Malpighian epithelia and papillomavirus infections]

Human papillomaviruses (HPV) are a large group of DNA viruses, with over 60 types identified to date, which can cause the development of benign tumors in the skin and mucosal squamous epithelia. Most of these tumors regress spontaneously but some, especially in the mucosal membranes, become malignant. HPV types with a high risk for inducing malignancies (e.g. 16 and 18) are the subject of increasing interest. HPVs are both host-specific and tissue-specific: some types preferentially infect specific epithelia, giving rise to lesions with distinct topographic characteristics. HPVs are difficult to study because they do not replicate in available in vitro models. In vivo, HPVs replicate well in epithelial cells undergoing terminal differentiation, e.g. in keratinized cells. Some 40 different types have been reported in epidermal keratinocytes, the most common being types 1 and 2 which produce large amounts of viral antigens and viral particles. In contrast, HPVs replicate poorly in the weakly keratinized squamous epithelia which line the digestive, respiratory, and genital tracts. Junctional epithelia, e.g. on the uterine cervix, are especially prone to HPV infection. The most prevalent HPV types in benign genital lesions are types 6 and 11, whose characteristic features include extrachromosomal DNA and production of only small amounts of viral antigens. The profound nuclear and cytoplasmic changes induced by HPVs lead to the formation of ko?locytes which are found mainly in the granular layer of epithelia and have been especially well described in the uterine cervix and vagina. HPV epithelial tumors are squamous cell carcinomas that often harbor HPV types 16 and 18; this is especially true of cervical intraepithelial neoplasias.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA replication specificity and functional E2 interaction of the E1 proteins of human papillomavirus types 1a and 18 are determined by their carboxyl-terminal halves

Replication of most papillomaviruses (PVs) requires the viral-encoded E1 and E2 proteins that bind to the origin of replication (ori) containing the E1- and E2-binding sites and help recruit host replication factors during the initiation of DNA replication. We studied the ability of heterologous E1 and E2 proteins to interact in vivo and support replication, using the human papillomavirus (HPV) types 1a and 18 as model systems. The E1 protein of HPV-1a in combination with HPV-18 E2 supported high-level replication of various ori plasmids. In contrast, the HPV-18 E1 protein interacted weakly with HPV-1a E2 during the replication of ori plasmids. We have previously shown that the E1 protein of HPV-1a alone is sufficient for replication of HPV-1a ori plasmids, whereas HPV-18 replication requires both the E1 and E2 proteins. However, in the latter case, E2-binding sites alone in the absence of the E1-binding site can function as the minimal ori. Based on the above observations, we generated hybrids between HPV-1a and HPV-18 E1 proteins in an effort to identify their "replication specificity" domains using a transient replication assay. These hybrids were also used to localize the domains in the E1 proteins that are involved in their functional interaction with the E2 protein during replication. Our results suggest that the "replication specificity" and functional E2 interaction domains of the HPV-1a and HPV-18 E1 proteins are located in their carboxyl-terminal halves.     

Prediction of conserved microRNAs from skin and mucosal human papillomaviruses

Eight human papillomavirus (HPV) types including four cutaneous HPV types (HPV-5, HPV-8, HPV-20 and HPV-38) and four mucosal HPV types (HPV-6, HPV-11, HPV-16 and HPV-18) were selected for this miRNA study. Pre-miRNAs were predicted using a computer programme, and the conserved mature miRNAs were compared to currently known miRNAs. Predicted HPV miRNAs related to miR-466, -467 and -669 were common and specific to the mucosal HPV types. Northern blot hybridization confirmed a predicted miRNA in HPV-positive cervical cancer cell lines encoded by mucosal HPVs. HPV-38 was predicted to express an miRNA conserved to human let-7a and the expression of let-7a, in HPV-38-positive non-melanoma skin cancer (NMSC) biopsies was 10-fold higher than those with HPV-positive (for other types except HPV-38) and HPV-negative NMSCs, suggesting that let-7a expression might be related to HPV-38 infection. Potential gene targets of the predicted miRNA that may aid HPV in infection and pathogenesis were also analysed.     

Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix

A total of eight human epithelial cell lines derived from the carcinoma of the cervix were examined for the presence of human papillomaviruses (HPVs) types 16 and 18 DNA sequences. Six out of eight cell lines contain sequences hybridizing to the DNA of these viruses. Two of the cell lines contain sequences hybridizing specifically to HPV 16. One of these two cell lines contains all of the HPV 16 sequences and the other cell line is missing fragments containing early regions E2 and E4 and some of the late regions. Four of the cell lines contain sequences hybridizing specifically to HPV 18. All these cell lines are missing fragments containing early regions E2, E4, and E5. Interestingly, all the cell lines contain sequences corresponding to early regions E1, E6, and E7.     

Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines.

A series of human carcinoma cell lines was examined for human papillomavirus (HPV) DNA sequences with the use of HPV-6, HPV-11, HPV-16, and HPV-18 DNA probes. Six of eight cell lines derived from human cervical carcinomas were shown to contain integrated HPV DNA sequences. In five of these six lines, HPV-specific polyadenylated RNA species could also be identified. The expression of HPV sequences was detected in three lines with a HPV-18 DNA probe and in two lines with a HPV-16 DNA probe. Of the two lines which contained HPV-16 specific RNA, one contained HPV DNA sequences which hybridized only to an HPV-16 probe, and the other contained HPV DNA sequences which hybridized to both HPV-16 and HPV-18 DNA probes. Six cell lines established from human squamous-cell carcinomas of the bladder, pharynx, lung, esophagus, and vulva were negative for HPV-6, HPV-11, HPV-16, and HPV-18 DNA sequences under stringent hybridization conditions.     

Detection of human papillomavirus type 16 DNA in cervical swabs and formalin-fixed, paraffin-embedded squamous cell carcinomas of non-genital tissues using a synthetic oligodeoxynucleotide probe

The potential of using a chemically synthesized oligodeoxynucleotide as a diagnostic probe to detect human papillomavirus type 16 (HPV-16) in genital infections was evaluated by comparing it with a cloned full-length HPV-16 probe in dot-blot DNA hybridizations. An oligonucleotide sequence, 20 bases in length from the E6 region of HPV-16 (E6 oligo) and different from the DNA sequences of HPV types 6, 11 and 18 by at least 2 base pairs, was chosen for chemical synthesis. The oligoprobe, which was 5'-end labelled with [32P]dATP, was found to be specific, but approximately ten times less sensitive than the full-length radiolabelled probe of HPV-16, in dot-blot hybridizations with the DNA of HPV-6, -11, -16 and -18, HPV positive and negative cell-lines. From 36 cervical or vulval scrapes two samples were found positive with both cloned HPV-16 and oligoprobe hybridization. Of 21 samples of formalin-fixed, paraffin-embedded squamous cell carcinomas originating from anus, oesophagus, penis, colon, breast and skin only 4 anal squamous cell carcinomas were positives when hybridized with cloned HPV-16 DNA or with the oligoprobe. This study confirms that HPV-16, which is frequently associated with squamous cell carcinoma of the cervix is also strongly associated with squamous cell carcinoma of the anus.     

Purification and characterisation of the E7 oncoproteins of the high-risk human papillomavirus types 16 and 18

E7 proteins are major oncoproteins of human papillomaviruses (HPVs) which play a key role in virus-associated cervical carcinogenesis. The E7 oncoprotein of HPV-16 has been shown to interact with a variety of cellular target proteins and these interactions are considered essential for the transforming properties of this oncoprotein. Several additional HPV types associated etiologically to cervical cancer have been described, the second most common being HPV-18. Less is known about the biochemical functions and interactions of HPV-18 E7. As a first step to determine biochemical properties common to the E7 proteins of the high-risk HPV types 16 and 18 these E7 proteins were expressed in bacteria and purified to homogeneity. Purified E7 proteins were used to investigate the in vitro interaction with the pocket protein p107 and insulin-like growth factor-binding protein-3 (IGFBP-3) that are known to interact with the amino-terminal and the carboxyl-terminal part of IGFBP-3, respectively. Both purified E7 proteins interacted strongly with p107 and, as demonstrated here for the first time, HPV-18 E7 was capable of binding to IGFBP-3, albeit to a lesser extent than HPV-16 E7. These findings suggest that the purified recombinant E7 proteins retain, at least in part, their biochemical activities.     

