Characterization of Novel Cutaneous Human Papillomavirus Genotypes HPV-150 and HPV-151

DNA from two novel HPV genotypes, HPV-150 and HPV-151, isolated from hair follicles of immuno-competent individuals, was fully cloned, sequenced and characterized. The complete genomes of HPV-150 and HPV-151 are 7,436-bp and 7,386-bp in length, respectively. Both contain genes for at least six proteins, namely E6, E7, E1, E2, L2, L1, as well as a non-coding upstream regulatory region located between the L1 and E6 genes: spanning 416-bp in HPV-150 (genomic positions 7,371 to 350) and 322-bp in HPV-151 (genomic positions 7,213 to 148). HPV-150 and HPV-151 are phylogenetically placed within the Betapapillomavirus genus and are most closely related to HPV-96 and HPV-22, respectively. As in other members of this genus, the intergenic E2-L2 region is very short and does not encode for an E5 gene. Both genotypes contain typical zinc binding domains in their E6 and E7 proteins, but HPV-151 lacks the regular pRb-binding core sequence within its E7 protein. In order to assess the tissue predilection and clinical significance of the novel genotypes, quantitative type-specific real-time PCR assays were developed. The 95% detection limits of the HPV-150 and HPV-151 assays were 7.3 copies/reaction (range 5.6 to 11.4) and 3.4 copies/reaction (range 2.5 to 6.0), respectively. Testing of a representative collection of HPV-associated mucosal and cutaneous benign and malignant neoplasms and hair follicles (total of 540 samples) revealed that HPV-150 and HPV-151 are relatively rare genotypes with a cutaneous tropism. Both genotypes were found in sporadic cases of common warts and SCC and BCC of the skin as single or multiple infections usually with low viral loads. HPV-150 can establish persistent infection of hair follicles in immuno-competent individuals. A partial L1 sequence of a putative novel HPV genotype, related to HPV-150, was identified in a squamous cell carcinoma of the skin obtained from a 64-year old immuno-compromised male patient

Dual role of tumor suppressor p53 in regulation of DNA replication and oncogene e6-promoter activity of epidermodysplasia verruciformis-associated human papillomavirus type 8

AbstractHuman papillomavirus 8 (HPV8) is a representative of Epidermodysplasia verruciformis (EV)-associated viruses. Transient assays in the human skin keratinocyte cell line RTS3b have shown that its replication depends in trans on expression of the viral proteins E1 and E2, similarly to other HPVs. Using deletion mutants and cloned subfragments of the noncoding region (NCR) of HPV8 we identified a 65-bp sequence in the 3′ part of the NCR to be necessary and sufficient to support replication in cis. The origin of replication (ori) of HPV8 is composed of the sequence motifs “CCAAC” (nt 57–73) and M29 (nt 84–112), which are highly conserved among the majority of EV HPVs. Analysis of M29 revealed an unconventional binding site of the E2 protein and an overlapping DNA recognition site of the tumor suppressor protein p53. Both these factors competitively bind to M29. In transient replication assays p53 acted as a potent inhibitor of ori activity, most probably in a DNA-binding-dependent fashion. The minimal ori sequences are also functionally critical for the E6 oncogene promoter P175. In contrast to its effect on replication, p53 stimulated promoter activity depending on its interaction with M29. Our observations suggest that p53 is involved in controlling the balance between DNA replication and gene expression of HPV8

Isolation of three novel rat and mouse papillomaviruses and their genomic characterization.

