The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) cooperates in cervical carcinogenesis and in epithelial transformation deregulating cell growth, survival and differentiation through the modulation of growth factor receptors. Among the epithelial receptor tyrosine kinases, the keratinocyte growth factor receptor/ fibroblast growth factor receptor 2b (KGFR/FGFR2b) is a major paracrine mediator of epithelial homeostasis and appears to have an unique and unusual role in epithelial tissues, exerting a tumor-suppressive function in vitro and in vivo. With the aim to better elucidate the molecular events involved in the pathological activity of 16E5, we investigated if the viral protein would be able to affect the KGFR expression, signaling and turnover by interference with its degradative and recycling endocytic pathways. Quantitative reverse transcriptase-PCR and biochemical approaches on human keratinocytes transfected with 16E5-HA showed that E5 protein is able to induce KGFR down-modulation at both transcript and protein levels. Immunofluorescence microscopy in double-transfected cells expressing both E5 and KGFR revealed that the viral protein alters the receptor endocytic trafficking and triggers its endosomal sorting to the indirect juxtanuclear recycling pathway. The shift from lysosomal degradation to recycling at the plasma membrane correlates with a reduced phosphorylation of the fibroblast growth factor receptor substrate-2a tyrosine 196, the major docking site for Grb2-Cbl complexes responsible for receptor ubiquitination and degradation. 5 0 -Bromo-deoxyuridine incorporation assay demonstrated that expression of 16E5 induces a decrease in the growth response to the receptor ligands as a consequence of KGFR down-modulation, suggesting that 16E5 might have a role on HPV infection in perturbing the KGFR-mediated physiological behavior of confluent keratinocytes committed to differentiation.

Mus musculus papillomavirus 1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not in common laboratory strains. To facilitate the study of immune control, we sought an outbred and immunocompetent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) with MmuPV1 by scarification on their tail resulted in three clinical outcomes: (i) persistent (>2-month) papillomas (∼20%); (ii) transient papillomas that spontaneously regress, typically within 2 months (∼15%); and (iii) no visible papillomas and viral clearance (∼65%). SKH1 mice with persistent papillomas were treated by using a candidate preventive/therapeutic naked-DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and mE7 early proteins and residues 11 to 200 of the late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and a failure of disease to recur after CD3 T cell depletion. Vaccination induced strong mE6 and mE7 CD8+ T cell responses in all mice, although they were significantly weaker in mice that initially presented with persistent warts than in those that spontaneously cleared their infection. A human papillomavirus 16 (HPV16)-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with an HPV16 pseudovirus. Thus, MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearances of HPV-related disease.IMPORTANCE High-risk-type human papillomaviruses (hrHPVs) cause 5% of all cancer cases worldwide, notably cervical, anogenital, and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Furthermore, an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16, suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.

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Persistent infection by high-risk (HR) human papillomavirus (HPV) types is a prerequisite for progression to cancer. HR-HPVs may lead to a deregulation of innate immunity by interfering with the epithelial type I interferon (IFN) response, whereas very little is known about type III IFNs, a key component of the mucosal antiviral response. This study reports a first attempt to evaluate the activation of type III IFN genes (IFN lambda 1-3), IFN lambda receptor genes (IFN-lambdaR1 and IL10R2), and IFN-induced genes (MxA, ISG15, ISG56) in HPV-positive and HPV-negative cervical cells from 154 women attending the gynecological unit of a university hospital in Rome. Despite an increased individual variability, a coordinated expression of several IFN lambda-related genes was observed. Furthermore, IFN lambda 1 and IFN-lambdaR1 genes were expressed at higher levels in cervical cells positive to low-risk (LR) HPV compared to HR-HPV and HPV-negative cells. Consistently, ISG15 expression was significantly higher in LR-HPV-infected women than in the other groups. Moreover, IFN lambda 1 expression decreased significantly with abnormal cytological results. This study is the first to show the activation of a type III IFN response in LR-HPV-positive cervical cells and suggests that the lack of this response in HR-HPV infection may be related to lesion progression.