of the replication origin on the viral genome. Under physiological conditions, papillomavirus replication requires sufficient levels of both E1 and E2 in trans and an intact origin in cis (67). Since both the transcriptional control elements for HPV gene expression and the replication origin are located within the URR, this domain plays a central role in viral regulation. Depending on transfection efficiency and other assay variations, the replication levels of HPV16 prototype genomes (E and W12) are 5- to 50-fold lower than that of HPV31 (W. G. Hubert, unpublished data). This low replication phenotype may be responsible for the earlier assessment that HPV16 genomes did not replicate when permissive cells were transfected transiently (14). Sufficient signal can now be obtained, however, and replication of HPV16 determined reliably with assay protocols adapted from studies with HPV31 (31).
To test the hypothesis that the transcriptional phenotypes exhibited by a panel of variant HPV16 URRs (36) could differentially modulate transient viral replication, hybrid HPV16 DNAs were constructed which contain variant URR sequences in a common background of the replication-competent HPV16 W12 genome. This strategy focuses the experimental analysis only on URR-mediated effects, excluding the potential contributions of sequence variations in other parts of the viral genome. The URR fragments of HPV16 variants Af1aB2, Af1aD1, Af2aE1, Af2aF1, Af2aF3, AAc, and NA1 (36), which were originally collected for an epidemiologic study (73), and the URR of the European (E) prototype were transferred to the HPV16W12 genome as described in Materials and Methods. Transient replication assays were then performed by transfecting HPV-negative SCC13 cells (53) with recircularized viral genomes and analyzing de novo replication. As shown in Fig. 2 A, the replication levels of the European prototypes (hybrid W12-URR-E and parental W12, lanes a and i) and most African URR variants (W12 hybrids b through e) were relatively low. In contrast, both American variants exhibited high replication levels (Fig. 2 A, lanes g and h) and were about 20-fold more efficient than prototype E (Table 1, mean relative replication). HPV16 DNAs defective in either E1 or E2 protein translation (E1TTL, E2FS; Materials and Methods) were replication defective in control transfections (Table 1), which is consistent with the physiological requirement for both replication factors (67).
HPV DNA replication is regulated in a dose-dependent manner by the abundance of the viral replication factors (10, 31, 41, 44, 45, 51, 74). Since the short-term replication assay with entire HPV genomes analyzes the efficiency of replication factor expression, as well as origin function, the differences in autonomous transient replication of the HPV16 DNAs could have been the result of both cis- and trans-acting changes. To more specifically test how E1 and E2 expression modulates replication, viral genomes were cotransfected with expression vectors for both viral replication factors (Materials and Methods), which had previously been shown to support replication of HPV16 origin plasmids (58). In the presence of heterologous E1 and E2, prototype (hybrid W12-URR-E and parental W12, a and i) and most African variant (W12 hybrids b through e) genomes exhibited similar replication levels (up to 1.53-fold, Fig. 2 B and Table 2, mean relative replication). Replication of variants Af2aF3, AAc, and NA1 exceeded that of prototype E by as much as 3.9-fold, possibly due to a combination of high levels of endogenous and exogenous E1 and E2. These data indicate (i) that HPV16 origin efficiency is not strongly affected by variant URR sequences and (ii) that, consistent with the behavior of HPV31 mutants with defects in replication factor expression (31), the low-establishment phenotypes of European prototypes and African URR variants of HPV16 are primarily the consequence of inefficient E1 and E2 expression.
Maintenance levels of URR variants are similar to their establishment efficiencies. Maintenance phase is the second phase of the viral life cycle, during which HPV plasmids replicate as frequently as the cellular genome on average (26), resulting in a stable copy number in an HPV-positive cell population. In the stable replication assay, which mimics the viral maintenance phase, primary HFKs are transfected with HPV genomes. It is performed over a 4- to 6-week time course, and the major viral parameters which determine replication behavior are (i) expression of the viral replication factors, (ii) viral origin function, and (iii) expression of the viral oncogenes (32, 66).
To test the hypothesis that the transcriptional phenotypes exhibited by variant HPV16 URRs (36) could differentially modulate viral maintenance levels, the panel of hybrid HPV16 genomes was analyzed in stable replication assays (Materials and Methods). HFKs were transfected with recircularized viral genomes and a selectable marker. After selection and expansion of drug-resistant colonies as mass culture cell lines, total cellular DNA was isolated and analyzed by Southern hybridization. Normal viral plasmid replication yields characteristic autoradiographic bands which indicate the presence of closed circular (cc) and relaxed circular (rc) monomers. As shown in Fig. 3 A, the replication levels of the European prototypes (hybrid W12-URR-E and parental W12, lanes a and i) and the African URR variant (W12 hybrid, f) were relatively low. In contrast, both American variants consistently replicated at high levels (Fig. 3A, lanes g and h, and Table 3, mean absolute replication). Interestingly, strong variations in the relative levels of the inefficient replicators were observed in multiple experiments which were performed with different primary cell isolates and transfection agents (Table 3, rows i and a through e, mean relative replication). These findings suggest that inefficient viral DNA replication is also modulated by other factors (see Discussion). Consistent with their behavior in transient assays (Fig. 2A. and Table 1), the E1 and E2 mutants of HPV16W12 were also found to be defective for stable replication (Table 3).
