Genomic instability of the host cell induced by the human papillomavirus replication machinery
2007; Springer Nature; Volume: 26; Issue: 8 Linguagem: Inglês
10.1038/sj.emboj.7601665
ISSN1460-2075
AutoresMeelis Kadaja, Alina Sumerina, Tatjana Verst, Mari Ojarand, Ene Ustav, Mart Ustav,
Tópico(s)Hepatitis B Virus Studies
ResumoArticle29 March 2007free access Genomic instability of the host cell induced by the human papillomavirus replication machinery Meelis Kadaja Meelis Kadaja Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Search for more papers by this author Alina Sumerina Alina Sumerina Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Search for more papers by this author Tatjana Verst Tatjana Verst Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Search for more papers by this author Mari Ojarand Mari Ojarand Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Search for more papers by this author Ene Ustav Ene Ustav Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Mart Ustav Corresponding Author Mart Ustav Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Meelis Kadaja Meelis Kadaja Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Search for more papers by this author Alina Sumerina Alina Sumerina Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Search for more papers by this author Tatjana Verst Tatjana Verst Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Search for more papers by this author Mari Ojarand Mari Ojarand Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Search for more papers by this author Ene Ustav Ene Ustav Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Mart Ustav Corresponding Author Mart Ustav Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Author Information Meelis Kadaja1, Alina Sumerina1, Tatjana Verst1, Mari Ojarand1, Ene Ustav2 and Mart Ustav 1,2 1Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia 2Institute of Technology, University of Tartu, Tartu, Estonia *Corresponding author. Department of Biomedical Technology, Institute of Technology, University of Tartu and Estonian Biocentre, Nooruse 1, Tartu 50411, Estonia. Tel.: +372 737 4800; Fax: +372 737 4900; E-mail: [email protected] The EMBO Journal (2007)26:2180-2191https://doi.org/10.1038/sj.emboj.7601665 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Development of invasive cervical cancer upon infection by ‘high-risk’ human papillomavirus (HPV) in humans is a stepwise process in which some of the initially episomal ‘high-risk’ type of HPVs (HR-HPVs) integrate randomly into the host cell genome. We show that HPV replication proteins E1 and E2 are capable of inducing overamplification of the genomic locus where HPV origin has been integrated. Clonal analysis of the cells in which the replication from integrated HPV origin was induced showed excision, rearrangement and de novo integration of the HPV containing and flanking cellular sequences. These data suggest that papillomavirus replication machinery is capable of inducing genomic changes of the host cell that may facilitate the formation of the HPV-dependent cancer cell. Introduction Papillomaviruses are small species-specific DNA tumor viruses that establish latent infection in the basal cells of the differentiating epithelium with the help of viral oncoproteins and maintain their small, about 8 kb, circular double-stranded (ds) genomes as episomal multicopy nuclear plasmids in proliferating transformed cells (Howley and Lowy, 2001) via action of the viral replication proteins E1 and E2 (Ustav and Stenlund, 1991; Chiang et al, 1992; Sverdrup and Khan, 1994). The E1 protein is the replication origin recognition factor and viral helicase (Yang et al, 1993; Sedman and Stenlund, 1998), which in cooperation with E2 facilitates recognition and effective loading of the host cell replication complexes at the papillomavirus origin in the upstream regulatory region (URR) (Ustav et al, 1991, 1993; Remm et al, 1992; Russell and Botchan, 1995; Stenlund, 2003). Increase of E1 concentration above a certain level results in overly frequent loading of the viral hexameric helicase and cellular replication complexes at the viral origin and induction of ‘onion skin’-type