Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma.

We have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1,284 and 4,471 (numbering system from K. Seedorf, G. Kr?mmer, M. Dürst, S. Suhai, and W. G. R?wekamp, Virology 145:181-185, 1985), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1,137 and 1,138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene described by Seedorf et al. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene.     

Analysis of integrated human papillomavirus type 16 DNA in cervical cancers: amplification of viral sequences together with cellular flanking sequences.

We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed.     

成都市人乳头瘤病毒16型感染与宫颈病变的相关性分析

本研究旨在探讨人乳头瘤病毒(human papillomavirus,HPV)16感染与宫颈病变的关系,为宫颈癌防治提供科学依据。通过核酸杂交法进行HPV感染分型,纳入1 057例HPV阳性且行组织切片病理学检查的患者,对各级别宫颈病变中HPV16构成比、不同年龄组HPV16阳性患者中宫颈上皮内瘤样病变(cervical intraepithelial neoplasia,CIN)Ⅱ及以上病变的患病率,以及HPV16单一与多重感染患者中CINⅡ及以上病变的患病率进行分析。结果显示,在1 057例HPV阳性患者中,352例感染HPV16,CINⅢ中HPV16构成比最高,各级别病变中HPV16构成比差异有统计学意义。随着病变级别增加,HPV16构成比有增高趋势(P0.05)。不同年龄组HPV16阳性患者中CINⅡ及以上病变的患病率差异有统计学意义(P0.05),且随年龄增加而升高(P0.05)。HPV16单一、双重与三重以上感染患者中,CINⅡ及以上病变的患病率差异有统计学意义(P0.05),且随着感染型别种类增加,患病率降低(P0.05)。本研究显示,HPV16与高级别宫颈病变有较明显的相关性,老年HPV16阳性患者检出宫颈癌的概率更高。因此,应高度重视HPV16持续性感染,做到及时诊断与治疗,以减少宫颈高级别病变和宫颈癌的发生。     

Interferon inhibition of neoplastic phenotype in cell lines harbouring human papillomavirus sequences.

The transformed phenotype is markedly affected by Interferon (IFN) long term treatment in HeLa and Siha cell lines. Two months of continuous exposure to 100 and 1000 International Units (IU)/ml of human recombinant alpha IFN causes a drastic reduction of anchorage independent growth in soft-agar. No effect on in vitro growing potential was observed at the same concentrations. This antiproliferative effect in soft-agar was more pronounced in Siha than in Hela cells and was concentration and time dependent. The Human Papillomavirus (HPV) sequences inserted in the cell genome apparently were unaffected as their RNA expression, indicating that IFN may modulate aspects of transformed phenotype regulated by genes other than HPV sequences.     

Detection of human papillomavirus gene sequences in cell lines derived from laryngeal tumors

The role of Human Papillomaviruses (HPV) in laryngeal carcinomas has been studied with conflicting results. To evaluate the etiologic relationship between HPV infection and epithelial malignancy of the larynx we studied five laryngeal carcinoma cell lines obtained from patients undergoing surgery for laryngeal tumors. The paraffin embedded biopsy samples of the original tumor and different passages of the new established cell lines were investigated by PCR with consensus primers specific for HPV DNA. The findings indicate that HPV infection is associated with some larynx carcinomas. The positive association has been enhanced when a method of enrichment of epithelial cells from fresh tumor samples was used. All tumor cells enriched smears were positive for HPV DNA not only by PCR but also by in situ hybridization (ISH). Investigated by PCR, different passages of larynx tumor cell lines maintained expression of HPV DNA. At subsequent passages ISH gives constantly no signals suggesting a minimal amount of viral harbored sequences. In one cell line propagated more than 60 population doublings, the chromosomal frequency distribution shifted from modal number 46 at the 5^th passage to 63 at the 60^th passage. The mechanisms by which persistent HPV infection maintains continuous cell proliferation were discussed.     

Sequence duplication and internal deletion in the integrated human papillomavirus type 16 genome cloned from a cervical carcinoma.

Integrated human papillomavirus type 16 (HPV16) sequences were cloned from a cervical carcinoma and analyzed by restriction mapping and nucleotide sequencing. The viral integration sites were mapped within the E1 and E2 open reading frames (ORFs). The E4 and E5 ORFs were entirely deleted. An internal deletion of 376 base pairs (bp) was found disrupting the L1 and L2 ORFs. Sequencing analysis showed that an AGATGT/ACATCT inverted repeat marked the deletion junction with two flanking direct repeats 14 and 8 bp in length. A 1,330-bp sequence duplication containing the long control region (LCR) and the E6 and E7 ORFs was also found. The duplication junction was formed by two 24-bp direct repeats with 79% (19 of 24) homology located within the LCR and the E2 ORF of the prototype viral genome, respectively. This observation leads us to propose that the initial viral integration involved an HPV16 dimer in which the direct repeats in tandem units recombined, resulting in reiteration of only a portion of the original duplication. A guanosine insertion between nucleotides 1137 and 1138 created a continuous E1 ORF which was previously shown to be disrupted. Results from this study indicate that sequence reiteration and internal deletion in the integrated, and possibly in the episomal, HPV16 genome are influenced by specific nucleotide sequences in the viral genome. Moreover, reiteration of the LCR/E6/E7 sequences further supports the hypothesis that the E6/E7 ORFs may code for oncogenic proteins and that regulatory signals in the LCR may play a role in cellular transformation.     

Mutational analysis of human papillomavirus type 16 E7 functions.

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.     

Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines.