The additional HPV topologies present in total DNA samples are referred to as high molecular weight (hmw, Fig. 3A). hmw signals generally indicate (i) integrated viral DNA, which migrates as part of the randomly sheared cellular DNA, (ii) viral plasmid concatemers, or (iii) trapped monomeric or multimeric HPV plasmids. To resolve this potential mixture, unsheared total cell DNA was digested with a restriction enzyme which linearizes HPV plasmid monomers (lin). As shown in Fig. 3B, all analyzed cell populations harbor detectable amounts of unit length viral DNA. Additional bands of larger or smaller size than linear HPV16 DNA (non-unit length, nul) are also detected. nul bands indicate the presence of junction fragments containing both cellular and viral DNA but could also result from replicating or integrated HPV genomes which are rearranged. However, in light of the fact that no replication-competent multimeric plasmid species, including rearranged partial multimers, were detected in earlier experiments with HPV31 (31) or HPV16 (Fig. 3A), nul hybridization signals likely indicate only rearranged, integrated viral DNA. Such HPV16 DNA species have also been observed in cloned cell populations from the W12 biopsy cell line (34). As hmw hybridization signals (Fig. 3A) and nul-specific bands (Fig. 3B) are present in most sample lanes, integration of viral DNA appears to occur with both prototypes and URR variants of HPV16.
URR variant and prototype genomes replicate vegetatively. Vegetative HPV replication is the final phase of the viral life cycle. Typically, HPV plasmid DNA is amplified from a low, stable copy number to several thousand in competent, suprabasal keratinocytes. Viral DNA amplification is required for HPV capsid gene expression (25), which precedes virion assembly and egress. Vegetative replication only occurs in differentiated keratinocytes, a process which can be induced by suspending epithelial cells in methylcellulose-containing growth medium to prevent their adherence to the substrate (29). During in vitro differentiation, a subset of HPV-positive keratinocytes are competent to amplify viral plasmid DNA in 24 to 48 h, which, in the case of HPV31, increases the plasmid copy number two- to fivefold in a treated cell population (31, 56). To test the capacity of HPV16 URR variants to amplify viral DNA, cell lines with detectable HPV16 replication were analyzed (Materials and Methods). The amounts of replicated DNA were obtained by combining the respective rc and cc signals at each time point, which were then normalized for DNA loading within each sample pair. Comparing viral replication in differentiated (24 h) cells to that in undifferentiated (0 h) cells, moderate DNA amplification ratios could be observed with the HPV16 prototype (parental W12, i) and URR variants Af2aF3, AAc, and NA1 (W12 hybrids f, g, and h, Fig. 4 and Table 4). While a relationship between URR variation and DNA amplification efficiency was not apparent in the two independent experiments, these data indicate that HPV16 DNA amplification can be analyzed with the in vitro differentiation assay. Since the 24-h amplification ratios of HPV16 appear disproportionally smaller than those observed with HPV31 in 48 h (31, 56), these data may also indicate that vegetative replication of HPV16 is less efficient overall.
DISCUSSION
Viral transcription and replication phenotypes are related. When the detected transient and stable replication levels of the HPV16 URR variants were normalized to the European prototype (hybrid W12-URR-E) and compared to the published P97 activities (36), the viral transcription and persistent replication phenotypes were found to correlate for most of the tested HPV16 DNAs (Table 5). The American variants (Table 5, rows g and h) exhibited the highest levels of replication in both establishment and maintenance phases, as well as the strongest P97 activity. In contrast, both phenotypes were more moderate with most African and European strains. The replication levels of prototype E (row a) and variants Af1aB2 and Af2aF3 (b and f) also correlated with their P97 activity. And the low P97 activities of variants Af2aE1 and Af2aF1 (d and e) are in line with their moderate transient replication and lack of stable replication. These data indicate that altered P97 activities are primarily responsible for the strong differences in replication levels observed with these HPV16 URR variants.
