Papillomaviruses and human oncogenesis.

During the past year, significant advances have been made in understanding the functions of the oncoproteins, E6 and E7, of human papillomaviruses that are associated with malignant genital tumors. In addition, important new information is now available on the responses of both the keratinocyte and of the individual following papillomavirus infection.

Targeted expression of the E6 and E7 oncogenes of human papillomavirus type 16 in the epidermis of transgenic mice elicits generalized epidermal hyperplasia involving autocrine factors.

The E6 and E7 early genes of human papillomavirus type 16 have been shown in vitro to play a central role in the transforming capability of this virus. To explore their effects on differentiating epithelial cells in vivo, we used a bovine cytokeratin 10 (K10) promoter to target the expression of E6 and E7 to the suprabasal layers of the epidermis of transgenic mice. In two different lines of mice efficiently expressing the transgene, animals displayed generalized epidermal hyperplasia, hyperkeratosis and parakeratosis in the skin and the forestomach, both known to be sites of K10 expression. Northern (RNA) blot analysis revealed high levels of E6 and E7 transcripts, and in situ hybridizations localized these transcripts to the suprabasal strata of epidermis. In vivo labeling of proliferating cells showed two distinct effects of E6 and E7 expression in the epidermis: (i) an increase in the number of growing cells in the undifferentiated basal layer and (ii) abnormal proliferation of differentiated cells in the suprabasal strata. The expression of c-myc in the skin of transgenics was higher than that in control animals. The induction of c-myc transcription by topical application of tetradecanoyl phorbol acetate was prevented by simultaneous treatment with transforming growth factor beta 1 in nontransgenic skin but not in transgenic skin. In addition, transforming growth factor alpha was found to be overexpressed in the suprabasal layers of the transgenic epidermis. These findings suggest that autocrine mechanisms are involved in the development and maintenance of epidermal hyperplasia. Animals of both lines developed papillomas in skin sites exposed to mechanical irritation and wounding, suggesting that secondary events are necessary for progression to neoplasia. Collectively, these results provide new insights into the tumor promoter activities of human papillomavirus type 16 in epithelial cells in vivo.

Antibodies against the human papillomavirus type 16 early proteins in human sera: correlation of anti-E7 reactivity with cervical cancer.

By Western blot technique, 519 samples of human sera were tested for the presence of antibodies to the human papillomavirus (HPV) type 16 proteins E4 and E7 that had been expressed in Escherichia coli as fusion proteins. Sera were obtained from patients attending the University hospitals for reasons unrelated to HPV infections (controls), from patients with HPV-associated lesions, as well as from patients suffering from cervical cancer. Within the control population, 18.1% of them had antibodies that reacted with the E4 protein, and 3.9% of them had antibodies that reacted with the E7 protein. No sex-specific difference in the antibody prevalence was observed. The highest proportion of anti-E4 antibody-positive individuals (40.7%) was observed in the age group between 11 and 20 years. The frequency of anti-E4-positive sera was threefold higher in patients with HPV-associated genital lesions than that in age-matched controls. Antibodies against the HPV16 E7 protein were found 14 times more frequently in patients with cervical cancer, compared with age- and sex-matched controls (P less than .00001). From these data, we concluded that anti-E4 antibodies may be correlated with virus replication and that anti-E7 antibodies may represent a marker for cervical cancer development.

Growth-regulating functions of human papillomavirus early gene products in cervical cancer cells acting dominant over enhanced epidermal growth factor receptor expression.

Squamous cell carcinomas of the human anogenital tract are usually associated with infection of specific types of human papillomaviruses (HPV 16, 18, 31, 33, 35). The intracellular concentration of human papillomavirus early gene products E6 and E7 has been directly linked to the proliferative capacity of cervical cancer cells. Since the expression rate of epidermal growth factor receptor correlates to growth properties in squamous carcinoma cell lines, it has been presumed that human papillomavirus early genes influence cell growth via enhanced epidermal growth factor receptor expression. This hypothesis implies that growth regulation by epidermal growth factor receptor overexpression dominates over a growth-regulatory influence of human papillomavirus early gene products in squamous carcinoma cells. To test this hypothesis epidermal growth factor receptor expression was analyzed in various clones of the C4-1 cervical cancer cell line which, upon dexamethasone treatment, express either increased or decreased levels of human papillomavirus 18 early gene products. In C4-1 clones expressing reduced levels of viral E6/E7 gene products upon glucocorticoid treatment expression of epidermal growth factor receptor was the same as in those clones displaying increased levels of papillomavirus proteins under identical culture conditions. The growth rate of the cells correlated with the level of viral gene products rather than with the expression of epidermal growth factor receptor. These findings suggest that unregulated overexpression of epidermal growth factor receptor is not the dominant mechanism of growth control in papillomavirus-positive carcinoma cells. Other, yet unknown pathways associated with papillomavirus early genes are essentially involved in growth control mechanisms of human cervical cancer cells.