Despite a growing knowledge about the biological diversity of papillomaviruses (PV), only little is known about non-human PV in general and about PV mice models in particular. We cloned and sequenced the complete genomes of two novel PV types from the Norway rat (Rattus norvegicus; RnPV2) and the wood mouse (Apodemus sylvaticus; AsPV1) as well as a novel variant of the recently described MmuPV1 (originally designated as MusPV) from a house mouse (Mus musculus; MmuPV1 variant). In addition, we conducted phylogenetic analyses using a systematically representative set of 79 PV types, including the novel sequences. As inferred from concatenated amino acid sequences of six proteins, MmuPV1 variant and AsPV1 nested within the Beta+Xi-PV super taxon as members of the Pi-PV. RnPV2 is a member of the Iota-PV that has a distant phylogenetic position from Pi-PV. The phylogenetic results support a complex scenario of PV diversification driven by different evolutionary forces including co-divergence with hosts and adaptive radiations to new environments. PV types particularly isolated from mice and rats are the basis for new animal models, which are valuable to study PV induced tumors and new treatment options

Analyse des onkogenen Potentials der frühen Gene E6 und E7 des humanen Papillomvirus Typ 8 durch die Etablierung transgener Mausmodelle.

Das kutane humane Papillomvirus 8 (HPV8) gehört zu den Hoch-Risiko Typen bei Epidermodysplasia verruciformis (EV)-Patienten. Mit Hilfe sensitiver Detektionstechniken konnte dieser EV-HPV auch in gesunder Haut und in nicht-melanozytärem Hautkrebs der Normalbevölkerung gefunden werden, vor allem bei immunsupprimierten Organ Transplantatempfängern. Durch die Etablierung transgener Mäuse mit der gesamten frühen Region (GFR) von HPV8 unter der Kontrolle des humanen Keratin-14 Promotors, welcher die Expression dieser Gene in basalen Hautkeratinozyten aktiviert, konnten wir das onkogene Potential von HPV8 in vivo demonstrieren. Da im HPV8-GFR Mausmodell alle frühen Gene von HPV8 gleichzeitig exprimiert werden, sollten im Rahmen dieser Dissertation die Gene E6 und E7 einzeln im transgenen Mausmodell untersucht werden, um deren Rolle in der Karzinogenese zu charakterisieren. Dabei stellte sich heraus, dass das E6 Protein alleine in der Lage ist singuläre oder multifokale gutartige Tumoren spontan zu induzieren. Diese Tumoren sind durch Papillomatose, Akanthose, Hyperkeratose und unterschiedliche Grade epidermaler Dysplasie gekennzeichnet und entarten in 6% der Fälle zu Plattenepithelkarzinomen, vergleichbar mit den Ergebnissen der HPV8-GFR Mäuse. Im Gegensatz dazu zeigten die HPV8-E7 transgenen Mäuse keinen offensichtlichen Phänotyp. Um synergistische Effekte des UV-Lichtes und der Wundheilung in diesen transgenen Tieren zu evaluieren, wurde die Haut der HPV8 Mäuse mit UVA und UVB bestrahlt oder mit Biopsiestanzen verwundet. Die UVA/B Bestrahlung und die Verwundung induzierten in den HPV8-GFR und -E6 transgenen Mäusen zwei bis drei Wochen nach der Behandlung Papillomatose. Die Bestrahlung mit UVA hatte keine induzierende Wirkung und die Bestrahlung mit UVB alleine erwies sich als schwacher Induktor der Papillomatose. In 40% der untersuchten PEK konnten Mutationen im Codon 61 des H-ras Gens identifiziert werden. Mutationen im p53 Gen wurden weder in benignen noch in malignen Tumoren von HPV8 positiven Mäusen gefunden. Immunhistochemische Färbungen von p53 zeigten gleiche Spiegel von p53 in der Epidermis HPV8 positiver und negativer Mäuse nach UV Bestrahlung. In der murinen Epidermis ist das E6 Protein von HPV8 das Hauptonkogen, notwendig und ausreichend, um eine spontane Tumorentwicklung bis hin zum Karzinom zu induzieren. Eine HPV8 Infektion, die über Dekaden, obgleich auf einem niedrigen Niveau, fortbesteht, könnte in Interaktion mit Prozessen der Wundheilung, eine relevante Ursache von Hautkarzinogenese beim Menschen sein

