Abstract
Association between non-European variants of human papillomavirus (HPV) type 16 (HPV-16) and HPV-18 and cervical lesions has been suggested. To compare the P105 promoter activity among 6 HPV-18 variants, their complete long control regions (LCRs), as well as that of HeLa cells, were cloned upstream of the luciferase gene and transiently transfected in C33 cells. Whereas the B18-2 European variant showed the lowest promoter activity, the Asian-Amerindian variant, B18-3, had the highest activity. All variants tested were more active than the P97 HPV-16 prototype promoter. Differences in the LCR that impact P105 promoter activity could be responsible for the observed variability in oncogenic potential
Human papillomavirus (HPV) infection is the main etiological factor in the development of cervical neoplasia [1]. Nucleotide sequencing of HPV isolates has characterized the intratype diversity of HPV type 16 (HPV-16) and HPV-18 [2]. HPV genomes are classified into molecular variants when they present >98% sequence similarity to the prototype in the L1 gene. However, variability within the long control region (LCR) can be as high as 5% among variants. HPV intratype variability has been used as an important tool in epidemiological studies of viral progression and persistence and of progression to clinically relevant lesions. We and others have suggested an association between non-European variants of HPV-16 and -18 and increased risk of cervical lesions [3, 4]
The LCR contains sequences important for regulation of viral replication and transcription of early genes. The LCR of HPV-18 and other genital HPV types contains 4 E2 viral binding sites in addition to a single E1 recognition sequence. The central segment of the LCR encloses an epithelial cell–specific enhancer that contains several binding sites for cellular transcription factors (AP-1, Sp-1, NF-1, Oct-1, TEF-1, YY-1, KRF-1, Skn-1a, and TFIID), as well as glucocorticoid-responsive elements. Most of these cellular factors stimulate P105 promoter activity, although YY-1 can either repress or stimulate activity [5]. P105 is located next to the E6 start codon and is the main early promoter of HPV-18. Sequence changes in the LCR of HPV-18 could modify the transcription of E6/E7 oncogenes and, thus, be biologically significant. Our aim was to analyze P105 promoter activity of HPV-18 molecular variants detected in the Ludwig-McGill cohort study, since we observed an enhanced oncogenic potential of non-European variants of HPV-16 and -18 [4]
PlasmidsWe amplified the complete LCR region (nucleotide positions 7137–104) of 6 HPV-18 isolates from the European and the Asian-Amerindian phylogenetic branches, representative of all HPV-18 isolates detected in our cohort study [6]. All patients gave informed consent. The LCRs of the HeLa HPV-18–positive cell line and of plasmids containing HPV-18 (GenBank accession no. X05015) or -16 (GenBank accession no. K02718) prototype sequence genomes (provided by E. M. de Villiers, Deutsches Krebsforschungszentrum, Heidelberg, Germany) were also analyzed. Molecular variants were classified as described elsewhere [2]. The amplified fragments were cut using the HindIII and KpnI restriction endonucleases (Biolabs), whose specific recognition sites were included in the primers. DEAE-cellulose–purified fragments were cloned in the precut pGL3-Basic vector (Promega) upstream of the luciferase gene. Variant clones derived from at least 2 independent polymerase chain reactions (PCRs) were confirmed by DNA sequencing
Promoter activity assaysC33 cells (derived from an HPV-negative human cervical carcinoma) (ATCC HTB-31) were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum and antibiotics. Cells were cotransfected with 4 μg of recombinant plasmid LCR-pGL3 of each variant and 1 μg of pCMV-β-Gal vector for internal control of transfection efficiency. Each LCR construct was tested in triplicate in 8 independent experiments. Transfections were performed using lipofectamine (Invitrogene), according to the manufacturer’s protocol. Twenty-four hours prior to transfection, 4×106 cells were plated in 9-cm-diameter culture dishes. Cells were harvested 48 h after transfection by addition of 900 μL of Reporter Lysis Buffer (Promega). The Promega Luciferase Assay system and the Promega β-Galactosidase Enzyme Assay system were used to measure the luciferase and β-galactosidase activity, respectively, of protein extracts, according to the manufacturer’s protocol. Relative luciferase activity measurements of the different molecular variants were normalized for β-galactosidase activity and protein content. The averages were based on the mean of triplicates of independent experiments. Kruskal-Wallis and Tukey’s tests were used to compare the P105 promoter activity among the different variants. P<.05 was considered to be significant. All statistical analyses were performed using the SPSS statistical package (version 11.0.0)