Host-specific factors and replication of HPV16. Stable replication levels of high-risk HPV genomes vary significantly among different isolates of primary cells. Such variability in supporting long-term HPV plasmid replication has been observed in other laboratories and are thought to be donor specific (W. G. Hubert, unpublished data; L. A. Laimins, personal communication; P. F. Lambert, personal communication; L. Turek, personal communication). Evidence for this variable property of HFKs is presented in Table 3. A threshold of a minimal replication efficiency appears to be required for attaining a stable copy number, as the levels of inefficiently replicating genomes (Table 3, rows a through c, stable experiment 3; rows d and e, stable experiment 1) are close to the detection limit. The URR variants AAc and NA1 consistently replicated at a high level in three experiments (Table 3, rows g and h, mean absolute replication). While such a threshold may be overcome by increased transfection efficiency in some cases, depending on the agent used (footnotes to Table 3), it may primarily be an intrinsic characteristic of an HFK isolate. Since the outcome of HPV-induced disease is likely affected by such donor-specific factors, future studies are necessary in this area.
Role of the major viral promoter. P97 activity, as well as transient- and stable-replication efficiencies, correlate for most of the tested URR variants (Table 5). These findings indicate that the strong activity of P97, which is known to be involved in the expression of E1 and E2 (32, 39), as well as E6 and E7 (13), during the persistent stage of infection, is also associated with high-establishment and -maintenance phenotypes. Restoring the replication signals of intrinsically low prototypes (hybrid W12-URR-E and parental W12, a and i) and African URR variants (c through e) to similar levels (Table 2) by overexpressing E1 and E2 from heterologous vectors confirms previous observations about the central role of P97 activity in the pathogenesis of HPV16. Similar to the intrinsic efficiencies of URR variants for expressing the viral replication factors, their capacity for viral oncogene expression is also expected to correlate with P97 activity. Since significant levels of E7 protein have already been observed with replicating HPV16 at high copy number in clonal W12 cell populations (34, 35), the severely oncogenic potential of the HPV16 variant AAc (6, 8) may be related to its strong transcription and replication phenotypes.
Transcription factor-specific regulation by the URR. The importance of the URR in the HPV life cycle was recognized quite early. Numerous studies have characterized the function of common factor binding sites in P97-mediated transcription of HPV16, such as AP1 (9), AP2/TEF2 (11), NF1 (2, 28), Oct1 (49), Sp1 (3, 65), TEF1 (33), and YY1 (16, 17, 46, 50). Our understanding of viral regulation can now be expanded by testing the importance of these sites for viral replication throughout the life cycle. Preliminary analysis of the variant changes in the URR has already shown that the low replication phenotypes of African variants may correlate with alterations of the P97– proximal Sp1 binding site. And the high-replication phenotype of the American variants may be aided by the acquisition of an additional AP1 site. Both Sp1 (3, 7, 15, 27, 59, 61) and AP1 (11, 12, 38, 42) are known to play complex activating roles in viral transcription and DNA replication. In addition, several URR variants also have a reduced number of YY1 binding sites which, depending on their sequence context, may increase transcriptional activation (5, 37) or repression (4, 46, 50). The effects of selected factor binding sites on viral replication will be analyzed in future studies.
ACKNOWLEDGMENTS
Thanks to Peter Howley, Lou Laimins, Paul Lambert, Lubos Turek, and Cosette Wheeler for providing essential plasmids for this study and to Paul Lambert and Lou Laimins for critically reading the manuscript.
This research was funded by grants from the American Cancer Society, Murphy Oil Corporation, and the University of Arkansas for Medical Sciences. W.G.H. is supported by a career development award from the Dermatology Foundation.
Mailing address: Department of Dermatology, MS576, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205. Phone: (501) 686-5110. Fax: (501) 686-7264. E-mail: wghubert@uams.edu.
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ABSTRACT
While the central role of the viral upstream regulatory region (URR) in the human papillomavirus (HPV) life cycle has been well established, its effects on viral replication factor expression and plasmid replication of HPV type 16 (HPV16) remain unclear. Some nonprototypic variants of HPV16 contain altered URR sequences and are considered to increase the oncogenic risk of infections. To determine the relationship between viral replication and variant URRs, hybrid viral genomes were constructed with the replication-competent HPV16 prototype W12 and analyzed in assays which recapitulate the different phases of normal viral replication. The establishment efficiencies of hybrid HPV16 genomes differed about 20-fold among European prototypes and variants from Africa and America. Generally, European and African genomes exhibited the lowest replication efficiencies. The high replication levels observed with American variants were primarily attributable to their efficient expression of the replication factors E1 and E2. The maintenance levels of these viral genomes varied about fivefold, which correlated with their respective establishment phenotypes and published P97 activities. Vegetative DNA amplification could also be observed with replicating HPV16 genomes. These results indicate that efficient E1/E2 expression and elevated plasmid replication levels during the persistent stage of infection may comprise a risk factor in HPV16-mediated oncogenesis.
Epidemiology.
