Abstract :
[en] Background
To date, more than 225 human papillomaviruses (HPV) genotypes have been fully characterized and these latter are clustered among five genera: α, β, γ, μ, and ν.
In the last 30 years, the very large majority (>90%) of published data focusing on HPV have been interested in α genotypes (most notably HPV16 and 18).
First identified during the 80s and comprising more than 50 strains, β-HPVs have recently been the subject of a growing interest. This is largely related to intriguing evidence showing that some β-HPV genotypes (e.g. HPV8, 38) could promote skin squamous cell carcinoma (SCC) development by potentiating the deleterious effects of ultraviolet (UV) radiation.
Aim
This project aims at deciphering the role of β-HPV infections in non-melanoma skin carcinogenesis and is articulated around two main objectives:
1) To study how E6/E7 from certain β-HPV strains (5, 8, 38, 49) potentiate the mutational effects of UVB irradiation. We hypothesized that viral proteins directly alter DNA damage and repair pathways by interacting with key host proteins, amplifying ultimately the mutational effect of UVB.
2) To determine the proportion of cutaneous preneoplastic lesions and SCC, displaying a transcriptionally active β-HPV infection and identify the different mutational signatures present in these (pre)neoplastic lesions.
Methods
In order to highlight these potential interactions (Aim 1), a high throughput 'Gaussia princeps Protein Complementation Assay' (GPCA) is used to perform a systematic and unbiased screening of direct binary interactions between viral proteins and DNA damage response proteins. To do so, we constituted a library of 180 cDNAs (made from ORFeome 7.1 and 8.1) in plasmid specialized for high throughput screening.
Results
Using GPCA, we highlighted 10 (e.g. RFC2, FANCE, CHEK2, TP53, etc…) potential interactions between E6 from various β genotypes (i.e. 5, 8, 38, 49) and the proteins involved in DNA repair pathways. 7 (e.g. RRM2B, ENDOV, CHEK2, TP53, etc…) potential interactions with the E7 oncoprotein were also determined.
Conclusion
Required for their viral replication, these latter promising results confirm the potential hijacking of DNA repair pathways by cutaneous HPVs. Validations by co-IP are currently ongoing. In parallel, in collaboration with Dr Nicolas Gillet’s group (University of Namur), normal keratinocytes stably transduced with E6 and E7 from various β-HPV were generated. Using these latter cells, we will then determine whether the interactions with E6/E7 alter the half-life, the cellular location and function of DNA repair proteins of interest (highlighted by GPCA).
In parallel, we are currently collecting a large cohort of cutaneous (pre)neoplastic lesions and the second goal of the present project will start in the next few weeks.
