Association of human papillomavirus types 16 and 18 E6 proteins with p53.

Human papillomavirus type 16 (HPV-16) is a DNA tumor virus that is associated with human anogenital cancers and encodes two transforming proteins, E6 and E7. The E7 protein has been shown to bind to the retinoblastoma tumor suppressor gene product, pRB. This study shows that the E6 protein of HPV-16 is capable of binding to the cellular p53 protein. The ability of the E6 proteins from different human papillomaviruses to form complexes with p53 was assayed and found to correlate with the in vivo clinical behavior and the in vitro transforming activity of these different papillomaviruses. The wild-type p53 protein has tumor suppressor properties and has also been found in association with large T antigen and the E1B 55-kilodalton protein in cells transformed by SV40 and by adenovirus type 5, respectively, providing further evidence that the human papillomaviruses, the adenoviruses, and SV40 may effect similar cellular pathways in transformation.

Multiple cutaneous papillomas and carcinomas that develop spontaneously in a mouse mutant, the repeated epilation heterozygote Er/+.

After a chance observation that multiple cutaneous papillomas and squamous cell carcinomas occurred in 2 adult mice heterozygous for the repeated epilation gene Er, we surveyed a panel of 10 +/+ (wild type) and 30 Er/+ (heterozygous) mice from birth to over 2 years of age. Homozygous Er/Er mice could not be included since their defect is lethal at birth. Whereas no cutaneous tumors developed in the +/+ mice, 20 of the Er/+ mice, males and females, had developed 1-5 cutaneous papillomas and at least 1 cutaneous invasive squamous cell carcinoma by 2 years of age. No lesions were seen in mice younger than 6 months old. Although almost all Er/+ mice died with their tumor burden, no metastases have yet been proven histologically. The Er/+ mouse should serve as a useful model for the exploration of genetic factors in cutaneous squamous cell carcinomas in humans.

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.

Demonstration of in vitro synthesis of human papilloma viral proteins from hand and foot warts.

HPV particles purified from [35S]-methionine labeled and unlabeled halves of single hand and foot warts have been fractionated into empty, light full, and heavy full particles by buoyant density gradient centrifugation, and their proteins analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE) and visualized by either fluorography or silver staining. The L1 coat protein (54 Kd) was found in trace amounts in unmodified and slightly modified forms in the labeled empty and light full particles but could not be detected in the labeled heavy particles. L1 appeared to exist in the three unlabeled particle types in differentially modified forms. A putative L2 protein was also found to be modified (74-80 Kd) and was found preferentially in the unlabeled heavy full particles. The commercial cross-reactive BPV antibody recognized a labeled 58-Kd protein found predominantly in the empty and light full particles and a pair of proteins (41-42 Kd) found unlabeled in the heavy full particles. Besides L1, there were several other proteins (IEF 40 Kd; NEPHGE 42, 38, and 36 Kd) which were detected labeled in the empty particles and in increasing unlabeled amounts in the light full and heavy full particles. Four proteins (IEF 66, 13 and 11 Kd, and NEPHGE 9 Kd) were found exclusively in the full particles and may be involved in packing the viral genome. These observations suggest that a virus particle assembly pathway exists from the empty particles, via the light full, to the mature heavy full particles.

Enhanced chemiluminescence for tissue antigen and cellular viral DNA detection.

The ability to use enhanced chemiluminescence (ECL) to detect horseradish peroxidase as a label for tissue antigens and cellular viral DNA was demonstrated. A liquid nitrogen-cooled charged-coupled device (CCD) was used to detect light output, which was visualized on a monitor or was quantitated using an attached microcomputer. In a tissue antigen model, equivalent sensitivity was observed between ECL and colorimetric detection.

High prevalence of genital tract papillomavirus infection in female adolescents.

