Institute of Virology, University of Cologne, Fürst-Pückler-Str. 56, 50935 Köln, Germany1
Epidemiology and Cancer Control Program, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA2
Author for correspondence: Herbert Pfister. Fax +49 221 478 3902. e-mail herbert.pfister{at}medizin.uni-koeln.de
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Abstract |
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Introduction |
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HPV types are defined by sequence variation of more than 10% between themselves, whereas intratype variants vary by up to only 2% in conserved regions of the genome, like E1 or L1, and by up to 5% in the LCR. The nucleotide sequences in just one region of the genome can be used to define HPV types and intratype variants. HPV-16 variants can be divided into six geographically clustered phylogenetic groups, the European (E) group, two African (Af) groups and the Asian (As), Asian American (AA) and North American (NA) variants (Wheeler et al., 1997 ; Yamada et al., 1995
, 1997
). There are certain indications that the HPV-16 prototype and HPV-16 variants exhibit differences in their biological and biochemical properties. Non-European HPV-16 variants can be detected more often in neoplasias of higher grade than the prototype HPV-16 (Londesborough et al., 1996
; Xi et al., 1997
, 1998
; Zehbe et al., 1998
), and AA variants of HPV-16 show differences in the degradation of p53 due to their E6 proteins (Stöppler et al., 1996
). An Asian American variant also exhibits increased P97 promoter activity compared with the prototype HPV-16 (Veress et al., 1999
).
In order to correlate sequence variations in the HPV-16 LCR and transcriptional activities of the P97 promoter, we investigated different HPV-16 variants by sequencing the complete LCRs and determining the P97 promoter activity.
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Methods |
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Site-directed mutagenesis.
Site-directed mutagenesis was performed with the AAc and NA1 variant clones by using the QuikChange site-directed mutagenesis kit (Stratagene) in order to change the nucleotide at position 7729 from a cytosine to an adenosine residue, as found in the European reference clone. The primers used comprised 34 base-pairs from bp 7715 to 7748 and contained the intended nucleotide exchange. After obtaining positive clones, the whole LCRs were sequenced to confirm the mutated nucleotide in the context of the former LCR sequences.
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Results |
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At six positions (7060, 7193, 7489, 7521, 7764 and 7786), the same nucleotide was found in all HPV-16 variants except for the European prototype variant. The T at nt 7193 and the A at nt 7521 can also be identified in some European isolates (Yamada et al., 1997 ; Veress et al., 1999
; unpublished observations). At the other four positions, more than 10 independent European isolates showed the same nucleotide as the European reference clone (unpublished results).
With this more extensive sequencing of the HPV-16 LCRs of variants Af1a, Af2a, NA1 and AAc, we identified new sequence alterations in each one compared with the European reference clone and with former published sequences (Table 1).
Analysis of the P97 promoter activity of the different HPV-16 LCR clones
Some of the nucleotide exchanges identified in the seven different HPV-16 LCR clones were located near to or within known binding sites for cellular or viral proteins (Fig. 1b). Therefore, the binding of cellular transcription factors to their recognition sites might be hindered or stimulated. There is also the possibility that new protein-binding sites were generated due to some of the nucleotide changes identified.
In order to determine whether sequence alterations can influence P97 promoter activity, the complete HPV-16 LCR fragments (nt 7009124) of the different variants were cloned into the pALuc vector in front of the luciferase gene (Fig. 1a). Cells of the C33A line were transfected and luciferase activities were analysed 48 h later. A significantly enhanced transcriptional activity of the P97 promoter was noted for the variants AAc and NA1. Here, the activity was stimulated 3·3- and 2·8-fold, respectively, when compared with the European reference clone, which was set at 1·0 in all assays (Fig. 2
). The variants Af1a B2 and Af1a D1, as well as Af2a E1, Af2a F1 and Af2a F3, exhibited more or less the same transcriptional activity as the reference clone.
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The results clearly indicated that the stimulation of P97 promoter activity was due to the 3' region of the LCRs of the more-active variants AAc and NA1. The pALuc clones Af1aAAc and Af1aNA1 showed 3·9- and 3·4-fold increases, respectively, in the transcriptional activity of the P97 promoter compared with the European reference clone (Fig. 3). In these experiments, the promoter activity of the recombined pALuc clones Af1aAAc and Af1aNA1 was greater than that of the original AAc and NA1 LCR fragments. No increase in promoter activity could be seen when the 3' region consisted of the Af1a variant and the 5' regions of the AAc or NA1 variant (Fig. 3
).