Correlation of modified human papilloma virus early gene expression with altered growth properties in C4-1 cervical carcinoma cells.

In cervical carcinomas and cell lines derived from these tumors the DNA of specific types of human papilloma viruses (HPVs) is integrated into the host cell genome. The two viral open reading frames E6 and E7 are consistently transcribed in tumors and cell lines, and the respective proteins were detected in cells cultured in vitro. As shown here, modulation of HPV 18 E6 and E7 gene expression in C4-1 cervical carcinoma cells is accompanied by an altered cell growth. HPV 18 E6 and E7 expression can be enhanced by glucocorticoid treatment of C4-1 cells, and an increased cell proliferation is observed. In contrast, after introduction of complementary RNA to the HPV 18 E6 and E7 open reading frames, their expression is inhibited, and decreased cell growth is observed. These results support the hypothesis that expression of HPV E6 and E7 open reading frames is directly involved in growth regulation of cervical carcinoma cells.

An amplification unit in human melanoma cells showing partial homology with sequences of human papillomavirus type 9 and with nuclear antigen 1 of the Epstein-Barr virus.

By partial homology with the DNA of human papillomavirus type 9 a cellular amplification unit was detected which is amplified in melanoma cells but not in Epstein-Barr virus-transformed B cells of two melanoma patients. A 2.4-kilobase EcoRI fragment of this amplification unit was cloned and designated mel/HPV9. At the chromosomal level we detected mel/HPV9 in homogeneously staining regions or in abnormally banded regions containing different marker chromosomes of both melanoma cell lines. DNA sequence analysis of a part of mel/HPV 9 revealed homology with the third internal repeat array of Epstein-Barr virus nuclear antigen 1.

Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA.

A proliferating population of human foreskin keratinocytes (presently in the sixtieth passage) has been obtained after transfection with human papillomavirus (HPV) type 16 DNA. In contrast, the control cultures did not survive beyond the sixth passage. Cytogenetic analysis of cells taken from the twelfth passage revealed a heteroploid male karyotype. In approximately 50% of the cells a common marker chromosome was found, suggesting a clonal origin for at least part of the population. This is further substantiated by Southern blot analysis of cellular DNA which revealed oligomeric HPV 16 genomes integrated at a single site within the host DNA. RNA transcribed from the early region of the HPV 16 genome was identified in the cytoplasm. The immortalizing effect of HPV 16 DNA on human keratinocytes could be reproduced in a second experiment. Such cell lines represent an unique system to study the interaction of HPV with its natural target cell in vitro.

The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells.

The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.

Assembly of human papillomavirus type 16 pseudovirions in Saccharomyces cerevisiae.

Studies of the encapsidation of papillomavirus (PV) DNA, and production of preparative amounts of PVs in vitro, have met with only limited success. To circumvent this problem we established a system in yeast to generate infectious HPV-16 pseudovirions. Saccharomyces cerevisiae strain 1699 was transformed with a construct to allow production of HPV-16 virus-like particles (VLPs). This strain was then transformed with a second construct (target plasmid), the same size as the HPV-16 genome and containing the HPV-16 upstream regulatory region (URR) and the HPV-16 E2 open reading frame. In addition, the target plasmid contained the green fluorescent protein gene to monitor delivery of the target plasmid into mammalian cells after infection. We conclude that this system allows HPV DNA encapsidation because (1) HPV-16 VLPs of two different types (heavy and light) were detected by CsCl gradient centrifugation, (2) DNase I-resistant DNA was detected by PCR/Southern blot analysis in fractions of CsCl gradients at a density corresponding to heavy VLPs, (3) in vitro infection of mammalian cells, including primary mouse splenocytes, with pseudovirions resulted in delivery of the reporter gene as demonstrated by FACS analysis for GFP expression, and (4) after injection of pseudovirions into mice, in vivo reporter gene expression was detected by confocal microscopy in sections of muscle tissue. We conclude that HPV-16 pseudovirions produced in yeast may be useful both for in vitro transduction and for gene delivery in vivo.

Papillomavirus infection of the anogenital region: correlation between histology, clinical picture, and virus type. Proposal of a new nomenclature.

