Identification of novel E6-E7 sequence variants of human papillomavirus 16

The rate of evolution of the human papillomavirus 16 (HPV16) genome is low. However, the ability of the E6 oncoprotein to interact with distinct p53 variants causes selective pressure on the E6 gene. In addition, intratypic recombination events in the HPV16 E6 and E7 genes have been characterized as extraordinary phenomena during the evolutionary history of virus. In the present study, we identified two new sequence variants through nucleotide analysis of the E6-E7 region of the HPV16 genome. Maximum-likelihood and empirical Bayesian methods were used in order to identify positive selection at particular residues of the E6 and E7 genes. Using the single recombination breakpoint (SBP) method, we found evidence of recombination events in the E6 ORF. Nucleotide sequence analysis showed that the new sequence variants are phylogenetically distant from the other members of the population. Our results indicate that new evolutionary intermediates of HPV16 might be formed either though positive selective pressure or through recombination events by multiple infections with distinct HPV16 variants.     

Identification of a novel human papillomavirus type 16 E1 gene variant with potentially reduced oncogenicity

The human papillomavirus (HPV) 16 genome has been studied extensively, although no study has focused on the E1 gene that is implicated in viral DNA replication. After analyzing the E1 region of HPV 16 genomes in 429 cervical samples, 11.2% were found to contain a 63 nucleotides duplication in this region. Sequence analysis of the E6 and the E7 regions has shown that all samples containing this duplication were related to E6‐G350 variant of the HPV 16 (Chi square test, P = 0.0012). A comparison of cervical lesion severity of the examinees having regular or variant E1 genes has shown that the variant group had a significantly (Fischer's exact test, P = 0.0401) lower percentage of high grade disease cases, suggesting that this particular duplication might reduce the oncogenic potential of HPV 16, and also might clarify the differences of E6‐G350 variant oncogenicity observed in European populations. Albeit, a decreased incidence of high grade cervical lesions can be linked to the prevalence of multiple HPV infection, the additional decrease of those cases with the variant E1 gene versus those without (10.5% and 18.6%, respectively) can only be ascribed to the effect of this particular HPV variant. Further research is needed to clarify the biology of these HPV 16 E1 variants. J. Med. Virol. 80:2134–2140, 2008. © 2008 Wiley‐Liss, Inc.     

Neuroepithelial carcinomas in mice transgenic with human papillomavirus type 16 E6/E7 ORFs.

The effect of the human papillomavirus (HPV) oncogenes E6 and E7 was examined in transgenic mice with a construct containing the human beta-actin promoter regulating HPV16 E6 and E7 open reading frames. In the sole line of mice that transmitted the transgene, neuroepithelial tumors appeared at 2.5 months of life, and by 10 months, 87 of 122 (71%) of the animals were dead from brain tumors. The most frequent type of tumor (74%) was an anaplastic neuroepithelial tumor associated with the ependyma of the third and fourth ventricles, which locally invaded adjacent brain tissue and spread for considerable distances along the ventricular surface. The other two types of tumor were well-differentiated choroid plexus carcinomas (26%) and rare pituitary carcinomas (8.7%). HPV16 E6 RNA and E7 oncoprotein expression were demonstrated in tumor tissue and primary cell lines derived from the tumors. Examination of two tumor suppressor gene products, the retinoblastoma protein and p53, known to bind to HPV16 E7 and E6 oncoproteins, respectively, showed both were expressed in the primary tumor cell lines. These data support a causative role for the HPV oncoproteins in epithelial carcinogenesis.     

Activities of human papillomavirus 16 E6 natural variants in human keratinocytes

Genetic variations in the E6 oncogene have been associated with different risk for cancer progression. In the present study, the functional significance of human papillomavirus (HPV) polymorphism in the E6 oncogene was investigated. Ten HPV16 E6 variants containing amino acid substitutions in the N-terminal region of E6 were evaluated for different biological and biochemical activities in human keratinocytes, the target cells for HPV infection. Western blot analyses of primary foreskin human keratinocytes or immortalized human keratinocytes, stably transduced with the E6 variants, revealed reduced p53 and Bax levels in all E6 expressing cultures. The reduction induced by most E6 proteins was at similar levels and comparable to the reduction induced by the E6 prototype. The ability of the proteins to induce serum/calcium-differentiation resistant colonies in primary keratinocytes was more variable. Overall activities of the variants ranged between 0.24- and 2.18-fold of the E6 prototype activity. The I27R/L83V variant showed the lowest activity whereas the R8Q variant showed the highest activity. The L83V polymorphism previously associated with risk for cancer progression in some populations, showed significant activity, comparable to that of the E6 prototype, in reducing p53 and Bax levels. Furthermore, this variant showed enhancement in the ability to induce colonies resistant to serum/calcium-triggered differentiation, however, the difference from the prototype was not statistically significant. This, and augmentation of other described functions might result in differences in L83V pathogenicity.     