To investigate clinical condyloma, abnormal cervical cytologic findings, and evidence of human papillomavirus infections, 89 adolescent girls were examined. Cellular DNAs extracted from exfoliated cervical cells were examined for human papillomavirus genomic sequences by Southern transfer hybridization using 32P-labeled human papillomavirus DNA probes. Human papillomavirus sequences were detected in 12 (13%) young women, abnormal cytologic specimens in 21 (24%), and vulvar condylomas in 12 (13%). The human papillomavirus types identified included HPV-6/11 (four instances), which is known to be associated with benign lesions, and HPV-16, -18, and -31 (eight instances) which are considered to have oncogenic potential. Two young women were infected with both HPV-16 and -31. Human papillomavirus sequences were found in 48% of the young women with abnormal cytologic findings and in 3% of patients with normal cytologic findings (P less than .0001). Condylomatous changes in the cervical smear were associated with the presence of HPV-6/11 and mild dysplasia with the presence of HPV-16, -18, and -31. The presence of vulvar condylomas correlated with condylomatous changes in the cervical smear and with the recovery of HPV-6/11 from the cervical epithelium. The results indicate that the prevalence of human papillomavirus infections in this population is high and that a majority of the infections are with viruses associated with lower genital tract malignancies.

Squamous papillary neoplasia of the adult upper aerodigestive tract.

Selected papillary squamous tumors of the upper aerodigestive tract (UADT) mucosa in adult patients do not have well-defined histologic criteria and the clinical behavior is poorly understood. To better characterize this spectrum of neoplasms, UADT papillary neoplasms were evaluated by routine histology, determination of cellular DNA content using Feulgen-stained tissue sections, and the typing of human papillomavirus (HPV) by in situ hybridization. Solitary papillomas were studied in two patients; there was no recurrence in either case, both had normal DNA content, and one was typed as HPV-6 while the other was typed as HPV-11. Seven adult patients with recurrent papillomatosis and at least one biopsy with dysplasia/atypia were identified (mean age at diagnosis, 13.3 years; mean age at last contact, 42.7 years). Six of seven patients had abnormal DNA cellular content in foci of epithelial atypia. In all biopsies evaluated, the papillomas of the seven patients were consistently typed as either HPV-6 or HPV-11. Six patients with malignant papillary neoplasms also had abnormal DNA cellular content, but none revealed evidence of HPV type 6, 11, 16, or 18 by in situ hybridization of tissue sections. In many of the recurrent papillomas, the degree of epithelial atypia encountered was pronounced and was commonly misdiagnosed as carcinoma in situ or papillary carcinoma. The aneuploid DNA content of these foci of atypia reflected the abnormal cellular appearance and partially explained the overdiagnosis of malignancy. However, none of the seven patients were treated for malignant disease and none progressed to invasive carcinoma, with an average follow-up period of almost 30 years. We conclude that histologic and cytologic atypia in HPV-containing papillomatosis may be appreciable. The aneuploid DNA content may represent premalignant conditions and the patient may be at an increased risk for the subsequent development of squamous cancer. However, none of the seven patients with recurrent papillomatosis developed any evidence of malignancy. In addition, none of the patients with papillary carcinomas had previous recurrent papillomatosis.

Identity of a newly isolated human polyomavirus from a patient with progressive multifocal leucoencephalopathy.

Isolation of a strain of polyomavirus, designated COL, from the brain of a patient with progressive multifocal leucoencephalopathy is described. The COL strain is antigenically similar to JC polyomavirus, previously isolated from a brain affected by progressive multifocal leucoencephalopathy and is unrelated to BK, another human polyomavirus isolated from urine. Immunological cross testing of viruses in the Polyomavirus genus shows that there are at least six antigenic types in this group.

Pruritic vulvar squamous papillomatosis: evidence for human papillomavirus etiology.

