HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes.

The human papillomavirus types (HPVs) most often associated with cancer of the cervix, such as HPV16, have been reported previously to immortalize normal human foreskin keratinocytes in vitro, while the types that are primarily associated with benign cervical lesions failed to do so. In this study we have determined the HPV16 genes that are responsible for the immortalizing activity of the viral genome. Transfection with a plasmid in which E6 and E7 were the only intact open reading frames (ORFs) induced an indefinite life-span in the keratinocytes with an efficiency similar to that of the entire early region of the viral DNA. Mutants in the E6E7 clone with inactivating lesions in E6 or E7 failed to induce immortalization. When transfected alone, E7 could induce hyperproliferation, but these cells eventually senesced. By itself, E6 exhibited no activity, Co-transfection of a plasmid with an intact E6 ORF and a second plasmid with an intact E7 ORF generated keratinocyte lines with indefinite growth potential. The E6 and E7 proteins were detected in the lines induced by the E6E7 DNA and by co-transfection of the E6 and E7 plasmids. Therefore, we conclude that HPV16 E6 and E7 cooperative to immortalize human keratinocytes in vitro. Changes in cellular gene expression are probably also required for immortalization since all of the keratinocyte lines examined were aneuploid. Serum and calcium resistant sublines were isolated from the E6E7 induced lines, indicating that other HPV genes do not play an obligatory role in the generation of resistance to differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16.

To examine the biological properties of the bovine papillomavirus type 1 (BPV) and human papillomavirus type 16 (HPV16) E5 genes, each was cloned separately into a retroviral expression vector and helper-free recombinant viruses were generated in packaging cell lines. The BPV E5 retroviruses efficiently caused morphologic and tumorigenic transformation of cultured lines of murine fibroblasts, whereas the HPV16 E5 viruses were inactive in these assays. In contrast, infection of the p117 established line of murine epidermal keratinocytes with either the BPV or the HPV16 E5 retrovirus resulted in the generation of tumorigenic cells. Pam212 murine keratinocytes were also transformed to tumorigenicity by the HPV16 E5 gene but not by the gene carrying a frameshift mutation. These results establish that the HPV16 E5 gene is a transforming gene in cells related to its normal host epithelial cells.     

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.     

E6 and E7 from human papillomavirus type 16 cooperate to target the PDZ protein Na/H exchange regulatory factor 1

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na(+)/H(+) exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3'-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.     

Mutational analysis of human papillomavirus type 16 E7 functions.

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.     

Immortalization of fetal mouse brain glial cells by human papillomavirus type 16 E7 genes.

Fetal mouse brain glial cells in primary cultures were immortalized by recombinant retroviruses containing human papillomavirus type 16 E7 genes, and named VR-2g cells. The presence and expression of E7 genes in VR-2g cells were demonstrated by the Southern and the Northern blot analyses. VR-2g cells did not form colonies in soft agar culture, indicating that VR-2g cells have no transforming phenotypes. By the karyotype analysis, VR-2g cells consisted of two cell populations, the pseudo-diploid and the pseudo-tetraploid. VR-2g cells were positive in immunostaining with mono- and polyclonal antibodies against glial fibrillary acidic proteins (glial-specific intermediate filaments). In addition, VR-2g cells secreted neurotrophic factors as assayed with primary cultures of fetal rat striatum neurons, although molecular characterization of the factor(s) was not yet determined. These results indicate that the present method for cell immortalization will be useful for establishing untransformed cell lines from primary cultures of fetal brain cells.     

Immortalization of human urothelial cells by human papillomavirus type 16 E6 and E7 genes in a defined serum-free system

Normal human epithelial cell cultures exhibit a limited (although different between tissues) lifespan in vitro. In previous studies, urothelial cell cultures were immortalized using retroviral transformation with human papillomavirus type 16 E6 and E7 genes, in undefined culture systems containing serum or bovine pituitary extract. OBJECTIVE: Due to the variability of results in such systems, we instead developed a procedure for the immortalization of urothelial cells using a defined, serum-free culture system. METHOD AND RESULTS: Immortalization through retroviral transformation with human papillomavirus type 16 E6 and E7 was successful, and transformation of urothelial cells conferred an extended over normal lifespan and restored telomerase activity. Transformed cells retained typical morphology and exhibited a similar growth rate, cytokeratin immunoreactivity pattern, and response to growth factors as observed in untransformed cells. Karyotype analysis revealed a gradual accumulation of genetic mutations that are consistent with previously reported mutations in epithelial cells transformed with human papillomavirus type 16 E6 and E7. CONCLUSION: The ability to extend the in vitro lifespan of cells holds the potential to reduce the continuous need for tissue samples and to enable complete investigations with one cell line.     

