Human papillomavirus type 16 variant analysis of E6, E7, and L1 [corrected] genes and long control region in [corrected] cervical carcinomas in patients in northeast China

Human papillomavirus type 16 (HPV 16) plays a cardinal role in the pathogenesis of cervical cancer. HPV 16 has intratypic variants which show different geographical distributions and different oncogenic potentials. To analyze the presence of sequence variations of HPV 16 variants in northeast China, 71 cervical carcinomas were identified by HPV typing. HPV 16-positive specimens were analyzed by PCR-directed sequencing in the E6, E7, and L1 genes and the LCR (long control region). The variation data were compared with those of neighboring districts. In this hospital-based study, HPV 16 was the most common type (73.24%). In HPV 16-positive specimens, 67.31% belonged to the European (E) lineage, while 32.69% were Asian (As) variants. The Asian-American (AA), African-1 (Af-1), African-2 (Af-2), and northern American (NA) lineages were not detected. The most frequently observed variation sites were T178G (32.69%) in E6; A647G (34.62%), G666A (38.46%), and T846C (32.69%) in E7; C6826T (36.17%) and G7060A (61.70%) in L1; and G7521A (98.08%) in the LCR. The most prevalent amino acid variations were D25E in E6 and N29S in E7. In addition, 28 novel variations of HPV 16 were reported. Some covariations between different genes were obtained. In this study, HPV 16 variants belonged to the European lineage and the Asian lineage. Compared with neighboring districts, the distribution of HPV 16 variants in northeast China had a typical pattern. As the first report on HPV 16 variants in northeast China, it should be helpful for designing a HPV vaccine and HPV vaccination program in China.     

98例宫颈癌人乳头状瘤病毒16型E6基因突变及多态性分析

目的 研究湖北地区宫颈癌患者人乳头状瘤病毒16型(HPV16)E6基因点突变分布及频率,与国内外其他研究人员发现的E6突变进行对比,分析E6突变在宫颈癌发生中的意义.方法 从宫颈癌患者手术切除标本中提取组织DNA,用HPV16 E6特异性引物进行PCR扩增,对扩增的E6基因片段进行测序分析.结果 在35例宫颈癌组织DNA中有18例发生E6基因178位核苷酸的突变,变突频率为51.43%,相应核苷酸改变为Asp→Glu,442位核苷酸有4例发生核苷酸序列的改变,突变频率为11.43%,另有10例发生1～2个核苷酸序列的改变.结论 湖北地区98例宫颈癌患者人乳头状瘤病毒E6基因存在高频率的178和442位核苷酸突变,一些为新发现的核苷酸序列的突变,这些突变在宫颈癌发生中的作用有待于进一步研究.     

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.     

Human papillomavirus 16 E6, L1, L2 and E2 gene variants in cervical lesion progression

The human papillomavirus (HPV) 16 E6 genome variant 350G has been found to be more prevalent in women with persistent infection and cervical disease progression than the HPV16 E6 prototype 350T. In this study, we examined whether women who progressed to a high-grade lesion, yet were infected with the prototype 350T, showed variants in other HPV genes such as L1, L2 and E2. Although we detected variants within these genes, they could not explain this phenomenon. Indeed they correlated similarly with variant 350G and prototype 350T. These data indicate that polymorphisms in HPV16 E6 rather than in the other analyzed genes play a role in determining the risk for cervical lesion progression and that additional factors are likely to be required as well.     

