Langerhans' cells and subtypes of human papillomavirus in cervical intraepithelial neoplasia.

There is strong circumstantial evidence that human papillomavirus is a cofactor in the development of cervical neoplasia. Systemic immunosuppression has also been implicated. A study was therefore carried out examining the relation between subtypes of human papillomavirus and local immunocompetent cells in the cervix. Colposcopically directed punch biopsy specimens were taken from normal cervix and from histologically proved cervical intraepithelial neoplasia for immunohistochemical studies. Human papillomavirus genome probing was performed on the abnormal specimens. A relation was apparent between decreased Langerhans'' cells and moderate to high copy numbers of human papillomavirus type 16. The reduction in Langerhans'' cells was significant for human papillomavirus type 18 even at low copy numbers. Conversely, the absence of human papillomavirus was associated with increased numbers of Langerhans'' cells in cervical intraepithelial neoplasia. These findings suggest that the proposed oncogenic potential of human papillomavirus type 16 and human papillomavirus type 18 in particular may be mediated by a specific effect on the afferent limb of the immune response.     

The polymerase chain reaction: a new epidemiological tool for investigating cervical human papillomavirus infection.

The polymerase chain reaction is an in vitro method for primer directed enzymatic amplification of specific target DNA sequences. The technique was used to detect human papillomavirus types 11 and 16 simultaneously in cellular DNA recovered from cervical smears in 38 women referred for colposcopy to evaluate cytological abnormality and 10 women with no history of cytological abnormality. The polymerase chain reaction was shown to be both specific and sensitive in detecting human papillomavirus DNA such that a single human papillomavirus molecule was detected in 10(5) cells. Of the 38 women with cytological abnormality, all were positive for human papillomavirus on testing with the polymerase chain reaction; 36 were infected with human papillomavirus type 16 and 22 dually infected with human papillomavirus types 11 and 16. Seven of the 10 women with no cytological abnormality were also infected with human papillomavirus type 11 or 16. The use of the polymerase chain reaction will facilitate epidemiological investigation of the aetiological role of human papillomavirus in cervical neoplasia. This preliminary analysis suggests that the prevalence of human papillomavirus infection is greater than previously reported.     

Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer.

The 7,909-nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer, has been determined and used to deduce the corresponding genome arrangement. Extensive sequence homologies and other genetic features are shared with the related oncogenic virus, human papillomavirus type 16, especially in the major reading frames. A surprising difference was found in the noncoding region of human papillomavirus type 33 as, unlike all other sequenced papillomaviruses, it contains a perfect 78-base pair tandem repeat.     

Human papillomavirus (HPV) type 50, a type associated with epidermodysplasia verruciformis (EV) and only weakly related to other EV-specific HPVs.

The cloning and partial characterization of the genome of human papillomavirus type 50 is presented. Alignment of the genomic map with that of human papillomavirus type 5, with which it is only weakly related, was permitted by partial DNA sequence analysis.     

mRNA Instability Elements in the Human Papillomavirus Type 16 L2 Coding Region

Human papillomavirus capsid proteins L1 and L2 are detected only in terminally differentiated cells, indicating that expression of the L1 and L2 genes is blocked in dividing cells. The results presented here establish that the human papillomavirus type 16 L2 coding region contains cis-acting inhibitory sequences. When placed downstream of a reporter gene, the human papillomavirus type 16 L2 sequence reduced both mRNA and protein levels in an orientation-dependent manner. Deletion analysis revealed that the L2 sequence contains two cis-acting inhibitory RNA regions. We identified an inhibitory region in the 5′-most 845 nucleotides of L2 that acted by reducing cytoplasmic mRNA stability and a second, weaker inhibitory region in the 3′ end of L2. In contrast, human papillomavirus type 1 L1 and L2 genes did not encode strong inhibitory sequences. This result is consistent with observations of high virus production in human papillomavirus type 1-infected tissue, whereas only low levels of human papillomavirus type 16 virions are detectable in infected epithelium. The presence of inhibitory sequences in the L1 and L2 mRNAs may aid the virus in avoiding the host immunosurveillance and in establishing persistent infections.     

