The role of the human papillomavirus type 18 E7 oncoprotein during the complete viral life cycle

The role of the human papillomavirus oncoprotein E7 in carcinogenesis has been extensively studied. While the role of HPV E7 in the viral life cycle has also been studied, certain disparities exist, indicating that genotype differences may influence the role that E7 plays in the viral life cycle. In this study, we investigated the role of HPV18 E7 in the viral life cycle in order to gain a further understanding of this issue. To determine the role that HPV18 E7 plays in the viral life cycle, a translation termination substitution mutant of E7 in the context of the full HPV18 genome was created. We introduced linearized HPV18 E7-deficient genomic DNA into primary keratinocytes, where it recircularized and was maintained episomally at a range of five to several hundred copies of HPV genomic DNA. The mutant genomes failed to amplify following epithelial stratification and differentiation in organotypic culture. Moreover, virion morphogenesis did not occur. We found that the expression of HPV16 or HPV18 E7 in trans was able to rescue the amplification defect but not the defect in virion morphogenesis. These studies indicate that HPV18 E7 plays a critical role in the productive stage of the viral life cycle. In addition, these studies add further proof to the hypothesis that genotype differences exist for the role of E7 during the viral life cycle.     

The full-length E1E4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1E4 protein. To determine the role of E1E4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1E4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1E4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1E4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1E4 expression, and to HPV16 where only C-terminal truncations in E1E4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1E4 proteins.     

Genetic diversity of human papillomavirus type 16 E6, E7, and L1 genes in Italian women with different grades of cervical lesions

High‐risk human papillomaviruses (HPVs) are risk factors for the development of cervical cancer. HPV 16 is the most common type, being present in about 60% of cervical cancers worldwide. Previous studies have reported upon the association between HPV 16 E6 variants and increased risk of cervical intraepithelial neoplasia and invasive cervical cancer. In this study, the presence of HPV 16 polymorphisms in the E6, E7, and L1 genes was investigated in relation to the presence of high‐grade lesions. Sequencing of the E6 gene revealed the presence of nucleotide mutations resulting in 15 amino acid changes. Of these, the G134D and C136R fall within the CXXC zinger finger domain important for p53 binding. In the E7 gene, four nucleotide variations were identified with two leading to the amino acid substitutions L15V and S31R. The L1 gene showed 13 nucleotide changes leading to 11 amino acid substitutions. Among these, the R364C and N367D are located at the base of the HI‐loop of the L1 protein, considered to be the immunodominant epitope of HPV 16. No significant relationship between HPV 16 variants and high‐grade lesions was found. Phylogenetic analysis showed that all the HPV 16 variants identified belonged to the European lineage, except one which was of the Asian‐American lineage. The European‐350G variant was detected most frequently (22 of 34, 64.7%). The study provides some new data on the genetic diversity of HPV 16 which may help to understand the oncogenic potential of the virus and to improve management of patients. J. Med. Virol. 81:1627–1634, 2009. © 2009 Wiley‐Liss, Inc.     

重组人乳头瘤病毒16型E6蛋白的表达、纯化和动物免疫效果分析

目的 诱导表达人乳头瘤病毒16型(HPV16)E6蛋白,制备可溶性蛋白,并通过动物免疫进行免疫效果评估.方法 采用IPTG诱导pQE30-HPV16E6/BL21(DE3)重组菌株,表达产物经SDS-PAGE和Western blot分析鉴定.提取包涵体进行变性处理,变性蛋白经Ni2+金属螯合层析纯化,将纯化产物用复性透析方法处理以获取可溶性蛋白.免疫Bal B/c小鼠,检测血清抗体、CD4+/CD8+和IFN-γ.结果 诱导后重组菌有相对分子量18 000蛋白质表达,以不溶性包涵体为主要表达方式.目的蛋白可与HPV16E6多克隆抗体识别,纯化和透析处理后得到可溶性的蛋白.小鼠免疫后血清抗体滴度升高,淋巴细胞CD4+/CD8+升高,IFN-γ未见升高.结论 成功表达了HPV16E6蛋白,同时处理包涵体并制备了可溶性目的蛋白.目的蛋白可刺激小鼠体内产生有效的免疫反应,该蛋白的成功制备为患者血清抗体的检测和肿瘤细胞杀伤的免疫研究奠定了坚实基础.     