Human papillomavirus type 16 E6-induced degradation of E6TP1 correlates with its ability to immortalize human mammary epithelial cells

Recent analyses have identified a number of binding partners for E6, including E6AP, ERC55, paxillin, hDlg, p300, interferon regulatory factor 3, hMCM7, Bak, and E6TP1. Notably, association with E6 targets p53, E6TP1, myc, hMCM7, and Bak for degradation. However, the relative importance of the various E6 targets in cellular transformation remains unclear. E6 alone can dominantly immortalize normal human mammary epithelial cells (MECs), permitting an assessment of the importance of various E6 targets in cellular transformation. Studies in this system indicate that E6-induced degradation of p53 and E6 binding to ERC55 or hDlg do not correlate with efficient immortalization. Here, we have examined the role of E6TP1, a Rap GTPase-activating protein, in E6-induced immortalization of MECs. We tested a large set of human papillomavirus type 16 E6 mutants for their ability to bind and target E6TP1 for degradation in vitro and in vivo. We observed a strict correlation between the ability of E6 protein to target E6TP1 for degradation and its ability to immortalize MECs. Recent studies have identified telomerase as a target of E6 protein. Previous analyses of E6 mutants have revealed this trait to closely correlate with MEC immortalization. We examined our entire panel of E6 mutants for rapid induction of telomerase activity and found in general a strong correlation with immortalizing ability. The tight correlation between E6TP1 degradation and MEC immortalization strongly supports a critical role of functional inactivation of E6TP1 in E6-induced cellular immortalization.     

Inhibition of growth, transformation, and expression of human papillomavirus type 16 E7 in human keratinocytes by alpha interferons.

We used a model system of normal human keratinocytes (HKc) and HKc immortalized with human papillomavirus type 16 DNA (HKc/HPV16) to investigate the effects of alpha interferons (IFN-alpha) on the growth of HPV16-immortalized human epithelial cells, on HPV16-mediated immortalization of normal HKc, and on HPV16 gene expression. Normal HKc and HKc/HPV16 were treated with several recombinant human IFN-alpha subtypes (IFN-alpha B, IFN-alpha D, and IFN-alpha B/D). These IFN-alpha subtypes inhibited proliferation of both normal HKc and HKc/HPV16 in a dose-dependent fashion; however, although 1,000 to 10,000 U of IFN-alpha per ml were required to inhibit growth of normal HKc, HKc/HPV16 were substantially growth inhibited by 100 U/ml. In addition, 100 U of IFN-alpha B/D per ml inhibited transformation of normal HKc by HPV16 DNA. Northern (RNA) blot analysis showed no effect of IFN-alpha on the mRNA levels of the HPV16 E6 and E7 open reading frames. However, immunofluorescence studies of the HPV16 E6 and E7 proteins with anti-E6 and anti-E7 monoclonal antibodies showed significant inhibition of E7 protein expression in cells treated with IFN-alpha, whereas E6 protein expression was not altered. The inhibition of E7 protein expression in cells treated with IFN-alpha was further confirmed by Western immunoblot analysis. These results suggest that IFN-alpha may inhibit HPV16-mediated transformation of HKc and proliferation of HKc/HPV16 through an inhibition of HPV16 E7 protein expression.     

Production of human papillomavirus type 16 E7 protein in Lactococcus lactis

The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.     

Human papillomavirus type 16 E6 and HPV-16 E6/E7 sensitize human keratinocytes to apoptosis induced by chemotherapeutic agents: roles of p53 and caspase activation

We and others have previously reported that human papillomavirus (HPV)-16 E6 protein expression sensitizes certain cell types to apoptosis. To confirm that this sensitization occurred in HPV's natural host cells, and to explore the mechanism(s) of sensitization, we infected human keratinocytes (HKCs) with retroviruses containing HPV-6 E6, HPV-16 E6, HPV-16 E7, or HPV-16 E6/E7. Apoptosis was monitored by DNA fragmentation gel analysis and direct observation of nuclei in cells stained with DAPI. Exposure of HKCs to etoposide, cisplatin, mitomycin C (MMC), atractyloside, and sodium butyrate, resulted in a time and dose-dependent induction of apoptosis. Expression of HPV-16 E6 and HPV-16 E6/E7, but not HPV-6 E6 or HPV-16 E7, enhanced the sensitivity of HKCs to cisplatin-, etoposide- and MMC-, but not atractyloside- or sodium butyrate-induced apoptosis. Expression of both HPV-16 E6 and HPV-16 E6/E7 decreased, but did not abolish, p53 protein levels relative to normal HKCs, and resulted in cytoplasmic localization of wt p53. P53 induction occurred in HPV-16 E6 and HPV-16 E6/E7 expressing cells after exposure to cisplatin or MMC, though never to levels found in normal untreated HKCs. P21 levels were decreased in HPV-16 E6 and HPV-16 E6/E7 expressing HKCs, and no induction of p21 was seen in these cells following exposure to cisplatin or MMC. Caspase-3 activity was found to be elevated in HPV-16 E6-expressing HKCs following exposure to cisplatin and MMC as documented by fluorometric and Western Blot analysis. Expression of wt CrmA or treatment of HPV-16 E6 expressing HKCs with the caspase-3 inhibitor DEVD.fmk prevented HPV-16 E6-induced sensitization in HKCs. These results suggest that HPV-16 E6 and HPV-16 E6/E7 expression sensitizes HKCs to apoptosis caused by cisplatin, etoposide and MMC, but not atractyloside or sodium butyrate. The data also suggest that wt p53 and caspase-3 activity are required for HPV-16 E6 and HPV-16 E6/E7-induced sensitization of HKCs to DNA damaging agents.     