The clinical and histologic picture of 84 anogenital condylomatous and condyloma-like lesions of both sexes were analyzed in an effort to establish a correlation to the different papillomavirus (PV) types. The presence of human papillomavirus (HPV)-specific DNA sequences was confirmed through molecular hybridization and the presence of PV structure antigens was verified in thin sections by means of a group-specific anti-PV-antiserum using the peroxidase-antiperoxidase (PAP) technique. Three distinct clinical forms harboring distinct HPV types were distinguished: (1) Condylomata acuminata in which HPV-6 DNA was present in 37 of 59 samples and HPV-11 DNA in only 13 of 59 samples. HPV-16 DNA was not detected at all and 9 condylomatous lesions remained unclassified. (2) Flat condyloma-like lesions, where HPV-6 and HPV-11 were associated with lesions of low epidermal atypia in 8 and in 2 of 18 cases, respectively, and where HPV-16 was associated exclusively with 6 of 18 such lesions with severe atypia, called bowenoid papulosis. (3) Pigmented papules where HPV-16 was detected twice in lesions of bowenoid papulosis and HPV-11 in 2 of the benign pigmented lesions. The fourth clinical manifestation of genital papillomavirus infections--the so-called condylomata plana--was not available for virologic analysis. Histologically 5 different koilocytotic features were determined which could not be correlated either with one of the clinical pictures or with a specific PV type. HPV-16, however, was found frequently in non-koilocytotic lesions exhibiting the features of severe epithelial atypia known in bowenoid papulosis. The existence of PV structure antigens in these lesions could not be verified using the indirect immunoperoxidase--PAP-technique--in contrast to the koilocytotic lesions where clear evidence of the presence of HPV was proved in 36 of 56 (64.3%) of the cases.

Correlation between human papillomavirus (HPV) type and histology of warts.

Forty warts from different patients and of different clinical type were examined histologically and virologically. Eight lesions were found to be associated with human papillomavirus type 1 (HPV 1), 15 tumors were induced by HPV 2, HPV 3 was detected 4 times, HPV 4 twice, and HPV 6 eleven times. HPV 3, HPV 4, and HPV 6 induced warts revealed a correlation between histology and virus type. They are characterized by the so called "edematous type clear cells". In HPV 3 associated flat warts pycnotic nuclei were mainly localized in the center of large vacuoles. In genital warts sickle shaped nuclei were pushed to the margin of the vacuolized cells. The histology of HPV 1 and HPV 2 induced warts was more heterogenous. With one exception HPV 1-induced lesions represented typical myrmecia warts, varying in the number and shape of inclusion bodies. HPV 2 associated common warts, however, revealed 3 very distinct histologic features: (1) Inclusion wart typical for HPV 1, (2) Classical common wart with marked condensation of keratohyalin granules, (3) Warts with extreme vacuolization of squamous and granular cells leading to a honeycomb-like picture.

Correlation between content of viral DNA and evidence of mature virus particles in HPV-1, HPV-4, and HPV-6 induced virus acanthomata.

In HPV-1 and HPV-4 induced warts as well as in HPV-6 positive condylomata acuminata the quantity of viral DNA encapsulated into virus particles was determined and compared to the total amount of viral DNA present in the papillomas. As shown by filter hybridization using 3H-labeled viral DNA molecularly cloned in Escherichia coli, the amount of total viral DNA found in HPV-1 or HPV-4 induced skin warts is similar. HPV-4 DNA, however, is encapsulated into virus particles with less efficiency. HPV-6 DNA can be detected only at minute amounts in condylomata acuminata and the percentage of DNA recovered from virions is extremely low.

Subclinical Epstein-Barr virus infection of both the male and female genital tract--indication for sexual transmission.

Epstein-Barr Virus (EBV) can infect B lymphocytes as well as epithelial cells of the oral cavity. Recently, infection of epithelial cells of the inflamed uterine cervix has been demonstrated, and EBV-DNA has been detected in urethral discharge of men suffering from genital infection. We investigated whether EBV can be found in the genital tract of both sexes independently from inflammatory disease states. Genital specimens of men and women of a sexually transmitted diseases outpatient clinic after excluding sexually transmitted diseases and clinically apparent signs of inflammation were investigated using the polymerase chain reaction to screen for EBV-DNA. In 13 of 47 samples (27.7%) swabbed from the uterine cervix, EBV-DNA could be detected. Similarly, 6 of 45 samples (13.3%) scraped from the sulcus coronarius contained EBV-DNA. Our study shows that the female genital tract and likewise the male genital tract can subclinically harbor EBV. These findings suggest i) that in addition to the oral cavity, the female and the male genital tract may be a reservoir for EBV and ii) that sexual transmission of this virus associated with an epidemiology different from that of oral infection may be possible.

Analysis of oral papillomas, leukoplakias, and invasive carcinomas for human papillomavirus type related DNA.