The origin of squamous papillae of the vulvar vestibule is controversial. Although some are considered as asymptomatic normal variants of pelvic anatomy, a review of 12 cases of vulvar squamous papillae in patients visiting the Infectious Diseases Clinic at UCLA reveals a distinctly symptomatic variety. A syndrome complex of premonitory vulvar vestibular pruritus, pain or burning, dyspareunia, and progressive development of squamous papillae was noted. Microscopic examination of tissue specimens of the areas of squamous papillae reveals the presence of koilocytic change suggestive of viral infection with human papillomavirus. Furthermore, immunoperoxidase stain revealed human papillomavirus capsid antigen in two cases, which has heretofore not been reported in the literature to the authors' knowledge. Evidence of partner infection on physical examination of sexual partners of these women revealed changes consistent with human papillomavirus in four of six partners who were available for examination. Treatment with podophyllin, cryotherapy, laser, or a combination seems to give predictable resolution of the condition and associated symptoms.

Multicentric human papillomavirus infections of the female genital tract: correlation of viral types with abnormal mitotic figures, colposcopic presentation, and location.

Human papillomavirus deoxyribonucleic acid (DNA) was identified by Southern blot hybridization in 21 of 24 patients with multicentric anogenital lesions and in 46 of 61 individual lesions. Type 6/11 was present in nine patients, type 16 in one, an undetermined type in one, and more than one type in ten patients. Mixed types were present in eight of 46 virus-positive individual lesions. Abnormal mitotic figures were found in 16, 87, and 75% of lesions associated with type 6/11, type 16, and mixed types, respectively. Colposcopic presentation or location of lesions was not predictive of viral types. The relatively high rate of mixed human papillomavirus types in multicentric lesions and in single lesions, and the lack of absolute correlation between viral types and abnormal mitotic figures, suggest that lesions should be removed to prevent viral transmission and possible progression to carcinoma.

Filter in situ hybridisation: an evaluation of the FISH technique for HPV detection in cervical swabs.

The filter in situ hybridisation (FISH) method for detection of HPV in cervical swabs was evaluated against the Southern blot technique on concomitant cervical biopsies. Of 73 biopsies, HPV 16 DNA sequences were found in 26 biopsies and HPV 18 sequences in 2 biopsies. Analysis by FISH of the corresponding smears detected 58 and 100% of these, respectively. Of the smears corresponding to the HPV-negative biopsies, 17% were HPV 16-positive and 3% were HPV-18 positive by FISH. Re-hybridisation with cold plasmid added for competition did not change these results. To estimate the risk of spurious hybridisation between vector remnants in the probe and bacterial DNA sequences present in smears, we have hybridised by FISH to preparations of the 19 most common vaginal microorganisms. Of these, E. coli, which is present in about 10% of cervical smears, hybridised strongly with a probe of the plasmid vector pBR322 and may be a significant cause of false positive FISH results. None of the bacteria hybridised with probes of purified HPV when cold, denatured plasmid was added for competition. Analysis by FISH with probes of purified pBR322 to 167 smears of a patient control group resulted in 6% positive reactions. In hybridisations with probes of HPV 16 and 18 to 2 or 3 different filter preparations of the same smear, identical results were obtained in 18 of 19 smears, indicating a good reproducibility by the FISH method. The high percentage of HPV negative smears is equivalent to the rates known from cytology and may reflect sampling errors.

Sensitive detection of nucleic acids and protein of human papillomavirus type 6 in respiratory and genital tract papillomata.

