Inherited MST1 deficiency underlies susceptibility to EV-HPV infections.

Epidermodysplasia verruciformis (EV) is characterized by persistent cutaneous lesions caused by a specific group of related human papillomavirus genotypes (EV-HPVs) in otherwise healthy individuals. Autosomal recessive (AR) EVER1 and EVER2 deficiencies account for two thirds of known cases of EV. AR RHOH deficiency has recently been described in two siblings with EV-HPV infections as well as other infectious and tumoral manifestations. We report here the whole-exome based discovery of AR MST1 deficiency in a 19-year-old patient with a T-cell deficiency associated with EV-HPV, bacterial and fungal infections. MST1 deficiency has recently been described in seven patients from three unrelated kindreds with profound T-cell deficiency and various viral and bacterial infections. The patient was also homozygous for a rare ERCC3 variation. Our findings broaden the clinical range of infections seen in MST1 deficiency and provide a new genetic etiology of susceptibility to EV-HPV infections. Together with the recent discovery of RHOH deficiency, they suggest that T cells are involved in the control of EV-HPVs, at least in some individuals.

[Biology of epidermodysplasia verruciformis-associated HPV]. / Biologie der Epidermodysplasia-verruciformis-assoziierten HPV.

The genus betapapillomavirus (betaPV) presently comprises more than 40 virus types including the so-called epidermodysplasia verruciformis (EV)-associated HPV, which were originally detected in EV-patients by Southern blot hybridization. BetaPV are ubiquitous in the general population and frequently establish themselves already during the first weeks of life. Hair follicles are regarded as natural reservoir. About 25% of betaPV detected in adults persist for at least 9 months. Due to very low virus production, seroconversion against betaPV starts sluggishly. Hyperproliferation of keratinocytes in psoriasis patients or after severe burns stimulates virus replication. Massive virus replication only occurs in EV-patients, associated with the induction of disseminated skin lesions with a high risk of malignant conversion. In 75% of EV-patients this can be put down to homozygous, inactivating mutations in the genes EVER1 or EVER2. A transgenic mouse model substantiated the crucial role of increased HPV8 oncogene expression, induced by UV-irradiation or wounding, for tumor induction.

A mouse model for studying epidermodysplasia-verruciformis-associated carcinogenesis.

Epidermodysplasia verruciformis (EV) is considered as a model of genetic cancer due to unusual susceptibility to EV-specific human papillomaviruses (HPVs). We established an in vivo experimental system for long-term propagation of EV tissue which should facilitate the study of tumor progression in EV. Skin fragments from benign and early pre-malignant lesions of 6 EV patients were grafted under the kidney capsule of athymic mice. After several months the grafted tissue formed epidermal cysts. These cysts showed typical EV cytopathic effect, in situ hybridization revealed the presence of HPV 5, 8, 9, 12 and 36 DNA, and immunoperoxidase staining detected papillomavirus-group-specific antigens in the permissive cells of the cysts. In some cysts, there were numerous mitoses and downward proliferation of the epidermis with slight dyskeratotic changes. The experimental system described constitutes a model for studies on the role of HPV and other co-factors in human cutaneous carcinogenesis.

Epidermodysplasia verruciformis in Africans.

BACKGROUND: Epidermodysplasia verruciformis (EV) is a rare cutaneous disorder characterised by persistent, refractory infection with human papillomaviruses (HPV). Although EV does not seem to have racial or geographic preference, there is a scarcity of reports on its occurrence in Africans. METHODS: Twenty Africans with EV were studied, and the literature on this condition in Africans was reviewed. Virologic studies were performed on 10 patients. RESULTS: Three types of lesions were observed: flat warts, pityriasis versicolor-like macules, and seborrheic keratosis-like changes. Malignant transformation occurred in only one patient. HPV-3 was isolated only from flat warts, HPV-5 and HPV-17 were isolated only from PV-like lesions, whereas an HPV-5-related type was found in all three types of changes. HPV-5-related type revealed DNA that was related but not identical to any of the viruses in the HPV-5 group. This particular type was isolated from all five South African patients with EV in whom virologic studies were performed. CONCLUSIONS: The benign nature of EV in dark-skinned Africans has been confirmed. Four HPV types have been isolated, of which HPV-related type was found in all South African patients with EV and in all types of skin changes, regardless of their morphology. African patients with EV frequently present seborrheic keratosis-like changes.