Infection with human papillomaviruses (HPVs) leads to hyperproliferative lesions in humans at specific anatomical sites which are determined by the tropism of individual viral genotypes. Among the more than 100 types of HPVs identified to date, a subset of mucosotropic viruses, which infect the epithelial lining of the anogenital tract and oral cavity, are causally associated with the great majority of cervical cancers worldwide. This group of high-risk HPVs includes HPV type 16 (HPV16), HPV18, HPV31, HPV33, and HPV35, with HPV16 being the most prevalent. In malignant anogenital lesions, high-risk HPV DNA is present in infected cells and the viral oncogenes are expressed detectably. While type 16 is also found in over 80% of HPV-positive head and neck cancers (39, 40, 54), and less frequently in HPV-positive cutaneous squamous cell carcinomas (64), its etiology in these diseases is still unclear. Over the past decade, numerous HPV16 variants have been identified which exhibit one or more single nucleotide changes compared to the European prototype E (19, 48, 60). Typically, L1, E6, and also upstream regulatory region (URR) sequences of HPV16 DNA from cervical cancers were analyzed (69, 70, 73) and variant classes established for different continents (73). Recent epidemiologic studies indicate that non-European variants of HPV16 exhibit increased oncogenicity (71, 72), specifically members of the Asian-American class AA (6). The underlying causes for the pathogenic differences of HPV16 variants, particularly those with altered URR sequences, are not well understood.
Viral life cycle, DNA replication, and oncogenesis.
The life cycle of papillomaviruses is closely linked to the differentiation program of their host cells. HPVs infect dividing basal keratinocytes in the stratified epithelium and replicate as nuclear plasmids at low copy number. During this persistent stage, only a subset of viral genes is expressed to ensure stable viral plasmid replication in dividing cells without generating progeny virions (43). The productive stage of infection ensues as HPV-positive cells differentiate and migrate to the upper layers of the epithelium. At this time, viral genomes are amplified to a high copy number per cell, capsid genes are expressed, and progeny virions are assembled (30). Persistent infections with high-risk HPVs, such as types 16, 18, 31, and 33, may last for years, during which the continued expression of the viral oncogenes induces genetic instability in host cells and initiates malignant conversion (18, 75). High-risk HPV-mediated oncogenesis is commonly associated with integration of the viral DNA into the host genome (20). In tumor cells with integrated HPV DNA, high levels of the viral oncoproteins have been observed. As a consequence, the proliferative capacity of such cells is increased, which leads to a preferential expansion during cell culture (1, 21, 34, 35, 57). Viral DNA integration, however, is not required for HPV16-induced oncogenesis, as cervical cancers which harbor extrachromosomal HPV16 genomes have been identified. In such biopsies, analysis of the URR showed that YY1 factor binding sites were altered or deleted, which increased the transcriptional activity of the major viral promoter P97 (17). At present, the molecular parameters which abolish normal viral plasmid replication and lead to integration of HPV16 DNA into the host chromosome are not known. However, the behavior of YY1 mutants of HPV16 indicates that long-term viral plasmid stability may depend on higher transcriptional activities than those provided by the HPV16 prototype URR. More recently, elevated P97 activities have also been observed with biopsy-derived variant URRs which contain multiple single-nucleotide changes (36, 68).
URR-dependent replication of HPV16.
Earlier efforts to genetically analyze the regulation of HPV16 DNA replication were hampered by the lack of a cell culture system which could support normal viral plasmid replication. Following the recent isolation of the HPV16 prototype W12 (22), however, stable replication of these viral genomes was demonstrated by different laboratories (22, 62). Using this replication-competent HPV16 prototype, the hypothesis was tested in this study that variant URR sequences, which alter P97 activity, also modulate viral DNA replication. To determine the viral establishment, maintenance, and vegetative replication phenotypes of HPV16, URR sequences from the European prototype (E) and five African (Af1aB2, Af1aD1, Af2aE1, Af2aF1, and Af2aF3) and two American (AAc and NA1) variants (36) were analyzed in the background of an HPV16W12 genome, adapting methodologies initially developed for the related HPV types 18 and 31 (23-25, 47). Comparison of the replication behavior of the HPV16 genomes with their published transcriptional phenotypes (36) indicates that the low replication levels observed with European and African hybrids correlate with inefficient expression of the viral replication factors E1 and E2. In contrast, American URR variants were found to replicate at much higher levels, which may be related to their elevated oncogenicity (8).