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.     

The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens.

The proteins encoded by the open reading frames of papillomavirus genomes and the minor polypeptides detected in purified virions are still poorly defined. We show here by its expression in Escherichia coli that the open reading frame L2 of human papillomavirus type 1a codes for a minor structural protein of Mr 76,000. Antisera raised against a truncated L2-beta-galactosidase fusion protein in which the conserved N-terminal region of L2 is missing are type specific for human papillomavirus type 1 virions and are reactive at high dilutions. Expression of the L2-encoded type-specific antigens thus provides a powerful new tool for the identification of papillomaviruses.     

Life cycle heterogeneity in animal models of human papillomavirus-associated disease

Animal papillomaviruses are widely used as models to study papillomavirus infection in humans despite differences in genome organization and tissue tropism. Here, we have investigated the extent to which animal models of papillomavirus infection resemble human disease by comparing the life cycles of 10 different papillomavirus types. Three phases in the life cycles of all viruses were apparent using antibodies that distinguish between early events, the onset of viral genome amplification, and the expression of capsid proteins. The initiation of these phases follows a highly ordered pattern that appears important for the production of virus particles. The viruses examined included canine oral papillomavirus, rabbit oral papillomavirus (ROPV), cottontail rabbit papillomavirus (CRPV), bovine papillomavirus type 1, and human papillomavirus types 1, 2, 11, and 16. Each papillomavirus type showed a distinctive gene expression pattern that could be explained in part by differences in tissue tropism, transmission route, and persistence. As the timing of life cycle events affects the accessibility of viral antigens to the immune system, the ideal model system should resemble human mucosal infection if vaccine design is to be effective. Of the model systems examined here, only ROPV had a tissue tropism and a life cycle organization that resembled those of the human mucosal types. ROPV appears most appropriate for studies of the life cycles of mucosal papillomavirus types and for the development of prophylactic vaccines. The persistence of abortive infections caused by CRPV offers advantages for the development of therapeutic vaccines.     

Papilloma formation in human foreskin xenografts after inoculation of human papillomavirus type 16 DNA.

A mouse model of high-risk human papillomavirus infection was developed in which human papillomavirus (HPV) type 16 DNA was inoculated into human foreskin grafted to the skin of severe combined immunodeficient (scid) mice. Grafted skin contained human epidermis and dermis and, like normal human skin, expressed involucrin in differentiating keratinocytes. HPV type 16 DNA, attached to gold particles, was delivered directly into human epidermal cells and induced exophytic papilloma with histologic features of papillomavirus infection, including koilocytosis and expression of papillomavirus capsid antigen. This model should be useful for determining in vivo the functions of viral genes and for developing strategies to prevent and treat HPV-associated disease. It may also be of value in developing animal models of other human skin diseases.     

Human papillomavirus type 18 DNA is integrated at a single chromosome site in cervical carcinoma cell line SW756.

SW756, a cervical carcinoma cell line, has multiple copies of human papillomavirus type 18 DNA sequences. The integration site of human papillomavirus type 18 DNA was localized by in situ hybridization to chromosome 12 at band q13. This single integration site corresponds to a heritable fragile site, which may have facilitated the integration of the viral DNA.     

Specific inactivation of inhibitory sequences in the 5' end of the human papillomavirus type 16 L1 open reading frame results in production of high levels of L1 protein in human epithelial cells

The expression of human papillomavirus type 16 late genes encoding virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We wish to understand the molecular mechanisms that determine the levels of expression of the human papillomavirus type 16 late genes. We have previously shown that the L1 coding region contains inhibitory sequences. Here we extend previous findings to show that the 5' end of the L1 gene contains strong inhibitory sequences but that the 3' end does not. We show that the first 514 nucleotides of the L1 coding region contain multiple inhibitory elements that act independently of one another and that the major inhibitory element is located within the first 129 nucleotides of the L1 gene. Introduction of point mutations in the inhibitory elements in the 5' end of the L1 gene which altered the RNA sequence without affecting the protein sequence specifically inactivated the inhibitory elements and resulted in production of high levels of human papillomavirus type 16 L1 mRNA and protein in human epithelial cells. Furthermore, we show that inhibitory sequences are present in the L1 coding regions of multiple human papillomavirus types, demonstrating that these elements are conserved among the human papillomaviruses, and suggest that they have an important function in the viral life cycle.     

