Siamaque Kazem

Seroprevalence of Trichodysplasia Spinulosa-associated Polyomavirus

We identified a new polyomavirus in skin lesions from a patient with trichodysplasia spinulosa (TS). Apart from TS being an extremely rare disease, little is known of its epidemiology. On the basis of knowledge regarding other polyomaviruses, we anticipated that infections with trichodysplasia spinulosa–associated polyomavirus (TSV) occur frequently and become symptomatic only in immunocompromised patients. To investigate this hypothesis, we developed and used a Luminex-based TSV viral protein 1 immunoassay, excluded cross-reactivity with phylogenetically related Merkel cell polyomavirus, and measured TSV seroreactivity. Highest reactivity was found in a TS patient. In 528 healthy persons in the Netherlands, a wide range of seroreactivities was measured and resulted in an overall TSV seroprevalence of 70% (range 10% in small children to 80% in adults). In 80 renal transplant patients, seroprevalence was 89%. Infection with the new TSV polyomavirus is common and occurs primarily at a young age.

Specific betapapillomaviruses associated with squamous cell carcinoma of the skin inhibit UVB-induced apoptosis of primary human keratinocytes.

Epidemiological studies have shown an association between infections by specific betapapillomaviruses, such as human papillomavirus (HPV) types 5 and 8, and cutaneous squamous cell carcinoma (SCC). The role of betapapillomaviruses in the development of cutaneous SCC is, however, still enigmatic. The ability to inhibit UVB-induced apoptosis, as demonstrated for HPV5 in vitro, may be important in this respect, as survival of DNA-damaged and mutated cells increases the risk of transformation. The aim of this study was to assess whether inhibition of UVB-induced apoptosis is a general property of betapapillomaviruses and to identify apoptotic factors that are potentially involved in this process. Primary human keratinocytes transduced with E6 and E7 of selected betapapillomaviruses (HPV5, HPV8, HPV15, HPV20, HPV24 and HPV38) were characterized and subjected to UVB irradiation. HPV8- and HPV20-expressing keratinocytes in particular showed fewer signs of apoptosis, as demonstrated by lower levels of active caspase 3, less enzymic caspase activity and less DNA fragmentation. The observed inhibition of UVB-induced apoptosis was mediated by E6 and coincided with reduced steady-state expression of the pro-apoptotic protein Bax. In conclusion, E6 of HPV8 and HPV20 reduces the apoptotic responses upon UVB irradiation when expressed in primary human keratinocytes. Infections with HPV8 and HPV20 may therefore augment the carcinogenic effect of UV radiation and potentially contribute to oncogenic transformation of the skin.

Trichodysplasia spinulosa is characterized by active polyomavirus infection

BACKGROUND Recently a new polyomavirus was identified in a patient with trichodysplasia spinulosa (TS), a rare follicular skin disease of immunocompromised patients characterized by facial spines and overgrowth of inner root sheath cells. Seroepidemiological studies indicate that TSPyV is ubiquitous and latently infects 70% of the healthy individuals. OBJECTIVE To corroborate the relationship between active TSPyV infection and TS disease by analyzing the presence, load, and precise localization of TSPyV infection in TS patients and in controls. STUDY DESIGN TS lesional and non-lesional skin samples were retrieved from TS patients through a PubMed search. Samples were analyzed for the presence and load of TSPyV DNA with quantitative PCR, and for expression and localization of viral protein with immunofluorescence. Findings obtained in TS patients (n=11) were compared to those obtained in healthy controls (n=249). RESULTS TSPyV DNA detection was significantly associated with disease (P<0.001), with 100% positivity of the lesional and 2% of the control samples. Quantification revealed high TSPyV DNA loads in the lesional samples (∼10(6)copies/cell), and low viral loads in the occasionally TSPyV-positive non-lesional and control samples (<10(2)copies/cell). TSPyV VP1 protein expression was detected only in lesional TS samples, restricted to the nuclei of inner root sheath cells over-expressing trichohyalin. CONCLUSIONS The high prevalence and load of TSPyV DNA only in TS lesions, and the abundant expression of TSPyV protein in the affected hair follicle cells demonstrate a tight relation between TSPyV infection and TS disease, and indicate involvement of active TSPyV infection in TS pathogenesis.

