E6-E7 Region Research Articles

Clinical validation of REALQUALITY RQ-HPV Screen according to the international guidelines for human papillomavirus DNA test requirements for cervical screening

BackgroundAccording to international guidelines, HPV DNA tests represent a valid alternative to Pap Test for primary cervical cancer screening, provided that they guarantee balanced clinical sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or more severe lesions. The aim of this study was to assess whether REALQUALITY RQ-HPV Screen, a new assay based on real time PCR that targets the E6-E7 region of 14 high-risk human papillomaviruses, meets the criteria for primary cervical cancer screening.MethodsAs required by guidelines, a non-inferiority test was conducted to compare the clinical performance of the test under evaluation with that of a clinically validated reference test (Hybrid Capture 2, HC2). The reproducibility of the device was assessed as well. The clinical samples used to test the hypothesis of non-inferiority and to asses reproducibility comprised 910 and 536 cervical specimens respectively. All specimens were originating from a population-based screening cohort.ResultsThe study demonstrates that both the clinical sensitivity and specificity of REALQUALITY RQ-HPV Screen are non-inferior to those of HC2. In addition, an adequate intra- and inter-laboratory reproducibility has been reached by the test.ConclusionsREALQUALITY RQ-HPV Screen fulfils all the requirements of the international guidelines and can be considered clinically validated for primary cervical cancer screening purposes.

Genetic Variability in E6 and E7 Oncogenes from Human Papillomavirus Type 58 in Mexican Women

Background/Aims: The study aimed to describe human papillomavirus (HPV) 58 genetic variability in E6 and E7 oncogenes from women in southeast Mexico and their phylogenetic relationships with the sequences from other geographical regions. Methods: The E6-E7 region was amplified by nested PCR, and sequenced for identification of polymorphisms, phylogenetic trees construction, and haplotype and fixation tests. Results: HPV58 positive samples were obtained from a repository, 54 were amplified, 47 sequences for the E6 gene, and 51 sequences for the E7 gene were obtained. Fifteen new E6 mutations were found; the most frequent were G279T (G57V; 29.78%), T249G (F47C; 34.04%), and A270G (Y54C; 34.04%), and previously reported c307t (63.82%). For E7, 17 known mutations were found, the most frequent were C632T (T20I), 35.30%, G760A (G63S), 35.30%, and t744g 74.50%. No significant association with the severity of the lesions was found. The polytomy in the E6 tree did not allow proposing phylogenetic relationship, and E7 tree presented defined branches. All sequences were presumably A lineage, most closely related to A1 and/or A3 sublineage. HPV58 variants are not specific for a geographical area. Population and fixation analyses suggest a possible Asian origin of HPV58 from Yucatan. The most frequent E7 haplotype in Yucatan groups with other populations of the world. Conclusion: The genetic variability of HPV58 from Mexico was described for the first time. E7 was more conserved than E6. New mutants present exclusively in Yucatan were identified.

Molecular and evolutionary analysis of HPV16 E6 and E7 genes in Greek women

Human papillomavirus type 16 (HPV16) non-European variants have been associated with persistent infection and cervical cancer development, while the L83V variant of the E6 gene has been correlated with the progression of cervical malignancy. The present study investigated the presence of the HPV16 L83V variant in Greek women. Molecular evolutionary analysis of the HPV16 E6 and E7 oncogenes was conducted in order to estimate the evolution of the HPV16 genome in the Greek population. The E6 L83V variant was found in 78.2 % of high- and 64.28 % of low-grade specimens. Moreover, the prototype and E6 L83V variants were both prevalent in high- and low-grade malignancies in Greek women. Selective pressure analysis of the individual amino acid residues of HPV16 sequences from the Greek population indicates that codon 83 of the E6 protein, as well as codon 85 of the E7 protein, are undergoing positive selection. Novel sequence variations were recorded within the E6 and E7 genes in cervical samples, characterized as (T350G) European variants. However, no signal of intratypic recombination event was identified within the E6-E7 region. Molecular and evolutionary analyses of HPV16 genomes from distinct geographical locations might provide valuable information about viral evolution and oncogenecity.