A novel interaction between the human papillomavirus type 16 E2 and E1^E4 proteins leads to stabilization of E2

The E4 (also called E1^E4) and E2 proteins of human papillomavirus type 16 are thought to be expressed within the same cells of a lesion, and their open reading frames overlap, suggesting that they may have a functional relationship. We have examined the effect of co-expression of these two proteins and found that each enhances the level of the other. We also identified the N-terminus of E2 as the first example of a viral protein that directly binds the HPV16 E1^E4 protein. This appears to result in the E2 becoming less soluble and promotes its relocation from the nucleus to the cytoplasm. In addition, the turnover of the E2 protein is decreased in the presence of E1^E4. All this raises the possibility that E1^E4 acts to influence E2 activity by varying the amount of available E2 in the cell.     

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 stability of the human papillomavirus E6 oncoprotein is E6AP dependent

Human papillomavirus (HPV) E6 oncoproteins target numerous cellular proteins for ubiquitin-mediated degradation. In the case of p53 this is mediated by the E6AP ubiquitin ligase. However, there are conflicting reports concerning how central E6AP is to the global function of the HPV-16 and HPV-18 E6 oncoproteins. To investigate this further we have analysed the effects of E6AP removal upon the stability of endogenously expressed E6 protein. We show that when E6AP is silenced in HPV-positive cells, E6 protein levels are dramatically decreased in a proteasome-dependent manner. Further, we show that when E6AP is depleted in HeLa cells, E6 has a greatly decreased half-life. In addition, overexpression of E6AP stabilises ectopically expressed HPV-16 and HPV-18 E6 in a manner that is independent of its ubiquitin ligase activity. These results demonstrate that the stability of HPV E6 is critically dependent upon the presence of E6AP.     

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.     

Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.     

The E6E7 oncoproteins of cutaneous human papillomavirus type 38 interfere with the interferon pathway

Non-melanoma skin cancer is the most frequent malignancy in Caucasian populations. Evidence suggests the involvement of cutaneous Human Papillomavirus (HPV) of the genus beta (beta) in this disease. The ability of E6 and E7 of mucosal HPV to promote cellular transformation and inhibit immune response-related pathways plays a key role in cervical carcinogenesis. beta HPV-38 E6 and E7 display transforming activities in in vitro and in vivo models, but their impact on immune surveillance is unknown. Here we show that HPV-38 E6 and E7 affect the IFN-induced up-regulation of MHC class I. Expression of the two viral proteins in HaCaT keratinocytes led to a decrease of MHC I levels. This down-regulation is associated with a reduction of expression of MHC I heavy chain, of the peptide chaperone TAP and of the STAT-1 downstream effector IRF-1. The down-regulation of these proteins is ultimately due to the inhibition of STAT-1 expression. Analysis of cells expressing either HPV-38 E6 or E7 suggests that these effects are primarily the result of E6 expression, although a contribution by E7 cannot be excluded. We conclude that HPV-38 encodes oncoproteins that potentially contribute to the evasion of host immune surveillance.     

Identification of a nuclear export signal sequence for bovine papillomavirus E1 protein

Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over- or under-expression of CRM1, the major cellular exportin, and export was strongly reduced by the CRM1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a CRM1-dependent process. Consistent with the in vivo functional results, E1 bound CRM1 in an in vitro pull-down assay. In addition, sumoylated E1 bound CRM1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wild-type E1, yet was defective for viral origin replication in vivo. However, NES2 exhibited no intrinsic replication defect in an in vitro replication assay, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state.     

人乳头状瘤病毒18型E6E7基因诱导胎儿食管永生化上 …

目的 ＳＨＥＥ细胞系是经人乳头状瘤病毒（ＨＰＶ）１８型Ｅ６Ｅ７基因诱导的永生化上皮细胞株,已传代超过５０代。研究胎儿食管上皮永生化的细胞 ＳＨＥＥ生物学特性,包括增殖,分化和调亡。方法 细胞于１９９培养基培养,用光镜、电镜和荧光显微镜研究其生长率、形态和染色体分析;用流式细胞仪研究其细胞增殖动力学;用免疫组织化学方法研究Ｋｉ６７和角蛋白和用末端转移酶标记（ＴＵＮＥＬ）凋亡细胞。结果 细胞培养呈单导     