修饰后中国山东地方株HPV16 E6E7基因的转化活性检测及免疫原性研究

目的 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（HPV16）E6E7基因，研制HPV16 DNA疫苗。方法 定点突变E6的终止密码，并保证E7读码框架不定；定点突变E7蛋白的Rb结合区中对其转化活性维持起关键作用的第24位氨基酸。突变修饰后的基因命名为fmE6E7。PCR扩增fmE6E7，重组人pLNCX载体，脂质体法转染3T3细胞，免疫荧光组织化学及Western blot检测转染细胞蛋白的表达。经软琼脂集落培养法和BALB／c裸鼠皮下接种法检测fmE6E7的转化活性。然后PCR扩增fmE6E7，构建pVR1012-fmE6E7真核表达质粒，于C57BL／6小鼠肌肉内直接进行裸DNA免疫，^51Cr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性，间接ELISA法检测免疫鼠血清中E7特异性抗体。结果 测序证实获得了预期的突变结果。pLNCX-fmE6E7转染细胞体外软琼脂培养3周未见集落形成；裸鼠皮下接种2月后未见移植瘤形成（0／3）。免疫鼠获得了较好的E7特异性的抗体E和抗原特异性的CTL。结论 修饰后E6E7基因可融合表达，转化活性消除的同时还可诱发特异的细胞免疫和体液免疫，表明中国山东地方株的E6E7基因可作为HPV16治疗性DNA疫苗的靶基因。     

Human papillomavirus type 18 E6 and E7 antibodies in human sera: increased anti-E7 prevalence in cervical cancer patients.

Antibody-reactive regions on the human papillomavirus type 18 (HPV-18) E6 and E7 proteins were identified with rabbit polyclonal anti-fusion protein sera by screening of an fd phage expression library containing subgenomic HPV-18 DNA fragments and by testing of overlapping decapeptides representing the E6 and E7 open reading frames. Peptides comprising the delineated regions (designated E6/1 to E6/4 and E7/1) were synthesized and used in an enzyme-linked immunosorbent assay (ELISA) to detect anti-HPV-18 antibodies in human sera. A total of 232 human serum samples (identical numbers of cervical cancer patients and age-matched controls) collected in Tanzania were tested. Similar prevalences (between 0.8 and 4.3%) of antibodies recognizing the different E6 peptides were found in the sera from tumor patients and controls. With a synthetic 28-mer peptide (designated pepE701) comprising the E7/1 region, a significant difference was found: 10 of 116 tumor serum samples but 0 of 116 control serum samples showed a specific reaction (P less than 0.001). This observation confirms earlier results with HPV-16 E7 fusion proteins (I. Jochmus-Kudielka, A. Schneider, R. Braun, R. Kimmig, U. Koldovsky, K. E. Schneweis, K. Seedorf, and L. Gissmann, J. Natl. Cancer Inst. 81:1698-1704, 1989). A lower prevalence of anti-HPV-18 E7 antibodies was observed when 188 human serum samples collected in Germany from tumor patients and controls were tested (3 of 94 positive in the cancer group; 0 of 94 positive in the control group). The type specificity of anti-HPV-18 E7 antibodies was demonstrated when the HPV type found by Southern hybridization in the cervical cancer biopsies was compared with seroreactivity: 4 of 8 serum samples obtained from HPV-18 DNA-positive but 0 of 16 serum samples from HPV-18 DNA-negative tumor patients reacted in the HPV-18 E7 ELISA. In addition, HPV-18-positive sera failed to react in a peptide ELISA with the homologous HPV-16 E7 region (M. Müller, H. Gausepohl, G. de Martinoff, R. Frank, R. Brasseur, and L. Gissmann, J. Gen. Virol. 71:2709-2717, 1990) and vice versa.     

Sequence analysis of human papillomavirus type 16 E6E7 gene from cervical carcinoma biopsies of Chinese patients in Shandong province

INTRODUCTION　　Humanpapillomavirustype16(HPV16)hasastrongassociationwithcervicalcarcinoma,Itrepresentsabout50%ofcervicalcancer-associatedHPVinfectionsworldwide.TheexpressionofitsearlyproteinsE6andE7contributestothetrans-formationprocessinvitro.Continuned…     

Human papillomavirus type 16 E6 and E7 oncoproteins act synergistically to cause head and neck cancer in mice