Identification of novel E6-E7 sequence variants of human papillomavirus 16

The rate of evolution of the human papillomavirus 16 (HPV16) genome is low. However, the ability of the E6 oncoprotein to interact with distinct p53 variants causes selective pressure on the E6 gene. In addition, intratypic recombination events in the HPV16 E6 and E7 genes have been characterized as extraordinary phenomena during the evolutionary history of virus. In the present study, we identified two new sequence variants through nucleotide analysis of the E6-E7 region of the HPV16 genome. Maximum-likelihood and empirical Bayesian methods were used in order to identify positive selection at particular residues of the E6 and E7 genes. Using the single recombination breakpoint (SBP) method, we found evidence of recombination events in the E6 ORF. Nucleotide sequence analysis showed that the new sequence variants are phylogenetically distant from the other members of the population. Our results indicate that new evolutionary intermediates of HPV16 might be formed either though positive selective pressure or through recombination events by multiple infections with distinct HPV16 variants.

HPV-16 and HLA-DRB1 Alleles Are Associated with Cervical Carcinoma in Mexican Mestizo Women

The aim of this report was to investigate the contribution of HLA-DRB1/DQB1 alleles to the expression of cervical cancer (CC) and squamous intraepithelial lesion (SIL) in Mexican patients. A total of 257 women were included in the study: 61with low-grade squamous intraepithelial lesions (LSIL), 30 with high-grade (HSIL), 73 with CC and 93 healthy females. All were Mexican Mestizos. For HLA class II typing, PCR-SSOP methodology was used. HPV-16 viral DNA was detected by PCR with specific primers for E6-E7 region. HPV-16 was found in 52% of the patients with CC as well as in 19% of women with HSIL and in 12.5% of females with LSIL. HLA-DRB1∗04:03 (OR = 5.88) was found increased in patients with HSIL as compared with controls, although significance ( p = 0.04) was lost after correction (pc = NS). HLA-DRB1∗04:03 seems to influence the risk for developing HSIL, disregarding the presence of HPV-16. HLA-DRB1∗01:01 (OR = 0.12; p = 0.01) may confer protection to the development of CC. An analysis performed stratifying by the presence of HPV-16 infection showed that the frequency of HLA-DRB1∗04:07 (OR = 2.71) was increased in CC patients infected with HPV-16, confirming that the HLA association is HPV dependent. These results shed light on the influence that this virus may have in the expression of CC in the susceptible host. Genetic background is, therefore, a crucial factor in understanding the etiopathogenesis of CC in HPV-positive patients.

Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types

Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra- and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intra-assay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Our real-time PCR methodologies are highly reproducible, sensitive, and specific over a sevenfold magnitude dynamic range.

Human papillomavirus type 16 E6 and E7 genotypes in head-and-neck carcinomas

Human papillomavirus type 16 (HPV16) is associated with squamous cell carcinomas of the head and neck (HNSCC) particularly from the Waldeyer’s tonsillar ring. A causal role of HPV16 in carcinogenesis is linked to the activity of the viral oncoproteins E6 and E7 which inactivate the cellular tumor suppressors p53 and pRB, respectively. Lack of E6 expression in HPV16-positive HNSCC has been reported, in some cases caused by disruption of the E6 gene. We have examined the status of the HPV16 E6–E7 gene region in tumor and metastasis samples of 24 HNSCC patients employing genomic PCR. No cases with a disrupted E6–E7 region could be identified. Sequence analysis of the E6–E7 segments revealed three different HPV16 E6–E7 genotypes: the HPV16 prototype (6 of 21 cases), the E6 variant T350G (8 of 21 cases), and the E6–E7 variant A131G/C712A (7 of 21 cases). The E6 variants T350G and A131G have been associated with increased oncogenic potential in cervical cancer patients depending on host genetic factors. Their high prevalence in the HNSCC samples studied indicates that they may be important also in HNSCC development.