Genetic heterogeneity of oncogenic human papillomavirus type 5 (HPV5) and phylogeny of HPV5 variants associated with epidermodysplasia verruciformis.

Variants of oncogenic human papillomavirus type 5 (HPV5), specifically associated with epidermodysplasia verruciformis, were recognized on the basis of the genetic heterogeneity of the E6 open reading frame (ORF). To further evaluate the genetic heterogeneity of HPV5, we sequenced the long control region (LCR), the E7 ORF, and the terminal parts of the E2 ORF of five previously characterized HPV5 variants and compared the data with the published HPV5a1 and HPV5b sequences. Alignment of the variants showed 140 (7.6%) variable nucleotides of 1,854 sequenced. Nucleotide substitution rates varied from 3.6% in the E7 ORF to 11% in the E6 ORF. By sequencing the variable region encompassing the LCR 3' part and the E6 ORF of isolates from six additional epidermodysplasia verruciformis patients, we identified three new variants and three already known variants, indicating the stability of HPV5 variants. This stability was further demonstrated by the identity of isolates obtained years later from benign and malignant lesions of three patients. Phylogenetic analysis of the 10 HPV5 variants distributed them into three groups, tentatively defining subtypes a, b, and c. The phylogenetic grouping shows no geographical dependence, a fact that may be related to the host restriction that characterizes HPV5 infections. No differences in the enhancer potential of the LCR or in the transactivating properties of the E2 protein assayed in vitro were observed among HPV5 variants. Whether HPV5 variants possess distinct biological properties in vivo remains to be determined.

Constitutive transcriptional activator of Epidermodysplasia verruciformis-associated human papillomavirus 8.

Human papillomavirus (HPV) 8 belongs to the HPV types frequently associated with skin cancers of Epidermodysplasia verruciformis (EV)-patients. There are 33 nucleotides (M33 motif) in the 5'-part of the non-coding regulatory region of HPV8, which appear highly conserved among EV-specific HPVs and are consistently followed by an AP1 binding site. These sequences were shown to constitute an essential activator of transcription driven by the HPV8 late promoter P7535. The M33/AP1 element displayed properties of a constitutive enhancer, being also able to stimulate the activity of the heterologous thymidine kinase promoter in a position-independent manner. No protein binding could be detected within the 5'-part of the M33/AP1 region, which contributed significantly to the overall activity of the HPV8 enhancer. As shown by DNasel-footprinting, the central and the 3'-part of the enhancer region were involved in interactions with nuclear proteins. Three specific complexes could be observed in gel retardation tests with nuclear extracts from epithelial cells. One of these interactions involved the AP1 protein. Analysis of deletion and point mutations revealed binding of the AP1 protein to be essential for transcriptional activation, but DNA-protein interactions within M33 were important for maximal stimulation. The response to the phorbol ester TPA also required a cooperation of M33 and AP1.

Epidermodysplasia verruciformis.

Epidermodysplasia verruciformis is a rare skin disease characterized by the disseminated, flat, wart-like lesions caused by human papillomavirus and a high frequency of various skin cancers. The clinical aspects, histological findings, genetic and immunological factors, and human papillomavirus types found in the disease and their roles in skin cancers are reviewed.

Human papillomaviruses and skin cancer.

Human papillomavirus (HPV)-induced skin warts are classically benign lesions. However an association between specific HPV types and skin cancer becomes obvious in epidermodysplasia verruciformis (EV). The analysis of this disease suggests that lesions infected with HPV types 5 and 8 carry a high risk of developing squamous cell carcinomas. The oncogenes of EV-viruses appear to be E6 and E2, rather than E7. The 'high risk' EV-viruses, HPV 5, 8, and 47, differ from related HPV types in the transforming activity of the E6 gene and in the density of positive transcription control elements in the non-coding region (NCR) of the genome. The extrachromosomal viral DNA in cancers may show deletions affecting regulatory sequences. EV-specific lesions occasionally occur in immunosuppressed patients and HPV 5 or 8 persist in some of the skin cancers to which these patients are prone. DNAs of HPV 2, 16, 34, or 41 were identified in few premalignant and malignant skin tumors of the general population.

Epidermodysplasia verruciformis as a model of human papillomavirus-induced genetic cancers: the role of local immunosurveillance.

