Isolation of a novel human papillomavirus (type 51) from a cervical condyloma.

We cloned the DNA from a novel human papillomavirus (HPV) present in a cervical condyloma. When DNA from this isolate was hybridized at high stringency with HPV types 1 through 50 (HPV-1 through HPV-50), it showed weak homology with HPV-6 and -16 and stronger homology with HPV-26. A detailed restriction endonuclease map was prepared which showed marked differences from the maps for other HPVs that have been isolated from the female genital tract. Reassociation kinetic analysis revealed that HPV-26 and this new isolate were less than 10% homologous; hence, the new isolate is a novel strain of HPV. The approximate positions of the open reading frames of the new strain were surmised by hybridization with probes derived from individual open reading frames of HPV-16. In an analysis of 175 genital biopsies from patients with abnormal Papanicolaou smears, sequences hybridizing under highly stringent conditions to probes from this novel HPV type were found in 4.2, 6.1, and 2.4% of biopsies containing normal squamous epithelium, condylomata, and intraepithelial neoplasia, respectively. In addition, sequences homologous to probes from this novel isolate were detected in one of five cervical carcinomas examined.     

Integration of human papillomavirus type 16 DNA sequences: a possible early event in the progression of genital tumors.

The keratinocyte line SK-v harbors only integrated human papillomavirus type 16 (HPV 16) DNA sequences, although it originated from vulvar Bowenoid papules predominantly containing multiple copies of free HPV 16 genomes. We have cloned a fragment of cell DNA that contains the integrated HPV 16 DNA sequences and have shown that integration interrupts the HPV 16 genome in open reading frames E2 and L2 and creates a deletion of 813 base pairs. This allows the expression of open reading frames E6 and E7, as actually substantiated by Northern (RNA) blot analysis of SK-v RNAs with subgenomic HPV 16 RNA probes. Using a unique flanking cellular DNA sequence as the probe, we have shown that the integration of HPV 16 sequences had already occurred in the premalignant lesions from which the SK-v cell line was derived.     

Molecular cloning of viral DNA from human genital warts.

The DNA of human papilloma virus type 6 (HPV 6) has been cloned in Escherichia coli K-12 by using pBR322 as vector. The DNA was cloned at the BamHI and EcoRI cleavage sites. This DNA was mapped by employing further restriction endonucleases and by terminal labeling. No major differences were noted as compared to HPV 6 DNA originating directly from a genital wart. The existence of at least two DNA subtypes (HPV 6a and 6b) became apparent.     

Characterization of a novel human papillomavirus DNA in the cervical carcinoma cell line ME180.

The human cervical carcinoma cell line ME180 was examined for human papillomavirus (HPV) DNA and RNA. The integrated DNA of a presumably new HPV type showing a relationship closer to HPV39 than to HPV18 was cloned and sequenced. HPV sequences from the E6-E7-E1 region are expressed as poly(A)+ RNAs.     

Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for proto-oncogene expression.

DNA sequences of specific human papillomavirus (HPV) types are found integrated in the cell genome in most invasive genital carcinomas. We have determined the chromosomal localization of integrated HPV type 16 (HPV-16) or HPV-18 genomes in genital cancers by in situ hybridization experiments. In three cancers, HPV sequences were localized in chromosome band 8q24.1, in which the c-myc gene is mapped, and in one cancer HPV sequences were localized in chromosome band 2p24, which contains the N-myc gene. In three of the four cases, the proto-oncogene located near integrated viral sequences was found to be structurally altered and/or overexpressed. These data indicate that HPV genomes are preferentially integrated near myc genes in invasive genital cancers and support the hypothesis that integration plays a part in tumor progression via an activation of cellular oncogenes.     