A cyclin-binding motif in human papillomavirus type 18 (HPV18) E1^E4 is necessary for association with CDK-cyclin complexes and G2/M cell cycle arrest of keratinocytes, but is not required for differentiation-dependent viral genome amplification or L1 capsid protein expression

The G2/M arrest function of human papillomavirus (HPV) E4 proteins is hypothesized to be necessary for viral genome amplification. Full-length HPV18 E1^E4 protein is essential for efficient viral genome amplification. Here we identify key determinants within a CDK-bipartite consensus recognition motif in HPV18 E1^E4 that are critical for association with active CDK-cyclin complexes and in vitro phosphorylation at the predicted CDK phosphorylation site (threonine 23). The optimal cyclin-binding sequence (⁴³RRLL⁴⁶) within this E4 motif is required for G2/M arrest of primary keratinocytes and correlates with cytoplasmic retention of cyclin B1, but not cyclin A. Disruption of this motif in the E4 ORF of HPV18 genomes, and the subsequent generation of stable cell lines in primary keratinocytes revealed that this motif was not essential for viral genome amplification or L1 capsid protein induction. We conclude that the HPV18 E4 G2/M arrest function does not play a role in early vegetative events.     

HPV16/18和HPV16E6/E7DNA在宫颈癌组织中的表达

目的检测HPV16/18和HPV16E6/E7 DNA在宫颈癌组织中的表达,探讨其在宫颈癌发病中的作用.方法应用PCR和琼脂糖凝胶电泳方法检测46例宫颈癌组织中HPV16/18和HPV16E6/E7DNA.结果 46例宫颈癌中56.5%(26/46)扩增HPV16/18 DNA,其中宫颈鳞癌25例,宫颈腺癌1例.正常对照组20例HPV16/18DNA均为阴性,与宫颈癌组相比差异有显著性(P<0.01).HPV16/18 DNA阳性拷贝对数值为4.32±2.45.HPV16E6,E7DNA分别有53.8%(14/26)、46.2%(12/26)扩增.结论 HPV16/18和HPV16E6/E7 DNA与宫颈癌的发生密切相关,是宫颈癌恶性转化的关键之一,预示着宫颈癌有较强的增殖能力和转移能力.     

Incidence of antibodies to human papillomavirus type 1 in patients with cutaneous and mucosal papillomas

A study of antibodies to human papillomavirus (HPV) 1 is reported in a series of sera from 126 patients with papilloma lesions of different clinical types (verruca plantaris, vulgaris, plana, condyloma acuminata, laryngeal papilloma) using a sensitive solid-phase enzyme-linked immunosorbent assay (ELISA). Our findings showed a significant prevalence of specific HPV 1 IgG antibodies in patients with plantar warts (65%). This incidence was considerably lower in patients with other papilloma lesions, in which positive sera may reflect a past infection with HPV 1. The ELISA test was evaluated as nearly 100 times more sensitive than the indirect immunofluorescence test, and IgG titers to HPV 1 in patients' sera were usually low. Serum samples taken at different times during the course of HPV infection in a few patients with refractory lesions did not show any significant change in their antibody titer.     

人乳头瘤病毒16型E5转化基因的克隆及原核表达

目的建立人乳头瘤病毒16型E5基因的无性繁殖系,为进一步研究其致癌机理作准备.方法用PCR技术从HPV16质粒DNA中扩增出E5基因序列,连接到pGEM-T载体,将阳性的重组pGEM-T用BamH Ⅰ/EcoRⅠ双酶切并回收目的片段连接到原核表达载体pGEX-2T,转化DH5α菌株.取工程菌,IPTG诱导表达,SDS-PAGE电泳鉴定.结果①经PCR、测序和双酶切鉴定,认为HPV16E5正确插入上述表达载体,建立了该转化基因的无性繁殖系.②经IPTG诱导的pGEX-2T-E5质粒表达出约42KD的融合蛋白,分析含有约16KD HPV16 E5蛋白,与预期含HPV16 E5的融合蛋白分子量相符.结论①采用PCR克隆技术,扩增HPV16 E5转化基因目的片段,将其克隆到pGEM-T载体在国内首次建立了HPV16 E5转化基因的无性繁殖子.②成功构建HPV16 E5基因的原核表达质粒,并表达出GST-E5融合表达蛋白.