Alternative fates of keratinocytes transduced by human papillomavirus type 18 E7 during squamous differentiation

The human papillomavirus type 18 (HPV-18) E7 protein promotes S-phase reentry in postmitotic, differentiated keratinocytes in squamous epithelium to facilitate vegetative viral DNA amplification. To examine the nature and fate of the differentiated cells that reenter S phase, organotypic cultures of primary human keratinocytes transduced with HPV-18 E7 were pulse-chase-pulse-labeled with (3)H-thymidine ((3)H-TdR) and bromodeoxyuridine (BrdU). The kinetics of the appearance of doubly labeled suprabasal cells demonstrate that E7 expression did not promote prolonged S phase. Rather, there was a considerable lag before a small percentage of the cells reentered another round of S phase. Fluorescence in situ hybridization analysis, indeed, revealed a small fraction of the cells with more than 4n chromosomes in the differentiated strata. Differentiated cells positive for (3)H-TdR, BrdU, or both often had enlarged nuclei or were binucleated. These results suggest that S phase is not followed by cell division, although nuclear division may occur. Interestingly, a significant fraction of differentiated cells that entered S phase subsequently accumulated p27kip1 protein with a kinetics preceding the accumulation of cyclin E. We conclude that E7-transduced, differentiated keratinocytes that enter S phase have two alternative fates: (i) a low percentage of cells undergoes endoreduplication, achieving higher than 4n ploidy, and (ii) a high percentage of cells accumulates the p27kip1, cyclin E, and p21cip1 proteins, resulting in arrest and preventing further S-phase reentry.     

Casein kinase II activities related to hyperphosphorylation of human papillomavirus type 16-E7 oncoprotein in epidermal keratinocytes

The present study describes hyper-phosphorylation of E7-oncoprotein of human papillomavirus (HPV) type 16 in epidermal keratinocytes. We found that highly phosphorylated E7-oncoprotein was present in epidermal keratinocytes but little in fibroblasts. The E7 oncoprotein contains serine residues (Ser-Ser-Glu-Glu-Glu) capable of being phosphorylated by casein kinase II (CK II). Extracts from various cell lines including human origins transformed by HPV 16 were examined for the casein kinase activity. The results showed that CK II activity was present at significantly high levels in keratinocytes but little or no detectable levels of the activity in human fibroblasts. These differential CK II activities in host cells may play a part in the differential transforming activity by E7-oncoprotein.     

A point mutational analysis of human papillomavirus type 16 E7 protein.

The E7 open reading frame of human papillomavirus type 16 (HPV16) has been shown to be selectively retained in cervical tumors and to encode both transforming and trans-activating functions in murine cells, supporting the notion that expression of E7 contributes towards the progression of premalignant cervical lesions. A comparison among E7 sequences of different HPV types reveals some homology at the amino acid level. Of particular interest are two regions, one which contains significant homology to a region of adenovirus E1a and simian virus 40 large T (LT), and a second region which contains two conserved Cys-X-X-Cys motifs. To determine the importance of these domains to the function of the E7 protein, a series of mutants carrying substitutions at amino acids in the region of E1a-LT homology and at the Cys-X-X-Cys motifs were constructed. The mutated E7 sequences were placed under the control of a strong heterologous promoter (Moloney long terminal repeat), and the activity of the mutants was assayed in NIH 3T3 cells, a cell line in which both the transforming function and the trans-activating function of E7 could be determined. A single amino acid substitution analogous to a mutation in E1a which destroys the transforming ability of this protein abolished both transformation and trans-activation by E7. Mutations at the Cys-X-X-Cys motifs demonstrated that this region contributes to the transforming potential of E7, although proteins in which both motifs were interrupted retained a low level of transforming activity. Mutations in the region of E1a-LT homology which occur within a recognition sequence for casein kinase II did not markedly affect transforming activity of E7 but severely reduced trans-activating ability. This indicates that efficient trans-activation is not required for transformation by HPV16 E7 in these cells.