Functional dissociation of transforming genes of human papillomavirus type 16

Human papillomavirus (HPV) type 16 is thought to be responsible for development of cervical carcinomas, but the mechanism of its carcinogenic action is unknown. To determine which viral genes are involved in cellular transformation, we constructed recombinant murine retrovirus DNAs containing various subgenomic fragments of the HPV 16 early region and examined their transforming activities. The results show that the E6 and E7 ORFs are the transforming genes of HPV 16; the former governs the tumorigenicity in nude mice and the latter influences cell growth properties such as saturation density and colony formation in soft agar. There may also be a tumor-suppressing gene in the E1-E2 ORF region, because the tumorigenic activity of recombinant DNA containing the E1 ORF and the 5' portion of the E2 ORF in addition to the E6 and E7 ORFs was much lower than that of recombinant DNA containing only the E6 and E7 ORFs.     

Expression of human papillomavirus type 16 E7 gene induces DNA synthesis of rat 3Y1 cells

Human papillomavirus type 16 (HPV 16) open reading frames (ORF) E6, E7, and E6E7, placed under the control of dexamethasone-inducible mouse mammary tumor virus long terminal repeat, were introduced into rat 3Y1 cells, an immortalized fibroblast line, with the aid of neomycin-selection. The cell clones containing inducible HPV 16 ORFs were selected and examined for DNA synthesis. Following induction of HPV mRNA synthesis by the hormone, DNA synthesis was stimulated in the cells containing E7 or E6E7 ORFs. The data indicate that expression of the HPV 16 E7 gene is mitogenic for rat 3Y1 cells.     

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。     

Molecular and phylogenetic analysis of the HPV 16 E4 gene in cervical lesions from women in Greece

The HPV16 E1(∧)E4 protein is thought to contribute to the release of newly formed viral particles from infected epithelia. In order to investigate amino acid mutations in the HPV16 E1(∧)E4 protein, the complete E4 ORF was amplified by PCR in 27 HPV16-positive cervical samples, and the amplicons were cloned. Fifteen nucleic acid variations were identified in the E4 ORF, including seven silent nucleic acid mutations. In addition, nine amino acid mutations (A7V, A7P, L16I, D45E, L59I, L59T, Q66P, S72F, H75Q) were detected in the E1(∧)E4 protein, and these were associated with the severity of cervical malignancy. A maximum-likelihood phylogenetic tree was constructed based on the E4 ORF, and nucleotide sequence analysis of the E4, E6 and E7 genes from the same samples was conducted in order to determine the phylogenetic origin of the cloned sequences from the amplified HPV16 E4. Based on the nucleotide sequence and phylogenetic analysis it was revealed that even though E4 ORF constitutes a small polymorphic portion of the viral genome (288?bp), it could provide valuable information about the origins of the HPV16 genome. In addition, molecular evolutionary analysis of the E4 coding region revealed that neutral selection is dominant in the overlapping region of the E4 and E2 ORFs.     

Integration of human papillomavirus type 16 into cellular DNA of cervical carcinoma: preferential deletion of the E2 gene and invariable retention of the long control region and the E6/E7 open reading frames

The integration patterns of human papillomavirus (HPV) type 16 in the cellular DNA of six cervical carcinoma samples were analyzed by the Southern blot procedure. None of the HPV integrants retained the entire viral genome. Double HPV integration was found in one case while all other cases were single integrants. In some samples, internal deletion and selective amplification of the viral sequences were observed. On integration, the E2 open reading frame (ORF) was invariably lost but the E6/E7 ORFs and the long control region of the HPV-16 genome were retained in all seven integrations analyzed and may play a role in cellular transformation and/or maintenance of the transformed phenotype.     

