Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18.

The E6-E7 region of human papillomavirus types 16 and 18 is selectively retained and expressed in cervical carcinoma cells. In cultured human keratinocytes, expression of the E6 and E7 open reading frames of human papillomavirus type 18, under the control of its homologous promoter, resulted in high-frequency immortalization. Furthermore, by using a system that allows for stratification of keratinocytes in vitro (raft system), we observed that the morphological differentiation of these E6-E7 immortalized cells was altered such that parabasal cells extended throughout most of the epithelium, with abnormal nuclei present in the upper regions. Examination of E6-E7-expressing cell lines in the raft system at a later passage revealed that complete loss of morphological differentiation had occurred. E7 alone was a much less effective immortalizing agent than E6 and E7 together and acted only minimally to alter morphological differentiation in vitro. No such activities were found for E6 alone. High-frequency transformation of human epithelial cells thus appears to require expression of both E6 and E7 gene products.     

Identification of B-epitopes in the human papillomavirus 18 E7 open reading frame protein.

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.     

Expression of human papillomavirus type 6 E1, E2, L1 and L2 open reading frames in Escherichia coli

Open reading frame (ORF) fragments (putative gene fragments) from human papillomavirus type 6b (HPV-6b) were inserted into the bacterial expression vector pHK413 to provide viral antigenic determinants. Approximately 86% of the entire L1 ORF, 82% of the E2 ORF, and 52% of the L2 ORF were expressed in Escherichia coli. The E1 ORF was cloned as two fragments. The constructions containing E1n (coding for the N-terminal region) and E1c (coding for the C-terminal region) expressed 27% and 16% of the E1 ORF, respectively. Protein encoded by the L1 ORF, but not that encoded by the L2 ORF, reacted with antibodies elicited by disrupted bovine papillomavirus. These reagents will be extremely useful in unravelling the HPV-6b replication cycle.     

Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells.

Human papillomavirus type 16 (HPV 16) DNA is capable of morphologically transforming rat 3Y1 cells. The expression plasmids, constructed from the simian virus 40-based expression vector pSV2-0 and specific DNA fragments from the putative early region of the HPV 16 genome, were tested for their transforming capacity. Among the various pSV2 plasmids, only those containing the intact E7 coding region were found to produce foci of the transformed rat cells which could grow in a soft-agar medium. The data indicate that expression of the HPV 16 E7 open reading frame is sufficient to induce focal transformation of rat cells.     

Lack of canonical E6 and E7 open reading frames in bird papillomaviruses: Fringilla coelebs papillomavirus and Psittacus erithacus timneh papillomavirus

Determination and analyses of the complete sequence of Fringilla coelebs papillomavirus and Psittacus erithacus timneh papillomavirus indicate that they represent a distinct and distant lineage of papillomaviruses. The lack of canonical E6-E7 open reading frames suggests that they serve adaptive functions during papillomavirus evolution.     

RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells

The human papillomavirus oncoproteins E6 and E7 promote cell proliferation and contribute to carcinogenesis by interfering with the activities of cellular tumor suppressors. We used a small interfering RNA molecule targeting the E7 region of the bicistronic E6 and E7 mRNA to induce RNA interference, thereby reducing expression of E6 and E7 in HeLa cells. RNA interference of E6 and E7 also inhibited cellular DNA synthesis and induced morphological and biochemical changes characteristic of cellular senescence. These results demonstrate that reducing E6 and E7 expression is sufficient to cause HeLa cells to become senescent.     

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.     

Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers.

The accumulation of human papillomavirus type 16 (HPV-16)-specific RNAs in tissue sections from biopsies of patients with genital precancers was studied by in situ hybridization with single-stranded 35S-labeled RNA. These analyses revealed that the most abundant early-region RNAs were derived from the E4 and E5 open reading frames (ORFs). RNAs homologous to the E6/E7 ORFs were also detected, whereas RNAs homologous to the intervening E1 ORF were not. This suggests that the E4 and E5 mRNAs are derived by splicing to the upstream E6/E7 ORFs, consistent with studies of HPV-11 in condylomata (L. T. Chow et al., Cancer Cells (Cold Spring Harbor) 5:55-72, 1987). Abundant RNAs homologous to the 5' portion of L1 were also detected. These RNAs were localized to the apical strata of the epithelium. HPV-16 RNAs accumulated in discrete regions of these lesions, and when present were most abundant in the upper cell layers of the precancerous epithelium. RNAs homologous to early ORFs were also detected in some germinal cells within the basal layer of the epithelium.     