MATERIALS AND METHODS
Plasmids. The parental HPV16 genome pBRmin3-HPV16W12 (542.203) was derived from pEFHPV16W12E (provided by P. Lambert, Madison, WI) (22), which had been isolated from a clonal population (34, 35) of the W12 biopsy cell line (63). The viral genome was excised from pEFHPV16W12E by BamHI digestion and cloned into pBRmin3, a minimal vector with bacterial origin, ?-lactamase, and rop genes, which was obtained from pBRmin-HPV31 (31) and adapted by inserting a BamHI linker at its unique EcoRI site. Nucleotide numbering for the W12 genome is based on its published sequence (22). Numbering for the non-W12 URRs is based on the revised HPV16R sequence (48) of the European prototype E (19, 60). Hybrid HPV16W12 genomes were prepared by transferring URR fragments (PmlI at nucleotide [nt] 7268 to PpuMI at nt 111) from the European prototype E (provided by L. Turek, Iowa City, IA) and variants Af1aB2, Af1aD1, Af2aE1, Af2aF1, Af2aF3, AAc, and NA1 (36) (pALuc reporter constructs provided by C. Wheeler, Albuquerque, NM) into pBRmin3-HPV16W12, which was opened at PmlI (nt 7266) and PpuMI (nt 111). Two control plasmids were also created in pBRmin3-HPV16W12. (i) E1TTL (543.301) contains a six-frame translational termination linker (52), 5'-d(gcgcCTAACCTAGGTTAG), with KasI-compatible ends at KasI (nt 1310), which terminates translation of E1 after codon 150. (ii) E2FS (808.103) was prepared by partial digestion of pBRmin3-HPV16W12 with BstXI, blunt ending with T4 DNA polymerase, and religation. The deletion of nt 2896 to 2899 results in a frameshift mutation in E2 which terminates translation after codon 75. pSK-E116 and pSK-E216 (58), provided by P. Howley, Boston, MA, are expression vectors which contain a human cytomegalovirus promoter and intact E1 or E2 coding sequences, respectively, derived from the European HPV16 prototype plasmid p1203 (55). Both vectors have been shown to support replication of HPV16 origin plasmids (58).
Short-term replication. Viral establishment efficiency was analyzed in transient replication assays essentially as previously described for HPV31 (31). Briefly, viral genomes were released from bacterial vector sequences and unimolecularly religated at a low DNA concentration. HPV-negative human squamous cell carcinoma 13 (SCC13) (53) cells (5 x 106) were transfected by electroporation either with ligated HPV genomes (5 μg of viral DNA) by themselves or together with equimolar amounts of pSK-E116 and pSK-E216 each. After expanding the transfected SCC13 cells on J2-3T3 fibroblast feeder layers for 5 days, low-molecular-weight DNA was extracted by a modified Hirt DNA procedure. Prior to gel electrophoresis, sample DNAs were digested to completion with NcoI to linearize HPV16 genomes and DpnI to remove bacterially methylated input DNA.
Long-term replication. Viral maintenance efficiency was analyzed in stable replication assays as published for HPV31 (31). Briefly, ligated viral genomes were prepared as described for the transient assay. Subconfluent, low-passage, primary human foreskin keratinocytes (HFKs; isolated from anonymous neonatal donors) were transfected with HPV16 genomes (5 μg of viral DNA) and a 0.5 molar equivalent of the marker plasmid pSV2neo using FuGENE 6 (Roche) or Lipofectamine (Invitrogen). Transfected HFKs were transferred to J2-3T3 fibroblast feeder layers and selected briefly with G418. Drug-resistant colonies from each transfection were pooled within 3 weeks and expanded as mass cultures. After removal of fibroblast feeders, total cellular DNA was isolated from HFKs 4 to 6 weeks posttransfection using proteinase K digestion and differential salt precipitation to minimize shearing (31). Gel electrophoresis was performed with DpnI-digested total cellular DNA samples (5 μg) which were either sheared or digested with the single-cut enzyme NcoI.
Vegetative replication. Viral DNA amplification efficiency was analyzed by in vitro differentiation of stably transfected cells as previously described for HPV31 (31, 56). Briefly, fibroblast feeders were removed from the expanded, HPV16-positive mass cultures (see above), and HFKs trypsinized. After collection, cell pellets were divided equally into two samples and suspended separately in methylcellulose (1.6%, wt/vol)-containing E medium. Cells were rinsed out from methylcellulose cultures immediately or after 24 h of incubation at 37°C. Total cellular DNA was isolated for the two time points, and sheared samples analyzed as in the stable replication assay (see above).
Southern hybridization and analysis. DNA samples from all replication assays were resolved on agarose gels (0.8%, wt/vol) and transferred to positively charged nylon membranes (Magna Probe; Osmonics) under alkali conditions as previously described for HPV31 (31). HPV16W12-derived DNA standards of 500, 25, 2.5, and 0.5 pg of genome size viral DNA were included on each blot. Prehybridization and hybridization were performed with dextran sulfate- and formamide-containing solutions at 42°C (31). HPV16-specific probe was prepared by labeling genome length viral DNA (W12 prototype) with [-32P]deoxycytosine triphosphate by random priming (Prime-It-RmT; Stratagene). After stringent washing, viral DNA was visualized by storage phosphor-mediated autoradiography (Molecular Dynamics). Autoradiograms were quantified with ImageQuant software (Molecular Dynamics) or IPLab gel (Signal Analytics), and figures assembled with Photoshop (Adobe) and Freehand (Macromedia), performing only linear adjustments.