Distinct patterns of alteration of myc genes associated with integration of human papillomavirus type 16 or type 45 DNA in two genital tumours

We previously described two genital carcinomas (IC2, IC4) containing human papillomavirus type 16 (HPV-16)- or HPV-18-related sequences integrated in chromosomal bands containing the c-myc (8q24) or N-myc (2p24) gene, respectively. The c-myc gene was rearranged and amplified in IC2 cells without evidence of overexpression. The N-myc gene was amplified and highly transcribed in IC4 cells. Here, the sequence of an 8039 bp IC4 DNA fragment containing the integrated viral sequences and the cellular junctions is reported. A 3948 bp segment of the genome of HPV-45 encompassing the upstream regulatory region and the E6 and E7 ORFs was integrated into the untranslated part of N-myc exon 3, upstream of the N-myc polyadenylation signal. Both N-myc and HPV-45 sequences were amplified 10- to 20-fold. The 3' ends of the major N-myc transcript were mapped upstream of the 5' junction. A minor N-myc/HPV-45 fusion transcript was also identified, as well as two abundant transcripts from the HPV-45 E6-E7 region. Large amounts of N-myc protein were detected in IC4 cells. A major alteration of c-myc sequences in IC2 cells involved the insertion of a non-coding sequence into the second intron and their co-amplification with the third exon, without any evidence for the integration of HPV-16 sequences within or close to the gene. Different patterns of myc gene alterations may thus be associated with integration of HPV DNA in genital tumours, including the activation of the protooncogene via a mechanism of insertional mutagenesis and/or gene amplification.

Detection and characterization of human papillomavirus type 45 DNA in the cervical carcinoma cell line MS751.

The cervical carcinoma-derived cell line MS751 was examined for human papillomavirus (HPV) DNA and RNA. A genomic fragment containing both viral and cellular sequences was cloned. Sequence analysis showed that MS751 cells contain a partially deleted HPV-45 genome integrated at a single chromosomal site. HPV sequences from the E6-E7 region are expressed as poly(A)+ RNA.

Sperm as a noninvasive gene delivery system for preimplantation embryos

To determine if sperm could be manipulated to be a noninvasive transport carrier for the delivery of gene fragments to the blastocyst.Sperm cells carrying foreign DNA fragments from human papillomavirus (HPV) types 16, 18, 31, and 33 were allowed to migrate from one end of an artificial reproductive tube and to come in contact with hatching mouse blastocysts at the other end of the tube. The blastocysts were then washed and analyzed for the presence of the foreign DNA fragments.Clinical and academic research environment.Detection of amplified products from transferred foreign DNA using the polymerase chain reaction and primers targeted at the E6-E7 region for different HPV types.Polymerase chain reaction analyses showed transference of DNA HPV type 18 to the blastocysts. Not all types of DNA fragments were transferred equally.The results suggested the possibility of using sperm as a noninvasive gene delivery system for passing on gene fragments to preimplantation embryos. It was demonstrated that certain DNA fragments were easier to deliver than others, indicating the necessity for exploring all the factors involved in the mechanism of the transference process. The study also serves to highlight the possibility of unintentional transmission of viral or bacterial DNA to the developing embryo via the sperm.

Biologic activity of human papillomavirus type 16 E6/E7 cDNA clones isolated from SiHa cervical carcinoma cell line.

Three species of E6/E7 cDNAs of human papillomavirus type 16 (HPV16) for the full-length E6/E7 and spliced E6*I/E7 and E6*II/E7 mRNAs were synthesized by reverse transcriptase-(RT-)PCR from RNA of the cervical carcinoma cell line SiHa. Two cDNA mutants carrying point mutations in either a splice donor site or acceptor site within the E6 open reading frame were also constructed. These HPV16 E6/E7 cDNAs were cloned under the SV40 enhancer/promoter and the MMTV LTR to examine the activities of ras-collaborative transformation and induction of cellular DNA synthesis, both of which depend on the E7 gene product. The E6*II/E7 cDNA and two mutated cDNAs deficient in the spliced mRNA transcription showed lower levels of both activities than the full-length E6/E7 and the E6*I/E7 cDNA. The rat cell lines carrying each of the E6/E7 cDNAs contained the E6/E7 mRNA species expected. A small amount of E6*I/E7-sized mRNA was transcribed from a splice-donor site mutant of the E6/E7 cDNA, which turned out to be a transcript derived from a cryptic splice donor site six bases upstream from the conventional site. Among NIH3T3 cells carrying one of the above-mentioned E6/E7 cDNAs, the cells expressing E6*I/E7 mRNA [cells carrying cF(wt) and c*I] produced an amount of E7 protein comparable with those carrying the E7 or E6E7 region. These results suggest that the E6*I/E7 is the mRNA that is important for the efficient expression of E7 product from the HPV16 E6/E7 region.