Human papillomaviruses: are we ready to type?

The issue of determining which human papillomavirus (HPV) is present in a clinical specimen (typing specimens for HPVs) is receiving attention because HPVs cause condyloma acuminata and are associated with the continuum of disease which ranges from dysplasia to invasive genital cancer. Morphological inspection of precancerous lesions is not sufficient to determine which lesions will progress and which will not. A number of research tools based primarily on deoxyribonucleic acid hybridization have been developed. These permit identification and typing of HPV in genital tract scrapings or biopsies. Some HPV types (e.g., HPV-16 and HPV-18) have been identified in high-grade dysplasias and carcinomas more commonly than other types (e.g., HPV-6) and have been designated "high risk" types for cervical cancer. Thus, the question arises whether HPV typing would improve patient management by providing increased sensitivity for detection of patients at risk or by providing a prognostic indicator. In this review, the available typing methods are reviewed from the standpoint of their sensitivity, specificity, and ease of application to large-scale screening programs. Data implicating HPVs in the genesis of genital tract cancers are reviewed, as is the association of specific HPV types with specific outcomes. We conclude that there is currently no simple, inexpensive assay for HPV types, although such assays may be developed in the future. Analysis of the typing data indicates that, while HPV types can be designated high risk and low risk, these designations are not absolute and thus the low-risk group should not be ignored. In addition, interpretation of the data is complicated by finding high-risk types in individuals with no indication of disease. Insufficient data exist to indicate whether knowledge of the presence of a given HPV type is a better prognostic indicator than cytological or histological results. Thus, more research is needed before it can be determined whether typing information will augment the method currently in use for deciding treatment regimen and whether it warrants widespread use.     

High prevalence of intermediate-risk human papillomavirus infection in uterine cervices of Kenyan women infected with human immunodeficiency virus

The aim of this study was to investigate an association between certain human papillomavirus (HPV) types and human immunodeficiency virus (HIV) infections. Sexually active females (n = 487; 19-61 years old) were enrolled in the study. Subjects underwent Pap testing and evaluations of HIV and HPV infection status on uterine cervical cell samples. HPV genotyping was performed using a Kurabo GeneSQUARE DNA microarray test. Overall, 23 HPV genotypes were detected, and the most prevalent HPV genotype was HPV-52, followed by HPV-39, -54, -45, -56, -53, -31, -42, -16, -68, and -51. HPV-30, -53, -54, -61, and -66, which are associated with abnormal cytology, are categorized as intermediate-risk in this study. Detection of both high- and intermediate-risk HPV types was significantly associated with cervical abnormality and HIV infection. Multivariate analysis revealed that some high-risk HPV types (HPV-31, -45, -51, -56, and -59) and most intermediate-risk HPV types were associated with HIV infection, while the high-risk types (HPV-16, -18, -33, -35, -39, -52, -58, and -68) were not. The oncogenic effect of the most malignant HPV types (e.g., HPV-16 and -18) appear to be lower, while that of intermediate-risk types are greater, in areas with a high prevalence of HIV infection.     

Worldwide genomic diversity of the human papillomaviruses-53, 56, and 66, a group of high-risk HPVs unrelated to HPV-16 and HPV-18