Integration sites of human papillomavirus 18 DNA sequences on HeLa cell chromosomes

Human papillomaviruses (HPV) 16 and 18 are closely linked with human genital cancer. In most cervical carcinomas, viral sequences are integrated into the host genome. HeLa, a cervical carcinoma cell line, has multiple copies of integrated HPV 18 DNA. In this study, in situ chromosome hybridization was used to assign the integration sites of HPV 18 DNA sequences on HeLa cell chromosomes. Four sites of hybridization were identified at 8q23----q24, 9q31----q34, p11----p13 on an abnormal chromosome 5, and q12----q13 on an abnormal 22. Three of these sites correspond with the locations of MYC, ABL, and SIS protooncogenes, and are at or in close proximity to fragile sites. The chromosomal localization of HPV 18 DNA may be useful in assessing the role of viral integration in the development of this malignancy.     

Uterine cervical carcinoma and human papillomaviruses]

For many years it has been thought that a significant proportion of cervical cancer could be attributed to sexually transmitted agents, such as sperm, smegma, Treponema pallidum, Gonococcus and herpes simplexvirus type 2. Recent advances of molecular biology, however, have revealed that human papillomavirus (HPV) might be the most causative virus of the disease. Since HPV type 16 DNA was found in a patient with cervical cancer in 1983, many HPV types have been cloned from cervical cancers, also from premalignant lesions (intraepithelial neoplasias). In Japan, we have found 6 new types of HPV (HPV 58, 59, 61, 62, 64, 67) in the female genital tract so far. Especially, HPV 58, which was cloned from a patient with cervical squamous cell carcinoma and was already fully sequenced, is thought to be an important agent for the development of cervical cancer as well as HPV 16. Now we are investigating extensively to clarify the real relationship between genital HPV infection and cervical cancer.     

Human papillomavirus DNA typing in the cervical specimens among women of Split and Dalmatian County

Human papillomavirus (HPV) infection is the main cause of cervical cancer, the second most common cancer in women worldwide. More than 200 types of HPV have been described, and within this range more than 40 types attack epithelium of genital tract. The types that were most commonly related to the development of cervical cancer are called high-risk types (HR HPV). There are very few studies about HPV prevalence in Croatia and there is an absolute lack of data for Split and Dalmatian County. Therefore, during a 6 month period, we routinely screened 570 women for HPV DNA at the Educational Public Health Institute of Split and Dalmatian County. HR HPV was detected in cervical exfoliated cells, by using Hybrid Capture 2 HPV DNA test. Out of the total number of tested women, 200 (35%) of them were positive to HR HPV Polymerase chain reaction (PCR) based assays were employed for HR HPV genotyping in positive specimens. The following frequency was observed: HPV 16 in 10%, HPV 18 in 6.1%, HPV 31 in 2.6%, HPV 33 in 1.9%, HPV 52 in 1.4%, HPV 59 in 0.7%, HPV 45 in 0.4% specimens, while 11.9% of tested specimens currently remained untyped. It is necessary to expand this study to a larger number of women, in order to better evaluate genital HPV types distribution among women in this region.     

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

This paper aimed at studying the transplacental transmission of HPV and looking at the epidemiological factors involved in maternal viral infection. The following sampling methods were used: (1) in the pregnant woman, (a) genital; (b) peripheral blood; (2) in the newborn, (a) oral cavity, axillary and inguinal regions; (b) nasopharyngeal aspirate, and (c) cord blood; (3) in the placenta. The HPV DNA was identified using two methods: multiplex PCR of human β-globin and of HPV using the PGMY09 and PGMY11 primers; and nested-PCR, which combines degenerated primers of the E6/E7 regions of the HPV virus, that allowed the identification of genotypes 6/11, 16, 18, 31, 33, 42, 52 and 58. Transplacental transmission was considered when type-specific HPV concordance was found between the mother, the placenta and the newborn or the mother and cord blood. The study included 49 HPV DNA-positive pregnant women at delivery. Twelve placentas (24.5%, n = 12/49) had a positive result for HPV DNA. Eleven newborn were HPV DNA positive in samples from the nasopharyngeal or buccal and body or cord blood. In 5 cases (10.2%, n = 5/49) there was HPV type-specific agreement between genital/placenta/newborn samples. In one case (2%, n = 1/49) there was type specific HPV concordance between genital/cord blood and also suggested transplacental transmission. A positive and significant correlation was observed between transplacental transmission of HPV infection and the maternal variables of immunodepression history (HIV, p = 0.011). In conclusion the study suggests placental infection in 23.3% of the cases studied and transplacental transmission in 12.2%. It is suggested that in future HPV DNA be researched in the normal endometrium of women of reproductive age. The possible consequence of fetal exposure to HPV should be observed.     

