The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells.

The contribution of the E6 and E7 open reading frames of human papillomavirus type 6b (HPV6b) and HPV16 to immortalization of human keratinocytes was evaluated by using amphotropic recombinant retroviruses. The HPV16 E7 gene could immortalize primary human keratinocytes without the cooperation of the viral E6 gene; however, E6 was able to contribute significantly to the efficiency of the E7 immortalizing function. Infection of HFE cells with retroviruses carrying the 16E6, 6bE6, or 6bE6E7 open reading frame did not result in immortalization.     

Regions of human papillomavirus type 16 E7 oncoprotein required for immortalization of human keratinocytes.

Binding of the retinoblastoma gene product (pRB) by viral oncoproteins, including the E7 of human papillomavirus type 16 (HPV 16), is thought to be important in transformation of cells. One of the steps in transformation is the immortalization process. Here we show that mutations in E7 within the full-length genome which inhibit binding of pRB do not abrogate the ability of the HPV 16 DNA to immortalize primary human epithelial (keratinocyte) cells. A mutation in one of the cysteines of a Cys-X-X-Cys motif which is contained in the carboxy half of the E7 and is part of a zinc finger arrangement completely eliminates the ability of HPV 16 DNA to immortalize cells. The results indicate the importance of E7 in the immortalization of primary keratinocytes but suggest that the binding of pRB is not essential.     

Mutant p53 can substitute for human papillomavirus type 16 E6 in immortalization of human keratinocytes but does not have E6-associated trans-activation or transforming activity.

Human papillomavirus type 16 (HPV16) E6 and E7 are selectively retained and expressed in HPV16-associated human genital tumors. E6 is active in several cell culture assays, including transformation of NIH 3T3 cells, trans activation of the adenovirus E2 promoter, and cooperation with E7 to immortalize normal human keratinocytes. Biochemically, the HPV16 E6 protein has been shown to bind to tumor suppressor protein p53 in vitro and induce its degradation in a rabbit reticulocyte lysate. To examine the relationship between the various biological activities of E6 and inactivation of p53, we tested the abilities of dominant negative mutants of p53 to substitute functionally for E6 in the three cell culture assays. While wild-type p53 inhibited keratinocyte proliferation, both mouse and human mutant p53s, in conjunction with E7, increased proliferation of the keratinocytes, resulting in generation of immortalized lines. However, in contrast to E6, mutant p53 was unable to induce transformation or trans activate the adenovirus E2 promoter in NIH 3T3 cells. These results suggest that inactivation of wild-type p53 is necessary for HPV-induced immortalization of human keratinocytes and that different or additional activities are required for E6-dependent transformation and trans activation of NIH 3T3 cells.     

Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38

The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter. Two distinct lines of HPV38 E6/E7-expressing transgenic mice that express the viral genes at different levels were obtained. In both lines, HPV38 E6 and E7 induced cellular proliferation, hyperplasia, and dysplasia in the epidermis. The rate of occurrence of these events was proportional to the levels of HPV38 E6 and E7 expression in the two transgenic lines. Exposure of the epidermis of nontransgenic mice to UV led to p21WAF1 accumulation and cell cycle arrest. In contrast, keratinocytes from transgenic mice continued to proliferate and were not positive for p21WAF1, indicating that cell cycle checkpoints are altered in keratinocytes expressing the viral genes. Although the HPV38 E6/E7-expressing transgenic mice did not develop spontaneous tumors during their life span, two-stage carcinogen treatment led to a high incidence of papillomas, keratoacanthomas, and squamous-cell carcinomas in HPV38 mice compared with nontransgenic animals. Together, these data show that HPV38 E6 and E7 display transforming properties in vivo, providing further support for the role of HPV38 in carcinogenesis.     

Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells.