The E7 oncoprotein of human papillomavirus type 16 interacts with F-actin in vitro and in vivo

We report here that E7 oncoprotein of human papillomavirus type 16 (HPV-16) forms a complex in vivo and in vitro with actin, one of the components of the cellular cytoskeleton. The in vivo interaction was detected by immunofluorescent staining and confocal microscopic examination of normal human oral keratinocytes (NHOK) and CV-1 cells after transient expression of E7 employing the vaccinia virus-T7 RNA polymerase system and by coimmunoprecipitation from an immortalized, nontumorigenic cell line obtained after transfecting NHOK with the cloned HPV-16 DNA genome. The in vitro interaction was detected by cosedimentation of bacterially expressed E7 phosphorylated with rabbit reticulocyte lysate or purified casein kinase II (CKII) prior to incubation with F-actin. This interaction was inhibited if E7 phosphorylation by the rabbit reticulocyte lysate was prevented with heparin, a CKII inhibitor, or if the amino acids Ser-31 and Ser-32 in E7, which are phosphorylated by CKII, were replaced with amino acids that cannot be phosphorylated. Interestingly, a decrease in the amount of polymerized actin occurred in cells expressing E7.     

特异siRNA抑制宫颈癌细胞中人乳头状瘤病毒16型E6表达的研究

目的 优化HPV-16 E6癌基因特异的U6质粒表达的siRNA，抑制HPV癌基因表达及其对子宫颈癌细胞生长繁殖的影响。方法 选择4个分别针对HPV-16 E6 mRNA外显子和内含子序列为靶序列，合成DNA链，构建表达HPV-16 E6短发卡样dsRNA的重组pSilencer1．0-U6载体，导入HPV-16DNA阳性的宫颈癌细胞株CaSki中，观察该细胞中HPV-16 E6、E7基因表达水平及其蛋白含量的变化，并观察细胞生长被抑制的情况。结果 4种HPV-16 E6 siRNA均能降低宫颈癌细胞CaSki的生长速率。通过细胞生长曲线观察到HPV-16 E6 shRNA表达质粒导入细胞0-96h内，可降低细胞生长速度。荧光定量RT-PCR检测HPV-16 E6 siRNA可使宫颈癌细胞株CaSki中HPV-16 E6、E7基因转录的mRNA水平降低，其中针对E6 mRNA内含子的重组shRNA只抑制E6基因的表达水平。Western blot分析表明，4个HPV-16 E6 siRNA作用72h后，未能检测到宫颈癌细胞中HPV-16 E6蛋白。结论 HPV-16 E6 siRNA能使宫颈癌细胞CaSki生长缓慢；选择针对E6内含子的siRNA作用位点，特异性抑制E6表达；而针对E6外显子的siRNA作用位点，可抑制E6和E7基因的表达，是用于治疗HPV阳性宫颈癌细胞的理想靶位。     

人乳头瘤病毒16型修饰后E6E7基因免疫原性增强

利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型(human papillomavirus type 16,HPVl6)E6E7融合基因(fmE6E7)，研制治疗HPVl6相关疾病的DNA疫苗。用PCR扩增fmE6E7基因后，插人真核表达质粒获得pVRl012-fmE6E7，瞬时转染Cos-7细胞，免疫荧光法检测证实其表达后，在C57BL／6小鼠后腿肌肉进行裸DNA免疫，5lCr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性Cytotoxic T lymphocyte，CTL)，间接ELISA法检测免疫鼠血清中E7特异性抗体。研究表明修饰后的中国地方株E6E7融合基因可诱导机体产生特异的抗体反应和CTL反应，与单独野生型E7基因免疫相比，E6E7融和基因可更好的活化CTT反应。表明修饰后消除转化活性的中国地方株E6E7融合基因可作为HPVl6治疗性DNA疫苗的靶基因。     

