Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.

The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes.

The E6 proteins of the high-risk human papillomaviruses (HPVs) have been shown to form a complex with and induce the degradation of human p53 in vitro. To determine whether p53 is degraded more rapidly in cells expressing E6 in vivo, the half-life of p53 was determined by pulse-chase analysis in early-passage normal human keratinocytes and fibroblasts, human keratinocytes immortalized with HPV type 16 (HPV16) E6 plus E7, and nonimmortal keratinocytes transfected with E6. The results of these experiments indicate that (i) the half-life of newly synthesized p53 is relatively long (4 h) in early-passage human keratinocytes and fibroblasts but short in keratinocytes expressing E6 (15 to 30 min), (ii) a similar increased rate of p53 degradation was measured in lines immortalized with HPV16 E6 plus E7 and senescent cells expressing E6, indicating that this increase is not simply the result of selection in the immortalized lines, and (iii) very low levels of expression of E6 result in a greatly decreased half-life of p53, suggesting that E6 acts in a catalytic manner.     

Involvement of human papillomavirus type 8 genes E6 and E7 in transformation and replication.

We investigated the transforming activity of human papillomavirus type 8 (HPV8) by expressing all early open reading frames from a heterologous promoter in different rodent fibroblast lines. Morphological transformation was observed only in G418-selected mouse C127 and Rat 1 cells containing an intact E6-coding region. E6 of HPV8 did not transform NIH 3T3 cells as did E6 of bovine papillomavirus type 1. C127 cells transformed by E6 were anchorage independent and had a reduced serum requirement but did not form tumors in nude mice. E7 of HPV8 showed no transforming potential, in contrast to E7 of HPV18 and HPV16. It was, however, able to complement an E7 mutant of bovine papillomavirus type 1 with a defect in high-copy-number DNA maintenance. The data indicate that the expression of the HPV8 E6 open reading frame is sufficient to induce morphological transformation in rodent fibroblasts, whereas E7 is involved in the replication of the viral DNA.     

Comparative analysis of transforming properties of E6 and E7 from different beta human papillomavirus types

The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.     

Degradation of p53, not telomerase activation, by E6 is required for bypass of crisis and immortalization by human papillomavirus type 16 E6/E7

Bypass of two arrest points is essential in the process of cellular immortalization, one of the components of the transformation process. Expression of human papillomavirus type 16 E6 and E7 together can escape both senescence and crisis, processes which normally limit the proliferative capacity of primary human keratinocytes. Crisis is thought to be mediated by telomere shortening. Because E6 stimulates telomerase activity and exogenous expression of the TERT gene with E7 can immortalize keratinocytes, this function is thought to be important for E6 to cooperate with E7 to bypass crisis. However, it has also been reported that E6 dissociates increased telomerase activity from maintenance of telomere length and that a dominant-negative p53 molecule can substitute for E6 in cooperative immortalization of keratinocytes with E7. Thus, to determine which functions of E6 are required to allow bypass of crisis and immortalization of keratinocytes with E7, immortalization assays were performed using specific mutants of E6, in tandem with E7. In these experiments, every clone expressing an E6 mutant capable of degrading p53 was able to bypass crisis and immortalize, regardless of telomerase induction. All clones containing E6 mutants incapable of degrading p53 died at crisis. These results suggest that the ability of E6 to induce degradation of p53 compensates for continued telomere shortening in E6/E7 cells and demonstrate that degradation of p53 is required for immortalization by E6/E7, while increased telomerase activity is dispensable.     

Human papillomavirus type 16 mutant E7 protein induces oncogenic transformation via up-regulation of cyclin A and cdc25A

A new mutant human papiUomavirus type 16 E7 gene, termed HPV16 HBE7, was isolated from cervical carcinoma biopsy samples from patients in an area with high incidence of cervical cancer (Hubei province, China). A previous study showed that the HPVI6 HBE7 protein was primarily cytoplasmic while wild-type HPV16 E7 protein, termed HPV16 WET, was concentrated in the nucleus. With the aim of studying the biological functions of HPV16 HBE7, the transforming potential of HPV16 HBE7 in NIH/3T3 cells was detected through observation of cell morphology, cell proliferation assay and anchorage-independent growth assay. The effect of HPVI6 HBE7 on cell cycle was examined by flow cytometry. Dual-luciferase reporter assay and RT-PCR were used to investigate the influence of HPVI6 HBE7 protein on the expression of regulation factors associated with GI/S checkpoint. The results showed that HPV16 HBE7 protein, as well as HPV16 WE7 protein, held transformation activity. NIH/3T3 cells expressing HPV16 HBE7 could easily transition from G1 phase into S phase and expressed high level of cyclin A and cdc25A. These results indicated HPV16 mutant E7 protein, located in the cytoplasm, induces oncogenic transformation of NIH/3T3 cells via up-regulation of cyclin A and cdc25A.     

