Splicing of a cap-proximal human Papillomavirus 16 E6E7 intron promotes E7 expression, but can be restrained by distance of the intron from its RNA 5' cap

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5' splice site and two alternative 3' splice sites, which produce E6(*)I and E6(*)II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6(*)I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector's exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5' cap, with an optimal distance of less than 307nt in order to facilitate better association of U1 small nuclear RNA with the intron 5' splice site. The same was true for splicing of human beta-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5' cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression.     

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.     

Cooperative transformation and coexpression of bovine papillomavirus type 1 E5 and E7 proteins

Productively infected bovine fibropapillomas were examined for bovine papillomavirus type 1 (BPV-1) E7 localization. BPV-1 E7 was observed in the cytoplasm of basal and lower spinous epithelial cells, coexpressed in the cytoplasm of basal cells with the E5 oncoprotein. E7 was also observed in nucleoli throughout the basal and spinous layers but not in the granular cell layer. Ectopic expression of E7 in cultured epithelial cells gave rise to localization similar to that seen in productive fibropapillomas, with cytoplasmic and nucleolar expression observed. Consistent with the coexpression of E7 and E5 in basal keratinocytes, BPV-1 E7 cooperated with E5 as well as E6 in an anchorage independence transformation assay. While E5 is expressed in both basal and superficial differentiating keratinocytes, BPV-1 E7 is only observed in basal and lower spinous epithelial cells. Therefore, BPV-1 E7 may serve to modulate the cellular response of basal epithelial cells to E5 expression.     

Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli beta-glucuronidase

BACKGROUND: Human papillomavirus type 16 (HPV16) E7 is an unstable oncoprotein with low immunogenicity. In previous work, we prepared the E7GGG gene containing point mutations resulting in substitution of three amino acids in the pRb-binding site of the HPV16 E7 protein. METHODS AND RESULTS: To increase E7GGG immunogenicity we constructed fusion genes of E. coli beta-glucuronidase (GUS) with one or three copies of E7GGG. Furthermore, a similar construct was prepared with partial E7GGG (E7GGGp, 41 amino acids from the N-terminus). The expression of the fusion genes was examined in human 293T cells. Quantification of GUS activity and the amount of E7 antigen showed substantially reduced GUS activity of fusion proteins with complete E7GGG that was mainly caused by decrease of their steady-state level in comparison with GUS or E7GGGpGUS. Still, the steady-state level of E7GGG.GUS was about 20-fold higher than that of the E7GGG protein. The immunogenicity of the fusion genes with complete E7GGG was tested by DNA immunisation of C57BL/6 mice with a gene gun. TC-1 cells and their clone TC-1/A9 with down-regulated MHC class I expression were subcutaneously (s.c.) inoculated to induce tumour formation. All mice were protected against challenge with TC-1 cells and most animals remained tumour-free in therapeutic-immunisation experiments with these cells, in contrast to immunisation with unfused E7GGG and the fusion with the lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1). Significant protection was also recorded against TC-1/A9 cells. Both tetramer staining and ELISPOT assay showed substantially higher activation of E7-specific CD8+ lymphocytes in comparison with E7GGG and Sig/E7GGG/LAMP-1. Deletion of 231 bp in the GUS gene eliminated enzymatic activity, but did not influence the immunogenicity of the E7GGG.GUS gene. CONCLUSIONS: The findings demonstrate the superior immunisation efficacy of the fusion genes of E7GGG with GUS when compared with E7GGG and Sig/E7GGG/LAMP-1. The E7GGG.GUS-based DNA vaccine might also be efficient against human tumour cells with reduced MHC class I expression.     

Relation between HPV-16 serology and clinico-pathological data in cervical carcinoma patients: prognostic value of anti-E6 and/or anti-E7 antibodies

 To investigate the clinical significance of the enhanced sensitivity of antibody detection by radio immunoprecipitation assays (RIPA), using in vitro translated HPV-16 E6 and E7 proteins, over synthetic-peptide enzyme-linked immunosorbent assay (ELISA), RIPA for HPV-16 E6 and E7 were performed. The results obtained with E6 and E7 RIPA were related to clinico-pathological data from cervical carcinoma patients positive for HPV type 16 DNA in their primary tumour. The data obtained with E6 and E7 RIPA were then compared to the results obtained using the E7/6-35 synthetic-peptide ELISA. The prevalence of antibodies to E6, E7, E6 and/or E7 and E6 and E7, as determined by RIPA, was significantly higher in cervical cancer patients than in both controls and cervical intraepithelial neoplasia patients. Odds ratios, calculated for cervical carcinoma patients versus controls, ranged from 7.4 to 15.4. Antibodies to E6 and/or E7 were largely restricted to patients with HPV DNA in their primary tumour. Analysis of the relation between prevalence of antibodies to E6 and E7 and clinico-pathological parameters was limited to 85 patients positive for HPV-16 DNA. The strongest relation with clinico-pathological data, such as lesion size, lymph node involvement, and prognosis, was found for E7 synthetic-peptide ELISA, whereas E6 and E7 RIPA did not reach significance. The significance of these findings is discussed. Received: 17 February 1997 / Accepted: 13 March 1997     

