Orientation-dependent toxic effect of human papillomavirus type 33 long control region DNA in Escherichia coli cells

Virus Genes - The functional analysis of human papillomavirus (HPV) sequence variation requires the molecular cloning of different genomic regions of virus variants. In this study, we report an...     

Transcriptional control of human papillomavirus type 18 oncogene expression in different cell lines: Role of transcription factor YY1

Binding of YY1 to the proximal fragment of the human papillomavirus type 18 (HPV-18) upstream regulatory region (URR) activates the oncogene expression of HPV-18 in HeLa cells, whereas in HepG2 cells this expression is repressed by YY1. In the present transient transfection study, we analyze the regulation of the HPV-18 URR by YY1 in an extended number of cell lines. Except for HeLa cells, YY1 represses or does not influence oncogene expression in all cell lines tested. In HeLa cells the activation of viral oncogene expression by YY1 is caused by the functional interplay between YY1 and a factor binding to the newly identified “switch region” of the HPV-18 URR. In this work we show that in HeLa cells, a 22 bp region located between the “switch region” and the promoter proximal fragment contributes to the modulation of the activity of YY1 and, in addition, regulates HPV-18 promoter activity independently of YY1.     

A T-helper cell epitope overlaps a major B-cell epitope in human papillomavirus type 18 E2 protein.

Cultivated CD4+ T-helper cells from two patients with cervical adenocarcinoma showed responses to a peptide EKTGILTVTYHSETQRTK derived from an E2 protein of human papillomavirus type 18 (HPV 18), but not to a corresponding HPV 16 peptide (HKSAIVTLTYDSEWQRDQ). Serum antibodies in the HPV 18 peptide were also demonstrated in these patients. The GILT motif resembles a common pattern present in many T-cell epitopes, and is located at the beginning of an 11-amino acid-long A-helix structure close to the carboxyterminal end of HPV 18 E2. We conclude that two epitopes (a T-helper cell epitope and a B-cell epitope) overlap in the HPV 18 E2.     

A human papillomavirus type 16 vaccine by oral delivery of L1 protein

To establish an edible HPV16 vaccine, we constructed a recombinant HPV16 L1-expressing Schizosaccharomyces pombe yeast strain (HPV16L1 yeast). A preliminary study revealed that freeze-dried yeast cells could be delivered safely, and were digested in the mouse intestine. The freeze-dried HPV16 L1 yeast was administered orally as an edible vaccine, with or without the mucosal adjuvant heat-labile toxin LT (R192G), to 18 female BALB/c mice. After the third immunization, none of the mice that received the edible HPV16 vaccine showed specific antibody responses, whereas all of the positive controls that were administered intranasally with 5 μg of HPV16-virus-like particles (VLP) had serum IgG, and genital IgA and IgG that reacted with HPV16-VLP in enzyme-linked immunosorbent assays (ELISAs). When a suboptimal dose (1 μg) of HPV16-VLP was administered to all the mice, including the negative control mice, 50% of the mice that were pre-immunized with the edible HPV16 vaccine showed positive serum IgG responses, while none of the negative controls showed any response. Vaginal IgG and IgA antibodies were also elicited in 33 and 39%, respectively, of the mice that were given with the edible HPV16 vaccine and the intranasal boost. All of the antibodies reacted more strongly to intact HPV16-VLP than to denatured HPV16-L1 protein suggesting that the edible vaccine primes for antibody responses against conformation-dependent epitopes. The inclusion of adjuvant in the vaccine formulation marginally increased the genital IgA response (P = 0.06). HPV16-L1 protein in the yeast might induce tolerance in the vaccinated animals that could be recovered by intranasal boosting with a suboptimal dose of HPV-VLP. This freeze-dried yeast system may be useful as an oral delivery of HPV 16 L1 protein.     

人乳头瘤病毒16型中国株L1基因的体外真核表达

目的 构建人类乳头瘤病毒16型(HPV16)中国株晚期基因L1的真核表达系统.方法 将HPV16中国株L1基因从pCR2.1/HPV16L1定向亚克隆至pTacer-CMV载体,构建重组质粒pTaeer-CMV/HPV16L1,将重组质粒用脂质体转染N1H3T3、HeLa细胞,用透射电镜、SDS-PAGE、蛋白印迹等方法观察HPV16 L1蛋白的表达.结果 NIH3T3、HeLa细胞经pTacer-CMV/HPV16L1转染后,SDS-PAGE、蛋白印迹等实验显示可表达HPV16 L1蛋白,电镜下可见病毒样颗粒(VLP).结论 pTacer-CMV/HPV16L1构建成功,可在体外表达HPV16中国株L1蛋白.     

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.     

HPV L1 C-末端保守序列短肽体液免疫学特性

目的 探讨一段位于HPV L1 C-末端、长30个氨基酸残基的保守序列的体液免疫学性质而进行此实验.方法 以此序列为基础人工合成短肽,以该短肽免疫小鼠和兔,获得抗血清,通过ELISA,Western Blot及免疫组织化学的方法,分别对含HPV6b,16及18 L1的细胞裂解物、重组蛋白或HPV阳性临床标本进行反应.结果 抗短肽抗血清能与含HPV6b,16及18 L1的细胞裂解物、重组蛋白发生针对HPV L1的反应,能使HPV6和16阳性的临床标本呈现阳性反应,而抗HPV16及抗重组HPV16 L1抗血清对此短肽的反应则较弱.结论结果表明由该保守序列短肽诱导的抗血清具有一定的型间交叉反应特征,这一特性可能对研究检测用广谱HPV L1抗体有一定的意义.该序列短肽抗血清对不同型HPV L1反应的差异性及抗血清对多型别HPV L1的反应是否具有中和性,尚需进一步的研究.     

Sequence variation in the L1 gene of human papillomavirus type 16 from Africa

Summary A 297 bp fragment of the HPV 16 L1 gene was sequenced from 35 isolates originating from normal cervical scrapes, cervical intraepithelial neoplasia biopsies and cervical carcinoma biopsies from South African women. A total of six point mutations that differed from the prototype HPV 16 sequence were detected in various combinations, giving rise to six distinct types of variants. Only one of the isolates had a sequence identical to the prototype. Our results indicate that the HPV L1 gene is conserved and there is no evidence that specific viral HPV L1 variants are associated with specific pathology.