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.     

Expression of human papillomavirus types 6b and 16 L1 open reading frames in Escherichia coli: detection of a 56,000-dalton polypeptide containing genus-specific (common) antigens.

The human papillomavirus (HPV) genome contains two large open reading frames (ORFs), designated L1 and L2. To characterize the antigenic properties of the L1 ORF-encoded proteins, we cloned the L1 ORFs of HPV6b and HPV16 in plasmids, and these were expressed in Escherichia coli. First, the HPV6b DNA, representing 85.2% of the L1 ORF, was cloned in pUC19 and expressed in E. coli JM83 and RB791 as a 160,000-molecular-weight (160K) fusion protein with E. coli beta-galactosidase (6bL1/beta-gal). Second, the HPV16 DNA, representing 89.8% of the L1 ORF, was cloned in pKK233-2 and expressed as a 56K protein (16L1) in strain RB791. Both the 6bL1/beta-gal and 16L1 proteins cross-reacted with anti-bovine papillomavirus type 1 (BPV1) antibody raised against disrupted BPV1 particles. An antibody raised against the 6bL1/beta-gal fusion protein reacted with the 16L1 protein and also with native papillomavirus antigens in human genital condyloma and bovine fibropapilloma tissues, as determined by biotin-streptavidin staining. Furthermore, the anti-6bL1/beta-gal antibody recognized a 54K protein which seemed to be a major capsid protein of BPV1 and also a 56K protein of biopsies harboring HPV6 or HPV11. From these results we concluded that the papillomavirus L1 gene product contains genus-specific (common) antigens and that the HPV6 and HPV11 L1 genes specify the 56K capsid protein.     

Comparative study of in situ hybridization and immunohistochemical techniques for the detection of human papillomavirus in lesions of the uterine cervix.

Among the techniques currently used for the detection of human papillomavirus (HPV) in genital lesions, only two correlate HPV with the histopathological findings of the lesion: immunohistochemistry and in situ hybridization. Consequently, we were prompted to carry out a comparative study on both techniques to check their utility and efficacy as routine diagnostic methods. 52 biopsy specimens of uterine cervix diagnosed histopathologically as condylomas and cervical intraepithelial neoplasia+koilocytosis were studied by immunohistochemical and in situ hybridization techniques using a polyclonal antibody against the common antigen of the HPV capsid and three biotinylated DNA probes specific to HPV types 6/11, 16/18 and 31/35/51. Immunohistochemistry detected 21 positive cases (40.38%), whereas in situ hybridization detected 40 positive cases (76.92%); of the latter, 30 were positive for HPV types 6/11, 3 for HPV types 16/18 and 11 for HPV types 31/35/51. The results suggest that in situ hybridization is a more sensitive technique than immunohistochemistry. However, we recommend the use of both techniques in the case of potentially malignant lesions since better prognostic information can be obtained from joint analysis of both results.     

Detection of human papillomavirus (HPV) in genital warts and carcinomas by DNA in situ hybridization in Chinese patients

A series of 51 genital biopsies from normal epithelium, condylomata acuminata, leucoplakia and squamous cell carcinoma from Chinese male and female patients were analysed for the presence of human papillomavirus (HPV) types 6, 11, 16 and 18 by DNA in situ hybridization. All of the nine genital condyloma acuminata were positive for HPV DNA, in which HPV 6 was found in six cases, HPV 11 in two cases and HPV 18 in one case. Twelve out of the 21 cases (57.1% of the total) of cervical squamous cell carcinoma were shown to contain HPV DNA; HPV 16 was found in nine cases, HPV 18 in two cases and HPV 16/18 in one case. Present results support the earlier concept that HPV 6/11 are closely associated with benign genital lesions, and HPV 16/18 are mostly confined to higher grade of intra-epithelial neoplasias and carcinoma.     