replication intermediates on the viral genome (Männik et al, 2002). Consequently, complicated topological structures of the ‘onion skin’-type replication intermediates as well as heterogeneous linear subgenomic fragments are generated. Based on tissue tropism, more than 100 different types of human papillomaviruses (HPVs) are divided into subgroups of mucosal or cutaneous papillomaviruses; individual viruses from each group are designated ‘high risk’ or ‘low risk’ according to the propensity of the infected transformed cells for malignant progression (zur Hausen, 2002; de Villiers et al, 2004; Munger et al, 2004). ‘Low-risk’ HPVs (LR-HPVs) like HPV6 and HPV11 are considered as rather normal microflora of the human epithelia, as they are generally limited to low-grade lesions or genital warts that rarely progress to malignancy. However, moderate and severe cervical dysplasia as well as invasive cervical carcinomas contain predominantly the ‘high-risk’ type of HPVs (HR-HPVs) such as HPV16 and HPV18. Development of invasive cervical cancer is a stepwise process, which is associated with integration of the HR-HPV DNA into the host cell chromosome, switching off the functions of the two viral replication proteins and upregulating the expression of viral oncoproteins E6 and E7 (Corden et al, 1999; Kalantari et al, 2001; Peter et al, 2006). Cells carrying integrated HR-HPV sequences have selective growth advantage due to the increased cell immortalization (Romanczuk and Howley, 1992; Jeon et al, 1995). The trigger and mechanism of the integration is unclear, but it seems to be a critical event in cervical carcinogenesis preceding the development of chromosomal abnormalities that drive the malignant progression (Pett et al, 2004, 2006). In the case of HPV18, HPV31 and HPV35, nearly 100% of the viral sequences show integration into the cancer cell genome. Integrated and episomal viral genomes are commonly found in the HPV16 DNA-positive cancers (Cooper et al, 1991; Kristiansen et al, 1994; Peitsaro et al, 2002b; Andersson et al, 2005; Arias-Pulido et al, 2006). Application of sensitive methods has shown that mixed (episomal/integrated) pattern is the most prevalent physical state of HPV16 already in PAP smears with normal morphology and in LG-SIL (Kulmala et al, 2006). The experiments with the HPV16-positive W12 cell line demonstrate the initial coexistence of episomal and integrated HPV16 DNA in the same cells in early passages (Pett et al, 2006), resulting in the loss of HPV plasmids in later passages. Similar results were obtained with the cell line carrying HPV33 (Peitsaro et al, 2002a). Coexistence of the replicating episomal viral genome and integrated HPV with potential viral replication origin raises the question about the functionality of the integrated origins. The papillomaviruses do not follow once-per-cell cycle replication mode (Ravnan et al, 1992; Piirsoo et al, 1996); therefore, multiple unscheduled initiation events at the functional integrated HPV origin could extend into the adjacent genomic locus and trigger rearrangements like deletions or duplications of the sequences of the cellular genome by repair/recombination machinery. Amplification of integrated polyomavirus genome in the presence of large T antigen (Botchan et al, 1979; Syu and Fluck, 1997) followed by recombination of the sequences suggests that such processes may take place also in the case of HPV. Furthermore, the integrated HPV regulatory sequences have been shown to be transcriptionally active in cervical cancer cells (Schneider-Gadicke and Schwarz, 1986; Baker et al, 1987). We studied the events that may contribute to the formation of invasive cancers in the case of HR-HPV infections by demonstrating the effective HPV E1- and E2-dependent DNA amplification of the integrated HPV18 and HPV16 origins in HeLa and SiHa cells, respectively. The replication forks initiated at the integrated HPV origins extend into the flanking regions of cellular DNA. These amplified genomic sequences, presumably resembling ‘onion skin’-type DNA replication intermediates, could be targets for the recombination and repair system that causes excisions of these sequences, resulting in