Evolutionary variation of papillomavirus E2 protein and E2 binding sites

Background: In an effort to identify the evolutionary changes relevant to E2 function, within and between papillomavirus genera, we evaluated the E2 binding sites (E2BS)s inside the long-control-region (LCR), and throughout the genomes. We identified E2BSs in the six largest genera of papillomaviruses: Alpha, Beta, Gamma, Delta, Lambda, and Xi-papillomaviruses (128 genomes), by comparing the sequences with a model consensus we created from known functional E2BSs (HPV16, HPV18, BPV1). We analyzed the sequence conservation and nucleotide content of the 4-nucleotide spacer within E2BSs. We determined that there is a statistically significant difference in GC content of the four-nucleotide E2BS spacer, between Alpha and Delta-papillomaviruses, as compared to each of the other groups. Additionally, we performed multiple alignments of E2 protein sequences using members of each genus in order to identify evolutionary changes within the E2 protein. Results: When a phylogenetic tree was generated from E2 amino acid sequences, it was discovered that the alpha-papillomavirus genera segregates into two distinct subgroups (α1 and α2). When these subgroups were individually analyzed, it was determined that the subgroup α1 consensus E2BS favored a spacer of AAAA, whereas subgroup α2 favored the opposite orientation of the same spacer; TTTT. This observation suggests that these conserved inverted linkers could have functional importance

Demonstration of new subtypes of adenovirus 7 in South Africa, and probing oesophageal carcinoma cell lines for evidence of adenovirus or of other oncogenic viruses

This study was carried out in 2 parts: 1. Genome analysis of human adenovirus species 7; 2. Search for a possible viral aetiology in oesophageal carcinoma. Sixteen laboratory isolates of adenovirus species 7, isolated in South Africa between 1975 and 1986, were characterized by restriction endonuclease analysis of their DNA genomes. Virus was propagated in human embryo fibroblast cells; genomic DNA, extracted and purified from cellular DNA extracts, was analyzed using 9 different restriction enzymes. Results of this study have demonstrated 2 new genome types of adenovirus 7c which have not previously been identified. The 2 novel strains, designated as genome types Ad7c1 and Ad7c2, were shown to differ from prototype Ad7 c according to restriction profiles with EcoRI; 2 new EcoRI sites were demonstrated in Ad7c1 and 1 in Ad7c2. The restriction sites were mapped on the viral genomes (at 3.68kb and 5.32kb from the left terminus) by double enzyme digestions, cloning of viral DNA, and nucleic acid hybridization using a cloned Ad7 probe. Strains resembling the prototype Ad7c and Ad7p (Gomen) genome types were also identified in the 1985 and 1986 Ad7 isolates. In order to investigate the possible role of a viral co-factor in the aetiology of oesophageal carcinoma, 18 probes, derived from potentially oncogenic viruses, were used to screen 3 human oesophageal carcinoma cell lines for the possible presence of integrated viral DNA. One of these, an Ad7 recombinant plasmid probe, was developed by cloning DNA from the transforming region of the Ad7cl strain into the plasmid vector pUC19. Cellular DNA, extracted from the 3 tumor lines HCU18, HCU33 and HCU39, was tested by means of both DNA dot hybridization and Southern blot hybridization for the presence of Epstein-Barr virus, human papillomavirus (types 1, 5, 6, 8, 11, 16, 18), human adenovirus (strains 5, 7, 12, 31) and human T-lymphotropic virus type I DNA. Both assays were demonstrated to be sensitive enough to detect 1 copy of viral DNA per cell. No hybridization between HPV, EBV, HTLV-I or adenovirus DNA probes, and the cellular DNA was detected. These findings indicate that the stable integration of these tumor viruses in host chromosomes did not play a role in the maintenance of the malignant phenotype of the 3 extensively passaged cell lines. Cells of the 3 oesophageal tumor lines were further examined by transmission electron microscopy, but the presence of virus particles in these cells was not observed

Immunohistochemistry in the diagnosis of cutaneous viral and bacterial infections

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Medicina. Fecha de lectura: 14-11-2014Tesis presentada como compendio de publicacione