High-risk human papillomaviruses (HPVs) contribute to cervical and other anogenital cancers, and they are also linked etiologically to a subset of head and neck squamous cell carcinomas (HNSCC). We previously established a model for HPV-associated HNSCC in which we treated transgenic mice expressing the papillomaviral oncoproteins with the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO). We found that the HPV-16 E7 oncoprotein was highly potent in causing HNSCC, and its dominance masked any potential oncogenic contribution of E6, a second papillomaviral oncoprotein commonly expressed in human cancers. In the current study, we shortened the duration of treatment with 4-NQO to reduce the incidence of cancers and discovered a striking synergy between E6 and E7 in causing HNSCC. Comparing the oncogenic properties of wild-type versus mutant E6 genes in this model for HNSCC uncovered a role for some but not other cellular targets of E6 previously shown to contribute to cervical cancer.     

Lineage analysis of human papillomavirus type 18 based on E6 region in cervical samples of Iranian women

Distinct human papillomavirus (HPV) 18 variants are thought to differ in oncogenic potential and geographic distribution. As such, understanding the regional variants of HPV 18 would be of great importance for evolutionary, epidemiological, and biological analysis. In this regard, the sequence variations of E6 gene were investigated to characterize more common variants of HPV 18 in normal cells, premalignant, and malignant samples collected from the cervix. In total, 99 samples of HPV 18 were analyzed by polymerase chain reaction and sequencing. In overall, lineages A was identified in all study subjects, among which sublineage A4 was dominant although the difference observed was not statistically significant with regard to different stages of disease. Sublineage A4 comprised 90.9% of samples and the remaining were belonged to sublineages A1, A2, A3, and A5 at the frequency of 6.1%, 1%, 1%, and 1%, respectively. In conclusion, our findings clearly highlight the sublineage A4 of HPV 18 as the most dominant variant in Iran.     

人乳头瘤病毒16型内变异株与宫颈病变

目的 研究患宫颈上皮内瘤变（Cervical intraepithelial neoplasia,CIN）的汉族妇女中人乳头瘤病毒（Human papillomavirus,HPV）16型内变异株的类型及其在临床上的意义.方法 随机收集中日友好医院妇科门诊就诊的77例感染HPV16的汉族患者的宫颈脱落细胞DNA,用PCR法扩增HPV16型包含E6和E7基因的DNA片段并测序.通过对测序得到的E6基因序列与GenBank下载的参考株的比对,研究77例汉族患者中HPV16变异株的类型并探讨其与CIN的关系.结果 纳入研究的77例患者中,最小年龄21岁,最大年龄56岁,平均年龄36.39±6.86岁.其中,CIN Ⅱ级以下病变16例（占比20.8%）,CIN Ⅱ级及以上病变61例（占比79.2%）.HPV16型内变异株只有亚洲株和欧洲株两种,亚洲株38例,欧洲株39例.经χ^2检验,χ^2=0.0034,P〉0.05,尚不支持亚洲株与欧洲株的致癌作用不同.结论 虽然本研究未发现HPV16型亚洲株与欧洲株的致癌作用不同,但本研究发现77例汉族患者感染的HPV16型内变异株以亚洲株和欧洲株为主,故我们有理由推测,HPV16型内变异株在我国汉族妇女中的分布以亚洲株和欧洲株为主,而其他变异株,特别是高致癌的亚美株并不常见.     

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。     

北京地区人乳头瘤病毒16型感染及其E6/E7基因变异与宫颈病变的相关性研究

目的探讨HPV16感染及其E6/E7基因变异与宫颈病变的相关性。方法采用导流杂交技术进行HPV感染分型检测,PCR扩增出80份HPV16阳性宫颈病变的E6/E7基因、克隆入pMD18-T载体,双向测序分析基因变异与宫颈病变相关性。结果HPV16在宫颈病变患者中的检出率最高为33.3%(154/463),与病变程度相关(P<0.05)。E6/E7基因72份测序成功,DNA序列变异发生率为88.9%(64/72)。氨基酸序列E6-D32E(T96G)和E7-N29S(A86G)位点突变同时伴随存在,D32E/N29S的检出率为38.9%(28/72),与宫颈病变程度相关(P<0.05)。结论HPV16是北京地区来源的宫颈病变中最常见的致病型,其D32E/N29S变异与病变程度相关。     

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.     