de novo rearrangements and recombinations within the cellular DNA. The data presented suggest that papillomavirus DNA replication machinery should not be considered as passive virus-specific activity alone, but also as one that can actively induce irreversible changes in the genomic make-up of the cell at sites of HPV origin integration. Results Regulation of the HPV E1 protein expression Expression of the HPV E1 protein is regulated by the activities of the promoters, pre-mRNA splicing, stability of the mRNA, unusual translation mechanism of the polycistronic mRNA, nuclear export and import, and by stability of E1 protein (Remm et al, 1999; Malcles et al, 2002; Deng et al, 2003). Based on preliminary experiments (M Kadaja et al, unpublished), the expression vectors for producing the HPV6b, -11, -16 and -18 E1 proteins in a wide range in host cells such as COS, CHO, C33A, 293, HeLa and SiHa, were constructed. The E1 protein expression was evaluated by Western blot (Materials and methods) using HA-tag-specific antibody in HeLa cells (Figure 1D) and in SiHa cells (Figure 3C). Expression of the 3F12 epitope-tagged E2 proteins, directed from pQM vectors under the CMV promoter, was also monitored using a monoclonal antibody of 4E4 (Figure 2D). Tagging of E1 and E2 did not interfere with the functions of the proteins (data not shown) and enabled comparable expression levels of all proteins (HPV6b, -11, -16 and -18). Figure 1.HPV E1 and E2 proteins initiate the replication from episomal and integrated HPV origins in HeLa cells. (A, B) HeLa cells were cotransfected with plasmids for expression of homologous E2 and E1 from HPV6b (lanes 1–4), HPV11 (lanes 5–8), HPV16 (lanes 9–12) and HPV18 (lanes 13–16) together with 0.5 μg of pUCURR-6b, -11, -16 and -18, respectively. As controls, either 5 μg pMHE1-18 and 5 μg pQMNE2-18 (lanes 17 and 18) or 0.5 μg pUCURR18 alone (lanes 19 and 20) was transfected. A 3 μg measure of total DNA extracted 24 and 48 h after transfection was digested with HindIII/DpnI and analyzed by Southern blotting with radiolabeled pUC probe (A) or all four HPV URR sequences (B). (C) Schematic presentation of the HPV6b, -11, -16 and -18 E1 expression constructs. (D) Analysis of expression of HA epitope-tagged E1 of HPV6b (lanes 1 and 2), HPV11 (lanes 3 and 4), HPV16 (lanes 5 and 6) and HPV18 (lanes 7 and 8) 48 h after transfection with 3F10-HRP antibody. (E) Schematic representation of the episomal HPV18 genome and the integrated HPV18 in HeLa cells (Lazo, 1987; Meissner, 1999). Cellular DNA is shown as dashed line and HPV18 DNA as solid line. Open arrows represent the viral ORFs and noncoding URR is shown as an open box. Download figure Download PowerPoint Figure 2.Replication of integrated HPV18 is dependent on E1 protein concentration, while E2 has no effect. (A, E) Increasing amounts of HPV18 E1 expression plasmids (from 0.5 to 10 μg) were cotransfected with 1 μg of HPV18 E2 expression vector. Total cellular DNA was extracted 24 h and 48 h after transfection and 3 μg of DNA was digested with HindIII (A) or BamHI (E) and analyzed by Southern blotting with 32P-labeled HPV18 URR probe. (B) Southern blot analysis of HeLa cells transfected with HPV18 E1 vector (5 μg) and an increasing amount of HPV18 E2 vector (0.5–10 μg). DpnI was used to remove input plasmids. (C) Western blot analysis of HPV18 E1 protein expression in HeLa cells 48 h after transfection with 3F10-HRP antibody. (D) Western blot analysis of HPV18 E2 protein with 4E4 antibody. (F) Total cellular DNA was extracted at various time points from cells transfected with 1 μg pQME2-18 and 10 μg pMHE1-18 (lanes 1–6) or from mock-transfected cells (lanes 7–12); 3 μg from each DNA sample was digested with BamHI/DpnI and subjected to Southern blotting analysis with 32P-labeled HPV18 URR-specific probe. Download figure Download PowerPoint Figure 3.Amplification of integrated HPV16 URR in SiHa cells transfected with HPV E1 and E2 expression vectors. (A) Schematic presentation of the integrated HPV16 genome in chromosome 13 in SiHa cells. Viral ORFs are shown as open arrows and URR is indicated by an open box. Numbers in italics indicate the nucleotides of viral genome at the