Sequencing results are presented in table 1. The nucleotide positions that differ from the prototype are shown. The HPV-18–containing plasmid available in our lab is identical to the prototype, except for a single substitution at position 7633. This substitution is not a result of PCR error, since we systematically cloned the products of at least 2 independent PCRs. We classified this sequence as being in the Asian-Amerindian phylogenetic branch. Both the prototype and the latter sequence were never detected in the present study. This HPV-18 LCR-pGL3 recombinant plasmid was further used in transcription activity studies. Among the samples from our cohort study, 4 variants were previously described and identified as SC18-2, B18-2, B18-6, and B18-3. The other 2 isolates exhibited substitutions at nucleotide positions not previously reported (positions 7677, 7707, and 7757), but both were classified as European because of their similarity to the SC18-2 sequence. Mutations found in European isolates were the most common findings. The transition at nucleotide position 7592 was observed in all HPV-18 variants studied. Nucleotide substitutions were detected throughout the LCR. Most nucleotide positions in which mutations were detected were located close to a known cellular transcription factor binding site. One can speculate that the binding of these proteins may be altered. Protein recognition sites may also have been created. The effect of these substitutions on the binding of transcription factors to adjacent recognition sequences is still unknown. The mutation at position 41 is the only one that overlaps a known binding site for the cellular transcription factor Sp-1. This mutation is commonly detected in samples isolated from Australians; it has been observed that not only is Sp-1 binding increased in isolates bearing the mutated sequence, but the transcriptional activity is also enhanced [7]. No deletions or insertions were detected among any isolates. Furthermore, none of our substitutions overlapped any of the E2 binding sites present in the HPV-18 LCR. The nucleotide sequence pattern obtained for the HPV-18 LCR in HeLa cells was as described elsewhere [7]
Sequence variability of the long control region and transcriptional activity of different human papillomavirus type 18 (HPV-18) molecular variants
Sequence variability of the long control region and transcriptional activity of different human papillomavirus type 18 (HPV-18) molecular variants
Comparison of promoter activitiesFirst, we compared the transcriptional activities of the HPV-16 and -18 main early promoters (P97 and P105, respectively). A 12-fold higher transcription activity was obtained from the P105 promoter (mean of triplicates, 932,099 relative luciferase units [RLUs]), compared with that of the P97 promoter (mean, 76,758 RLUs). We then compared the transcriptional activities of natural variants of HPV-18 detected in our study. In this analysis, the activity of the B18-2 LCR was arbitrarily defined as the reference, since this was the most prevalent HPV-18 European variant detected in our cohort study. Moreover, such European variants were suggested to have a decreased oncogenic potential relative to non-European variants. Transcriptional activities of all variants analyzed were 2.64–8.18 times greater than that for the B18-2 genotype (table 1). Overall, there was significant variability across molecular variants with respect to their transcriptional activities (P<.001), and, with the exception of the HeLa and the New-1 variants, all of the differences with respect to B18-2 were statistically significant. The most transcriptionally active isolate tested was B18-3, an Asian-Amerindian variant. Furthermore, this variant’s promoter was 43 times more transcriptionally active than the corresponding HPV-16 prototype promoter (data not shown)
The accumulated epidemiological and biological evidence indicates that HPV-16 and -18 are carcinogenic in humans. Nucleotide sequence comparison of HPV-positive samples collected worldwide suggests a correlation between viral diversity and ethnicity [2]. The Brazilian population has 3 ethnic origins: European, African, and indigenous American Indian. Not surprisingly, we have detected European, African, and Asian-Amerindian HPV-18 variants within this population (authors’ unpublished data and [4]). Within other admixed populations, the same variants have been observed [3]