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Discussion |
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Some new nucleotide changes identified in this study were specific for the individual Af1a and Af2a variant clones. The two Af1a clones B2 and D1 differed in nine nucleotide positions and the three Af2a clones E1, F1 and F3 exhibited nine, seven and four intratype variations, respectively. Comparing the intertype variations between the Af1a and Af2a LCR clones, they exhibited 1619 differences. An intertype comparison of the Af1a and Af2a variants with the E, AAc and NA1 variants also revealed 1620 sequence variations. This confirmed previous results, showing that the E, Af1a, Af2a and AAc/NA1 variants belong to different branches of the phylogenetic tree.
The variants AAc and NA1 exhibited several additional nucleotide changes relative to the reference sequence and some of these were distinct for the two variants. For instance, the nucleotide changes at positions 7339, 7507, 7827, 7886 and 7894 were only seen in the AAc variant and the nucleotide changes at positions 7227 and 7283 were specific to the NA1 variant. Whereas the sequence of the AAc variant studied here differed from that published by Veress et al. (1999) in five nucleotide positions, 11 nucleotide changes were identified between the AAc and NA1 variants. This indicates that the relationship between the AAc and NA1 variants is intermediate to those found with intra- and intervariant isolates and confirms a closer phylogenetic relationship of AAc and NA1 variants to each other than to the E, Af1a or Af2a group of variants (1620 nucleotide exchanges).
The HPV-16 LCR variants exhibited differences in promoter activity when the complete LCRs were cloned in front of the reporter gene luciferase. The variants AAc and NA1 showed a roughly 3-fold increase in P97 promoter activity, whereas the Af1a and Af2a variants were more or less as active as the reference clone. A 1·7-fold increase in P97 promoter activity of an AAc variant has also been observed by Veress et al. (1999) . It seems that enhanced P97 promoter activity can be attributed to the AAc variants in general, but minor sequence alterations affect P97 promoter activity. Investigations by Xi et al. (1997
, 1998
) demonstrated an increased tendency of progression in infections with non-prototype variants such as the AAc variant. Stöppler et al. (1996)
showed stimulated induction of differentiation-resistant colonies of human foreskin keratinocytes when studying an AA isolate. The assumed enhanced oncogenic potential of the Asian American variants could therefore be due both to differences in the biological activities of the E6 protein and to an upregulation of the P97 promoter, which controls the expression of E6.
The NA1 LCR increased the P97 promoter activity 2·8-fold. This result indicates that the AA and NA1 variants share biological properties in line with their close sequence relationship. One might speculate that this also reflects a higher oncogenic potential of North American variants. The results of transient-transfection experiments with recombined LCR fragments demonstrated that the enhanced transcriptional activity could be attributed to the 3' region comprising bp 7619124 of the LCRs of the AAc and NA1 variants (Fig. 3). The activities of the recombined LCRs Af1aAAc and Af1aNA1 were even higher (3·9- and 3·4-fold) than those of the original LCR fragments. The increased P97 promoter activity could not be attributed to the enhancer region located in the 5' region of the LCR of either variant. Similar results were also obtained by Veress et al. (1999)
, who showed that the enhanced transcriptional activity of the AAc variant could be ascribed to nucleotide changes in the 3' region of the LCR. Contrary to their results, which showed no enhanced P97 promoter activity of the chimeric LCR compared with the original AAc LCR, we demonstrated an increase in P97 promoter activity when the 3' region of our AAc variant was fused to the 5' region of the Af1a variant. This indicates that the overall sequence context of the LCR may be important for promoter activity.
We identified a single nucleotide substitution at position 7729 (A/C transition) that was characteristic for the more-active variants AAc and NA1. Electrophoretic mobility shift assays with C33A nuclear extracts did not exhibit differences in protein binding to oligonucleotides with either A or C at this position (unpublished results). With the help of site-directed mutagenesis, the A/C transition was reversed in the AAc and NA1 variants, generating Mut-AAc and Mut-NA1, with all other sequence variations unchanged. Luciferase expression experiments demonstrated that this single nucleotide exchange reduced the P97 promoter activity of the variants AAc and NA1 almost to that of the European reference clone. Therefore, the enhanced transcriptional activity of AAc and NA1 can be attributed mainly to the A/C transition at nt 7729.
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Acknowledgments |
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References |
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Received 25 February 2000;
accepted 5 April 2000.