Less than 10 years after publication of the hypothesis of a papillomavirus etiology of cancer of the cervix (zur Hausen 1975, 1976, 1977), the DNAs of the first cervical cancer-associated human papillomavirus (HPV) types were cloned and characterized. These DNAs are regularly and frequently found in biopsies obtained from cervical cancer patients throughout the world (Durst et al. 1983; Boshart et al. 1984).

Molecular pathogenesis of cancer of the cervix and its causation by specific human papillomavirus types.

cis-Acting elements involved in E2 modulation of human papillomavirus type 16 (HPV-16) P97 promoter activity and HPV-18 P105 promoter activity were examined. In transfected primary human keratinocytes, each promoter had a basal activity that could be repressed by the bovine papillomavirus type 1 E2 gene product. Mutational analysis of the E2-binding sites in the long control region upstream of each promoter revealed that E2 repression was mediated through the E2-binding sites proximal to each promoter. In the context of a mutated E2-binding site at the promoter proximal position, the HPV-16 P97 and HPV-18 P105 promoters could be transactivated by E2. E2-mediated repression of HPV-18 P105 promoter activity was shown to be a transcriptional effect. The interaction of E2 with promoter-proximal E2-binding sites is likely to be important for the controlled expression of viral genes transcribed from the HPV-16 P97 promoter and the HPV-18 P105 promoter in infected human genital epithelial cells.

Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters.

Infection with genital human papillomaviruses (HPVs) is the primary cause of cervical cancer. The infection is widespread, and little is known about the secondary factors associated with progression from subclinical infection to invasive carcinoma. Here we report that HPV genomes are efficiently targeted in vivo by CpG methylation, a well-known mechanism of transcriptional repression. Indeed, it has been shown previously that in vitro-methylated HPV type 16 (HPV-16) DNA is transcriptionally repressed after transfection into cell cultures. By using a scan with the restriction enzyme McrBC, we observed a conserved profile of CpG hyper- and hypomethylation throughout the HPV-16 genomes of the tumor-derived cell lines SiHa and CaSki. Methylation is particularly high in genomic segments overlying the late genes, while the long control region (LCR) and the oncogenes are unmethylated in the single HPV-16 copy in SiHa cells. In 81 patients from two different cohorts, the LCR and the E6 gene of HPV-16 DNA were found to be hypermethylated in 52% of asymptomatic smears, 21.7% of precursor lesions, and 6.1% of invasive carcinomas. This suggests that neoplastic transformation may be suppressed by CpG methylation, while demethylation occurs as the cause of or concomitant with neoplastic progression. These prevalences of hyper- and hypomethylation also indicate that CpG methylation plays an important role in the papillomavirus life cycle, which takes place in asymptomatic infections and precursor lesions but not in carcinomas. Bisulfite modification revealed that in most of the HPV-16 genomes of CaSki cells and of asymptomatic patients, all 11 CpG dinucleotides that overlap with the enhancer and the promoter were methylated, while in SiHa cells and cervical lesions, the same 11 or a subset of CpGs remained unmethylated. Our report introduces papillomaviruses as models to study the mechanism of CpG methylation, opens research on the importance of this mechanism during the viral life cycle, and provides a marker relevant for the etiology and diagnosis of cervical cancer.

https://jvi.asm.org/content/jvi/77/11/6227.full.pdf

CpG Methylation of Human Papillomavirus Type 16 DNA in Cervical Cancer Cell Lines and in Clinical Specimens: Genomic Hypomethylation Correlates with Carcinogenic Progression

The HPV E6/E7 Oncogenes: Key Factors for Viral Carcinogenesis and Therapeutic Targets

The papillomavirus E2 protein plays a central role in the viral life cycle as it regulates both transcription and replication of the viral genome. In this study, we showed that transient expression of bovine papillomavirus type 1 or human papillomavirus type 18 (HPV18) E2 proteins in HeLa cells activated the transcriptional activity of p53 through at least two pathways. The first one involved the binding of E2 to its recognition elements located in the integrated viral P105 promoter. E2 binding consequently repressed transcription of the endogenous HPV18 E6 oncogene, whose product has been shown previously to promote p53 degradation. The second pathway did not require specific DNA binding by E2. Expression of E2 induced drastic physiological changes, as evidenced by a high level of cell death by apoptosis and G1 arrest. Overexpression of a p53 trans-dominant-negative mutant abolished both E2-induced p53 transcriptional activation and E2-mediated G1 growth arrest, but showed no effect on E2-triggered apoptosis. These results suggest that the effects of E2 on cell cycle progression and cell death follow distinct pathways involving two different functions of p53.

/pdf/expression-of-the-papillomavirus-e2-protein-in-hela-cells-15d8g0rlp6.pdf

Expression of the papillomavirus E2 protein in HeLa cells leads to apoptosis.

The long control region of human genital papillomavirus type 18 harbors transcription regulatory elements, such as the E6 promoter and a cell type-specific enhancer independent of the E2 protein. By performing DNase I footprint experiments in vitro with protein extracts from different cell lines and tissues we searched for cellular factors interacting with the totality of the control region. We detected a total of eight different protected sites; most of them were found with all the extracts. Two of these sites, one in the enhancer and the other in the sequences proximal to the E6 cap site, interact with a member of the activator protein 1 family. However, in the absence of fine mutational analysis, we cannot readily discern an exact functional role for the different binding sites. We also characterized two regions, which are protected only in the presence of E2, corresponding to the perfect palindromes ACCGN4CGGT and present either 500 nucleotides or tandemly repeated about 70 nucleotides upstream of the E6 cap site. This last protected area covers a large part of the putative TATA box of the E6 promoter and could explain its repression by bovine papilloma virus type 1 E2, which could interfere with binding of the TFII D ubiquitous transcription factor to the TATA sequence of the promoter.

A Garcia-Carranca

Papers

Interplay of viral and cellular proteins along the long control region of human papillomavirus type 18.

type 18. long control region of human papillomavirus Interplay of viral and cellular proteins along the