Prospective seroepidemiological study of role of human papillomavirus in non-cervical anogenital cancers.

OBJECTIVE: To evaluate the association between infection with the major oncogenic types of human papillomavirus and the risk of developing non-cervical anogenital cancers in a cohort followed up prospectively. DESIGN: Data from two large serum banks to which about 700,000 people had donated serum samples were followed up for a mean of 8 years. People who developed non-cervical anogenital cancers during follow up were identified by registry linkage with the nationwide cancer registries in Finland and Norway. Within this cohort a nested case-control study was conducted based on the serological diagnosis of infection with human papillomavirus types 16, 18, and 33. SUBJECTS: 81 cases and 240 controls matched for sex, age, and storage time of serum samples. MAIN OUTCOME MEASURES: Odds ratios of developing non-cervical anogenital cancers in presence of IgG antibodies to specific micro-organisms. RESULTS: Subjects seropositive for human papillomavirus type 16 had an increased risk of developing non-cervical anogenital cancers (odds ratio 3.1 (95% confidence interval 1.4 to 6.9)). Subjects seropositive for type 33 also had an increased risk (odds ratio 2.8 (1.0 to 8.3)) but not significantly after adjustment for infection with type 16. Seropositivity for human papillomavirus type 16 was associated with an increased risk of developing vulvar and vaginal cancers (odds ratio 4.5 (1.1 to 22)) and a strongly increased risk of developing preinvasive vulvar and vaginal lesions (odds ratio infinity (3.8 to infinity)). Seropositivity for human papillomavirus type 18 increased the risk of developing preinvasive lesions (odds ratio 12 (1.2 to 590)). High, but non-significant odds ratios for types 16 and 33 were seen for penile cancers. CONCLUSIONS: This study provides prospective seroepidemiological evidence that infection with human papillomavirus type 16 confers an increased risk of developing non-cervical genital cancers, particularly vulvar and vaginal cancers.     

Human papillomavirus infection of the uterine cervix of women without cytological signs of neoplasia.

One hundred and six patients were studied whose cervical smears showed only non-specific inflammatory changes. Screening for genital pathogens yielded only a few positive cases. Histological examination of biopsy specimens taken by colposcopically directed tissue sampling showed cervical intraepithelial neoplasia in 13 of the women (12.3%). Deoxyribonucleic acid (DNA) hybridisation techniques were used to detect human papillomavirus, which was found in 24 patients (22.6%). In a second group of 104 patients with normal cervical cytology tissue biopsy samples were obtained and examined histologically but in no case was cervical intraepithelial neoplasia found. On DNA hybridisation, however, 12 patients (11.5%) were found to be positive for human papillomavirus. In this group finding human papillomavirus DNA was usually associated with a columnar ectopy. An association between human papillomavirus type 16 DNA and both cervical intraepithelial neoplasia and cervical cancer is well established. In this study it was type 16 which occurred most frequently in both groups.     

Presence of human papillomavirus DNA sequences in cervical intraepithelial neoplasia.

Twenty two patients referred to a district colposcopy clinic because of an abnormal cervical cytology report or a suspicious cervix and found to have a cervical epithelial abnormality were studied. The techniques of cytology, histology, immunohistochemistry, and DNA-DNA hybridisation were used to detect infection by human papillomavirus. Using an indirect immunoalkaline phosphatase technique human papillomavirus antigen was found in biopsy specimens from six of the 22 patients and DNA of papillomavirus type 6 in biopsy specimens from 13 of these women, including four out of six whose histological diagnosis was cervical intraepithelial neoplasia grade 3. In eight cases where cytological, colposcopical, and histological investigations all indicated the presence of wart virus infection, papillomavirus type 6 DNA was found in seven. Papillomavirus type 6 DNA was found in more than half of the proved cases of cervical intraepithelial neoplasia. The presence of this viral DNA in women with no cervical abnormality is to be studied.     