Molecular genetic studies on HeLa cell-derived nontumorigenic and tumorigenic hybrids have previously localized the HeLa cell tumor-suppressor gene to the long arm of chromosome 11. Extensive molecular and cytogenetic studies on HeLa cells have shown chromosome band 11q13 to be rearranged in this cell line. To determine whether q13 rearrangement is a nonrandom event in cervical carcinomas, six different human papilloma virus (HPV)-positive (HeLa, SiHa, Caski, C4-I, Me180, and Ms751) and two different HPV-negative (C33A and HT3) cell lines were studied. Long-range restriction mapping using a number of q13-specific probes showed molecular rearrangements within 75 kb of INT2 probe in three HPV-positive cell lines (HeLa, SiHa, and Caski) and in an HPV-negative cell line (HT3). FISH using an INT2 YAC identified a breakpoint within the sequences spanned by this YAC in two of the cell lines, HeLa and Caski. INT2 cosmid derived from this YAC showed deletion of cosmid sequences in two other cell lines, SiHa and C33A. These two cell lines, however, retained cosmid sequences of Cyclin D1, a probe localized 100 kb proximal to INT2. Deletions being the hallmark of a tumor-suppressor gene, we conclude that the 100-kb interval between the two cosmids might contain sequences of the cervical carcinoma tumor-suppressor gene.     

Computer-assisted analysis of molecular mimicry between human papillomavirus 16 E7 oncoprotein and human protein sequences

The immunology of human papillomavirus (HPV) infections has peculiar characteristics. The long latency for cervical cancer development after primary viral infection suggests mechanisms that may aid the virus in avoiding the host immunosurveillance and establishing persistent infections. In order to understand whether molecular mimicry phenomena might explain the ability of HPV to avoid a protective immune response by the host cell, sequence similarity between HPV16 E7 oncoprotein and human self-proteins was examined by computer-assisted analysis. Data were obtained showing that the HPV16 E7 protein has high and widespread similarity to several human proteins involved in a number of critical regulatory processes. In addition, multiple identical and different E7 peptide motifs are present in the same human protein. Thus, sharing of common motifs between viral oncoproteins and molecules of normal cells may be one cause underlying the scarce immunogenicity of HPV infections. The hypothesis is advanced that synthetic peptides harbouring viral motifs not and/or scarcely represented in the host's cellular proteins may represent a valuable immunotherapeutic approach for cervical cancer treatment.     

Production of human papillomavirus type 16 virions in a keratinocyte cell line.

Human papillomavirus type 16 (HPV-16) is strongly associated with carcinoma of the cervix, but the complete life cycle of the virus cannot be studied because no experimental system is available in which HPV-16 progeny are produced, and there is currently no source of HPV-16 virus particles. Most cell lines that harbor HPV-16 DNA contain the viral genome as integrated or concatenated DNA in which open reading frames are disrupted or deleted, but a human cervical keratinocyte cell line has been described which maintains HPV-16 DNA in monomeric episomal form (M.A. Stanley, H.M. Brown, M.W. Appleby, and A.C. Minson, Int. J. Cancer 43:672-676, 1989). This cell line was induced to form a stratified differentiating epithelium by grafting onto nude mice. Long-term grafts displayed the histological features of a low-grade cervical dysplasia, and terminally differentiated cells contained amplified levels of HPV-16 DNA, virus capsid antigen, and virus particles. This experimental system appears to permit the completion of the HPV-16 life cycle in virus-containing keratinocytes.     

Serologically diagnosed infection with human papillomavirus type 16 and risk for subsequent development of cervical carcinoma: nested case-control study.

OBJECTIVE--To study human papillomavirus type 16 in the aetiology of cervical carcinoma. DESIGN--Within a cohort of 18814 Finnish women followed up to 23 years a nested case-control study was conducted based on serological diagnosis of past infection with human papillomavirus type 16. SUBJECTS--72 women (27 with invasive carcinoma and 45 with in situ carcinoma) and 143 matched controls were identified during the follow up. MAIN OUTCOME MEASURE--Relative risk of cervical carcinoma in presence of IgG antibodies to human papillomavirus type 16. RESULTS--After adjustment for smoking and for antibodies to various other agents of sexually transmitted disease, such as herpes simplex virus type 2 and Chlamydia trachomatis, the only significant association was with infection with human papillomavirus type 16 (odds ratio 12.5; 95% confidence interval 2.7 to 57, 2P<0.001). CONCLUSION--This prospective study provides epidemiological evidence that infection with human papillomavirus type 16 confers an excess risk for subsequent development of cervical carcinoma.     

Whole genome sequencing and evolutionary analysis of human papillomavirus type 16 in central China

Human papillomavirus type 16 plays a critical role in the neoplastic transformation of cervical cancers. Molecular variants of HPV16 existing in different ethnic groups have shown substantial phenotypic differences in pathogenicity, immunogenicity and tumorigenicity. In this study, we sequenced the entire HPV16 genome of 76 isolates originated from Anyang, central China. Phylogenetic analysis of these sequences identified two major variants of HPV16 in the Anyang area, namely the European prototype (E(p)) and the European Asian type (E(As)). These two variants show a high degree of divergence between groups, and the E(p) comprised higher genetic diversity than the E(As). Analysis with two measurements of genetic diversity indicated that viral population size was relatively stable in this area in the past. Codon based likelihood models revealed strong statistical support for adaptive evolution acting on the E6 gene. Bayesian analysis identified several important amino acid positions that may be driving adaptive selection in the HPV 16 population, including R10G, D25E, L83V, and E113D in the E6 gene. We hypothesize that the positive selection at these codons might be a contributing factor responsible for the phenotypic differences in carcinogenesis and immunogenicity among cervical cancers in China based on the potential roles of these molecular variants reported in other studies.