From Stockholm to Malawi: recent developments in studying human polyomaviruses

Until a few years ago the polyomavirus family (Polyomaviridae) included a dozen viruses identified in avian and mammalian hosts. Two of these, the JC and BK-polyomaviruses isolated a long time ago, are known to infect humans and cause severe illness in immunocompromised hosts. Since 2007 an unprecedented number of eight novel polyomaviruses were discovered in humans. Among them are the KI- and WU-polyomaviruses identified in respiratory samples, the Merkel cell polyomavirus found in skin carcinomas and the polyomavirus associated with trichodysplasia spinulosa, a skin disease of transplant patients. Another four novel human polyomaviruses were identified, HPyV6, HPyV7, HPyV9 and the Malawi polyomavirus, so far not associated with any disease. In the same period several novel mammalian polyomaviruses were described. This review summarizes the recent developments in studying the novel human polyomaviruses, and touches upon several aspects of polyomavirus virology, pathogenicity, epidemiology and phylogeny.

The trichodysplasia spinulosa‐associated polyomavirus: virological background and clinical implications

Trichodysplasia spinulosa‐associated polyomavirus (TSPyV) is a new species of the family Polyomaviridae that was discovered in 2010. TSPyV infects humans and is associated with the development of a rare disease called trichodysplasia spinulosa. Trichodysplasia spinulosa is a skin disease of severely immunocompromised hosts characterized by follicular distention and keratotic spine formation especially on the face. Electron microscopy, immunohistochemistry, and viral load measurements indicate an etiological role of active TSPyV infection in the development of this disease. This review will address virological and pathogenic properties of TSPyV, as well as epidemiologic, diagnostic, and therapeutic aspects of TSPyV infection.

Polyomavirus-Associated Trichodysplasia Spinulosa Involves Hyperproliferation, pRB Phosphorylation and Upregulation of p16 and p21

Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS). TS probably results from active infection with the TS-associated polyomavirus (TSPyV), as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67) and cell cycle regulation (p16ink4a, p21waf, pRB, phosphorylated pRB), and the putatively transforming TSPyV early large tumor (LT) antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LT-antigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16ink4a and p21waf. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.

Characterization of T Antigens, Including Middle T and Alternative T, Expressed by the Human Polyomavirus Associated with Trichodysplasia Spinulosa

ABSTRACT The polyomavirus tumor (T) antigens play crucial roles in viral replication, transcription, and cellular transformation. They are encoded by partially overlapping open reading frames (ORFs) located in the early region through alternative mRNA splicing. The T expression pattern of the trichodysplasia spinulosa-associated polyomavirus (TSPyV) has not been established yet, hampering further study of its pathogenic mechanisms and taxonomic relationship. Here, we characterized TSPyV T antigen expression in human cell lines transfected with the TSPyV early region. Sequencing of T antigen-encoded reverse transcription-PCR (RT-PCR) products revealed three splice donor and acceptor sites creating six mRNA splice products that potentially encode the antigens small T (ST), middle T (MT), large T (LT), tiny T, 21kT, and alternative T (ALTO). Except for 21kT, these splice products were also detected in skin of TSPyV-infected patients. At least three splice products were confirmed by Northern blotting, likely encoding LT, MT, ST, 21kT, and ALTO. Protein expression was demonstrated for LT, ALTO, and possibly MT, with LT detected in the nucleus and ALTO in the cytoplasm of transfected cells. Splice site and start codon mutations indicated that ALTO is encoded by the same splice product that encodes LT and uses internal start codons for initiation. The genuineness of ALTO was indicated by the identification of acetylated N-terminal ALTO peptides by mass spectrometry. Summarizing, TSPyV exhibits an expression pattern characterized by both MT and ALTO expression, combining features of rodent and human polyomaviruses. This unique expression pattern provides important leads for further study of polyomavirus-related disease and for an understanding of polyomavirus evolution. IMPORTANCE The human trichodysplasia spinulosa-associated polyomavirus (TSPyV) is distinguished among polyomaviruses for combining productive infection with cell-transforming properties. In the research presented here, we further substantiate this unique position by indicating expression of both middle T antigen (MT) and alternative T antigen (ALTO) in TSPyV. So far, none of the human polyomaviruses was shown to express MT, which is considered the most important viral oncoprotein of rodent polyomaviruses. Coexpression of ALTO and MT, which involves a conserved, recently recognized overlapping ORF subject to positive selection, has not been observed before for any polyomavirus. As a result of our findings, this study provides valuable new insights into polyomavirus T gene use and expression. Obviously, these insights will be instrumental in further study and gaining an understanding of TSPyV pathogenicity. More importantly, however, they provide important leads with regard to the interrelationship, functionality, and evolution of polyomaviruses as a whole, indicating that TSPyV is a suitable model virus to study these entities further.

