Anatomy, taxonomy and evolution of papillomaviruses.
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Animals↗
Biomedical subjects
H Pfister
Publications and source records attributed to H Pfister.
Transcription of papillomavirus genomes.
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Animals↗
The predominant mRNA class in HPV16-infected genital neoplasias does not encode the E6 or the E7 protein.
Human papillomavirus (HPV) type 16 is strongly implicated in the development of progressive neoplasias of the uterine cervix. Its oncogenic potential is decisively determined by the activity of the early gene products E6 and E7. To look for changes in the expression of these genes during tumour progression we cloned subgenomic fragments of HPV16 into RNA expression vectors, which allowed the generation of 35S-labelled riboprobes specific for distinct mRNA classes. Four constructs were made to differentiate between transcripts starting upstream of the E6 ORF or the E1 ORF, and one probe was specific for unspliced E6/E7 region transcripts. Five other constructs were used to identify transcripts covering the E1, E2, E4, L1 and L2 regions. With the help of these constructs, we analyzed by in situ hybridization 2 low-grade intraepithelial neoplasias of the vulva, 1 high-grade neoplasia of the cervix as well as 4 vulvar and 3 cervical carcinomas. Transcripts from the E1, E2, E4, L1 and L2 region that were consistently detected in the differentiated layers of benign lesions were variably expressed in precancers and carcinomas. None of the investigated cases revealed detectable amounts of unspliced E6/E7 transcripts with a coding potential for a full-length E6 protein. In benign lesions, the E7 transcripts were confined to isolated nuclei of differentiated cells, whereas high-grade lesions and invasive cancers showed elevated levels of equally distributed E7-specific signals in the cytoplasm of all tumour cells. The most abundant transcripts observed in intraepithelial neoplasias and in invasive cancers appear to initiate within ORF E7 and therefore have no coding potential for full-length E6 and E7 proteins. Our data show that the actual level of E7-specific transcripts in cancers is lower than anticipated from earlier studies using an ORF E6/E7-specific probe that hybridizes with the 5'-ends of the abundant mRNA class.
Base Sequence↗
Specific rearrangements of human papillomavirus DNA provide molecular evidence for genetic heterogeneity of primary cervical cancers, recurrencies, and lymph node metastases in two patients.
Two different characteristic patterns of human papilloma virus (HPV) type 16 DNA were found by Southern blot hybridization in four pelvic and paraaortic lymph node metastases in a patient with FIGO stage IIIb cervical cancer. Both patterns added up to give the HPV 16 DNA pattern of the primary tumor. This strongly suggests that the tumor was composed of two distinct compartments, each spawning its own lymph node metastases. A second patient presented with a vaginal tumor 4 years after stage IIb cervical cancer had been treated with hysterectomy only. The vaginal tumor was removed and pelvic lymphadenectomy performed. Integrated HPV 16 DNA was found in the vaginal tumor whereas one involved and one free lymph node contained episomal HPV 16 DNA with a characteristic deletion. The apparent heterogeneity of the cancer cell population may indicate that the metastasis is not related to the vaginal tumor but that it is a late sequel of the cervical cancer. Alternatively the metastasis could have originated from an unsampled portion of the vaginal tumor.
Adult↗
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.
Base Sequence↗
Determination of the IgG index for the detection of intrathecal immunoglobulin synthesis in dogs using an ELISA.
The IgG index is a calculated quotient using IgG and albumin contents of cerebrospinal fluid (CSF) and serum to detect intrathecal IgG synthesis, which is important in the diagnosis of inflammatory infectious diseases of the central nervous system (CNS). An ELISA to measure IgG and albumin contents in blood and CSF was developed to determine the IgG index. Twenty-three normal dogs and 98 dogs with various neurological diseases were examined. In most dogs with infectious inflammatory diseases, with the exception of the acute form of nervous canine distemper, there was an elevation of the IgG index. Tumours of the CNS had an IgG index within the normal range. An elevation was found in lymphoid tumours or meningiomas with secondary cellular infiltration. Degenerative diseases, spinal cord compression and ischaemic myelopathy due to fibrocartilagenous embolism had normal or only slightly elevated IgG indices. In seven cases with inflammatory lesions there was no obvious CSF pleocytosis but the IgG index was elevated. In 17 cases in which infectious inflammatory disease was suspected, based on elevated CSF cell counts, inflammation could be excluded after determination of the IgG index. The reproducibility of the technique was found to be good and, considering the limited amount of time, expense and effort required, the determination of the IgG index in dogs is a useful extension of the conventional CSF examination.
Animals↗
Cloning and expression in mammalian cells of porcine tumor necrosis factor alpha: examination of biological properties.