Human papillomavirus type 18 variant lineages in United States populations characterized by sequence analysis of LCR-E6, E2, and L1 regions

While HPV 16 variant lineages have been well characterized, the knowledge about HPV 18 variants is limited. In this study, HPV 18 nucleotide variations in the E2 hinge region were characterized by sequence analysis in 47 control and 51 tumor specimens. Fifty of these specimens were randomly selected for sequencing of an LCR-E6 segment and 20 samples representative of LCR-E6 and E2 sequence variants were examined across the L1 region. A total of 2770 nucleotides per HPV 18 variant genome were considered in this study. HPV 18 variant nucleotides were linked among all gene segments analyzed and grouped into three main branches: Asian-American (AA), European (E), and African (Af). These three branches were equally distributed among controls and cases and when stratified by Hispanic and non-Hispanic ethnicities. Among invasive cervical cancer cases, no significant differences in the three HPV variant branches were observed among ethnic groups or when stratified by histopathology (squamous vs. adenocarcinoma). The Af branch showed the greatest nucleotide variability when compared to the HPV 18 reference sequence and was more closely related to HPV 45 than either AA or E branches. Our data also characterize nucleotide and amino acid variations in the L1 capsid gene among HPV 18 variants, which may be relevant to vaccine strategies and subsequent studies of naturally occurring HPV 18 variants. Several novel HPV 18 nucleotide variations were identified in this study.     

人乳头瘤病毒16型E6E7反义RNA抑制宫颈癌细胞恶性表型的作用

目的研究癌基因的特异性反义ＲＮＡ对癌细胞生长繁殖和恶性程度的影响。方法用逆转录病毒载体将人乳头瘤病毒（ＨＰＶ）－１６Ｅ６Ｅ７反义ＲＮＡ导入ＨＰＶ－１６ＤＮＡ阳性的宫颈癌细胞株ＣａＳｋｉ中，观察该细胞在导入反义ＲＮＡ后其表型特征和在裸鼠体内致癌能力的变化。结果ＨＰＶ－１６Ｅ６Ｅ７反义ＲＮＡ能降低宫颈癌细胞ＣａＳｋｉ的生长速率，抑制其在软琼脂上的集落形成能力，并能明显地抑制其在裸鼠体内的致癌能力。Ｗｅｓｔｅｒｎｂｌｏｔ分析发现ＨＰＶ－１６Ｅ６Ｅ７反义ＲＮＡ能使宫颈癌细胞中病毒ＨＰＶ－１６Ｅ６基因的表达水平降低。结论ＨＰＶ－１６Ｅ６Ｅ７反义ＲＮＡ能使宫颈癌细胞ＣａＳｋｉ恶性表型逆转；由其引起的癌细胞中ＨＰＶ－１６癌基因表达水平的降低可能是癌细胞表型逆转的原因之所在；ＨＰＶ－１６癌基因的表达水平对维持癌细胞的恶性表型起着重要作用。     