junction with cellular DNA. Acc65I restriction sites at nt 880 and nt 5378 of the viral genome are shown (B) SiHa cells were transfected with expression vectors for E1 and E2 as follows: 5–20 μg of HPV6b E1 (lanes 1–3); 5–20 μg of HPV11 E1 (lanes 4–6); 1.2–5 μg of HPV16 E1 (lanes 7–9) and 1.2–5 μg of HPV18 E1 (lanes 10–12). A 5 μg measure of homologous E2 expression vectors was added for each transfection. For controls, either 5 μg pMHE1-16 alone (lane 13) or 5 μg pQMNE2-16 alone (lane 14) was transfected into the cells. The total DNA was isolated 24 h after transfection and 3 μg was digested with Acc65I/DpnI and analyzed by Southern blotting using radiolabeled HPV16 URR probe. (C) Analysis of E1 protein expression in SiHa cells 24 h after transfection using Western blot. Download figure Download PowerPoint Expression of E1 and E2 proteins induces amplification of episomal and integrated HPV18 origins in HeLa cells Replication of HPV plasmids in cervical carcinoma-derived cell lines has been shown to be strongly influenced by the presence of integrated HPV DNA, as these cells poorly support E1- and E2-dependent HPV-transient DNA replication (Chiang et al, 1992; Lin et al, 2000). To test our E1 expression constructs and to identify the conditions conducive to viral origin replication, the origin plasmids (pUC/URRs) of all HPVs, together with the homologous expression vectors for E1 and E2, were cotransfected into HeLa cells. The DpnI-resistant replication signal from the transient assays was examined by Southern blot analysis of total DNA with common plasmid probe (Figure 1A). Replication of all origin plasmids was clearly detected in HeLa cells. Western blot analysis to monitor E1 protein levels in transfected cells (Figure 1D) confirms its effective and comparable expression in HeLa cells. E2 levels were kept constant and considerably low in order to support replication without suppression of E6 promoter. HPV16 and HPV18 E1 proteins seem to be the most efficient in initiation of DNA replication within the used expression range, while HPV6B and HPV11 E1 proteins are clearly less efficient. In addition to the unit-sized plasmid, a smear of newly synthesized products with considerably larger and smaller size than input was detected, especially in the case of HR-HPV replication proteins (Figure 1A, lanes 9–12 and 13–16). The same kind of smearing characterized the ‘onion skin’ type of replication mode, as identified by 2D analysis (Männik et al, 2002). Probing of the same blots with URR-specific probes showed additional replication signals of different intensity in every lane across the panel (Figure 1B, compare with panel A). These signals may result from the multiple integrated HPV18 sequences located in at least five different sites of the host genome (Lazo, 1987; Meissner, 1999). It has been demonstrated that the URR with origin is present in addition to the coding sequences for viral oncoproteins E6 and E7 (schematically shown in Figure 1E). According to Lazo and Meissner, the HindIII digest generates three fragments of 8.4 kb (1A), 7.9 kb (1B) and 5.8 kb (2), carrying HPV18 sequences (Figure 1E). The URR-specific probe for HPV18 detected amplification of exactly these fragments (Figure 1B). The intensities of these bands correlated with the E1 expression levels and were the strongest in the lanes of HR-HPV E1 expression constructs (Figure 1B, lanes 9–18). LR-HPV E1 proteins were less efficient for replicating integrated HPV18 origin, which may be explained by lower specificity toward the HPV18 origin, although expression level of the E1 proteins was comparable. Intensive replication signal was detected also in the control lanes, where only the viral expression vectors were transfected, suggesting that integrated HPV18 origin is fully competent for initiation of DNA replication (Figure 1B, lanes 17 and 18). These results show that papillomavirus replication proteins are capable of mobilizing integrated HPV origin and that simultaneous DNA replication of episomal and integrated HPV origins may occur in HeLa cells. E1 protein is the major determinant of the efficiency of DNA replication Titration of the HPV18 E1 and E2 proteins in the