Analyse fonctionnelle des polymorphismes du virus du papillome humain de type 33

The infection by the oncogenic human papillomavirus (HR-HPV) is associated with the development of cervical cancer. The expression of the E6 and E7 viral oncogenes favors progression towards cancer and is driven by the long control region (LCR) which contains binding sites for both cellular factors and the viral E2 protein. The expression of these oncogenes is often upregulated in cervical cancer due to the integration of the viral genome which frequently disrupts the open reading frame of the repressor E2 gene. Several isolates or variants of HPV type 33 (HPV33) with a slightly different nucleotide sequence were previously identified in clinical samples from women infected with HPV33 and presenting squamous intraepithelial lesions of the uterus. Two polymorphisms, a 79-bp deletion and the C7732G transversion, were previously associated with an increased risk of persistence of infection and of developing high-grade lesions, respectively. The functional effect of polymorphisms in the HPV33 LCR on viral gene expression has never been assessed in particular for the 79-bp deletion and C7732G. In the first study presented here, the transcriptional activity of the HPV33 LCR variants were compared to that of the prototype in C33A and HeLa cell lines using gene reporter assays following their introduction upstream of the Renilla luciferase. Our results indicate that the LCR activity of the variants reflects their phylogenetic classification. As such, variants from the A2-sublineage were the most active in C33A cells when compared to the A1-sublineage prototype. Mutational analysis showed that the increased in LCR activity was the result of several weakly-acting variations, including the 79-bp deletion and C7732G. In HeLa cells, the lower transcriptional activity of the A2 variants was caused by A7879G which induces repression of the LCR in these cells. These results show that the combination of weakly-acting variations in the LCR significantly alters viral gene expression.  In the second study, we show that the mutation of important regulatory elements also significantly enhances the transcriptional activity of the LCR. The rare HPV33 LCR10 variant from the B lineage exhibits increased transcriptional activity compared to the prototype. Mutational analysis shows that the T7791C variation is responsible for this phenotype. The stimulating effect of this variation is caused by the disruption of a repressor C/EBPβ site as determined in an in vitro DNA-binding assay and gene reporter assays. A second site necessary this time for the activation of the LCR was also identified.  Finally, in the third study, we show that C/EBPβ is a common regulator of the oncogenic HPV gene expression. Several C/EBPβ sites were found in the enhancer region of the LCR of these HPV types as determined by our prediction using both energy- and frequency-based matrices. Some of these sites were found to be similar to the repressor and activating sites in the HPV33 LCR. Taken together, our studies highlight the importance of characterizing individual variations in the LCR in order to improve our knowledge on the mechanisms involved in the regulation of viral gene expression. We also show that the activating and inhibitory functions of C/EBPβ are important for the modulation of viral transcription.

Keyphrases: LCR, C/EBPb, HPV33, variant, polymorphism, fluorescence polarization, energy-based matrix, frequency-based matrix