Initiation of DNA synthesis by human papillomavirus E7 oncoproteins is resistant to p21-mediated inhibition of cyclin E-cdk2 activity.

The E6 and E7 proteins from the high-risk human papillomaviruses (HPVs) bind and inactivate the tumor suppressor proteins p53 and Rb, respectively. In HPV-positive cells, expression of E6 proteins from high-risk types results in increased turnover of p53, which leads to an abrogation of p21-mediated G1/S arrest in response to DNA-damaging agents. In contrast, keratinocytes which express E7 alone have increased levels of p53 but, interestingly, also fail to undergo a G1/S arrest. We investigated the mechanism by which E7 bypasses this p21 arrest by using both keratinocytes which stably express E7 as well as U20S cells which stably or transiently express E7. We observed that E7 does not affect the induction of p21 synthesis by p53. While glutathione S-transferase (GST)-E7 bound a low level of in vitro-translated p21, we were unable to detect E7 and p21 in the same complex by GST-E7 binding assays or immunoprecipitations from cell extracts. Furthermore, E7 did not prevent p21-mediated inhibition of cyclin E kinase activity. In keratinocytes expressing E7, increased levels of p53, p21, and cyclin E, as well as increased cyclin E kinase activity, were observed. To determine if this increase in cyclin E activity was necessary for E7's ability to overcome p21-mediated G1/S arrest, we examined U20S cells in which cyclin E levels are not increased in response to E7 expression. U20S cells which stably express E7 were found to initiate DNA synthesis in the presence of DNA-damaging agents despite the inhibition of cyclin E activity by p21. In transient assays, cotransfection of E7 or E2F-1 along with p21 into U20S cells rescued G1 arrest and resulted in S-phase entry, as measured by the ability to incorporate bromodeoxyuridine. These data indicate that E7 is able to overcome G1/S arrest without directly affecting p21 function and likely acts through deregulation of E2F activity.     

Fusion of CTLA-4 with HPV16 E7 and E6 Enhanced the Potency of Therapeutic HPV DNA Vaccine

Preventive anti-HPV vaccines are effective against HPV infection but not against existing HPV-associated diseases, including cervical cancer and other malignant diseases. Therefore, the development of therapeutic vaccines is urgently needed. To improve anti-tumor effects of therapeutic vaccine, we fused cytotoxic T-lymphocyte antigen 4 (CTLA-4) with HPV16 E7 and E6 as a fusion therapeutic DNA vaccine (pCTLA4-E7E6). pCTLA4-E7E6 induced significantly higher anti-E7E6 specific antibodies and relatively stronger specific CTL responses than the nonfusion DNA vaccine pE7E6 in C57BL/6 mice bearing with TC-1 tumors. pCTLA4-E7E6 showed relatively stronger anti-tumor effects than pE7E6 in therapeutic immunization. These results suggest that fusing CTLA-4 with E7E6 is a useful strategy to develop therapeutic HPV DNA vaccines. In addition, fusing the C-terminal of E7 with the N-terminal of E6 impaired the functions of both E7 and E6.     

Induction of apoptosis in cervical carcinoma cells by peptide aptamers that bind to the HPV-16 E7 oncoprotein.

Human papillomaviruses (HPV) of the high-risk type are causally involved in human tumors, in particular cervical carcinoma. Expression of the viral oncogenes E6 and E7 is maintained in HPV-positive tumors, and it was shown that E6 and E7 of HPV-16 can immortalize human keratinocytes, the natural host cells of the virus. Expression of the viral genes is also required for maintenance of the transformed phenotype. The oncogenic activity of the E6 and E7 oncoproteins is mediated by their physical and functional interaction with cellular regulatory proteins. To knock out the function of the E7 protein in living cells, we have developed peptide aptamers with high specific binding activity for the E7 protein of HPV-16. We show here that E7-binding peptide aptamers induce programmed cell death (apoptosis) in E7-expressing cells, whereas E7-negative cells are not affected. Furthermore, E7-binding peptide aptamers induce apoptosis in HPV-16-positive tumor cells derived from cervical carcinoma. The data suggest that E7-binding peptide aptamers may be useful tools to specifically eliminate HPV-positive tumors.     