ヒトパピローマウイルスと癌

More than twenty genotypes of human papillomaviruses (HPVs) are associated with mucosal lesions and more than thirty genotypes with cutaneous lesions. We established a method using consensus polymerase chain reaction (PCR) with restriction endonuclease analysis of PCR products, which can detect and distinguish mucosal high risk HPV types 16, 18, 31, 33, 52b and 58 (J. Gen. Virol. 72, 1039, 1991). By using this method, we analyzed 39 cases of cervical carcinomas and 27 cases of cervical intraepithelial neoplasias (CIN). It was found that the HPV16 was most prevalent in malignant (19 cases) and premalignant (19 cases) cervical lesions, but other types including HPV18, 31, 33, 52b and 58 were also detected especially in cervical carcinomas. We also established a highly sensitive method for specific detection of HPV types 16, 18, and 33 (Jpn. J. Cancer Res., 81, 1, 1990) . The method detected HPV16 DNA in only 5 out 92 cases of cytologically or histologically normal cervices (Jpn. J. Cancer Res., 82, 532, 1991) . HPV16 was also predominantly detected in penile (68 out of 111 cases) and tongue (8 out of 24 cases) squamous cell carcinomas.HPV16 E6E7 region produces at least three different species of E6E7 mRNAs (unspliced E6-E7, spliced E6* I and E6* II) in the HPV16-containing carcinoma cells. We synthesized each of the E6E7 cDNAs and studied on their biological activities. The E6*I cDNA showed highest activities of ras-collaborative transformation of primary rat fibroblasts and induction of cellular DNA synthesis. We analyzed HPV16 E6E7 mRNAs in three cervical carcinomas and six CINs which contained HPV16 DNA by PCR with reverse transcription (RT-PCR) . The major transcript from HPV16 E6E7 region was E6* I except for one CIN tissue where only the full-length E6-E7 mRNA was detected.Different from E7 genes of mucosal high-risk HPVs, E7 genes of HPV5 and HPV8, which are associated with epidermodysplasia verruciformis (EV) were inactive for transforming ability in cell lines. We cloned HPV5 and 8 E7s separately into the expression vector pcD2Y or pGEM3Zf and examined their biological and biochemical activities. Although HPV5 and 8 E7s failed to immortalize primary baby rat kidney cells, they collaborated with activated ras gene to transform primary rat embryo fibroblasts (Jpn. J. Cancer Res., 82, 1340, 1991) and baby rat kidney cells. In vitro binding experiment of retinoblastoma tumor suppressor protein (RB) detected the complex formation of HPV5 and 8 E7s with RB but with reduced affinities compared to HPV16 E7.

Isolation of two keratinocyte cell lines derived from HPV-positive dysplastic vaginal lesions.

Explant cultures were started from human papillomavirus (HPV)-infected genital lesions in order to isolate and propagate abnormally differentiating cells from squamous intraepithelial neoplasia. A medium with high calcium concentration was used to induce terminal differentiation of cells from surrounding normal epithelium. Two cell lines with extended life-spans were established. The UT-DEC-1 cell line was derived from an HPV-33-positive mild vaginal dysplasia (VAIN I). In cultured UT-DEC-1 cells, HPV 33 DNA was detected with Southern-blot hybridization and the polymerase chain reaction (PCR) technique. The restriction pattern of HPV 33 changed during early passages and flow cytometric analysis detected a decrease in chromosomal DNA content. HPV 33 RNA from the E6-E7 region could be amplified by PCR at late passage. UT-DEC-2 cell line was derived from an HPV-16-positive moderate vaginal dysplasia (VAIN II). HPV 16 DNA was also detected in cultured cells by the PCR technique. The senescence of normal keratinocytes and growth selection in favor of aneuploid cells was observed by flow cytometric analysis at subsequent passages. Karyotype analysis showed clonal chromosomal abnormalities in both cell lines. To date, UT-DEC-1 cells have undergone 40 and UT-DEC-2 cells 25 passages. This study shows that the isolation of HPV-infected dysplastic cells can be achieved by culturing the cells in a medium with high calcium concentration. The cell lines presented provide the opportunity of evaluating the early stages of squamous-cell carcinogenesis.

