Unit: Genetics, Paillomavirus and Human Cancer
Director: Michel FAVRE
The identification of susceptibility genes for infectious agents constitutes a major objective of current research programs. We identified two genes (EVER1 and EVER2) for the susceptibility to infection with human papillomavirus (HPV) genotypes specific for epidermodysplasia verruciformis (EV), especially the oncogenic HPV5 genotype. Our on-going research is aimed at the understanding of the function(s) of EVER1 and EVER2 proteins in both normal and HPV5-infected cells.
Certain HPV genotypes are the causative agents of the carcinoma of the uterine cervix (HPV16 and HPV18), the third woman cancer worldwide, and of the skin carcinomas (HPV5) developing in EV, a rare disease. EV is a genetic condition resulting in an abnormal susceptibility to a specific group of HPV (EV HPV), including HPV5 that causes widespread inapparent infections. EV is our model to study both the susceptibility to HPV infection and the genetic mechanisms underlying the malignant transformation associated with oncogenic HPV.
We have previously found that mutations in EVER1 and EVER2 genes are linked to the sensitivity to HPV5 and other EV HPV. EVER proteins may play a role in the permissivity of keratinocytes to EV HPV infection or in the triggering of immune responses responsible for the eradication of EV HPV-induced lesions. Our first objective is to determine the functions of EVER proteins and their role in viral infection. A second objective is to understand the mechanisms involved in the malignant transformation associated with oncogenic HPV5. Our strategy is to analyze the impact of HPV5 infection in keratinocyte cultures obtained from patients or healthy individuals and to identify the cellular targets of EVER proteins and HPV5 E6 and E7 oncoproteins by using a yeast two-hybrid method .
1) EVER protein function(s)
We are currently studying the impact of mutations in EVER proteins on the expression patterns of cytokins and cellular proteins in lymphoblastoid cell lines and keratinocytes obtained from patients and healthy relatives in families with EV. Our strategy is the use of protein arrays.
2) EVER proteins and HPV infection
We have successfully established the conditions to obtain keratinocyte cultures from hair follicles and to induce their terminal differentiation in vitro. We have obtained keratinocyte cultures from patients with EV and from healthy individuals. This will enable the study of the impact of mutations in EVER genes on the first stages of HPV5 infection (entry of HPV5 virus-like particles, transport of the viral genome to the nucleus) and transcription of the viral genome (E1, E2, E6, and E7 genes). The late steps of the viral life cycle (viral DNA replication, virion production) will be studied in differentiating keratinocytes (raft cultures).
We have also shown that EVER1 and EVER2 proteins associate into a complex with ZNT1, a protein involved in zinc homeostasis. We are currently studying the impact of mutations in EVER proteins on this signaling pathway and investigating whether EV HPV E6 and E7 proteins affect this pathway. Our preliminary results indicate that the EVER/ZNT1 complex negatively regulates the Zn2+-activated PI3K/JNK1 signaling pathway, and that HPV5 and HPV9 E6 proteins specifically interact with a MAP kinase of this pathway.
3) Signaling pathways targeted by early proteins of oncogenic HPVs
Our aim is to unravel the strategy used by HPVs to replicate and, for oncogenic HPV types, to induce the malignant transformation of infected cells.
3.1) Interactome of HPV5 E6 and E7 oncoproteins
By using a yeast two-hybrid method, we have shown that HPV5 E6 and E7 proteins interact with proteins of the TGF-α 1 (E6), TNFα (E7), and calcium (E6 and E7) signaling pathways and with the cell cycle regulation (E6 and E7). The observed interactions are studied by cell biology techniques (confocal microscopy, co-immunoprecipitation, "GST pull-down") or molecular biology methods based on tandem purification of protein complexes (TAP-TAG technology) coupled to mass spectrometry analysis (LC-MS/MS technology). Functional analysis of these signaling pathways involves study of expression of a reporter gene (luciferase) driven by promoters specific for these pathways. Our first results revealed an interaction between HPV5 E6 protein and SMAD3, a protein of the TGF-α 1 signaling pathway. TGF-α 1 induces the synthesis of proteins blocking cell transition from G1 phase to S phase. Interestingly, the switching off of the block constitutes a necessary step for viral DNA replication.
3.2) Interaction of the E1, E2, E6 and E7 proteins of skin and genital oncogenic HPV
Our approach is based on a global comprehensive study of the interacting networks between the viral proteins of the different oncogenic or non oncogenic HPV genotypes and cell proteins. This approach requires the cloning and expression of open reading frames (ORFeome) of the different HPV genotypes. By using a two-hybrid method, we started to determine the interactome of the early proteins of eleven cutaneous (HPV1, 3, 5, 8, 9) and mucous (HPV6, 11, 16, 18, 33, 39) HPV genotypes with a low risk (HPV1, 3, 6, 9, 11) or a high risk (HPV5, 8, 16, 18, 33, 39) for the development of skin or genital carcinomas. This current study is performed in collaboration with the group of Marc Vidal (Dana Farber Cancer Institute, Boston, USA)
4) Future prospects
The identification and the characterization of the interactome of EVER proteins and of the early proteins of the different HPV genotypes should lead i) to determine the function(s) of EVER proteins, ii) to identify the physiologic pathways affected by viral proteins and susceptible to be under the control of EVER proteins, and iii) to compare the signaling pathways preferentially or specifically modified by low risk and high risk HPV proteins.
These studies should allow a better understanding of the viral strategies involved in skin and genital carcinogenesis and to define new approaches for the control of these infections that are widespread.
Figure 1 : Search for cellular proteins interacting with HPV5 E6 protein by the two-hybrid mating technique
Keywords: virology, human papillomavirus, genital and cutaneous carcinomas, genetic predisposition, epidermodysplasia verruciformis, EVER1 and EVER2 genes, signaling pathways