Human papillomavirus types 16 E6 and E7 contribute differently to carcinogenesis

High-risk human papillomaviruses (HPVs) are etiologically implicated in human cervical cancer. Two viral genes, E6 and E7, are commonly found expressed in these cancer cells. We have previously shown that mice transgenic for the HPV-16 E6 gene or E7 gene, in which the E6 or E7 was expressed in the basal layer of epithelia, developed skin tumors. The spectrum of tumors derived from E6 and E7 mice differed, however; although most tumors derived from the E7-transgenic mice were benign, the majority of the tumors from the E6-transgenic mice were malignant. These findings led us to hypothesize that E6 and E7 play different roles in carcinogenesis. To assess at what stages in carcinogenesis E6 and E7 act, we treated the skin of K14E6- and K14E7-transgenic mice with chemical carcinogens known to contribute to distinct stages in carcinogenesis. Both E6 and E7 were found to synergize with chemical carcinogens in causing tumor formation. E6 was found to act weakly at the promotion stage of carcinogenesis in the formation of benign tumors but strongly at the progression stage which involves the malignant conversion of benign tumors. In contrast, E7 primarily affected the promotion stage of carcinogenesis. These results provide direct evidence that E6 and E7 contribute differently to carcinogenesis; E7 promotes the formation of benign tumors, and E6 acts primarily to accelerate progression of these benign tumors to the malignant stage. Consistent with this model, we found E6 and E7 to cooperate in inducing tumor formation in mice expressing both oncogenes.     

Independent association of antibodies against human papillomavirus type 16 E1/E4 and E7 proteins with cervical cancer.

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an E1/E4 fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac-E1/E4 and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti-E1/E4 antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti-E1/E4 and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.     

T-cell response to human papillomavirus type 52 L1, E6, and E7 peptides in women with transient infection, cervical intraepithelial neoplasia, and invasive cancer

The E6 and E7 proteins encoded by human papillomaviruses (HPV) are prime targets for therapeutic vaccine development. Ninety-five women with HPV 52 infection (33 transient infections, 17 cervical intraepithelial neoplasia grade II, 15 cervical intraepithelial neoplasia grade III, and 30 invasive cervical cancers) were examined for T-cell responses using interferon-γ enzyme-linked immunospot (IFN-γ ELISPOT) assay. Of the 29 peptides (13 L1, 10 E6, and 6 E7) screened positive by an in vitro peptide-binding assay, 14 were positive by the IFN-γ ELISPOT assay. Positive epitopes for HLA A11 were located at amino acid positions 103-111, 332-340, 342-350, and 373-381 of the L1 protein; and at 27-35 and 86-94 of the E6 protein; and at 1-9 and 27-35 of the E7 protein. A24-specific epitopes included 60-68 and 98-106 of the L1 protein, 42-50 and 59-67 of the E6 protein, and 24-32 of the E7 protein. Only one epitope (99-107) of the E6 protein showed positive responses for HLA A2 subjects. Overall, T-cell responses against L1 were observed mainly in subjects who had cleared infection; whereas responses against E6 and E7 were confined mainly to subjects who had developed cervical neoplasia. The proportion of subjects showing detectable T-cell responses was low across all grades of cervical neoplasia suggesting that immune evasion mechanisms had set on early in the course of disease progression. This study provides the first set of T-cell epitopes mapped for HPV 52, which can be considered for further evaluation as targets for immunotherapy.     

Independent association of antibodies against human papillomavirus type 16 and E7 proteins with cervical cancer

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac- and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti- antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti- and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.     

人乳头瘤病毒16型E6E7反义RNA抑制宫颈癌细胞恶性？…

目的 研究癌基因的特异性反义ＲＮＡ对癌细胞生长繁殖和恶性程度的影响。方法 用逆转录病毒载体将人乳头瘤病毒（ＨＰＶ）－６Ｅ６Ｅ７反义ＲＮＡ导入ＨＰＶ－１６ＤＮＡ阳性的宫颈癌细胞株ＣａＳｋｉ中，观察该细胞在导入反义ＲＮＡ后其表型特征和在裸鼠体内致癌能力的变化。结果 ＨＰＶ－１６ Ｅ６Ｅ７反义ＲＮＡ能降低宫颈癌细胞ＣａＳｋｉ的生长速率，抑制其在软琼脂上的集落形成能力，并能明显地抑制其在裸鼠体内的致癌能力