DNA sequence and genome organization of genital human papillomavirus type 6b.

The complete nucleotide sequence of the circular double-stranded DNA of the genital human papillomavirus type 6b (HPV6b) comprising 7902 bp was determined and compared with the DNA sequences of human papillomavirus type 1a (HPV1a) and bovine papillomavirus type 1 (BPV1). All major open reading frames are located on one DNA strand only. Their arrangement reveals that the genomic organization of HPV6b is similar to that of HPV1a and BPV1. The putative early region includes two large open reading frames E1 and E2 with marked amino acid sequence homologies to HPV1a and BPV1 which are flanked by several smaller frames. The internal part of E2 completely overlaps with another open reading frame E4. The putative late region contains two large open reading frames L1 and L2. The L1 amino acid sequences are highly conserved among analyzed papillomavirus types. By sequence comparison, potential promoter, splicing and polyadenylation signals can be localized in HPV6b DNA suggesting possible mechanisms of genital papillomavirus gene expression.     

The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.     

A negative regulatory element in the human papillomavirus type 16 genome acts at the level of late mRNA stability.

A negative regulatory element present in the human papillomavirus type 16 genome has been characterized. Deletion analysis has localized the 5' end of the element to the late region of the genome at the extreme 3' end of the coding region of the L1 open reading frame, around the L1 stop codon, with the element extending into the L1 3' untranslated region. For the cell lines used, the element's function was independent of cell type, tissue, or species of origin, unlike papillomavirus infection, which is very dependent on such factors. By using an mRNA decay assay, we have determined that polyadenylated RNA containing the element is much less stable than polyadenylated RNA lacking the element. This indicates that the element acts as an mRNA instability element. The significance of A-rich, GU-rich, and AUG-rich sequences for the functioning of this human papillomavirus type 16 instability element is discussed.     

Human papillomavirus type 28 (HPV-28), an HPV-3-related type associated with skin warts.

The cloning and partial characterization of the genome of human papillomavirus type 28 (HPV-28) is presented. The virus is a distinct type and by hybridization analyses is most closely related to HPV-3 and HPV-10.     

Cloning and characterization of a papillomavirus associated with papillomas and carcinomas in the European harvest mouse (Micromys minutus).

Individuals in a colony of European harvest mice (Micromys minutus) were diagnosed with a variety of skin tumors including papillomas, trichoepitheliomas, and sebaceous carcinomas. Papillomavirus group-specific antigens and viruslike particles were detected in the papillomas. A 7.6-kilobase supercoiled circular DNA, which was cleaved once by EcoRI, was visualized in papilloma extracts by low-stringency Southern blot hybridization with a bovine papillomavirus type 2 probe. The molecule was cloned in pUC18, and a restriction map was generated. The molecule was shown to be colinear with the genome of human papillomavirus type 1a by partial sequence analysis. The DNA hybridized to human papillomavirus type 1a, rabbit oral papillomavirus, and the genome of Mastomys natalensis papillomavirus at Tm - 33 degrees C but not to the DNAs of 13 other papillomaviruses. Transformation of NIH 3T3 or C127I cells by tail papilloma extracts or transfected viral DNA was not observed. All 17 tumors examined contained large amounts of viral DNA in a supercoiled, unintegrated form as revealed by Southern blot hybridization. Furthermore, many extracts (25 of 35) from normal organs and skin of individuals with lesions elsewhere on their bodies contained viral DNA. This represents the first reported molecular cloning of a papillomavirus genome from a mouse species.     

Tissue site-specific enhancer function of the upstream regulatory region of human papillomavirus type 11 in cultured keratinocytes.

The human papillomavirus type 11 regulatory region was cloned upstream of a reporter complex and microinjected into nuclei of individual primary human keratinocytes. Genital and laryngeal keratinocytes, normal host cells for this virus, exhibited higher levels of expression than cutaneous keratinocytes. We conclude that a papillomavirus enhancer(s) shows preferences among epithelial cells from different tissue sites.