Transcription of the E6 and E7 genes of human papillomavirus type 6 in anogenital condylomata is restricted to undifferentiated cell layers of the epithelium

The E6 and E7 genes of human genital papillomaviruses (HPVs) appear to transform cells by different mechanisms. They seem to act synergistically but are not equally important when tested under diverse experimental conditions. We were therefore tempted to investigate the E6- and E7-specific transcription pattern in HPV6-infected condylomas separately, by in situ hybridization. Recent studies have identified three promoters within the E6-E7 region of HPV6 and HPV11 by applying S1, exonuclease VII, and cDNA analyses. On the basis of these data, we cloned subgenomic fragments of HPV6 into plasmid pBS to obtain riboprobes that differentiated between transcripts starting upstream of the E6 and E7 open reading frames, respectively. These different species of mRNAs were analyzed in serial thin sections of eight HPV6-positive anogenital condylomas. The E6 probe (nucleotides 7862 to 241) led to weak signals within the basal layer. In three cases, rather strong signals were confined to a few basal cells. The E7 probe (nucleotides 242 to 534) gave rise to a more pronounced labeling of all cells within the two to three lowest epidermal layers. In situ hybridization with a riboprobe for human c-fos revealed an expression pattern similar to that observed with the E7 probe. In contrast to the preferential expression of the transforming E6 and E7 genes in the lower epithelium, the major transcriptional activity of the virus was detected in the middle and upper third by probes colinear with the 3' moiety of the early region.

Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction

A multiplex polymerase chain reaction (PCR) based on the simultaneous amplification of human papillomavirus (HPV) types 6 11 , 16 and 18 in a single-step procedure was developed, using primers chosen in the E6–E7 region. The specificity and sensitivity of this technique have been proved by amplifying mixtures or various amounts of plasmid-containing HPV DNA; it allowed the detection of as few as 5–25 HPV DNA copies. Application of the multiplex PCR to 71 clinical samples showed that HPV DNA was detected in 80% ( 45 57 cases) of mucosal biopsies and 35% ( 5 14 cases) of cutaneous specimens. HPV 16 was predominant in high-grade CIN whereas HPV 6 and 11 were detected more frequently in genital condylomas and laryngeal papillomas. In cutaneous Bowen's disease HPV 16, 18 or 6 11 + 16 were detected and in squamous cell carcinomas HPV 6 11 or 16 were found. After sequence amplification with primers of one HPV type, the clinical samples displayed the same HPV types but the frequency of positive and coinfected lesions increased. Thus, multiplex PCR is a valuable technique for typing HPV DNA but coinfections may be underestimated.

Presence of Human Papilloma Virus Type 16 DNA Sequences in Human Nonmelanoma Skin Cancers

The presence of human papillomaviruses (HPV) has been shown to be associated with the development and progression of invasive cancers of the genital tract, skin, and head and neck. In this study we analyzed 37 human nonmelanoma skin cancers (21 squamous cell carcinomas and 16 basal cell carcinomas) for the presence of HPV sequences. The polymerase chain reaction (PCR) was employed using primers designed to amplify DNA encoding the E6-E7 region of HPV types 6b/11, 16, and 18. HPV type 6b/11 and 18 sequences were absent from the DNA of all 37 tumors examined. However, HPV type 16 sequences were detected in four of 21 squamous cell carcinomas (SCC) (19%) and three of 16 basal cell carcinomas (BCC) (19%) as indicated on agarose gel electrophoresis by the presence of a single specific DNA band of predicted length. Furthermore, HPV type 16 sequences were absent in the DNA of normal skin from these seven skin cancer patients. The presence of HPV type 16 sequences in the seven skin tumors was confirmed by dot blot hybridization of PCR-amplified material to 32P-labeled HPV type 16, but not to HPV type 6/11 or 18-specific probes. These data indicate that HPV type 16, but not 6b/11 or 18, is associated with development of some human nonmelanoma skin cancers.

HPV-16 viral transcripts in vulvar neoplasia: Preliminary studies

Specific human papillomavirus (HPV) types are strongly associated with intraepithelial neoplasia and invasive cancer of the cervix. In contrast, the role of HPVs in the pathogenesis of invasive carcinoma of the vulva is poorly understood. We have employed in situ hybridization for the detection of subgenomic transcripts in four vulvar specimens to elucidate the role of HPV type 16 in the development and progression of vulvar cancer. These analyses revealed that the transcripts of the E6–E7 region were more abundant than those of the L1–L2 region in vulvar neoplastic tissues. The transcripts from early and late region of HPV-16 continued to increase with the differentiation of the epithelial cells in both the warty and the basaloid types of vulvar precancerous lesions. This pattern persisted in invasive warty carcinoma but not in basaloid invasive carcinoma; the transcripts in basaloid carcinoma were distributed in an even and discrete pattern. In contrast to earlier studies, L1–L2-region transcripts, as well as viral capsid protein, were detected in focal areas of well-differentiated cells of invasive warty carcinoma. These findings suggest that expression of HPV-16 is regulated by the degree of cellular differentiation.