E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene.

We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low-molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not.     

Identification of the E5 open reading frame of human papillomavirus type 16.

Sequencing of the E5 open reading frame (ORF) of human papillomavirus type 16 revealed an additional nucleotide, a thymidine residue, at position 3903 compared with the original sequence (Seedorf et al., Virology 145:181-185, 1985). The additional T had two effects; first, in reading frame 2, in which the original E5 ORF was predicted, the additional T changed the reading frame downstream of position 3903 to create an ORF, which we designated E5, that terminated at position 4018 and potentially encoded a 52-amino-acid polypeptide. Secondly, in reading frame 3, a new ORF was created (positions 3807 to 4097), which we propose is the authentic papillomavirus type 16 E5 ORF. It contained a methionine residue and encoded an additional 82 amino acids. Both ORFs have been cloned into bacterial expression vectors (pATH), and the fusion proteins have been used to generate polyclonal antibodies in rabbits.     

HPV18 E7致癌蛋白的高效表达、纯化和鉴定

目的为进一步研究人乳头状瘤病毒18（Human papillomavirus18,HPV18）E7蛋白的结构与功能。方法构建HPV18 E7的谷胱甘肽S-转移酶融合蛋白质粒pGEX-6P-1-GST-HPV18 E7,重组质粒转入大肠埃希菌BL21进行可溶性融合蛋白的高效表达。结果柱上切除法去除GST标签,表达产物经glutathione Sepharose 4B亲和层析纯化,获得了SDS-PAGE和HPLC-ESI-MS纯度的HPV18 E7均质蛋白,非变性PAGE和凝胶过滤表明HPV18 E7以稳定的单体形式存在于水溶液中。高压液相色谱-电喷雾质谱（HPLC-ESI-MS）分析得到HPV18 E7精确分子量为12865．0 Da,与其理论值吻合。纯化蛋白经HPLC-ESI-MS／MS鉴定为目的产物,鉴定出的9个匹配肽段覆盖率为HPV18 E7整个氨基酸序列的96．5％。结论本文所建立的技术可以有效地大量制备HPV18 E7,为进一步研究其结构与功能和致癌机制奠定了重要的物质基础。     

Open reading frames E6 and E7 of bovine papillomavirus type 1 are both required for full transformation of mouse C127 cells.

A series of mutations in open reading frames (ORFs) E6 and E7 of bovine papillomavirus type 1 (BPV1) was constructed to analyze the roles of these ORFs in transformation of mouse C127 cells. The mutations were designed to prevent synthesis of specific proteins encoded by these genes. None of the mutations caused a decrease in the focus-forming activity of the full-length viral genome or in the ability of the viral DNA to replicate as a high-copy-number plasmid. Analysis of these mutants in the absence of a functional BPV1 E5 gene revealed a weak focus-forming activity encoded by ORF E6. Mutations preventing synthesis of the E6 protein did cause defects in anchorage-independent growth and tumorigenicity of transfected and transformed cells. However, a frameshift mutation between the first and second ATG codons of ORF E6 did not inhibit induction of colony formation, suggesting that translation from the first methionine codon is not required. Mutations that inactivated ORF E7 or E6/E7 individually did not inhibit induction of colony formation in agarose. However, a defect in this activity was caused by simultaneous disruption of both ORF E7 and ORF E6/E7 when they were expressed from the full-length viral genome but not when they were expressed under the control of a retrovirus long terminal repeat. These results suggest that translation of both ORF E6 and the 3' end of ORF E7 is required for efficient induction of anchorage-independent growth by the intact BPV1 genome.     

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.