We have shown previously that introduction of the human papillomavirus type 16 (HPV16) or HPV18 genome into human mammary epithelial cells induces their immortalization. These immortalized cells have reduced growth factor requirements. We report here that transfection with a single HPV16 gene E6 is sufficient to immortalize these cells and reduce their growth factor requirements. The RB protein is normal in these cells, but the p53 protein is sharply reduced, as shown by immunoprecipitation with anti-p53 antibody (pAB 421). We infer that the E6 protein reduces the p53 protein perhaps by signalling its destruction by the ubiquitin system. The HPV-transforming gene E7 was unable to immortalize human mammary epithelial cells. Thus, cell-specific factors may determine which viral oncogene plays a major role in oncogenesis.     

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.     

The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes.

The early human papillomavirus type 16 genes that directly participate in the in vitro transformation of primary human keratinocytes have been defined. In the context of the full viral genome, mutations in either the E6 or E7 open reading frame completely abrogated transformation of these cells. Mutations in the E1, E2, and E2-E4 open reading frames, on the other hand, had no effect. Thus, both the full-length E6 and E7 genes were required for the induction of keratinocyte immortalization and resistance to terminal differentiation. The E6 and E7 genes expressed together from the human beta-actin promoter were sufficient for this transformation; mutation of either gene in the context of this recombinant plasmid eliminated the ability to induce stable differentiation-resistant transformants.     

The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.

Previous studies have shown that the E7 gene of human papillomavirus (HPV) type 16 or 18 alone was sufficient for immortalization of human foreskin epithelial cells (HFE) and that the efficiency was increased in cooperation with the respective E6 gene, whereas the HPV6 E6 or E7 gene was not active in HFE. To detect weak immortalizing activities of the HPV6 genes, cells were infected with recombinant retroviruses containing HPV genes, alone and in homologous and heterologous combinations. The HPV6 genes, alone or together (HPV6 E6 plus HPV6 E7), were not able to immortalize cells. However the HPV6 E6 gene, in concert with HPV16 E7, increased the frequency of immortalization threefold over that obtained with HPV16 E7 alone. Interestingly, 6 of 20 clones containing the HPV16 E6 gene and the HPV6 E7 gene were immortalized, whereas neither gene alone was sufficient. Thus, the HPV6 E6 and E7 genes have weak immortalizing activities which can be detected in cooperation with the more active transforming genes of HPV16. Acute expression of the HPV6 and HPV16 E6 and E7 genes revealed that only HPV16 E7 was able to stimulate the proliferation of cells in organotypic culture, resulting in increased expression of the proliferative cell nuclear antigen and the formation of a disorganized epithelial layer. Additionally, combinations of genes that immortalized HFE cells (HPV16 E6 plus HPV16 E7, HPV16 E6 plus HPV6 E7, and HPV6 E6 plus HPV16 E7) also stimulated proliferation.     

人乳头瘤病毒16型长控制区上YY1结合位点的破坏可增强病毒诱导细胞永生化能力

细胞转录调节因子 Ｙ Ｙ１ 可抑制人乳头瘤病毒１６ 型（ Ｈ Ｐ Ｖ １６） 癌基因启动子 Ｐ９７ 的活性, Ｙ Ｙ１ 位点的突变和缺失不仅可诱导 Ｐ９７ 活性增强而且可在全基因组内增强 Ｅ６ 癌基因转录,同时使病毒对啮齿类动物纤维细胞的转化能力增强。为了观测人乳头瘤病毒１６ 型长控制区（ Ｈ Ｐ Ｖ１６ Ｌ Ｃ Ｒ） 序列上 Ｙ Ｙ１ 蛋白特异性结合位点破坏在完整基因组范围内对人原代包皮角源细胞永生化能力的影响,将 Ｈ Ｐ Ｖ １６ Ｙ Ｙ１ 位点突变株和野毒株转染至人原代包皮角源细胞。筛选结果表明,突变株可诱导形成永生化细胞,永生化能力明显高于野毒株。对４ 株永生化细胞系 Ｄ Ｎ Ａ检测发现,均含有呈整合状态的 Ｈ Ｐ Ｖ １６ Ｄ Ｎ Ａ,其中３ 株的 Ｅ１／ Ｅ２ 区域有缺失。 Ｒ Ｎ Ａ 检测显示,４株细胞内均有 Ｅ６／ Ｅ７ ｍ Ｒ Ｎ Ａ 的转录。这表明, Ｈ Ｐ Ｖ １６ Ｌ Ｃ Ｒ 上 Ｙ Ｙ１ 蛋白特异性结合位点的破坏,可在完整基因组范围内增强病毒使人原代包皮角源细胞永生化的能力。     