Human papillomavirus type 31 uses a caveolin 1- and dynamin 2-mediated entry pathway for infection of human keratinocytes

Papillomaviruses are species-specific and epitheliotropic DNA viruses that cause tumors in their natural hosts. Certain infections with genital human papillomavirus (HPV) types are causally related to cervical cancer development. Most papillomaviruses are thought to infect cells via a clathrin-dependent pathway, yet no studies have determined the entry route in permissive host epithelial cells. Employing fluorescently labeled and native virions, we tested the effects of dominant-negative and biochemical inhibitors of cellular endocytosis pathways. Infections of human keratinocytes, a natural host cell type for HPVs, were assessed visually and by infectious entry assays. We found that HPV type 31 (HPV31) entry and initiation of early infection events require both caveolin 1 and dynamin 2 and occur independently of clathrin-mediated endocytosis. Treatment with chlorpromazine and filipin had opposing effects on HPV31 and HPV16 infection. HPV31 entry was remarkably slow, with a half-time of approximately 14 h, whereas the entry half-time of HPV16 was 4 h. Consistent with a caveola-mediated entry pathway for HPV31, the virions associated with detergent-resistant lipid rafts. During a 16-h microscopic tracking of HPV31 and HPV16 virions, no colocalization of the two viral types was observed. These data suggest that HPV31 and HPV16 virions use distinct routes for host epithelial cell entry.     

Detection of viral DNA and RNA by in situ hybridization

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.     

Mechanisms of tumor suppressor protein inactivation by the human papillomavirus E6 and E7 oncoproteins

There are now several examples where experimental and epidemiologic data have implied a causative role for viruses in human cancer. Human papillomavirus (HPV) DNA is found in approximately 90% of cervical cancers. Only a subset of the HPV types that infect the anogential tissues, however, are associated with cancer. Interestingly, only the cloned DNA of this subset is capable of immortalizing human primary genital keratinocytes in culture. The oncoproteins of the HPVs are encoded by the E6 and E7 genes. Analogous to the oncoproteins of certain other DNA tumor viruses, the E6 and E7 proteins have been shown to functionally inactivate the tumor suppressor proteins p53 and pRB, respectively. We will review what is known of the mechanisms by which the E6 and E7 proteins inactivate these tumor suppressors and the evidence that these activities are related to the transforming capabilities of the HPVs associated with cancer.     

Identification of multiple HPV types on spermatozoa from human sperm donors

Human papillomaviruses (HPV) may cause sexually transmitted disease. High-risk types of HPV are involved in the development of cervical cell dysplasia, whereas low-risk types may cause genital condyloma. Despite the association between HPV and cancer, donor sperm need not be tested for HPV according to European regulations. Consequently, the potential health risk of HPV transmission by donor bank sperm has not been elucidated, nor is it known how HPV is associated with sperm. The presence of 35 types of HPV was examined on DNA from semen samples of 188 Danish sperm donors using a sensitive HPV array. To examine whether HPV was associated with the sperm, in situ hybridization were performed with HPV-6, HPV-16 and -18, and HPV-31-specific probes. The prevalence of HPV-positive sperm donors was 16.0% and in 66.7% of these individuals high-risk types of HPV were detected. In 5.3% of sperm donors, two or more HPV types were detected. Among all identified HPV types, 61.9% were high-risk types. In situ hybridization experiments identified HPV genomes particularly protruding from the equatorial segment and the tail of the sperm. Semen samples from more than one in seven healthy Danish donors contain HPV, most of them of high-risk types binding to the equatorial segment of the sperm cell. Most HPV-positive sperm showed decreased staining with DAPI, indicative of reduced content of DNA. Our data demonstrate that oncogenic HPV types are frequent in men.