Identification of a naturally processed HLA A0201-restricted viral peptide from cells expressing human papillomavirus type 16 E6 oncoprotein

Human papillomavirus (HPV) DNA encoding the oncogenic proteins E6 and E7 is usually retained in cervical carcinomas, implicating these proteins as potential target antigens for immune recognition in this virally associated tumor. We have characterized endogenously processed peptides eluted from major histocompatibility complex class I molecules in cells infected with a recombinant vaccinia expressing the HPV-16 E6 oncoprotein. The reverse-phase chromatography profile of peptides eluted from isolated HLA-A0201 molecules in cells expressing the E6 oncoprotein differs from that of cells not expressing E6. Sequential Edman degradation of novel peaks found in the peptide profiles from cells expressing HPV-16 E6 led to the identification of a naturally processed HLA-A0201-restricted E6 peptide of sequence KLPQLCTEL. This approach has allowed the identification of a viral peptide which is processed and presented by cells expressing the E6 oncoprotein and is a likely target for cytotoxic T lymphocyte recognition in HLA-A0201-positive patients.     

Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein

Despite extensive studies in plant virus-host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N-terminal portion. The potential significance of the NSP-NIK interaction is discussed.     

Microarray analysis identifies differentiation-associated genes regulated by human papillomavirus type 16 E6

In this study, we used oligonucleotide microarray analysis to determine which cellular genes are regulated by the human papillomavirus type 16 (HPV-16) E6 oncoprotein. We found that E6 causes the downregulation of a large number of cellular genes involved in keratinocyte differentiation, including genes such as small proline-rich proteins, transglutaminase, involucrin, elafin, and cytokeratins, which are normally involved in the production of the cornified cell envelope. In contrast, E6 upregulates several genes, such as vimentin, that are usually expressed in mesenchymal lineages. E6 also modulates levels of genes involved in inflammation, including Cox-1 and Nag-1. By using E6 mutants that differentially target p53 for degradation, we determined that E6 regulates cellular genes by both p53-dependent and independent mechanisms. The microarray data also indicate that HPV-16 E6 modulates certain effects of HPV-16 E7 on cellular gene expression. The identification of E6-regulated genes in this analysis provides a basis for further studies on their role in HPV infection and cellular transformation.     

Identification of recombinant human papillomavirus type 16 variants

Intratypic diversity of human papillomavirus (HPV) genome is generally characterized by point mutation, insertion, and/or deletion. Using PCR-based cloning and sequencing, we detected concurrent infection with 8 HPV16 variants in a woman enrolled in the ASCUS-LSIL Triage Study. The European variant was the major variant; each of the 7 minor variants had partial DNA sequences identical to the European variant and another part identical to the African 2 variant. At a follow-up visit, only an HPV16 African 2 variant was detected. Results from the present study suggest presence of intratypic recombination of HPV genome in natural infection.     

人乳头瘤病毒E6、E7的表达对足叶乙甙作用下细胞凋亡的影响

目的探讨人乳头瘤毒(HPV)16早期基因E6和E7的表达对NIKS细胞凋亡表型的影响。方法用含有HPV16的E6、HPV16E7、HPV16E6E7病毒癌基因的逆转录病毒感染角质生成细胞株NIKS,puromycin筛选稳定表达细胞;基因组PCR和Western blot方法验证E6和E7的表达;转染后的NIKS细胞用不同浓度的足叶乙甙处理,流式细胞仪和Annexin V染色检测细胞凋亡情况。结果经逆转录病毒感染后,建立表达E6、E7及E6E7的NIKS细胞株,基因组PCR证明E6和E7整合入NIKS细胞基因组;Western blot证实表达的E6、E7具有生物学活性,能够分别降解p53和pRB;在足叶乙甙处理后,E6、E7以及E6E7表达细胞发生明显的细胞死亡,E6和E7具有叠加作用,且具有剂量依赖性,Annexin V染色证实细胞发生凋亡,凋亡率分别为(33.5±3.2)%、(38.3±2.4)%和(56.7±4.3)%。结论人乳头瘤病毒E6和E7的表达都可以促进细胞对DNA损伤药物的敏感性,提示乳头瘤病毒感染状态有可能影响肿瘤细胞对化疗的敏感性。     