transient assays showed that the efficiency of DNA replication initiation depends on the E1 protein concentration (Figure 2A), while modulation of E2 concentration in a quite wide range had little effect on the efficiency of initiation of the DNA replication of the integrated HPV (Figure 2B). The replication reached a plateau at 0.5 μg of transfected E2 expression plasmid (Figure 2B, lanes 6 and 7) and changed little at higher vector concentrations. Therefore, replication assays were run at low E2 levels to avoid suppression of E6 and E7 mRNA transcription from integrated copies and induction of apoptosis as well as cellular senescence (Desaintes et al, 1997; Wells et al, 2000). The E2 expression levels in our replication assays did not induce senescence or apoptosis of the transfected cells, as analyzed by different methods (data not shown). High E1 levels caused smearing of the integrated HPV18 origin replication signals characteristic of the ‘onion skin’ type of replication mode. To prove that in HeLa cells high E1 levels do not cause unspecific initiation of DNA replication (Bonne-Andrea et al, 1995, 1997) and that origin-specific replication initiation is assured at any E1 concentration, we further analyzed the replication products. Cleavage of the integrated HPV18 sequences in HeLa cells (Figure 2E and F) with BamHI generates a 1 kb URR fragment, which includes the complete functional HPV replication origin and 5.5 kb URR fragment containing nonfunctional origin linked to cellular sequence. The kinetics of accumulation of amplification products with time was studied from 24 h to 96 h after transfection (Figure 2E and F). The amplification level of functional HPV18 URR was estimated up to 90 times at the 72 h time point compared with controls. Moderate amplification of 5.5 kb fragment was detected at later time points, which most likely indicates that the replication forks that initiated from functional origins reach the nonfunctional origins in the 5.5 kb fragment. Interestingly, the 5.5 kb band is three times stronger than the 1 kb fragment in mock-transfected cells, which indicates that less than a quarter of the HPV18 URRs in HeLa cells carry functional replication origins. The amplification of 6–8 kb viral/cellular fragments in the presence of E1 and E2 (Figure 2A) implies that DNA replication from HPV origin can extend into the flanking cellular sequences. However, it was difficult to assess the size of viral amplicon in HeLa cells because of multiple integration sites and limited information about the surrounding sequences of the different HPV18 integration locuses. Therefore, the alternative cell line, SiHa, carrying a single copy of the integrated HPV16 genome, was further used to study the replication of the integrated HPV origin. HPV E1 and E2 proteins efficiently initiate replication from integrated HPV16 origin in SiHa cells SiHa cell line derived from the cervical carcinoma of a 55-year-old Japanese female is aneusomic and has been found to contain 66–72 chromosomes. However, this cell line has been shown to be disomic with respect to chromosome 13, which contains one copy of the HPV16 genome (Meissner, 1999; Szuhai et al, 2000). Integration of the HPV16 genome has occurred, with disruption in the E2 and E4 ORFs at nucleotides 3132 and 3384 of HPV16 genome (Figure 3A). Expression plasmids for the E1 and E2 proteins were transfected into SiHa cells and replication of integrated HPV16 origin DNA was analyzed using HPV16 URR probe. The E2 expression plasmid concentration was kept constant at the level where replication of integrated HPV16 had reached a plateau (data not shown). Increasing amounts of E1 expression plasmids were used in replication assays. In order to reach comparable levels of E1 expression, much higher amounts of HPV6b and HPV11 E1 expression plasmids had to be used in the transfection mixture (Figure 3B and C). The results were interpreted using a previously derived physical map of the integration site of HPV16 (Meissner, 1999), shown schematically in Figure 3A. Untransfected SiHa cells as well as cells transfected with HPV16 E1 or E2 plasmids alone gave the faint, similar-strength signal of 3.4 kb after