Epidermodysplasia verruciformis (EV) presents a genetically determined, unusual susceptibility to infection with EV-specific human papillomaviruses (HPVs) related to abrogation of immunosurveillance exclusively against these viruses. The cutaneous viral carcinogenesis depends upon potentially oncogenic HPVs, the cocarcinogenic effect of ultraviolet irradiation, and genetic host factors, presumably a defect of anti-oncogenes or alleles of major histocompatibility complex and tumor necrosis factor locus involved in antigen presentation.

Late promoter of human papillomavirus type 8 and its regulation.

Human papillomavirus type 8 (HPV8) belongs to the HPV types associated with skin carcinomas of patients with epidermodysplasia verruciformis (EV). Its noncoding regulatory sequences (NCR) were shown to drive the expression of the reporter gene chloramphenicol acetyltransferase (cat) in transient assays with human epithelial cells (HT3 cells). This constitutive activity could be enhanced by coexpression of the HPV8 transactivator protein E2. The analysis of 5' deletions of the NCR showed that the EV-specific sequence motif M33 and the neighboring AP1 site are essential for the promoter activity, whereas 44 nucleotides located immediately upstream of M33 are strongly inhibitory. The same effects were observed in simian virus 40-immortalized fetal keratinocytes (SV61 cells) and spontaneously immortalized skin keratinocytes (HaCaT cells). By using primer extension and RNase protection analyses two promoters could be identified within the HPV8 NCR. A nested set of weak signals, corresponding to start sites between positions 175 to 179, represented the previously described E6 promoter. The vast majority of transcripts was initiated at position 7535 and shown to undergo processing at an NCR-internal splice donor (positions 1 to 8). The promoter P7535 is similar to late promoters of other skin-associated papillomaviruses as far as localization, transcript structure, and sequence characteristics are concerned. To confirm that P7535-initiated transcripts proceed indeed to the L1 gene for the major capsid protein, viral mRNAs from an HPV8-induced lesion of a patient with EV were characterized by RNase protection and sequence analysis of polymerase chain reaction-amplified cDNAs. The NCR leader (positions 7535 to 4) appeared in two messages with three exons each. The third exon started with the second ATG codon of L1 in both cases; the short central exons from the 3' part of the early coding region were defined by a common splice acceptor site (position 3303) and different splice donor sites (positions 3443 and 3704).

Human papillomavirus (HPV) types specific of epidermodysplasia verruciformis detected in warts induced by HPV3 or HPV3-related types in immunosuppressed patients.

Epidermodysplasia verruciformis (EV) is characterized by an abnormal genetic predisposition to infection with specific types of human papillomavirus (HPV). Specific defects of the cell-mediated immunity and/or of the control of HPV infection in keratinocytes are assumed to be involved in the development of the disease. As a model to test this hypothesis, we have studied the prevalence of EV-specific HPV in skin warts of 56 immunosuppressed patients. All main types of cutaneous HPV (HPV1, 2, 3, 4, 10, and 28) responsible for skin warts in the general population were detected by blot hybridization. EV-specific HPV (HPV5, 20, and 23) were detected in three patients. Four additional patients were found infected with HPV49, first characterized in the course of this study, and found to be related to EV HPV. A most important finding was that HPV5, 20, 23, and 49 were always codetected with HPV3 or the related types HPV10 and 28. None of the specimens showed the typical clinical morphology of EV lesions. In none of these specimens was the specific cytopathic effect of EV recognized; instead that of HPV3 and related types was seen. No evidence for productive EV HPV DNA replication was obtained for the three specimens that could be further analyzed by in situ hybridization. Our data suggest that HPV3 infection favors infection with EV HPV in immunosuppressed patients but that the full expression of EV HPV is usually restricted as in the general population.

Differences in transforming activity and coded amino acid sequence among E6 genes of several papillomaviruses associated with epidermodysplasia verruciformis.