Sequence analysis. The published HPV16R (48) sequence was used as a base for variant URRs Af1aB2, Af1aD1, Af2aE1, Af2aF1, Af2aF3, AAc, and NA1 (36). The approximate positions of variant nucleotides within the URR are shown in Fig. 1B. The search for potential factor binding sites in the URR was performed with DNA Strider (Christian Marck, CEA, France) as described in the legend to Fig. 1.
Research compliance. All experiments with human cells and human papillomavirus DNAs were in compliance with federal, state, and institutional regulations and were approved by the chairman of the Institutional Review Board at the University of Arkansas for Medical Sciences.
RESULTS
Establishment efficiencies of URR variants are strongly modulated by E1 and E2 expression. The viral establishment phase is the first phase of the HPV life cycle during which a stable low plasmid copy number is attained within an infected cell population. In this phase, viral plasmids replicate more frequently than the host genome for multiple cell division cycles. For a transient replication assay which mimics the viral establishment phase, permissive human epithelial cells are transfected with HPV genomes and their replication levels are quantified after 5 days. In this assay, the expression efficiency and functionality of the viral replication factors E1 and E2 can be analyzed, as well as the function of the replication origin on the viral genome. Under physiological conditions, papillomavirus replication requires sufficient levels of both E1 and E2 in trans and an intact origin in cis (67). Since both the transcriptional control elements for HPV gene expression and the replication origin are located within the URR, this domain plays a central role in viral regulation. Depending on transfection efficiency and other assay variations, the replication levels of HPV16 prototype genomes (E and W12) are 5- to 50-fold lower than that of HPV31 (W. G. Hubert, unpublished data). This low replication phenotype may be responsible for the earlier assessment that HPV16 genomes did not replicate when permissive cells were transfected transiently (14). Sufficient signal can now be obtained, however, and replication of HPV16 determined reliably with assay protocols adapted from studies with HPV31 (31).
To test the hypothesis that the transcriptional phenotypes exhibited by a panel of variant HPV16 URRs (36) could differentially modulate transient viral replication, hybrid HPV16 DNAs were constructed which contain variant URR sequences in a common background of the replication-competent HPV16 W12 genome. This strategy focuses the experimental analysis only on URR-mediated effects, excluding the potential contributions of sequence variations in other parts of the viral genome. The URR fragments of HPV16 variants Af1aB2, Af1aD1, Af2aE1, Af2aF1, Af2aF3, AAc, and NA1 (36), which were originally collected for an epidemiologic study (73), and the URR of the European (E) prototype were transferred to the HPV16W12 genome as described in Materials and Methods. Transient replication assays were then performed by transfecting HPV-negative SCC13 cells (53) with recircularized viral genomes and analyzing de novo replication. As shown in Fig. 2 A, the replication levels of the European prototypes (hybrid W12-URR-E and parental W12, lanes a and i) and most African URR variants (W12 hybrids b through e) were relatively low. In contrast, both American variants exhibited high replication levels (Fig. 2 A, lanes g and h) and were about 20-fold more efficient than prototype E (Table 1, mean relative replication). HPV16 DNAs defective in either E1 or E2 protein translation (E1TTL, E2FS; Materials and Methods) were replication defective in control transfections (Table 1), which is consistent with the physiological requirement for both replication factors (67).
HPV DNA replication is regulated in a dose-dependent manner by the abundance of the viral replication factors (10, 31, 41, 44, 45, 51, 74). Since the short-term replication assay with entire HPV genomes analyzes the efficiency of replication factor expression, as well as origin function, the differences in autonomous transient replication of the HPV16 DNAs could have been the result of both cis- and trans-acting changes. To more specifically test how E1 and E2 expression modulates replication, viral genomes were cotransfected with expression vectors for both viral replication factors (Materials and Methods), which had previously been shown to support replication of HPV16 origin plasmids (58). In the presence of heterologous E1 and E2, prototype (hybrid W12-URR-E and parental W12, a and i) and most African variant (W12 hybrids b through e) genomes exhibited similar replication levels (up to 1.53-fold, Fig. 2 B and Table 2, mean relative replication). Replication of variants Af2aF3, AAc, and NA1 exceeded that of prototype E by as much as 3.9-fold, possibly due to a combination of high levels of endogenous and exogenous E1 and E2. These data indicate (i) that HPV16 origin efficiency is not strongly affected by variant URR sequences and (ii) that, consistent with the behavior of HPV31 mutants with defects in replication factor expression (31), the low-establishment phenotypes of European prototypes and African URR variants of HPV16 are primarily the consequence of inefficient E1 and E2 expression.