We have cloned a full length complementary DNA (cDNA) of the porcine tumor necrosis factor alpha (pTNF-alpha) gene and expressed it in porcine and murine cells. Total RNA obtained from lipopolysaccharide (LPS) stimulated porcine peripheral blood mononuclear cells was reverse transcribed with a specific antisense pTNF-alpha primer to generate a single stranded cDNA which was subsequently amplified by the polymerase chain reaction utilizing an additional pTNF-alpha specific sense primer. The resulting double stranded cDNA was introduced into the pBMGNeo expression vector and transfected by electroporation in porcine (PK(15)) and murine (L929) cell lines. TNF-alpha bioactivity was detected in the supernatant of the transfected cells using a standard L929 bioassay or a PK(15) bioassay. The activity was zinc inducible as expected for a gene controlled by a metallothionein promoter. The bioactivity was not lowered by an anti-mouse TNF-alpha antiserum neutralizing murine, but not human TNF-alpha and a broad immunoreactive band of 17-19 kD was detected using an anti-mouse TNF-alpha serum suitable for immunoblotting. This newly developed tool will allow us to investigate the role of TNF-alpha in pathogenesis of viral infections and gram-negative sepsis.
Animals↗
Evolution of human papillomavirus type 18: an ancient phylogenetic root in Africa and intratype diversity reflect coevolution with human ethnic groups.
Papillomaviruses are an ideal model system for the study of DNA virus evolution. On several levels, phylogenetic trees of papillomaviruses reflect the relationship of their hosts. Papillomaviruses isolated from remotely related vertebrates form major branches. One branch of human papillomaviruses (HPVs) includes an ape and two monkey papillomaviruses, possibly because the diversification of the viruses predated the separation of the infected-primate taxa. This hypothesis predicts that the root of the evolution of some if not all HPV types should point to Africa, since humans evolved from nonhuman primates in this continent. We tested this hypothesis and compared the genomic sequences of HPV type 18 (HPV-18) isolates from four continents. Diversity within HPV-18 correlates with patterns of the evolution and spread of Homo sapiens: HPV-18 variants, just like HPV-16 variants, are specific for the major human races, with maximal diversity in Africa. Outgroup rooting of the HPV-18 tree against HPV-45, which is closely related to HPV-18, identifies African HPV-18 variants at the root of the tree. The identification of an African HPV-45 isolate further reduces the evolutionary distance between HPV-18 and HPV-45. HPV-18 variants from Amazonian Indians are the closest relatives to those from Japanese and Chinese patients and suggest that a single point mutation in the phylogenetically evaluated genomic segment represents at least 12,000 years of evolution. We estimate that diversity within HPV-18 and probably within other HPV types evolved over a period of more than 200,000 years and that diversity between HPV types evolved over several million years.
Africa↗
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.
Cloning, Molecular↗
Human papillomavirus type 13 and pygmy chimpanzee papillomavirus type 1: comparison of the genome organizations.
Human papillomavirus type 13(HPV-13) is associated with oral focal epithelial hyperplasia (FEH) in humans. A recent epidemic of a FEH-like disease in a pygmy chimpanzee (Pan paniscus) colony allowed us to clone a novel papillomavirus genome. To assess the homology between HPV-13 and the pygmy chimpanzee papillomavirus type 1 (PCPV-1), the complete nucleotide sequences of both FEH-related viruses were determined. In both viruses, all eight major open reading frames were located on one strand and the genomic organization was similar to that of other mucosal papillomaviruses. The genomes of PCPV-1 and HPV-13 showed extensive overall sequence homology (85%). They could be classified, using phylogenetic analysis, together with HPV types 6, 11, 43, and 44 in a group associated with benign orogenital lesions. These data indicate that two phylogenetically related papillomaviruses can elicit similar pathology in different primate host species, reflecting viral genomic similarities.
Animals↗
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).
Adult↗
Transcription of the E6 and E7 genes of human papillomavirus type 6 in anogenital condylomata is restricted to undifferentiated cell layers of the epithelium.
The E6 and E7 genes of human genital papillomaviruses (HPVs) appear to transform cells by different mechanisms. They seem to act synergistically but are not equally important when tested under diverse experimental conditions. We were therefore tempted to investigate the E6- and E7-specific transcription pattern in HPV6-infected condylomas separately, by in situ hybridization. Recent studies have identified three promoters within the E6-E7 region of HPV6 and HPV11 by applying S1, exonuclease VII, and cDNA analyses. On the basis of these data, we cloned subgenomic fragments of HPV6 into plasmid pBS to obtain riboprobes that differentiated between transcripts starting upstream of the E6 and E7 open reading frames, respectively. These different species of mRNAs were analyzed in serial thin sections of eight HPV6-positive anogenital condylomas. The E6 probe (nucleotides 7862 to 241) led to weak signals within the basal layer. In three cases, rather strong signals were confined to a few basal cells. The E7 probe (nucleotides 242 to 534) gave rise to a more pronounced labeling of all cells within the two to three lowest epidermal layers. In situ hybridization with a riboprobe for human c-fos revealed an expression pattern similar to that observed with the E7 probe. In contrast to the preferential expression of the transforming E6 and E7 genes in the lower epithelium, the major transcriptional activity of the virus was detected in the middle and upper third by probes colinear with the 3' moiety of the early region.