Using the polymerase chain reaction technique, we cloned and sequenced DNA fragments containing the E6 genes of the epidermodysplasia verruciformis (EV)-associated HPVs 5, 8, 14, 20, 21, 25, and 47, of which only the sequences of HPVs 5, 8, and 47 have previously been reported. Based on the deduced amino acid sequence homology (57.3 to 83.0%), these HPVs could be divided into two clusters: HPVs 5, 8, and 47, and HPVs 14, 20, 21, and 25. The E6 genes of three HPVs from each cluster were examined for transforming activity toward a cultured rat fibroblast cell line, 3Y1, using the retrovirus-mediated gene transfer technique, and all were found to induce morphological transformation. However, the E6 genes of the first cluster were more potent than those of the second. Since HPVs 5 and 8 are the most frequently found HPVs in malignant lesions of EV patients, the observed in vitro transforming activities of the E6 genes may reflect their oncogenic potential in humans.

In vitro expressed HPV 8 E6 protein does not bind p53.

The oncogene E6 of human papillomavirus 8, which is associated with skin cancers in epidermodysplasia verruciformis, was transcribed and translated in vitro. The resulting 17 kDa protein did not bind to the cellular p53 in contrast to E6 of HPV16.

Unique topography of DNA-protein interactions in the non-coding region of epidermodysplasia verruciformis-associated human papillomaviruses.

The non-coding region (NCR) of the epidermodysplasia verruciformis (EV)-associated human papillomavirus 8 (HPV-8) has been investigated for sequence-specific DNA-protein interactions with nuclear proteins from epithelial HeLa cells. Using DNase I protection analysis 18 footprints could be found within the HPV-8 NCR, covering altogether over 60% of both DNA strands. Several footprints coincided with the known binding sites of transcription factors (NF1, AP1, octamer and PEA3 consensus sequences); the other displayed no obvious similarities in this regard. The overall distribution of sequences involved in DNA-protein interactions differed clearly from the binding patterns reported for other HPVs. Parts of the two binding sites for the viral trans-activator protein E2 were shown to interact with non-E2 factors. The EV-specific NCR motifs M33, M29 and A/T box were all involved in protein binding. By comparing the footprints within the respective motifs of the closely related types HPV-8 and -19, quantitative and qualitative differences were detected for M33 and the A/T box.

Genetic heterogeneity among human papillomaviruses (HPV) associated with epidermodysplasia verruciformis: evidence for multiple allelic forms of HPV5 and HPV8 E6 genes.

In order to get some insight into modifications of human papillomavirus (HPV) genomes which could play a role in tumor progression in epidermodysplasia verruciformis (EV), we studied three EV patients infected by HPV5 and one by HPV8, with cancers containing mostly or only episomal viral genomes with a deletion. The mutants were compared with the full-length genomes present in the benign lesions of each patient. Deletions affected the L1 and/or L2 open reading frames (ORFs), and extended in the 5' end of the long control region in two cancers. The isolates studied showed a polymorphism of restriction endonuclease cleavage sites and variations in the nucleotide sequence of the E6 ORF and the regions flanking the deletions. However, except for one patient infected by two distinct HPV5 variants, no difference was observed in the nucleotide sequence of isolates cloned from the benign lesions and the cancer of the same patient. This may suggest that point mutations are not involved in tumor progression. Comparison of nucleotide sequence data revealed an unexpectedly high number of nucleotide substitutions among the four HPV5 variants and the HPV8 variant, as compared with HPV5 and HPV8 published sequences. Changes involved 49 of the 457 nucleotides of HPV5 E6 ORF and 14 of the 465 nucleotides of HPV8 E6 ORF. This corresponds to amino acid substitutions affecting 17 of the 157 amino acids of HPV5 E6 proteins and 7 of the 155 amino acids of HPV8 E6 proteins. Half of the substitutions represent nonconservative changes. The variants showing the highest degree of sequence variation were detected in additional EV patients by PCR. This points to the existence of a set of HPV5 and HPV8 stable variants, encoding for multiple allelic forms of the transforming E6 gene.

A subtype of human papillomavirus 5 (HPV-5b) and its subgenomic segment amplified in a carcinoma: nucleotide sequences and genomic organizations.

A subtype of human papillomavirus 5 (HPV-5b) is closely associated with carcinomas in the disease epidermodysplasia verruciformis (EV). The complete genome was cloned from virus particles in benign lesions of a patient with EV and sequenced: it was 7779 nucleotides long and consisted of six open reading frames (ORFs) (E6, E7, E1, E2, E4, and E5) in the early region, three ORFs (L2, L3, and L1) in the late region, and a noncoding region, all existing on one DNA strand. The 40% segment of the HPV-5b genome specifically amplified in carcinomas was cloned from a primary carcinoma of the same EV patient and sequenced: it was 3143 nucleotides long and corresponded to a segment of the original HPV-5b genome containing the entire sequences of E6, E7, and the noncoding region and portions of E1 and L1. Compared to the whole genomic DNA, no mutations were detected in this probable malignancy-associated viral subgenomic segment cloned from carcinoma. These results suggest that amplification of the viral segment containing E6, E7, and the noncoding region may play a role in the malignant conversion of HPV-5b-infected benign lesions and that mutations in these genes or regions are not necessarily required.