Cutaneous human papillomavirus type 38 E7 regulates actin cytoskeleton structure for increasing cell proliferation through CK2 and the eukaryotic elongation factor 1A

We previously reported that the oncoproteins E6 and E7 from cutaneous human papillomavirus type 38 (HPV38) can immortalize primary human keratinocytes in vitro and sensitize transgenic mice to develop skin cancer in vivo. Immunofluorescence staining revealed that human keratinocytes immortalized by HPV38 E6 and E7 display fewer actin stress fibers than do control primary keratinocyte cells, raising the possibility of a role of the viral oncoproteins in the remodeling of the actin cytoskeleton. In this study, we show that HPV38 E7 induces actin stress fiber disruption and that this phenomenon correlates with its ability to downregulate Rho activity. The downregulation of Rho activity by HPV38 E7 is mediated through the activation of the CK2-MEK-extracellular signal-regulated kinase (ERK) pathway. In addition, HPV38 E7 is able to induce actin fiber disruption by binding directly to eukaryotic elongation factor 1A (eEF1A) and abolishing its effects on actin fiber formation. Finally, we found that the downregulation of Rho activity by HPV38 E7 through the CK2-MEK-ERK pathway facilitates cell growth proliferation. Taken together, our data support the conclusion that HPV38 E7 promotes keratinocyte proliferation in part by negatively regulating actin cytoskeleton fiber formation through the CK2-MEK-ERK-Rho pathway and by binding to eEF1A and inhibiting its effects on actin cytoskeleton remodeling.     

The E6 Oncoproteins of High-Risk Papillomaviruses Bind to a Novel Putative GAP Protein, E6TP1, and Target It for Degradation

The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high-risk HPV E6 protein to immortalize human mammary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation. In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization. However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein. Using the yeast two-hybrid system, we have isolated a novel E6-binding protein. This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP. The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway.     

Serum- and calcium-induced differentiation of human keratinocytes is inhibited by the E6 oncoprotein of human papillomavirus type 16.

Transfection of the E6 and E7 genes of the high-risk human papillomaviruses (HPVs) into primary genital keratinocytes generates colonies of proliferating cells which are resistant to calcium- and serum-induced terminal differentiation. To genetically map the HPV gene(s) responsible for this cellular phenotype, we cloned cDNAs for full-length E6, full-length E7, four truncated E6 splice variants (E6I to E6IV), and a series of E6 C-terminal truncation mutants into a simian virus 40 expression vector. The E6 proteins were tagged with the AU1 epitope at the C terminus to verify their expression in COS cells by immunoprecipitation and immunofluorescence. Transfection of the full-length E6 protein, either wild type or epitope tagged, induced calcium- and serum-resistant keratinocyte colonies, but the truncated E6 variants and full-length E7 protein did not. E6-induced colonies, while altered in response to serum and calcium, could not be established into cell lines without the combined presence of the E7 protein, further exemplifying the independent roles of E6 and E7 in cell differentiation and cell proliferation. The E6 C-terminal deletion mutants defined two distinct functional domains between amino acids 120 and 151. Amino acids 120 to 151 contained an apparent bipartite nuclear localization signal, whereas amino acids 132 to 141 were required for the induction of resistance to calcium- and serum-induced differentiation and for immortalization of human keratinocytes in conjunction with E7.