The Canine Papillomavirus and Gamma HPV E7 Proteins Use an Alternative Domain to Bind and Destabilize the Retinoblastoma Protein

The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb.     

RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53

The simultaneous expression of human papillomavirus type 16 (HPV16) E6 and E7 oncogenes is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether HPV16 E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a small interfering RNA (siRNA) targeting HPV16-E7 region to degrade either E6, or truncated E6 (E6*) and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. HPV16-transformed SiHa and CaSki cells were used as a model system; RT-PCR, Western Blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay and flow cytometry were applied to examine the effects of pSIREN-16E7. Our results indicated that HPV16-E7 specific shRNA (16E7-shRNA) induced selective degradation of E6 and E7 mRNAs and proteins. E6 silencing induced accumulation of cellular p53 and p21. In contrast, E7 silencing induced hypophosphorylation of retinoblastoma (Rb) protein. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death selectively in HPV-positive cancer cells, compared with the HPV-negative ones. We demonstrated that 16E7-shRNA can induce simultaneous E6 and E7 suppression and lead to striking apoptosis in HPV16-related cancer cells by activating cellular p53, p21 and Rb. Therefore, RNAi using E7 shRNA may have the gene-specific therapy potential for HPV16-related cancers.     

The papillomavirus E7 oncoprotein is ubiquitinated by UbcH7 and Cullin 1- and Skp2-containing E3 ligase

Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2(-/-) mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.     

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.     

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)     

DNA-damage response control of E2F7 and E2F8

Here, we report that the two recently identified E2F subunits, E2F7 and E2F8, are induced in cells treated with DNA-damaging agents where they have an important role in dictating the outcome of the DNA-damage response. The DNA-damage-dependent induction coincides with the binding of E2F7 and E2F8 to the promoters of certain E2F-responsive genes, most notably that of the E2F1 gene, in which E2F7 and E2F8 coexist in a DNA-binding complex. As a consequence, E2F7 and E2F8 repress E2F target genes, such as E2F1, and reducing the level of each subunit results in an increase in E2F1 expression and activity. Importantly, depletion of either E2F7 or E2F8 prevents the cell-cycle effects that occur in response to DNA damage. Thus, E2F7 and E2F8 act upstream of E2F1, and influence the ability of cells to undergo a DNA-damage response. E2F7 and E2F8, therefore, underpin the DNA-damage response.     

UV-vulnerability of human papilloma virus type-16 E7-expressing astrocytes is associated with mitochondrial membrane depolarization and caspase-3 activation

The human papilloma virus-type 16 (HPV-16) E6 and E7 proteins interact with the p53 and pRb tumor suppressor proteins, respectively. The effect of E6 or E7 expression on UV irradiation (5 and 20 J/m2)-induced genotoxic injury of confluent primary murine astrocytes was determined. Retroviral vectors were used to overexpress E6 and E7. Astrocytes expressing E7 showed increased vulnerability to UV-induced apoptosis while E6 over expressing astrocytes were protected from the same insults. Cell death in the E7 overexpressing cells was apoptotic because it showed DNA ladders, activation of caspase-3, formation of apoptotic bodies and decreased DNA content to less than the G0 level. After UV-irradiation the level of E2F1 in E7-expressing astrocytes was higher than E6-, LXSN- or mock-infected cells, and caspase-3 was activated to a greater extent. E7-expressing astrocytes showed the highest levels of Bax under normal growth conditions. The mitochondrial membrane potential of E7-expressing astrocytes was depolarized by 90% after UV-irradiation while the depolarization in control cells was about 50%. E6 overexpression decreased while E7 overexpression increased UV-induced astrocyte apoptosis.     

Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.     

转染人乳头瘤病毒16型E6E7重组基因及感染模型细胞建立

人乳头瘤病毒(human papillomavirus,HPV)是性传播疾病的主要病原之一,高危型人乳头瘤病毒感染和宫颈癌的关系已被肯定[1],在约50%~90%的宫颈癌组织中可检出HPV16 DNA。为了解与HPV相关疾病的的分子生物学致病基础,尚待深入研究其生物特性和病理特征,方能逐步解决临床简便易行的试验诊断方法和抗HPV感染的治疗问题。目前已经证实HPV16 E6/E7基因是转化基因,它们编码的蛋白可分别使抑癌蛋白P53和PRb失活,在宫颈癌的发生中起着重要作用[2],被认为是宫颈癌的始动因子。本文选择HPV16 E6E7作为研究对象,利用基因重组技术构建EGFP-…