Epidermodysplasia verruciformis-associated papillomavirus infection complicating human immunodeficiency virus disease.

Three males infected with the human immunodeficiency virus (HIV) were noted to have extensive flat warts of the face and/or body. In two there were also pityriasis versicolor-like lesions. Biopsies showed foamy, basophilic, distended cytoplasm in granular layer keratinocytes, characteristic of the human papillomavirus types seen in epidermodysplasia verruciformis. DNA hybridization techniques demonstrated the presence of HPV-type 8 in one patient and HPV 5 and 8 in another. Patients with immune suppression due to HIV infection may demonstrate the clinical features of epidermodysplasia verruciformis with the same potentially oncogenic HPV types.

[Papillomaviruses pathogenic to human and their relationship to oncogenesis]. / Humanpathogene Papillomviren und deren Beziehung zur Onkogenese.

In recent times, modern gene technology has led to a new understanding of the role of the human papillomaviruses in the development of some malignant tumours in humans. As a result, models based on molecular-biological analysis of the interactions between papillomaviruses and tumour cells have been developed. Cancer appears to be the result of inadequate control of the host cell over persisting viral genes. Virus infection is the necessary precondition for malignancy, but it is not sufficient to trigger the growth of a tumour. This can only occur if the cellular control genes are impaired possibly as the result of a mutagenic effect of simultaneous factors such as recurrent infections, UV radiation, X-rays, or chemical carcinogens.

Human papillomaviruses: pediatric perspectives on a family of multifaceted tumorigenic pathogens.

As summarized here human papillomaviruses are associated with a wide spectrum of epithelial lesions, ranging from benign warts to invasive carcinomas. They have been difficult to study in part because they have not yet been propagated in tissue culture. Fortunately advances in molecular biology have allowed characterization of HPV genomes and identification of some HPV gene functions. In addition to their clinical importance HPVs represent an important tool for exploring virus-cell interactions, gene expression, cellular differentiation and cancer. HPV infections are not only common but also difficult to treat and prevent. Depending on the HPV type and location, the modes of HPV transmission may involve casual physical contact, sexual contact and perinatal vertical transmission. HPV DNA genomes replicate at a low copy number in basal cells and, as most clinicians know, are difficult to eradicate. There is often a long latent period and subclinical infections, and HPV DNA can be found in normal tissue adjacent to lesions. HPVs can cause widely disseminated lesions, especially in the immunocompromised host and in epidermodysplasia verruciformis. Aside from the rare carcinomas, the most serious life-threatening HPV-induced illness in children is recurrent respiratory papillomatosis. Somewhat surprisingly in malignant lesions HPV DNA is also found as fragments incorporated into the cellular genome. Unlike retroviruses such as human immunodeficiency virus which integrate into the cellular genome as part of their life cycle, HPV integration is a terminal event for viral replication. Such integration may be critical, however, for viral-induced abnormal cell growth. Perhaps the most important implication of the finding that some anogenital cancers are in part sexually transmitted infectious diseases is that they may be preventable. The data overwhelmingly suggest that avoidance of exposure to HPV via abstinence or monogamy in both partners markedly reduces the risk of cervical cancer. A more realistic goal, however is prevention of HPV transmission by the use of barrier method contraceptives, which may be protective against development of cervical carcinoma. The America Association of Pediatrics Committee on Adolescents has outlined the obligation of pediatricians to be actively involved in adolescent education on sexually transmitted diseases. Certainly a fundamental knowledge of HPV epidemiology, the risks of HPV-related sequelae and prevention of HPV infection are important considerations for adolescent sexuality. Although helpful, such awareness alone falls far short of making an impact on sexual behaviors. A significant reduction in HPV infection rates could be achieved only by inundating adolescents at an early age with a highly visible society-wide campaign directed at these issues.