人乳头瘤病毒16型早期基因E6、E7的克隆及表达

高危人乳头瘤病毒16型的感染与宫颈癌的发病密切相关。HPV16E6和E7蛋白在大多数HPV16相关宫颈癌及其癌前病变中持续表达,因此E6和E7蛋白可作为制备HPV16相关肿瘤及其癌前病变治疗性疫苗的靶抗原〔2〕。采用套式PCR方法,从宫颈癌患者组织中扩增出E6、E7基因并成功构建了包含E6、E7的表达质粒PET-E6、PET-E7。SDS聚丙烯酰胺凝胶电泳和Western-blotting分析结果表明,外源基因E6,E7在T7启动子控制下可获得稳定表达。     

Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins.

The E6 and the E7 proteins of the oncogenic human papillomavirus types 16 and 18 can stably associate with p53 and the retinoblastoma protein, respectively. The E6-p53 interaction results in the accelerated degradation of p53 in vitro via the ubiquitin-dependent proteolysis system. In this study we demonstrate that a fusion protein consisting of the N-terminal half of the HPV-16 E7 protein and the full length HPV-16 E6 protein promotes the in vitro degradation of the retinoblastoma protein. This indicates that the property of the HPV-16 E6 protein to stimulate the degradation of p53 can be targeted to other proteins. Unlike the HPV-16 or HPV-18 E6 protein, the E6 proteins of HPV-6 and 11 do not bind to p53 and consequently do not target p53 for degradation. Analogous E7-E6 fusion proteins using the E6 proteins of HPV-6 and HPV-11, however, also have the ability to promote the degradation of the retinoblastoma protein, indicating that the property to target associated proteins for degradation is shared by the anogenital specific HPV E6 proteins.     

The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53

The E6 protein encoded by the oncogenic human papillomavirus types 16 and 18 is one of two viral products expressed in HPV-associated cancers. E6 is an oncoprotein which cooperates with E7 to immortalize primary human keratinocytes. Insight into the mechanism by which E6 functions in oncogenesis is provided by the observation that the E6 protein encoded by HPV-16 and HPV-18 can complex the wild-type p53 protein in vitro. Wild-type p53 gene has tumor suppressor properties, and is a target for several of the oncoproteins encoded by DNA tumor viruses. In this study we demonstrate that the E6 proteins of the oncogenic HPVs that bind p53 stimulate the degradation of p53. The E6-promoted degradation of p53 is ATP dependent and involves the ubiquitin-dependent protease system. Selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant-acting oncoproteins.     

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line.     

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.     

Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines.

Human cervical carcinoma cell lines that harbor human papillomavirus (HPV) have been reported to retain selectively and express HPV sequences which could encode viral E6 and E7 proteins. The potential importance of HPV E6 to tumors is suggested further by the observation that bovine papillomavirus (BPV) E6 can induce morphologic transformation of mouse cells in vitro. To identify HPV E6 protein, a polypeptide encoded by HPV-16 E6 was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 18-kd protein in two human carcinoma cell lines known to express HPV-16 RNA and in mouse cells morphologically transformed by HPV-16 DNA. The 18-kd E6 protein was distinct from a previously identified HPV-16 E7 protein. The HPV-16 E6 antibodies were found to be type specific in that they did not recognize E6 protein in cells containing HPV-18 sequences and reacted weakly, if at all, to BPV E6 protein. The results demonstrate that human tumors containing HPV-16 DNA can express an E6 protein product. They are consistent with the hypothesis that E6 may contribute to the transformed phenotype in human cervical cancers that express this protein.     

Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene.

The human papillomavirus type 16 (HPV-16) genome is commonly present in human cervical carcinoma, in which a subset of the viral genes, E6 and E7, are expressed. The HPV-16 E6 and E7 gene products can associated with and inactivate the tumor suppressor proteins p53 and Rb (the retinoblastoma susceptibility gene product), and in tissue culture cells, these viral genes display oncogenic properties. These findings have led to the hypothesis that E6 and E7 contribute to cervical carcinogenesis. This hypothesis has recently been tested by using transgenic mice as an animal model. HPV-16 E6 and E7 together were found to induce cancers in multiple tissues in which they were expressed, including squamous cell carcinoma, the cancer type most commonly associated with HPV-16 in the human cervix. We have extended these studies to investigate the in vivo activities of HPV-16 E7 when expressed in squamous epithelia of transgenic mice. Grossly, E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In lines of mice expressing higher levels of E7, we observed stunted growth and mortality at an early age, potentially caused by an incapacity to feed. Histological analysis demonstrated that E7 causes epidermal hyperplasia in multiple transgenic lineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratin 10-positive layer of cells and was associated with hyperkeratosis. Hyperplasia was found at multiple sites in the animals in addition to the skin, including the mouth palate, esophagus, forestomach, and exocervix. In multiple transgenic lineages, adult animals developed skin tumors late in life with low penetrance. These tumors arose from the squamous epithelia and from sebaceous glands and were characterized histologically to be highly differentiated, locally invasive, and aggressive in their growth properties. On the basis of these phenotypes, we conclude that HPV-16 E7 can alter epithelial cell growth parameters sufficiently to potentiate tumorigenesis in mice.     