Five papillomas, five leukoplakias, and six carcinomas were investigated for the presence of papillomavirus group-specific antigens and viral DNA. Viral proteins were identified with genus-specific papillomavirus antibodies. Cloned human papillomavirus (HPV) 11 and 16 DNA were used as probes in Southern blot hybridization at conditions of different stringency in order to determine viral DNA. Four of five papillomas, four of five leukoplakias, and three of six carcinomas reacted with HPV DNA probes and revealed some stained cells after exposure to HPV antibodies. HPV type 16 was found in one carcinoma and HPV type 11 was demonstrated in another case of carcinoma.

The human papillomavirus type 11 upstream regulatory region triggers hair-follicle-specific gene expression in transgenic mice.

We have generated transgenic mice carrying the URR of the human papillomavirus type 11 ligated in front of the Escherichia coli beta-galactosidase coding region sequence. Using X-Gal staining to demonstrate beta-galactosidase production, we observed a hair-specific transcription of the reporter gene. This transcription was limited to the epithelial cells of the hair bulge region. The transgene was developmentally regulated, as no LacZ staining was demonstrated during embryogenesis and specific staining was first observed after birth. Surprisingly, dexamethasone and ultraviolet B, but not phorbol myristate acetate or progesterone treatment of the animals resulted in an increase in number and intensity of hair follicles expressing the reporter gene.

Presence of human papillomavirus in genital tumors.

Human papillomavirus (HPV) types 16 and 18 have been identified in two different human cervical carcinomas. The viral DNAs were molecularly cloned and used as probes to screen a large number of genital tumors by Southern blot analysis. HPV-16 or HPV-18 sequences, respectively, were found in a high percentage of cervical carcinomas, but only in a small number of condylomata acuminata or flat condylomas. The majority of the latter lesions, however, contained HPV-6 or HPV-11 sequences, respectively, which in contrast were detected only rarely in carcinomas in situ or invasively growing carcinomas. A similar distribution of the different papillomaviruses was observed when cell swabs taken from the cervix were tested by in situ hybridization.

Detection of Epstein-Barr virus-DNA in tongue epithelium of human immunodeficiency virus-infected patients.

Oral hairy leukoplakia is a lesion on the lateral part of the tongue that contains replicating Epstein-Barr virus (EBV) and presages progression from human immunodeficiency virus (HIV) infection to AIDS. To clarify the role of EBV in the development of the lesions, we used filter in situ DNA hybridization to determine the prevalence of EBV and of human papillomavirus (HPV) in epithelial cells obtained on swabs from the tongue of HIV-infected patients who had hairy leukoplakia, HIV-infected patients who did not have hairy leukoplakia, and healthy uninfected control persons. In samples collected from the 35 uninfected control persons, EBV DNA could not be detected except at low concentrations in three people. In contrast, all but one of the samples from 11 HIV-infected patients who had hairy leukoplakia contained EBV DNA. Of greatest interest, in 19 of 32 HIV-infected patients who had no signs of hairy leukoplakia, EBV DNA was also detected on the epithelium of the tongue. DNA filter in situ hybridization for the detection of HPV serotypes 6, 11, 16, and 18 in all cases yielded negative results. Statistical analysis showed that the presence of EBV DNA was significantly correlated with the clinical status of the HIV-infected persons, as determined by Walter Reed staging classification, whereas hairy leukoplakia was not. It is concluded that detection of EBV DNA in oral epithelium may be an earlier and more powerful predictor of progression to AIDS than is hairy leukoplakia.

A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates.

DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79 . The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage lambda L47 . The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA- Escherichia coli hosts.

Cervical carcinoma cells transfected with the CD80 gene elicit a primary cytotoxic T lymphocyte response specific for HPV 16 E7 antigens.

Cervical carcinoma is strongly associated with human papillomavirus (HPV) type 16, and the transforming viral genes E6 and F7 are steadily expressed by the tumor cells. Therefore these viral oncogenes may be regarded as tumor-associated antigens. Our previous studies showed that cervical cancer cells after introduction of the CD80 gene activated allogeneic cytotoxic T lymphocytes (CTLs). In this study, we tested whether HPV 16+ cervical tumor cells (CaSki) expressing CD80 were able to activate CTLs recognizing HPV 16 E7 antigen. To this end, CD80+ CaSki cells (HLA-A*0201+) were used to stimulate peripheral blood T lymphocytes from HLA-A*0201+ healthy donors. We found that the activated T cells were able to lyse parental CaSki cells as well as Epstein-Barr virus-immortalized autologous B cells loaded with HLA-A*0201-restricted E7 peptides (amino acids 11-19, 82-90, 86-93). In contrast, no lysis was observed against target cells loaded with a control HIV-reverse transcriptase peptide (amino acid 476-484, HLA-A 0201-restricted). Our data, for the first time, provide evidence that CD80-expressing cervical cancer cells are able to activate tumor-specific CTLs using HPV 16 E7 as tumor-associated antigens.