Nucleotide polymorphisms of the human papillomavirus 16 E1 gene

The E1 ORF is one of the most conserved regions in the human papillomavirus (HPV) genome. The complete E1 gene of the HPV16 genome was amplified with four overlapping primer sets in 16 high-grade (CIN II, III) and 13 low-grade cervical (CIN I) intraepithelial neoplasias as well as in one cervical cancer case. Sequence analysis of the E6 and E7 genes was also carried out in the same cervical samples in order to confirm the association between nucleotide sequence variations in the HPV16 E1 ORF and HPV16 variant lineages. Analysis of the E1 ORF revealed 27 nucleotide changes, and these changes were correlated with those found in HPV16 Asian American and African type II variants. Of these nucleotide variations, A1668G, G2073A, T2169C, T2189C, A2453T, C2454T, A2587T and G2650A were identified only in high-grade dysplasia cases. A phylogenetic tree of the E1 ORF and nucleotide sequence analysis of the E1, E6 and E7 genes revealed that intratypic nucleotide sequence polymorphisms located in the E1 ORF can be used to identify the major phylogenetic branch to which a HPV16 genome belongs. Moreover, amplification of the E1 ORF revealed a disruption between nucleotides 878 and 1523 in five high- and two low-grade cervical cases, indicating that integration of HPV DNA occurs at an early stage of viral infection.     

Generation of a DNA microarray for determination of E6 natural variants of human papillomavirus type 16

Infection with high-risk types of human papillomavirus (HPV) is necessary for the development of cervical cancer. However, the majority of the HPV infections are efficiently cleared by the immune system and only a minority persist and induce the development of malignant lesions. Several studies provided evidence that intratype genetic variations are implicated in determining the clinical outcome of HPV infections. In this study, we describe a DNA chip based on arrayed primer extension (APEX) for the analysis of the natural variants of HPV16, the most frequently detected type in cervical cancer world-wide. We show that HPV16 E6 variants are detected efficiently by APEX. In addition, APEX is a valid assay for the simultaneous detection of different HPV16 variants. Thus, it could be used for clinical studies aiming to analyse multiple infections with different HPV16 subtypes. Finally, since APEX combines the advantages of dideoxy DNA sequencing with the high-throughput potential of the microarray, it is suitable for the simultaneous analysis of a number of variable regions of HPV16 genome in large-scale studies.     

Immunization against human papillomavirus type 16 tumor cells with recombinant vaccinia viruses expressing E6 and E7.

Papillomaviruses are etiological agents of epithelial proliferative disease. In man, neoplastic transformation of the uterine cervix has been linked to infection with specific subtypes of human papillomavirus, particularly types 16 and 18. We previously reported that live vaccinia virus recombinants expressing early transforming proteins of other tumor viruses can immunize against challenge with cognate tumor cells and we have extended this approach to HPV16. Neoplastic transformation by papillomaviruses involves expression of early open reading frames (ORFs) E5, E6, and E7, and we report the construction of vaccinia recombinants separately expressing ORFs E5-E7 of HPV16. Primary rat cell lines cotransformed with HPV16 and an activated ras oncogene were established in order to evaluate the potential of the recombinants to elicit antitumor immunity. We report that inoculation of rats with vaccinia recombinants expressing E6 or E7 retarded or prevented tumor development in a proportion of animals challenged by subcutaneous seeding of tumor cells whereas the recombinant expressing E5 was inactive.     

逆转录病毒载体介导HPV16 E6E7基因转化人胚食管纤维细胞的研究

目的 研究HPV16型病毒感染与食管癌发生的关系。方法 包装含有HPVl6E6E7基因重组逆转录病毒，以重组病毒感染人胚食管纤维细胞，在TPA协同下诱导SCID小鼠成瘤。结果 重组病毒感染人胚食管纤维细胞可以诱导SCID小鼠形成肉瘤，可以检测到E6E7基因的存在及表达，流式细胞仪检测从瘤组织培养出来的纤维细胞，确定为异倍体；但未能诱导人胚食管组织成瘤。结论 建立的重组逆转录病毒系统可以成功介导HPV16E6E7基因的转移，可以应用于HPV致瘤性研究。