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IRF5.2 binds to the M29 and NRE motifs of HPV8. Gel shift assays were done with purified His-IRF5.2 protein, which was preincubated with poly(dI-dC). In 1–3 the binding activity of IRF5.2 was confirmed by using IRF-BS as an oligonucleotide containing a consensus sequence for IRF proteins. The IRF5.2 binding to M29 (5) was efficiently competed by a 400-fold molar excess of both unlabeled specific competitors M29 (7) and IRF-BS (8), but not by the heterologous oligonucleotide Sp1 (9). In lanes 10–12, it is shown that IRF5.2 also binds to the NRE region of HPV8. Supershift experiments (3, 6, 12) were performed using antibodies directed against IRF5.2
Source publication
Human papillomavirus type 8 (HPV8) poses a high risk for malignant conversion of skin lesions in patients with Epidermodysplasia verruciformis (Ev). Its oncogene promoter P(175) contains the conserved sequence motifs CCAAC, M29, and an A/T-rich region, which are common in many Ev-associated viruses. In human keratinocytes P(175) shows very weak act...
Citations
... Dermal up-regulation of IRF5 was found in macrophage-and fibroblast-like cells and plasma cells. Expression of IRF5 detected in cultured keratinocytes (41) supports our findings in skin specimens. In addition, expression of IRF5 has been detected in B-cells and plasmacytoid dendritic cells (42). ...
Please cite this paper as: Tyrosine kinase 2 and interferon regulatory factor 5 polymorphisms are associated with discoid and subacute cutaneous lupus erythematosus. Experimental Dermatology 2010; 19: 123–131.
Abstract: Lupus erythematosus (LE) is a heterogeneous disease ranging from skin-restricted manifestations to a progressive multisystem disease. The specific skin lesions include chronic cutaneous, subacute cutaneous and acute cutaneous LE. Both genetic and environmental factors are involved in the development of LE. However, reports on the genetic background of cutaneous lupus erythematosus (CLE) forms, namely discoid (DLE) and subacute cutaneous lupus erythematosus (SCLE), are sparse. We investigated whether the known systemic LE (SLE) susceptibility genes also predispose to CLE. Altogether, 219 Finnish patients with DLE or SCLE and 356 healthy controls were recruited. Single nucleotide polymorphisms tagging reported risk genes were genotyped. Tyrosine kinase 2 (TYK2) rs2304256 was associated with increased risk of DLE (P = 0.012, OR = 1.47, 95% CI = 1.01–1.98). Expression of TYK2 was demonstrated by immunohistochemistry in macrophage-like cells and neutrophils and interferon regulatory factor 5 (IRF5) in macrophage- and fibroblast-like cells of DLE, SCLE and SLE skin. IRF5 rs10954213 showed association with DLE (P = 0.017, OR = 1.40, 95% CI = 1.06–1.86) and SCLE (P = 0.022, OR = 1.87, 95% CI = 1.09–3.21). A haplotype of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) showed association with DLE (P = 0.0065, OR = 2.51, 95% CI = 1.25–5.04). Our results show that the TYK2, IRF5 and CTLA4 genes previously associated with SLE also confer risk for DLE and SCLE, suggesting that different LE subphenotypes may share pathogenetic pathways.
Actinic keratoses and cutaneous squamous cell carcinomas are associated with infections with human papillomavirus of genus beta (betaHPV) in immunosuppressed patients. To date, targeted therapy against betaHPV-associated skin cancer does not exist due to the large number of betaHPV without defined high-risk types. Here, we hypothesized that the activation of innate antiviral immunity in skin, asymptomatically infected with betaHPV, induces an anti-tumor response by in situ autovaccination and prevents the formation of betaHPV-associated skin cancer. To test this, we used the preclinical K14-HPV8 transgenic mouse model, which develops skin tumors after mechanical wounding. Remarkably, treatment with antiviral immune response activating poly(I:C) completely prevented cutaneous tumor growth. Induction of the interferon-induced-genes CXCL10 and IFIT1 by poly(I:C) depended on MDA5 activation. Increased numbers of total and activated CD4 and CD8 T cells were detected in poly(I:C) treated skin. T cells were found in the skin of poly(I:C)-treated mice but not in skin tumors of untreated mice. T cell depletion demonstrated a predominant role of CD4 T cells in poly(I:C)-mediated tumor prevention. Our findings identify the MDA5 ligand poly(I:C) as a promising candidate for in situ autovaccination approaches, which might serve as novel treatment strategy against betaHPV-related skin diseases.
The genus β human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patients with epidermodysplasia verruciformis, and evidence for its protumorigenic potential in the general population increases. To date, strategies to suppress genus β HPV infections are limited. Interferon regulatory factors IRF-3 and IRF-7 play key roles in the activation of the innate immune response to viral infections. In this study, we show for the first time that both IRF-3 and IRF-7 regulate transcription of a papillomavirus, but with opposing effects. IRF-7, expressed in the suprabasal layers of human epidermis, increased HPV8 late promoter activity via direct binding to viral DNA. UV-B light-induced activation of the HPV8 promoter involved IRF-7 as a downstream effector. In contrast, IRF-3, expressed in all layers of human epidermis, induced strong HPV8 suppression in primary keratinocytes. IRF-3-mediated suppression prevailed over IRF-7-induced HPV8 transcription. Unlike the E6 oncoprotein of the mucosal high-risk HPV16, the HPV8 E6 protein did not bind to IRF-3 and only weakly antagonized its activity. Strong antiviral activity was also observed, when keratinocytes were treated with potent IRF-3 activators, poly(I:C) or RNA bearing 5' phosphates. In conclusion, we show that IRF-3 activation induces a state of cell-autonomous immunity against HPV in primary human keratinocytes. Our study suggests that local application of IRF-3-activating compounds might constitute an attractive novel therapeutic strategy against HPV8-associated diseases, particularly in epidermodysplasia verruciformis patients.
Infections by human papillomaviruses (HPVs) are the most frequently occurring sexually transmitted diseases. The crucial role of genital oncogenic HPV in cervical carcinoma development is now well established. In contrast, the role of cutaneous HPV in skin cancer development remains a matter of debate. Cutaneous beta-HPV strains show an amazing ubiquity. The fact that a few oncogenic genotypes cause cancers in patients suffering from epidermodysplasia verruciformis is in sharp contrast to the unapparent course of infection in the general population. Our recent investigations revealed that a natural barrier exists in humans, which protects them against infection with these papillomaviruses. A central role in the function of this HPV-specific barrier is played by a complex of the zinc-transporting proteins EVER1, EVER2, and ZnT-1, which maintain cellular zinc homeostasis. Apparently, the deregulation of the cellular zinc balance emerges as an important step in the life cycles not only of cutaneous but also of genital HPVs, although the latter viruses have developed a mechanism by which they can break the barrier and impose a zinc imbalance. Herein, we present a previously unpublished list of the cellular partners of EVER proteins, which points to future directions concerning investigations of the mechanisms of action of the EVER/ZnT-1 complex. We also present a general overview of the pathogenesis of HPV infections, taking into account the latest discoveries regarding the role of cellular zinc homeostasis in the HPV life cycle. We propose a potential model for the mechanism of function of the anti-HPV barrier.