We have developed a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in biopsy specimens of genital and respiratory tract lesions by using in situ hybridization and immunoperoxidase assays on sections of plastic-embedded tissue. This modified in situ hybridization technique, using ultrathin sections and strand-specific 3H-labelled riboprobes, offers the advantages of superior morphological preservation and detection of viral genomes at low copy number with good resolution. This modified immunocytochemistry provides better sensitivity when compared to previous methods using paraffin-embedded materials. In respiratory tract lesions, immunoperoxidase assay detected only a few capsid antigen positive cells, while in the genital tract lesions, there were more capsid antigen positive cells. Southern transfer analyses and in situ hybridizations demonstrated the presence of more viral nucleic acids in genital tract papillomata than respiratory tract papillomata. Epithelial cells throughout the papillomata were infected by HPV-6 as evidenced by positive hybridization, with more viral DNA present in superficial cells. Our results suggest that genital tract epithelium is more permissive for HPV-6 replication than respiratory tract epithelium. Using stand-specific probes synthesized from subgenomic fragments of the HPV-6 genome in conjunction with nuclease digestions, we were able to demonstrate that HPV-6 transcripts specific to open reading frames (ORFs) E6, E7, E1, L1, and L2 occur in maturing superficial cells. In contrast, transcripts specific to ORFs E1, E2, E4, E5a, and E5b could be detected throughout the whole of the epithelium with more signals noted at the basal cell areas. In addition, the distribution of HPV-6 nucleic acids and protein in a carcinoma in situ of the larynx was analyzed. In comparison to benign respiratory tract papillomata, more viral DNA was found in the malignant lesion, but the pattern and distribution of transcription and capsid antigen was similar.

Detection of human papillomavirus DNA sequences in conjunctival papilloma.

Two histologically proven conjunctival papillomas (one from a 33-month-old boy and the other from a 28-year-old woman) were examined for the presence of papillomavirus by DNA molecular hybridization. The first case, a recurrent tumor known to be positive for papillomavirus structural antigen, demonstrated human papillomavirus DNA sequences that cross-hybridized to a human papillomavirus type 11 DNA probe. The second case, an initial tumor, which was negative for papillomavirus structural antigen, demonstrated no viral DNA sequences by hybridization.

Laryngeal papillomatosis: clinical, histopathologic and molecular studies.

The clinical course and pathology of 57 patients with laryngeal papillomatosis were reviewed. Tissues from 26 patients were analyzed for human papillomavirus (HPV) DNA by Southern blot hybridization. Histopathologic evaluation of the papillomas showed no correlation with age of onset or clinical pattern of remission and recurrence. The pathology was characterized by abnormal squamous maturation with parakeratosis, retardation of superficial cell maturation, papillomatosis, and basal hyperplasia. HPV DNA was present in all lesions, with 92% containing either HPV-6 or 11. Latent HPV DNA was detected in clinically uninvolved tissues of 11 of 14 (78.5%) patients studied. There was no correlation between HPV type, histopathology and/or clinical pattern. Despite homogeneity of pathology, the clinical expression of laryngeal HPV infection varied widely. A mechanism for the pathogenesis of laryngeal papillomatosis, based on the concept of maturational arrest, is proposed.

Electron microscopy of cells showing viral cytopathic effects in Papanicolaou smears.

A technique is described whereby cells showing cytologic changes suggestive of virus infection by light microscopy can be processed further for examination in the electron microscope so that virus particles present in the cell can be visualized directly. We present the results of electron microscopy of over 100 Papanicolaou-stained smears processed this way. The morphologic changes in the cells and the ultrastructural appearances of the virus particles are demonstrated. This technique is particularly valuable for retrospective studies of mounted cytologic or histologic material. It has also proven to be a valuable research tool in the study of human polyomavirus and human wart virus infection.

Sequences of papillomavirus DNA in equine sarcoids.

DNA was extracted from 14 equine sarcoids, electrophoresed and hybridised with a radioactively labelled probe of bovine papillomavirus type I (BPV 1) DNA under conditions of low stringency. Twelve sarcoids contained sequences of DNA that hybridised with the probe and that comigrated with BPV 2 DNA. The viral DNAs in four of these sarcoids differed from BPV 1 and BPV 2 DNA on restriction endonuclease analysis. One of four cell lines derived from sarcoids also contained BPV 1 related DNA. The results confirm the frequent presence in equine sarcoids of unintegrated papillomaviral DNA and suggest a role for papillomavirus infection in this disease.