人类风湿性关节炎滑膜细胞系(RASB)的建立和生物学特性的观察

目的　建立人类风湿性关节炎滑膜细胞永生细胞系。　方法　用重组有HPV16病毒E6 E7基因阅读框架的逆转录病毒载体转染原代培养的人类风湿性关节炎滑膜细胞 ,经G418筛选 ,获取细胞克隆RASB ,并从形态学、生长特性、核型组成、致瘤性和分泌功能等多方面对建系细胞RASB进行生物学观察。　结果　实验和观察证实 ,转化滑膜细胞染色体整合HPV病毒DNA ,表达HPVE6蛋白 ,基本保留了B型滑膜细胞特征形态、细胞骨架和分泌功能 ,倍增时间缩短一半 ,对裸鼠无致瘤性 ,软琼脂培养形成细小集落。已稳定传代大于 10 0代。　结论　建立了能长期体外稳定传代的人类风湿性关节炎滑膜细胞系。此细胞系的建立将为类风湿关节炎致病机理的研究和治疗提供极有意义的体外细胞模型。     

Oligonucleotide primers for DNA amplification of the early regions 1, 6, and 7 from human papillomavirus types 6, 11, 16, 18, 31, and 33

Summary Human papillomavirus (HPV) type-specific sequences required for polymerase chain reaction (PCR) mediated amplification of HPV DNA sequences are presented. One primer pair within the E1 open reading frame (ORF) was shared by HPV 6, HPV 11, HPV 16, and HPV 31, whereas the other primer pair within the E1 ORF was specific for HPV 16. Eight primer pairs from the E6 and E7 ORFs specifically detected HPV 6, HPV 16, HPV 18, and HPV 33 sequences. This system has been used for detection of HPV DNA in biopsies, cytological smears and sections of formalin-fixed tissues.     

Human papillomavirus (HPV) types 101 and 103 isolated from cervicovaginal cells lack an E6 open reading frame (ORF) and are related to gamma-papillomaviruses

Complete genomes of HPV101 and HPV103 were PCR amplified and cloned from cervicovaginal cells of a 34-year-old female with cervical intraepithelial neoplasia grade 3 (CIN 3) and a 30-year-old female with a normal Pap test, respectively. HPV101 and HPV103 contain 4 early genes (E7, E1, E2, and E4) and 2 late genes (L2 and L1), but both lack the canonical E6 ORF. Pairwise alignment similarity of the L1 ORF nucleotide sequences of HPV101 and HPV103 indicated that they are at least 30% dissimilar to each other and all known PVs. However, similarities of the other ORFs (E7, E1, E2, and L2) indicated that HPV101 and HPV103 are most related to each other. Phylogenetic analyses revealed that these two types form a monophyletic clade, clustering together with the gamma- and pi-PV groups. These data demonstrated that HPV genomes closely related to papillomaviruses identified from cutaneous epithelia can be isolated from the genital mucosal region. Moreover, this is the first report of HPVs lacking an E6 ORF and phylogenetic evidence suggests this occurred subsequent to their emergence from the gamma-/pi-PVs.     

含人乳头瘤病毒16型E6E7反意基因的假型逆转录病毒对人乳头瘤病毒16型转化细胞生长的抑制作用

构建了一个含人乳头瘤病毒１６型Ｅ６Ｅ７反意基因片断的逆转录病毒载体ｐＨ１９。用Ｌｉｐｏｆｅｃｔｉｎ将ｐＨ１９导入病毒包装细胞ｐＡ３１７，使之产生假型逆转录病毒Ｈ１９。Ｈ１９病毒感染人乳头瘤病毒１６型转化的细胞后，使转化细胞的生长速率和裸鼠体内形成肿瘤的能力均明显下降。     

The primary structure of major viral RNA in a rat cell line transfected with type 47 human papillomavirus DNA and the transforming activity of its cDNA and E6 gene

A transformed cell line (RC335) showing higher saturation cell density was obtained from 3Y1 cells (a fibroblastic cell line of rat) transfected with DNA of human papillomavirus type 47 (HPV-47), an epidermodysplasia verruciformis-associated virus, which our laboratory reported previously. The cell line was found to produce a major and several minor species of viral RNAs. The primary structure of the major viral RNA in RC335 was extensively studied and found to consist of two exons and contain open reading frames (ORFs) E6, E7, and a fused ORF, E1/E4. The major RNA was indicated to play an important role in the transformation of RC335 by an experiment with the recombinant retrovirus designed to produce the RNA containing these ORFs, i.e., the recombinant virus induced transformation similar to that in RC335 upon infection of 3Y1 cells. Furthermore the experiments with recombinant viruses carrying a nonsense mutation or large deletion in the above ORF(s) indicated that E6 was necessary and sufficient for the transformation.     