Adolescent↗
[Condylomata acuminata in children--detection of HPV 6/11 and 2. Local therapy with interferon-beta hydrogel].
Four cases of genital warts in children (girls) are reported. HPV 6/11-DNA was identified in two cases, and HPV 2-DNA in one. In one case no virus identification was possible. The clinical features of the HPV 2-induced genital warts showed the typical morphology of condylomata acuminata. The mode of transmission of the virus, in absence of sexual contact, could not be explained. The HPV 2-associated genital warts might have been transmitted by autoinoculation from warts on the hands. Topical treatment with IFN-beta-hydrogel was applied over 8 weeks, either as single-agent therapy (1 case) or as adjuvant therapy after removal of the condylomata (3 cases). No remission was seen with the single-agent therapy. In one case the genital warts reappeared after adjuvant therapy, but in the other two cases no recurrence was seen.
Administration, Topical↗
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.
Basal Cell Carcinoma↗
Lipopolysaccharide synergizes with tumour necrosis factor-alpha in cytotoxicity assays.
Lipopolysaccharide (LPS) from Escherichia coli was found to synergize with human recombinant tumour necrosis factor-alpha (TNF-alpha) in the lysis of L929 and WEHI 164 (clone 13) murine fibroblasts, two cell lines classically used in TNF-alpha bioassays. The effect was noted with TNF-alpha at low (sublytic or lightly lytic) concentrations and was significant for LPS concentrations in the ng range. The LPS effect could be inhibited by polymyxin B, and was not observed when the TNF-alpha assay was performed in the absence of actinomycin D. Enhancement of TNF-alpha lysis by LPS occurred in several assays for determining TNF-alpha, including MTT cleavage, crystal violet staining and lactate dehydrogenase release. Synergism was obtained only when LPS and TNF-alpha were added to cells simultaneously, but not when applied in sequence. The reported synergism may be relevant for TNF-alpha determinations by bioassay, and for the understanding of pathophysiology of Gram-negative sepsis.
Animals↗
CD36-mediated signal transduction in human monocytes by anti-CD36 antibodies but not by anti-thrombospondin antibodies recognizing cell membrane-bound thrombospondin.
Mononuclear cells (MNC) treated with anti-CD36 Fab or F(ab')2 fragments and then stimulated with anti-rabbit (F(ab')2 displayed an oxidative burst, suggesting that the crosslinking of CD36 promotes signal transduction in the absence of an Fc receptor involvement. Moreover, intact anti-TSP mediates a weak oxidative burst in MNC, which was strongly enhanced upon pretreatment of monocytes (but not lymphocytes) with TSP. This response, however, was mediated by Fc receptors, not by an involvement of CD36. Other means of crosslinking cell-bound TSP and exposure of MNC to surface-bound TSP failed to promote an oxidative burst. Crosscompetition tests confirmed that the interaction site(s) of TSP with monocytes are distinct from the signal-promoting sites recognized by polyclonal and 3 monoclonal anti-CD36 antibodies.
ADP-ribosyl Cyclase↗
Antibody-dependent cellular cytotoxicity in antimyelin antibody-induced oligodendrocyte damage in vitro.
Treatment of dissociated murine brain cell cultures with an antibody recognizing galactocerebroside (GalC) led to degeneration of oligodendrocytes with loss of their cell processes. F(ab')2 fragments prepared from this antibody showed no effect. The anti-GalC antibody--but not its F(ab')2 fragments b2 was able to stimulate macrophages in these cultures as seen in a chemiluminescence assay. Therefore, antibodies bound to oligodendrocytes stimulated nearby macrophages by interacting with their Fc receptors. The oligodendroglial damage coincided with the release of toxic compounds by the stimulated macrophages, since treatment of the cultures with the anti-GalC antibody and a variety of other macrophage stimulating agents led to secretion of reactive oxygen species and--in some experiments--tumor necrosis factor, both known to be toxic for oligodendrocytes. These in vitro experiments show evidence that antibody-dependent cellular cytotoxicity may be an important mechanism of tissue destruction in inflammatory demyelinating diseases.
Animals↗
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.
Base Sequence↗