Among more than 200 human papillomavirus (HPV) types presumed to exist, 18 "high-risk" HPV types are frequently found in anogenital cancer. The best studied types are HPV-16 and 18, which are only distantly related to one another and form two separate phylogenetic branches, each including six closely related types. HPV-30, 53, 56, and 66 form a third phylogenetic branch unrelated to HPV-16 and 18. Worldwide comparison of HPV-16 and 18 isolates revealed a distribution of variant genomes that correlated with the geographic origin and the ethnicity of the infected cohort and led to the concept of unique African, European, Asian, and Native American HPV-16 and 18 variants. Here, we address the question whether similar phylogenies are found for HPV-53, 56, and 66 by determining the sequence of the long control regions (LCR) of these HPVs in samples from Europe, Asia, and Africa, and from immigrant societies in North and South America. Phylogenetic trees calculated from point mutations and a few insertions/deletions affecting 2-4.2% of the nucleotide sequences were distinct for each of the three HPVs and divergent from HPV-16 and 18. In contrast to the "star-phylogenies" formed by HPV-16 and 18 variants, 44 HPV-53 isolates represented nine variants, which formed two deep dichotomic branches reminiscent of the beginning split into two new taxa, as recently observed for subtypes of HPV-44 and 68. A total of 66 HPV-56 isolates represented 17 variants, which formed three branches preferentially containing European, Asian, and African variants. Variants of a fourth branch, deeply separated from the other three, were characterized by a 25 bp insertion and created a dichotomy rather than star-like phylogeny. As it contained isolates from cohorts in all continents, it may have evolved before the spread of humans into all continents. 18 of 31 HPV-66 isolates represented the prototype clone, which was found in all parts of the world, while the remaining 13 clones formed 11 branches without any geographic association. Our findings confirm the notion of a quantitatively limited genomic diversity of each HPV type with some correlation to the geographic origin of the sample. In addition, we observed in some variants of these three HPV types mutations that affect the amino acid sequence of the E6 oncoproteins and the L1 capsid protein, supporting the possibility of immunogenic and oncogenic diversity between variants of any HPV type.     

CD4+ T cell immunity against the human papillomavirus-18 E6 transforming protein in healthy donors: identification of promiscuous naturally processed epitopes

Infection by 'high-risk' human papillomaviruses (HPV) is associated with the development of neoplastic lesions. HPV-18 is responsible for a very aggressive form of cancer and poor survival. As for other HPV types, immune surveillance has probably a role in the control of the infection. However, very little is known on HPV-18 immunogenicity. CD4(+) T cells from 16 healthy donors were tested ex vivo for reactivity to synthetic peptides corresponding to 3 sequences on the HPV-18 E6 transforming protein predicted by bioinformatics as promiscuous HLA-DR ligands, and to the recombinant E6 protein. We found 3 donors with CD4(+) T cells that specifically proliferated in the presence of HPV-18 E6 antigens and produced IFN-gamma in the presence of the E6 protein. We then propagated CD4(+) T cell lines and clones from the responsive subjects to better characterize the recognized sequences. We show that E6(52-66) and E6(97-111) are indeed promiscuous and, most importantly, they contain naturally processed epitopes. Collectively, our data indicate that healthy donors may develop spontaneous CD4 immunity against HPV-18 E6 epitopes, thus strongly suggesting the potential for this protein to elicit in the host a natural productive immune response.     

Human papillomavirus infection as a risk factor for squamous-cell carcinoma of the head and neck

BACKGROUND: Oncogenic human papillomaviruses (HPVs), especially HPV type 16 (HPV-16), cause anogenital epithelial cancers and are suspected of causing epithelial cancers of the head and neck. METHODS: To examine the relation between head and neck cancers and HPVs, we performed a nested case-control study within a joint Nordic cohort in which serum samples were collected from almost 900,000 subjects. Samples collected at enrollment from 292 persons in whom squamous-cell carcinoma of the head and neck developed, on average, 9.4 years after enrollment and from 1568 matched controls were analyzed for antibodies against HPV-16, HPV-18, HPV-33, and HPV-73 and for cotinine levels as a marker of smoking habits. Polymerase-chain-reaction (PCR) analyses for HPV DNA were performed in tumor tissue from 160 of the study patients with cancer. RESULTS: After adjustment for cotinine levels, the odds ratio for squamous-cell carcinoma of the head and neck in subjects who were seropositive for HPV-16 was 2.2 (95 percent confidence interval, 1.4 to 3.4). No increased risk was observed for other HPV types. Fifty percent of oropharyngeal and 14 percent of tongue cancers contained HPV-16 DNA, according to PCR analysis. CONCLUSIONS: HPV-16 infection may be a risk factor for squamous-cell carcinoma of the head and neck.