A C-terminal hydrophobic,solvent-protected core and a flexible N-terminus are potentially required for human papillomavirus 18 E7 protein functionality

The oncogenic potential of the high-risk human papillomavirus (HPV) relies on the expression of genes specifying the E7 and E6 proteins. To investigate further the variation in oligomeric structure that has been reported for different E7 proteins, an HPV-18 E7 cloned from a Hispanic woman with cervical intraepithelial neoplasia was purified to homogeneity most probably as a stable monomeric protein in aqueous solution. We determined that one zinc ion is present per HPV-18 E7 monomer by amino acid and inductively coupled plasma-atomic emission spectroscopy analysis. Intrinsic fluorescence and circular dichroism spectroscopic results indicate that the zinc ion is important for the correct folding and thermal stability of HPV-18 E7. Hydroxyl radical mediated protein footprinting coupled to mass spectrometry and other biochemical and biophysical data indicate that near the C-terminus, the four cysteines of the two Cys-X₂-Cys motifs that are coordinated to the zinc ion form a solvent inaccessible core. The N-terminal LXCXE pRb binding motif region is hydroxyl radical accessible and conformationally flexible. Both factors, the relative flexibility of the pRb binding motif at the N-terminus and the C-terminal metal-binding hydrophobic solvent-protected core, combine together and facilitate the biological functions of HPV-18 E7.     

The E7 proteins of the nononcogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinoblastoma protein binding and other properties.

The E7 early viral protein of the oncogenic human papillomavirus type 16 (HPV-16) has been strongly implicated in the maintenance of the malignant phenotype in cervical cancers and cancer-derived cell lines. HPV-16 E7 is a nuclear phosphoprotein that can cooperate with ras to transform baby rat kidney cells, transactivates the adenovirus E2 promoter, and binds to the retinoblastoma (RB) protein. The E7 phosphoprotein of the nononcogenic HPV-6b, which is generally associated with benign genital warts, is similar to the HPV-16 E7 in amino acid sequence but differs dramatically in migration in sodium dodecyl sulfate-polyacrylamide gels, sedimentation in nondenaturing glycerol gradients, and the ability to bind the RB protein. Our results indicate that the RB protein preferentially binds the phosphorylated form of HPV-6b E7, which comprises a minor fraction of the total E7 expressed in transiently transfected COS-7 cells. These characteristics may help to explain the difference in the oncogenic potential of the oncogenic and nononcogenic types of genital papillomaviruses.     

云南HPV-16型E6、E7基因的突变分析

人乳头瘤病毒(Human papillomavirus,HPV)16型(HPV-16)是引起宫颈癌的一种主要高危型病毒,其2个致癌基因E6和E7的核酸序列变异可能会影响其对宿主细胞的致癌性,已有研究表明其序列突变呈现地域差异性。因此,研究不同地域HPV-16这2个基因的变化情况是宫颈癌流行病学调研的主要内容,也可为研究E6和E7的致癌性积累数据。研究以NCBI登录号为NC_001526.2的HPV-16型病毒的序列为参照,采用Neighbor-joining方法对云南地区74例HPV-16样本的E6、E7的DNA序列构建进化树,结果显示:只有亚洲和欧洲变异亚型,而没有发现非洲1、非洲2、亚-美洲和北美洲这4种变异亚型。DNA序列分析显示:E6的碱基突变以T178G(D25E,59.46%)和T350G(L83V,8.11%)为主,E7的碱基突变主要以A647G(N29S,59.46%)和T846C(同义突变,60.81%)为主。发现E6的新突变有A95G(同义突变,1.35%)和A135G(K11R,1.35%);E7的新突变有C625T(L22F,1.35%)、C627T(同义突变,12.16%)、G689A(G43E,1.35%)、T748G(S63A,1.35%)。此外还发现有一个共突变现象:T178G(D25E,59.46%)-A647G(N29S,59.46%)-T843C(同义突变,21.62%)-T846C(同义突变,60.81%)。     

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-…     

Identification of human papillomavirus type 18 transforming genes in immortalized and primary cells.

The selective retention and expression of the E6-E7 region of human papillomavirus (HPV) types 16 and 18 in cervical carcinomas suggests that these viral sequences play a role in the development of genital neoplasia. Each of three possible gene products, E6, E6*, and E7, from this region of HPV-18 were examined for transforming properties in several types of rodent cells. We have found that in immortalized fibroblasts, both E6 and E7 (but not E6*) are capable of inducing anchorage-independent growth. In rat embryo cells, the HPV-18 E7 open reading frame was an effective immortalizing agent and complemented an activated ras oncogene for transformation. In both immortalized and primary cells, transformation was observed when the HPV-18 sequences were expressed from either the HPV-18 promoter or a heterologous promoter. The E6-E7 region is not, however, the sole transforming domain of HPV-18, since another portion of the early region, possibly E5, also exhibited transforming capability in immortalized fibroblasts. The development of human cervical carcinomas may therefore involve a series of steps involving multiple viral and cellular gene products.