HPV-16 and -18 account for ∼65% of all cervical cancer and are associated predominantly with squamous cell carcinoma and adenocarcinoma, respectively. The latter neoplasia has a more aggressive nature, is less differentiated, has a poor prognosis, and is also more frequently associated with cancer recurrence and lymph node metastasis [8]. It has been observed that HPV-18 is ∼10–50-fold more efficient in transforming keratinocytes than is HPV-16 [9] and that E6/E7 early genes of both HPV types immortalize primary human keratinocytes with the same efficiency when expressed from a heterologous promoter [10]. We observed that the HPV-18 prototype was more active than the HPV-16 prototype under our experimental conditions, which could, in part, explain not only the results obtained in the studies cited above but also the link between the detection of HPV-18 and the occurrence of adenocarcinoma [11]. With a few exceptions, the same cellular factors appear to interact with the LCR of both HPV types. However, the target sites for these factors are arranged differently in the regulatory regions of the 2 viruses. Furthermore, it has been suggested that this difference could be attributed to the fact that the binding of keratinocyte-specific transcriptional activator is specific to HPV-18 [5]
Epidemiologically, the oncogenicity of HPV-16 molecular variants appears to vary not only by geographic region, possibly because of the different prevalence of the molecular variants around the world, but also with the ethnic origin of the population under study. Several studies conducted in admixed populations, such as those of Brazil, Costa Rica, Mexico, and the United States, have confirmed the association between non-European variants of HPV-16 and -18 and risk of persistent infection and cervical lesions [3]. However, in populations from different parts of Europe, in which the majority of variants detected are European, no differences in oncogenic potential between variants from different phylogenetic branches are observed [12]. These results may have implications for ascertaining the risk of cervical lesions in different geographic regions. With regard to the analysis of genome fragments of HPV-18 samples, variations in the E2 gene of HPV-18 have indicated the existence of a subtype with decreased oncogenic potential [13]. Furthermore, the analysis of the LCR sequence variability among HPV-18–positive frozen biopsy specimens from Mexican patients suggested an association between specific variants and the histopathology of cervical disease [11,14]. However, other studies analyzing nucleotide variability in the LCR and in the E2 and E6/E7 genes found no correlation between HPV-18 genotypes and lesion grade [3, 7]
The highest P105 promoter activity was exhibited by the B18-3 Asian-Amerindian variant, the only isolate in which we detected a substitution at position 7528. However, we can attribute the increased activity to this position only if the reversion of this mutation leads to elimination of the increase. The B18-2 European variant, which had the lowest transcriptional activity among all variants tested, is identical to the SC18-2 variant, with the exception of the transition at position 7717. Nevertheless, the SC18-2 variant had a considerably higher activity. These functional results are consistent with our epidemiological observations that indicate a stronger association with cervical neoplasia for the non-European variants. Other studies conducted with HPV-16 isolates revealed not only similar promoter activities among European isolates and enhanced P97 promoter activity by non-European variants but also that not all LCR polymorphisms cause significant changes in promoter activity [15]. The oncogenic potential of non-European variants could also be influenced by polymorphisms in other viral regions. In fact, analysis of HPV-16 E6 natural variants revealed biological and biochemical differences [16]. The observed differences could also be attributed to differences in viral load. However, so far we have been unable to find any statistically significant correlation between a specific variant and copy number [4]
To our knowledge, this is the first report describing differences in promoter activity among naturally occurring variants of HPV-18. Research in this area is anticipated to provide important information concerning the biological significance of HPV-18 intratype genomic variability, which ultimately could be used to address the control of these infections
Acknowledgments
We are grateful to Anna Christina Sallim for the sequencing of the recombinant vectors and to Karina Ribeiro from Centro de Pesquisa e Tratamento do Hospital do Câncer for statistical analysis