Inactivation of p53 Rescues the Maintenance of High Risk HPV DNA Genomes Deficient in Expression of E6

The human papillomavirus DNA genome undergoes three distinct stages of replication: establishment, maintenance and amplification. We show that the HPV16 E6 protein is required for the maintenance of the HPV16 DNA genome as an extrachromosomal, nuclear plasmid in its natural host cell, the human keratinocyte. Based upon mutational analyses, inactivation of p53 by E6, but not necessarily E6-mediated degradation of p53, was found to correlate with the ability of E6 to support maintenance of the HPV16 genome as a nuclear plasmid. Inactivation of p53 with dominant negative p53 rescued the ability of HPV16 E6STOP and E6SAT mutant genomes to replicate as extrachromosomal genomes, though not to the same degree as observed for the HPV16 E6 wild-type (WT) genome. Inactivation of p53 also rescued the ability of HPV18 and HPV31 E6-deficient genomes to be maintained at copy numbers comparable to that of HPV18 and HPV31 E6WT genomes at early passages, though upon further passaging copy numbers for the HPV18 and 31 E6-deficient genomes lessened compared to that of the WT genomes. We conclude that inactivation of p53 is necessary for maintenance of HPV16 and for HPV18 and 31 to replicate at WT copy number, but that additional functions of E6 independent of inactivating p53 must also contribute to the maintenance of these genomes. Together these results suggest that re-activation of p53 may be a possible means for eradicating extrachromosomal HPV16, 18 or 31 genomes in the context of persistent infections.     

Transforming Activity of a Novel Mutant of HPV16 E6E7 Fusion Gene

An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintain...     

Transforming activity of a novel mutant of HPV16 E6E7 fusion gene

An optimized recombinant HPV16 E6E7 fusion gene(HPV16 ofE6E7)was constructed according to codon usage for mammalian cell expression,and a mutant of HPV16 ofE6E7 fusion gene(HPV16 omfE6E7)was generated by site-directed mutagenesis at L57G,C113R for the E6 protein and C24G,E26G for the E7 protein for HPV16 ofE6E7 [patent pending(CN 101100672)].The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice,but also maintained very good stability and antigenicity.These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors.     

Conservation of E6 and E7 regions of human papillomavirus types 16 and 18 present in cervical cancers

The E6 and E7 regions of human papillomavirus (HPV) type 16 were present in the DNA samples from cervical cancer cell lines, SKG-IIIa and SKG-IIIb, and those from cervical cancer tissues of three different patients. T601 cells, an NIH3T3 transformant obtained by transfection of DNA from a surgical specimen of a cervical cancer, also contained the E6 and E7 regions. The E6 region of HPV type 16 was expressed as mRNA in SKG-IIIa, SKG-IIIb and T601 cells. The E6 and E7 regions of HPV type 18 were present in the DNA samples from cervical cancer cell lines, SKG-I and SKG-II, and those from cervical cancer tissues of two different patients. SKG-I and SKG-II cells expressed the E6 region of HPV type 18 as mRNAs. These results strongly suggest that the E6 and E7 regions or the sequence surrounding these regions are important for maintaining malignant phenotype of cervical cancer cells.     

Genetic analysis of high-risk e6 in episomal maintenance of human papillomavirus genomes in primary human keratinocytes

Papillomaviruses possess small DNA genomes that encode five early (E) proteins. Transient DNA replication requires activities of the E1 and E2 proteins and a DNA segment containing their binding sites. The E6 and E7 proteins of cancer-associated human papillomavirus (HPV) transform cells in culture. Recent reports have shown that E6 and E7 are necessary for episomal maintenance of HPV in primary keratinocytes. The functions of E6 necessary for viral replication have not been determined, and to address this question we used a recently developed transfection system based on HPV31. To utilize a series of HPV16 E6 mutations, HPV31 E6 was replaced by its HPV16 counterpart. This chimeric genome was competent for both transient and stable replication in keratinocytes. Four HPV16 E6 mutations that do not stimulate p53 degradation were unable to support stable viral replication, suggesting this activity may be necessary for episomal maintenance. E7 has also been shown to be essential for episomal maintenance of the HPV31 genome. A point mutation in the Rb binding motif of HPV E7 has been reported to render HPV31 unable to stably replicate. Interestingly, HPV31 genomes harboring two of the three p53 degradation-defective E6 mutations combined with this E7 mutation were maintained as replicating episomes. These findings imply that the balance between E6 and E7 functions in infected cells is critical for episomal maintenance of high-risk HPV genomes. This model will be useful to dissect the activities of E6 and E7 necessary for viral DNA replication.