Human papillomavirus 8 E6 disrupts terminal skin differentiation and prevents pro-Caspase-14 cleavage.

Expression of the betapapillomavirus (betaPV) E6/E7 genes has been shown to impair both keratinocyte differentiation and apoptosis. Especially late-terminal keratinocyte differentiation shares certain aspects with apoptosis, such as fragmentation of DNA and activation of caspases. Here we investigated the disruption of keratinocyte differentiation in organotypic skin (raft) cultures of primary (PHK) and immortalized (N/TERT) human keratinocytes, in particular by human papillomavirus (HPV)8. Immunohistochemical analysis of HPV5 and HPV8 E6/E7-expressing PHK revealed thickening of the rafts and complete absence of stratum corneum formation, even after 18 days of culture. This phenotype was confirmed in N/TERT raft cultures. When expressed separately, the aberrant morphology was observed only in rafts expressing E6, not E7. Immunofluorescence analysis of HPV8 E6 PHK rafts showed an increase in number and size of Filaggrin- and Caspase-14-positive cells in the granular layer. In raft lysates analyzed by western-blot, the presence of pro-Caspase-14 in the differentiated keratinocytes was confirmed, but in the HPV8 E6 rafts none of the Caspase-14 subunits were detected. In conclusion, in the raft system, HPV8 E6 prevented late-terminal keratinocyte differentiation resulting in an accumulation of Filaggrin and pro-Caspase-14-positive cells in the absence of stratification. This differentiation arrest was accompanied by the failure to express Caspase-14 subunits, suggesting absence of Caspase-14 activation and probable abrogation of Filaggrin maturation in HPV8 E6-expressing keratinocytes.

Human Papillomavirus Type 8 E6 Oncoprotein Inhibits Transcription of the PDZ Protein Syntenin-2

ABSTRACT The E6 proteins from high-risk alpha human papillomavirus (HPV) types (e.g., HPV16) are characterized by the presence of a PDZ-binding motif through which they interact with a number of cellular PDZ domain-containing substrates and cooperate in their degradation. The ability of these E6 proteins to bind to PDZ domain proteins correlates with the oncogenic potential of the virus. The E6 proteins of oncogenic HPV from the genus Betapapillomavirus (betaPV, e.g., HPV8) do not encode a PDZ-binding motif. We found that the PDZ domain protein syntenin-2 is transcriptionally downregulated in primary human epidermal keratinocytes (PHEK) by HPV8 E6. The mRNA levels of the known HPV16 E6 PDZ protein targets Dlg, Scribble, Magi-1, Magi-3, PSD95, and Mupp1 were not changed by HPV8 E6. Decreased protein levels of syntenin-2 were observed in cell extracts from PHEK expressing HPV5, -8, -16, -20, and -38 E6 but not in HPV1 and -4 E6-positive keratinocytes. Surprisingly, HPV16 E6 also repressed transcription of syntenin-2 but with a much lower efficiency than HPV8 E6. In healthy human skin, syntenin-2 expression is localized in suprabasal epidermal layers. In organotypic skin cultures, the differentiation-dependent expression of syntenin-2 was absent in HPV8 E6- and E6E7-expressing cells. In basal cell carcinomas of the skin, syntenin-2 was not detectable, whereas in squamous cell carcinomas, expression was located in differentiated areas. Short hairpin RNA-mediated knockdown of syntenin-2 led to an inhibition of differentiation and an increase in the proliferation capacity in PHEK. These results identified syntenin-2 as the first PDZ domain protein controlled by HPV8 and HPV16 at the mRNA level.

Absence of the trichodysplasia spinulosa-associated polyomavirus in human pilomatricomas

ejd.2011.1395 Auteur(s) : Jean Kanitakis1 jean.kanitakis@univ-lyon1.fr, Siamaque Kazem2, Els Van Der Meijden2, Mariet Feltkamp2 1 Department of Dermatology & Dermatopathology Laboratory, Ed. Herriot Hospital Group, 5 Place d’Arsonval, 69437 Lyon, France 2 Department of Medical Microbiology, University Medical Center, Leiden, The Netherlands Although the association of polyomaviruses (PyV) with tumors in animals has been known for decades, the involvement of PyV in human oncogenesis was substantiated [...]