Gene expression profiles reveal an upregulation of E2F and downregulation of interferon targets by HPV18 but no changes between keratinocytes with integrated or episomal viral genomes

Persistent infections with human papillomaviruses type 18 can result in the development of cervical cancer. HPV18 genomes persist extrachromosomally in low-grade and precancerous lesions but are always integrated in cervical cancers, and this might contribute to the progression of HPV18-induced lesions. To address whether integration induces additional changes in host cells, several keratinocyte lines with wild type and replication-deficient E1 mutant HPV18 (E1C-TTL) genomes were analyzed with high density oligonucleotide arrays. In comparison to normal keratinocytes, wild type and integrated E1C-TTL HPV18 genomes deregulate the expression of 280 annotated genes. However, the comparison of wild type with E1C-TTL cell lines did not reveal any significant differences, indicating that neither the loss of E1 nor viral integration induces additional gene expression changes in low passage HPV18-positive keratinocytes. Half of the deregulated genes have been described as targets of the p16/Rb/E2F, p53, interferon or NFkappaB pathways consistent with the functions ascribed to the viral E6 and E7 oncoproteins, but the other half can currently not be ascribed to certain pathways.     

Expression of the human papillomavirus type 6b L2 open reading frame in Escherichia coli: L2-beta-galactosidase fusion proteins and their antigenic properties

Human papillomavirus (HPV) type 6b genome contains two large open reading frames (ORFs), designated L1 and L2, in a putative late region. These ORFs are expected to code for viral structural proteins. To examine antigenic properties of a L2 gene product, we constructed two plasmids which contain N-terminal (L2-N) and internal (L2-I) regions of the HPV6b L2 ORF and then each region was expressed in Escherichia coli as a fusion protein with E. coli beta-galactosidase (beta-Gal). Both L2-N/beta-Gal fusion proteins reacted with anti-beta-Gal antibody, but did not react with the antibody prepared against bovine papillomavirus type 1 (BPV1), in contrast with a high reactivity of HPV6b L1-beta-Gal fusion protein with the anti-BPV1 antibody. Antibody raised against the L2-I/beta-Gal protein in a rabbit reacted with viral antigens in the nuclei of cells in superficial epithelium of the condyloma acuminatum tissue, but did not react with the antigens in the bovine papilloma tissue. This antibody recognized a protein from condyloma acuminata which migrates to the position of mol wt 70K-76K on an electrophoresed SDS-polyacrylamide gel. These results suggested that the L2 ORF of HPV6b codes for a capsid protein which is less cross-reactive than the L1 antigen with anti-BPV1 antibody.     

Expression of HPV16 E5 produces enlarged nuclei and polyploidy through endoreplication

Anogenital cancers and head and neck cancers are causally associated with infection by high-risk human papillomavirus (HPV). The mechanism by which high-risk HPVs contribute to oncogenesis is poorly understood. HPV16 encodes three genes (HPV16 E5, E6, and E7) that can transform cells when expressed independently. HPV16 E6 and E7 have well-described roles causing genomic instability and unregulated cell cycle progression. The role of HPV16 E5 in cell transformation remains to be elucidated. Expression of HPV16 E5 results in enlarged, polyploid nuclei that are dependent on the level and duration of HPV16 E5 expression. Live cell imaging data indicate that these changes do not arise from cell-cell fusion or failed cytokinesis. The increase in nuclear size is a continual process that requires DNA synthesis. We conclude that HPV16 E5 produces polyploid cells by endoreplication. These findings provide insight into how HPV16 E5 can contribute to cell transformation.     

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.