the digestion of an equal amount of total DNA samples with Acc65I (Figure 3B, lanes 13–15). Cotransfection of the E2 plasmid with increasing amounts of the E1 vector resulted in a significant increase in the HPV16 URR-specific signal in the case of all four HPVs. Expression vectors for the HPV16 (lanes 7–9) and HPV18 E1 proteins (lanes 10–12) induced most efficient initiation of DNA replication of the integrated HPV16 origin when compared with LR-HPV (lanes 1–3 and lanes 4–6). Remarkably, at higher concentrations of E1 protein of HR-HPVs (lanes 8 and 9, 11 and 12), a heterogeneous mixture of fragments that did not migrate in the gel as a specific band was generated. Analysis of the replication products by 2D gel indicated formation of ‘onion skin’-type replication intermediates and linear dsDNA fragments as shown for BPV1 (Männik et al, 2002) and HPV16 in SiHa (data not shown). This confirms that integrated replication origins of HR-HPV types are functionally active for the HPV E1- and E2-dependent initiation of replication. Estimation of the size of the integrated HPV replicon The replication competence of the integrated HPV origins directed by viral replication proteins brings up an intriguing possibility that flanking cellular sequences on both sides of viral integration could be coamplified. To test this possibility, we determined the size of the replicon in SiHa cells by measuring the amplification levels of cellular sequences at various distances from integrated viral DNA replication origin. The HPV16 genome is integrated at chromosome 13q21–31 in SiHa cells. The integrated HPV16 as well as the flanking cellular DNA have been sequenced (Baker et al, 1987; Meissner, 1999). The 5′ and 3′ flanking cellular DNA sequences, determined by Baker et al, were subjected to BLAST search against the NCBI 36 assembly of human genome (released in November 2005). According to the results, 5′ viral–cellular junction in SiHa was located at 72 686 871 bp and 3′ viral–cellular junction at 72 984 815 bp of chromosome 13. This suggests that the distance between these junctional sequences is normally ∼300 kb, indicating that considerable loss of genomic DNA has taken place in the process of integration of HPV16 DNA. Coordinates of the viral–cellular junctions, determined by BLAST search, were used to identify and amplify cellular sequences at various distances from integrated HPV16 using PCR. Sequence blocks with the furthermost nucleotide at 5.4 and 12.6 kb upstream (SL1, SL2 in Figure 4A) and 4.7 and 7.9 kb downstream of HPV16 ori (SR1, SR2 in Figure 4A), together with URR sequence itself, were used as probes in the following quantitative Southern blot analysis. The goal was to determine the amplification level of genomic sequences at different distances on both sides of the integrated HPV origin in SiHa cells, cotransfected with expression plasmids for E1 and E2 proteins of either HPV16 (Figure 4B, row 2) or HPV18 (Figure 4B, row 3). The control transfection was performed with the carrier DNA alone (Figure 4B, row 1). The cellular DNA from transfected cells was digested with appropriate combinations of enzymes (lanes 1–5 in Figure 4B; location of restriction sites is depicted in the drawing in Figure 4A) and three analogous blots (with SiHa DNA, +HPV16 E1 and E2, and +HPV18 E1 and E2) were probed with four appropriate radiolabeled cellular sequences or URR probe (shown at the bottom of lanes 1–5 in Figure 4B). The hybridization signals of two independent experiments were quantified by Phosphorimager and normalized to the carrier-transfected SiHa cells (Figure 4A). The data show that DNA replication, initiated from the HPV16 origin, will extend into the flanking cellular sequences on both sides and that the DNA replication fork travels a distance of at least 12.6 kb from the HPV16 origin, on average, as analyzed at 24 h after transfection. That makes the total size of the amplicon to be more than 25 kb. We estimated that, under the experimental conditions used, the transfection efficiency was approximately 40%. This means that more than half of the signal comes from the cells that do not have E1- and E2-induced amplification. This suggests that at