The E7 protein of human papillomavirus 8 Is a nonphosphorylated protein of 17 kDa and can be generated by two different mechanisms

The E7 protein of human papillomavirus (HPV) 8 shows no in vitro transforming activity, in contrast to E7 of the genital HPVs, but seems to be involved in the control of viral DNA replication. To determine whether functional differences between the E7 proteins of HPV16 and HPV8 were reflected in differences in their biochemical properties, we characterized the E7 protein of HPV8 expressed from the late simian virus 40 promoter in COS 7 cells. An E7-specific antiserum was obtained by immunizing rabbits with a β-gal-E7 fusion protein containing all of the E7 polypeptide. This antiserum specifically precipitated from [ 35S]cysteine but not from 32PO 4-labeled transiently transfected COS 7 cells a protein with a low mobility of 17 kDa in SDS-PAGE. The high sedimentation rate in nondenaturing glycerol gradients pointed to an interaction with other proteins or to the existence of E7 oligomers. An association with the retinoblastoma protein could, however, be excluded. The Fends of HPV8 transcripts derived from the E6–E7 region in G418 selected rodent cells, which were mapped by nuclease S1 digestion, are located upstream of ORFs E6 and E7, respectively. No evidence for an E6∗I-like mRNA was found. In COS 7 cells transfected with a plasmid expressing the E6–E7 region of HPV8 under the control of the late SV40 promoter, however, the E7 protein was translated from a polycistronic mRNA potentially encoding E6 and E7. These data indicate that the E7 protein of HPV8 may be expressed in two ways: (i) through translation of an E7-specific mRNA, as with HPV6 and HPV11, or (ii) through internal initiation of translation at the ATG of E7.

Expression of the human papillomavirus type 16 genome in SK-v cells, a line derived from a vulvar intraepithelial neoplasia

The SK-v cells, established from a premalignant vulvar lesion, contain human papillomavirus type 16 (HPV-16) sequences integrated at a single cellular site and derive from a cell clone present in vivo. Transcription of the HPV-16 genome in SK-v cells was analysed by cDNA heteroduplex mapping and sequencing, and by RNase mapping. Viral sequences were shown to be transcribed into virus-cell fusion messengers. The two major transcripts have a coding capacity for a truncated E6 protein, an E7 protein and an E1-E4 fusion protein, but differ in their 3' virus-cell junction. Minor transcripts have a coding capacity for a full-length E6 protein and another truncated version of E6. The transcription pattern in the E6-E7 region was found to be the same both in SK-v cells and in CaSki cells, a line derived from an invasive cervical carcinoma. Immunoprecipitation experiments showed that the E6 protein (18K) and, predominantly, the E7 protein (20K) are expressed in SK-v cells as in CaSki cells. The E7 protein was found in a two- to threefold lower amount in SK-v cells, but showing the same half-life (about 1 h).

Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18

The E6-E7 region of human papillomavirus types 16 and 18 is selectively retained and expressed in cervical carcinoma cells. In cultured human keratinocytes, expression of the E6 and E7 open reading frames of human papillomavirus type 18, under the control of its homologous promoter, resulted in high-frequency immortalization. Furthermore, by using a system that allows for stratification of keratinocytes in vitro (raft system), we observed that the morphological differentiation of these E6-E7 immortalized cells was altered such that parabasal cells extended throughout most of the epithelium, with abnormal nuclei present in the upper regions. Examination of E6-E7-expressing cell lines in the raft system at a later passage revealed that complete loss of morphological differentiation had occurred. E7 alone was a much less effective immortalizing agent than E6 and E7 together and acted only minimally to alter morphological differentiation in vitro. No such activities were found for E6 alone. High-frequency transformation of human epithelial cells thus appears to require expression of both E6 and E7 gene products.