Unit: Genetics, Paillomavirus and Human Cancer
Director: Michel FAVRE
In our previous studies, two genes (EVER1 and EVER2) were identified which control the susceptibility to infection with the human papillomaviruses specifically associated with epidermodysplasia verruciformis (EV HPVs), in particular the oncogenic HPV5. Our aim is to determine the function of EVER proteins and to define their mechanism of action and their role in the control of EV HPV infections. This should open new perspectives to the study of the susceptibility to oncogenic cutaneous or genital HPVs.
Epidermodysplasia verruciformis (EV) is a rare genodermatosis, with an autosomal recessive transmission, that is characterized by an abnormal susceptibility to related HPVs (EV HPVs) and to the development of skin carcinomas associated with HPV5. We showed that homozygous mutations in EVER1 or EVER2 gene confer the susceptibility to EV. EVER genes encode transmembrane proteins (photo) with unknown function.
Function of EVER proteins
Our first objective was to determine the tissue and cell types expressing EVER genes and the signal (cytokines, chemokines) regulating their expression. Our preliminary data suggest distinct modes of regulation of the expression of EVER1 and EVER2 genes. Indeed, we found a high level of expression of EVER genes in different cell lines derived from the immune system. In contrast, whereas high levels of EVER1 transcripts were detected in different keratinocyte lines, EVER2 transcripts were only detected by a sensitive RT-PCR method.
A second objective was to identify the cellular proteins interacting with EVER proteins, using a two-hybrid method. We constructed a cDNA library obtained from the human keratinocyte HaCaT line in Saccharomyces cerevisiae. Two cellular proteins interacting with the EVER2 protein have been already identified. We are currently studying the biological significance of these interactions.
A third objective is to identify the signaling pathway(s) altered by a mutation in one of either EVER genes, by using the lymphoblastoid cell lines we established from EV patients and healthy relatives.
Role of EVER proteins in the interaction between EV HPVs and keratinocytes
Our aim is to find out whether EVER proteins are involved in the permissivity of keratinocytes to infection with EV HPVs or in the establishment of innate immune responses leading to the eradication of the lesions induced by these viruses. We are currently establishing the conditions for keratinocyte culture and differentiation in vitro in order to analyze the impact of EVER1 or EVER2 mutations (keratinocytes of EV patients) on the first stages of HPV5 infection (virion entry, transport of the viral genome to the nucleus) and on the HPV5 genome transcription (E1, E2, E6, and E7 genes). The late stages of the virus life cycle (viral DNA replication and virion production) will be studied in differentiating keratinocyte raft cultures. Comparative analysis of the transcriptomes of infected keratinocytes from patients or healthy relatives should allow the identification of the genes or signaling pathways differentially expressed.
Cellular proteins and signaling pathways targeted by EV HPVs
Our aim is to identify cellular protein targets of the early proteins of HPV5 that could account for the distinct biological properties of oncogenic (HPV5) and non oncogenic (HPV9) EV HPVs. Our first approach has been to identify the cellular proteins interacting with HPV5 E6 and E7 oncoproteins by the two-hybrid method in yeast. We identified several proteins involved in the TNFα , TGF-α 1 and calcium signaling pathways or in the cell cycle regulation. Our second approach will be to use the TAP-TAG method coupled to mass spectrometry (MALDI-TOF method). We are currently studying the interference of the E6 protein with the TGF-α 1 signaling pathway. This cytokine induces the synthesis of proteins blocking the cellular transition G1-S. The release of this block is a necessary step to allow the viral genome replication.
Further study of these interacting proteins and their involvement in cellular pathways should lead to a better understanding of the cellular mechanisms altered by the viral oncoproteins and, more generally, of the transformation processes induced by oncogenic HPVs.
EVER2 protein (red) is a membrane protein located in the cytoplasm in the endoplasmic reticulum. No EVER2 protein is detected in the cytoplasmic membrane (green).
Keywords: Human papillomavirus, genetic predisposition, epidermodysplasia verruciformis, EVER1 and EVER2 genes, human cancers, signaling pathways, virology