the cellular level amplification as well as the replicon size is likely to be more than the estimated values. Figure 4.Amplification of the integrated HPV16 origin and flanking cellular sequences induced by HR-HPV E1 and E2 proteins. (A) Graphical representation of amplification of HPV16 URR and flanking sequences of two independent experiments, as described in (B). A 5 μg portion of pMHE1-16 and 5 μg pQMNE2-16 (dashed line in panel A and second row in panel B) or 5 μg pMHE1-18 and 5 μg pQMNE2-18 (solid line in panel A and third row in panel B) were transfected into SiHa cells. A 3 μg portion of total cellular DNA was extracted 24 h after transfection, digested with different enzyme combinations (indicated at the top in panel B, restriction sites shown in panel A) and then subjected to Southern blot analysis. Filters were probed with either radiolabeled HPV16 URR (lane 3 in panel B) or with cellular sequences (panel B, lanes 1, 2, 4 and 5) from various distances from both sides of the viral integration (SL1, SL2, SR1 and SR2). The replication signals were quantified on PhosphorImager and normalized to the signal from the mock-transfected SiHa cells. The average increase in copy numbers of different sequences is calculated (shown by vertical italic numbers in the graph in panel A). (C) Graphical representation of the results from two independent experiments, where 5 μg pQMNE2-18 together with increasing amounts of pMHE1-18 (2.5 μg, dotted line; 5 μg, dashed line; 10 μg, solid line) was transfected into SiHa cells. The replication signals were analyzed as described above and the average copy numbers corresponding to different sequences (URR-16, SL1, SL2, SR1, andSR2) are shown in the table (D). Download figure Download PowerPoint The replication proteins of HPV18 were more efficient in initiating DNA replication of the HPV16 URR compared with HPV16 proteins (solid line compared with dashed line in graph in Figure 4A). However, there are almost no differences in the amplification of distal cellular sequences (SL1, SL2, SR1 and SR2) between these sets of replication proteins. Furthermore, we observed that even if the replication of integrated HPV16 origin is highly dependent on E1 concentration (Figure 4C and D), the E1 concentration dependence of amplification of distal sequences decreases with the distance from the replication origin. There was a 10 times difference in the amplification signal of the HPV16 URR when 2.5 or 10 μg of HPV18 E1 plasmid was used, but less than 1.5 times difference in amplifying cellular sequences that are about 10 kb away (SL2 and SR2). These data suggest that overexpression of E1 protein may generate replication intermediates, locked for elongation, owing to the topological constraints in the ‘onion skin’ structures that would prevent traveling of replication forks further into the genome. Therefore, elongation of replication forks at considerable distances from the initiation site is determined presumably more by proper configuration of the template and replication complex functioning at it and less by the E1 level. These data also suggest that low E1 and E2 protein levels, for example from episomal HPV genome, and respective extension of the E1-driven replication into the flanking sequences may be highly relevant for induction of amplification of the HPV integration locus in HPV-infected cells. Coreplication of integrated and episomal HPV18 in HeLa cells HeLa cells were transfected with the plasmid carrying functional full-size HPV18 genome cloned into pUC19 plasmid. Extrachromosomal supercoiled plasmids from the transfected cells were extracted at certain time points by alkaline lysis. Analysis of the episomal replication products (provided in Figure 5A) shows that DpnI-resistant and HindIII-linearized HPV18 viral genome replication could be detected in HeLa cells at low level (Figure 5A, lanes 2 and 3), indicating that viral genome is basically functional and directs the expression of E1 and E2 replication proteins in some cells, although at low level. Cotransfection of HPV18 plasmid with E2 expression vector did not increase replication signal (data
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