Maintenance levels of URR variants are similar to their establishment efficiencies. Maintenance phase is the second phase of the viral life cycle, during which HPV plasmids replicate as frequently as the cellular genome on average (26), resulting in a stable copy number in an HPV-positive cell population. In the stable replication assay, which mimics the viral maintenance phase, primary HFKs are transfected with HPV genomes. It is performed over a 4- to 6-week time course, and the major viral parameters which determine replication behavior are (i) expression of the viral replication factors, (ii) viral origin function, and (iii) expression of the viral oncogenes (32, 66).
To test the hypothesis that the transcriptional phenotypes exhibited by variant HPV16 URRs (36) could differentially modulate viral maintenance levels, the panel of hybrid HPV16 genomes was analyzed in stable replication assays (Materials and Methods). HFKs were transfected with recircularized viral genomes and a selectable marker. After selection and expansion of drug-resistant colonies as mass culture cell lines, total cellular DNA was isolated and analyzed by Southern hybridization. Normal viral plasmid replication yields characteristic autoradiographic bands which indicate the presence of closed circular (cc) and relaxed circular (rc) monomers. As shown in Fig. 3 A, the replication levels of the European prototypes (hybrid W12-URR-E and parental W12, lanes a and i) and the African URR variant (W12 hybrid, f) were relatively low. In contrast, both American variants consistently replicated at high levels (Fig. 3A, lanes g and h, and Table 3, mean absolute replication). Interestingly, strong variations in the relative levels of the inefficient replicators were observed in multiple experiments which were performed with different primary cell isolates and transfection agents (Table 3, rows i and a through e, mean relative replication). These findings suggest that inefficient viral DNA replication is also modulated by other factors (see Discussion). Consistent with their behavior in transient assays (Fig. 2A. and Table 1), the E1 and E2 mutants of HPV16W12 were also found to be defective for stable replication (Table 3).
The additional HPV topologies present in total DNA samples are referred to as high molecular weight (hmw, Fig. 3A). hmw signals generally indicate (i) integrated viral DNA, which migrates as part of the randomly sheared cellular DNA, (ii) viral plasmid concatemers, or (iii) trapped monomeric or multimeric HPV plasmids. To resolve this potential mixture, unsheared total cell DNA was digested with a restriction enzyme which linearizes HPV plasmid monomers (lin). As shown in Fig. 3B, all analyzed cell populations harbor detectable amounts of unit length viral DNA. Additional bands of larger or smaller size than linear HPV16 DNA (non-unit length, nul) are also detected. nul bands indicate the presence of junction fragments containing both cellular and viral DNA but could also result from replicating or integrated HPV genomes which are rearranged. However, in light of the fact that no replication-competent multimeric plasmid species, including rearranged partial multimers, were detected in earlier experiments with HPV31 (31) or HPV16 (Fig. 3A), nul hybridization signals likely indicate only rearranged, integrated viral DNA. Such HPV16 DNA species have also been observed in cloned cell populations from the W12 biopsy cell line (34). As hmw hybridization signals (Fig. 3A) and nul-specific bands (Fig. 3B) are present in most sample lanes, integration of viral DNA appears to occur with both prototypes and URR variants of HPV16.
URR variant and prototype genomes replicate vegetatively. Vegetative HPV replication is the final phase of the viral life cycle. Typically, HPV plasmid DNA is amplified from a low, stable copy number to several thousand in competent, suprabasal keratinocytes. Viral DNA amplification is required for HPV capsid gene expression (25), which precedes virion assembly and egress. Vegetative replication only occurs in differentiated keratinocytes, a process which can be induced by suspending epithelial cells in methylcellulose-containing growth medium to prevent their adherence to the substrate (29). During in vitro differentiation, a subset of HPV-positive keratinocytes are competent to amplify viral plasmid DNA in 24 to 48 h, which, in the case of HPV31, increases the plasmid copy number two- to fivefold in a treated cell population (31, 56). To test the capacity of HPV16 URR variants to amplify viral DNA, cell lines with detectable HPV16 replication were analyzed (Materials and Methods). The amounts of replicated DNA were obtained by combining the respective rc and cc signals at each time point, which were then normalized for DNA loading within each sample pair. Comparing viral replication in differentiated (24 h) cells to that in undifferentiated (0 h) cells, moderate DNA amplification ratios could be observed with the HPV16 prototype (parental W12, i) and URR variants Af2aF3, AAc, and NA1 (W12 hybrids f, g, and h, Fig. 4 and Table 4). While a relationship between URR variation and DNA amplification efficiency was not apparent in the two independent experiments, these data indicate that HPV16 DNA amplification can be analyzed with the in vitro differentiation assay. Since the 24-h amplification ratios of HPV16 appear disproportionally smaller than those observed with HPV31 in 48 h (31, 56), these data may also indicate that vegetative replication of HPV16 is less efficient overall.