Heterogeneity of human papilloma viruses.

Two types of human papilloma viruses (HPV-1 and HPV-4) were characterized, which show no relationship as far as the following criteria are concerned: the pattern of restriction enzyme fragments of their DNA, DNA-cRNA hybridization, and complement fixation with specific rabbit antisera. Furthermore, the molecular weights of the major proteins differed significantly. Both types of viruses could be isolated from plantar warts and from verrucae vulgares. This paper gives the molecular weights of the major proteins of a third type of HPV from skin warts, which reacts neither with HPV-1 nor with HPV-4 rabbit antiserum.

The human papillomaviruses.

Recent biochemical and serological studies have shown the existence of at least four distinct types of human papillomaviruses (HPVs) causing benign skin lesions. These viruses show hardly no antigenic relationships; their DNAs differ by their sensitivity to restriction endonucleases, and show little, if any, sequence homology, as detected by molecular hybridization using complementary RNAs transcribed in vitro. Data on the pathogenicity of HPVs are still incomplete but indicate that some types of benign skin lesions (plantar warts, common warts, flat warts) may be preferentially associated with some types of HPV. Most interesting is that epidermodysplasia verruciformis has been found associated with two types of virus, and that malignant conversion of some lesions has been observed in all the patients infected with one of them. This suggests that at least a HPV may have a higher oncogenic potential, as do rabbit (Shope) papillomavirus and bovine alimentary tract papillomavirus. Much remains to be known on human papilloma-viruses and further studies may lead to the characterization of additional types of HPVs, especially in genital condylomata acuminata and laryngeal papillomas whose malignant conversion, although rare, may be observed. Progress in this field has been and remains hampered by the lack of cell culture systems allowing replication of these highly host and tissue specific viruses, and by the widely variable virus content of the different human lesions known to be associated with a papillomavirus. Further studies are warranted by the possible role of these widespread and epitheliotropic viruses in the origin of some carcinomas in man.

Detection of human papillomavirus (HPV) structural antigens and DNA types in inverted papillomas and squamous cell carcinomas of the nasal cavities and paranasal sinuses.

To assess the suggested etiological role of human papillomavirus (HPV), biopsies from 14 patients operated on for an inverted papilloma (11 cases) and squamous cell carcinoma (3 cases) of the nasal cavity and paranasal sinuses were analysed for light microscopical evidence of HPV, by indirect immunoperoxidase (IP-PAP) to demonstrate HPV structural proteins, and using in situ DNA-hybridization to disclose the DNA of HPV types 6, 11 and 16. The majority of the inverted papillomas contained areas of metaplastic squamous cells, including koilocytes as well as dysplastic changes consistent with intra-epithelial neoplasia as described in uterine cervix. In 3 patients, frankly invasive squamous cell carcinomas were found, originating from dysplastic squamous epithelium. Of the 14 lesions, 7 (50%) expressed HPV antigens, usually confined to only a few cells close to the surface of the epithelium. None of the malignant lesions expressed HPV antigens. In situ DNA-hybridization disclosed HPV 11 DNA alone in 5 lesions, but none of the lesions contained HPV 6 DNA. HPV 16 DNA was found in 2 lesions as a single HPV type, and in 3 other lesions concomitant with HPV 11. All three carcinomas contained HPV 16 DNA. The HPV DNA distribution proved to be different from that found in the genital tract HPV lesions; HPV DNA was less abundant in the nasal papillomas, and it was also found in the basal and suprabasal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection of papillomaviruses by in situ hybridization with sulfonated probes]. / Détection par hybridation in situ des papillomavirus avec des sondes marquées par sulfonation.

A technique of detection by in situ hybridization of human papillomavirus in sections of condylomatous lesions is described. The probes are labeled and modified by sulfonation and the hybrids are revealed by immunohistochemistry, using alkaline phosphatase.