DISCUSSION
Viral transcription and replication phenotypes are related. When the detected transient and stable replication levels of the HPV16 URR variants were normalized to the European prototype (hybrid W12-URR-E) and compared to the published P97 activities (36), the viral transcription and persistent replication phenotypes were found to correlate for most of the tested HPV16 DNAs (Table 5). The American variants (Table 5, rows g and h) exhibited the highest levels of replication in both establishment and maintenance phases, as well as the strongest P97 activity. In contrast, both phenotypes were more moderate with most African and European strains. The replication levels of prototype E (row a) and variants Af1aB2 and Af2aF3 (b and f) also correlated with their P97 activity. And the low P97 activities of variants Af2aE1 and Af2aF1 (d and e) are in line with their moderate transient replication and lack of stable replication. These data indicate that altered P97 activities are primarily responsible for the strong differences in replication levels observed with these HPV16 URR variants.
Host-specific factors and replication of HPV16. Stable replication levels of high-risk HPV genomes vary significantly among different isolates of primary cells. Such variability in supporting long-term HPV plasmid replication has been observed in other laboratories and are thought to be donor specific (W. G. Hubert, unpublished data; L. A. Laimins, personal communication; P. F. Lambert, personal communication; L. Turek, personal communication). Evidence for this variable property of HFKs is presented in Table 3. A threshold of a minimal replication efficiency appears to be required for attaining a stable copy number, as the levels of inefficiently replicating genomes (Table 3, rows a through c, stable experiment 3; rows d and e, stable experiment 1) are close to the detection limit. The URR variants AAc and NA1 consistently replicated at a high level in three experiments (Table 3, rows g and h, mean absolute replication). While such a threshold may be overcome by increased transfection efficiency in some cases, depending on the agent used (footnotes to Table 3), it may primarily be an intrinsic characteristic of an HFK isolate. Since the outcome of HPV-induced disease is likely affected by such donor-specific factors, future studies are necessary in this area.
Role of the major viral promoter. P97 activity, as well as transient- and stable-replication efficiencies, correlate for most of the tested URR variants (Table 5). These findings indicate that the strong activity of P97, which is known to be involved in the expression of E1 and E2 (32, 39), as well as E6 and E7 (13), during the persistent stage of infection, is also associated with high-establishment and -maintenance phenotypes. Restoring the replication signals of intrinsically low prototypes (hybrid W12-URR-E and parental W12, a and i) and African URR variants (c through e) to similar levels (Table 2) by overexpressing E1 and E2 from heterologous vectors confirms previous observations about the central role of P97 activity in the pathogenesis of HPV16. Similar to the intrinsic efficiencies of URR variants for expressing the viral replication factors, their capacity for viral oncogene expression is also expected to correlate with P97 activity. Since significant levels of E7 protein have already been observed with replicating HPV16 at high copy number in clonal W12 cell populations (34, 35), the severely oncogenic potential of the HPV16 variant AAc (6, 8) may be related to its strong transcription and replication phenotypes.
Transcription factor-specific regulation by the URR. The importance of the URR in the HPV life cycle was recognized quite early. Numerous studies have characterized the function of common factor binding sites in P97-mediated transcription of HPV16, such as AP1 (9), AP2/TEF2 (11), NF1 (2, 28), Oct1 (49), Sp1 (3, 65), TEF1 (33), and YY1 (16, 17, 46, 50). Our understanding of viral regulation can now be expanded by testing the importance of these sites for viral replication throughout the life cycle. Preliminary analysis of the variant changes in the URR has already shown that the low replication phenotypes of African variants may correlate with alterations of the P97– proximal Sp1 binding site. And the high-replication phenotype of the American variants may be aided by the acquisition of an additional AP1 site. Both Sp1 (3, 7, 15, 27, 59, 61) and AP1 (11, 12, 38, 42) are known to play complex activating roles in viral transcription and DNA replication. In addition, several URR variants also have a reduced number of YY1 binding sites which, depending on their sequence context, may increase transcriptional activation (5, 37) or repression (4, 46, 50). The effects of selected factor binding sites on viral replication will be analyzed in future studies.
ACKNOWLEDGMENTS
Thanks to Peter Howley, Lou Laimins, Paul Lambert, Lubos Turek, and Cosette Wheeler for providing essential plasmids for this study and to Paul Lambert and Lou Laimins for critically reading the manuscript.
This research was funded by grants from the American Cancer Society, Murphy Oil Corporation, and the University of Arkansas for Medical Sciences. W.G.H. is supported by a career development award from the Dermatology Foundation.
Mailing address: Department of Dermatology, MS576, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205. Phone: (501) 686-5110. Fax: (501) 686-7264. E-mail: wghubert@uams.edu.
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